SMALL CELL LUNG CANCER CELLS: STIMULATION BY MULTIPLE AND INHIBITION BY BROAD SPECTRUM ANTAGONISTS.

A thesis submitted for the degree of Doctor of Philosophy in the University of London

by

TARIQJ. SETHI

July 1993

Growth Regulation Laboratory Department of Cell Biology Imperial Cancer Research Fund University College London London ProQuest Number: 10017254

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Human small cell lung cancer (SCLC) constitutes 25% of lung cancers and follows an aggressive clinical course. SCLC is characterised by the presence of intracytoplasmic neurosecretory granules and by its ability to secrete many hormones and neuropeptides. Only bombesin-like , which include -releasing (GRP), have been shown to act as autocrine growth factors for certain SCLC cell lines. This thesis focused on other neuropeptides and particularly their ability to mediate SCLC growth. The neuropeptides , (CCK), GRP, and at nanomolar concentrations stimulated an increase in the intracellular concentration of calcium ([Ca^+Jj), inositol phosphate hydrolysis, and increased colony formation in semi-solid medium in responsive SCLC cell lines. These results suggest that multiple Ca^+-mobilising neuropeptides stimulate SCLC growth by an extensive network of autocrine and paracrine interactions. With tumour progression these neuropeptides have increased potency. a 29 amino-acid peptide opposes Ca^+ signals and modulates the action of other neuropeptides in various cellular systems. Thus the effect of galanin in SCLC cell lines was investigated. Surprisingly, galanin increased rather than decreased [Ca2+]j, and stimulates the production of inositol phosphates in certain SCLC cell lines. In view of the Ca^+-mobilizing actions of galanin, the effect on SCLC growth was tested. Galanin stimulates clonal growth in SCLC cells, further supporting the proposition that SCLC growth is stimulated by multiple autocrine/paracrine interactions involving Ca^+ mobilizing neuropeptides. This is the first time that galanin is shown to evoke inositol phosphate, Ca^+ mobilisation and growth responses in any cell type. Gastrin, CCK and CCK-related peptides have identical carboxy-terminal amino-acid structure. SCLC cells are shown to express two distinct functional CCK receptor subtypes, CCK& and gastrin/CCKg receptors, both of which increase [Ca2+]j and stimulate clonal growth. The cDNA of the gastrin/CCKg receptor was cloned and sequenced. Northern blot analyses revealed a single transcript of 2.4 kb in SCLC. The levels of RNA expression match directly with the ability of these SCLC cells to respond to gastrin. This is the first time that CCK& and gastrin/CCKg receptors are shown to stimulate growth outside the gastointestinal system. The broad-spectrum antagonists [D-Arg** .D-Phe^, D- Trp^'^.Leu"! and [Arg®,D-Trp^»®,MePhe®] substance P (6 - 11) block bradykinin, CCK, galanin, gastrin, GRP, neurotensin and vasopressin mediated signals and growth in SCLC cell lines, and inhibit SCLC growth in vitro and in vivo. Thus, broad spectrum neuropeptide antagonists constitute potential anticancer agents. CONTENTS

CHAPTER 1 1.1 INTRODUCTION ...... 1 1.2 CELL PROLIFERATION VIA THE G-PROTEIN PATHWAY...... 3 1.2.a G protein-coupled receptors ...... 3 1.2.b G-proteins and phospholipase C ...... 5 1.2.C Inositol Phosphates and Calcium ...... 6 1.2.d Inositol phosphate metabolism and lithium ...... 8 1.3. NEUROPEPTIDES AND CELL GROWTH...... 10 1.3.a Bombesin/Gastrin-releasing peptide ...... 10 1.3.b Bombesin/GRP as a growth factor ...... '...... 12 1.3.C Bombesin/GRP in development ...... 12 1.4. BOMBESIN/GRP SIGNAL TRANSDUCTION...... 13 1.4.a Bombesin/GRP Receptors ...... 13 1.4.b Phosphatidyl inositol turnover, CaZ+ mobilisation and activation of protein kinase C...... 14 1.4.C Arachidonic acid release and prostaglandin synthesis: ...... 16 1.4.d Bombesin induction of the proto-oncogenes c-fos and c-myc ...... 16 1.4.e Neuropeptide stimulation of tyrosine kinase activity...... 17 1.4.f Regulation of cellular responsiveness to bombesin-stimulated ...... mitogenesis ...... 18 1.5. OTHER NEUROPEPTIDE GROWTH FACTORS...... 19 1.5.a Vasopressin ...... 19 1.5.b Bradykinin...... 20 1.5.C Tachykinins...... 21 1 .S.d Vasoactive intestinal peptide ...... 22 1.6 . SIGNAL TRANSDUCTION BY TYROSINE KINASE RECEPTORS...... 23 1.6 .a Structural features of receptor tyrosine kinases...... 23 1.6 .b Receptor activation ...... 24 1.6.C Phosphatidylinositol 3-kinase ...... 25 1.6 .d Phospholipase C-y ...... 26 1.5.e GTPase-activating protein ...... 27 1.6 .f SH2 domains mediate the interaction of signalling molecules to recep to rs ...... 28 1.7. POLYPEPTIDE GROWTH FACTORS AND CANCER...... 28 1.8. LUNG CANCER ...... 3 0 1.8.a INTRODUCTION ...... 30 1.8.b Classification of Lung Cancers...... 31 1.8.C SMALL CELL LUNG CANCER: Clinical features...... 32 1.9. Causative agents ...... 3 5 1.10. GENETIC CHANGES...... 35 1.10.a Hereditary predisposition - lung cancer families ...... 36 1.10.b Chromosomal abnormalities ...... 36 1.11. RECESSIVE ONCOGENES...... 37 1.11.a Retinoblastoma gene ...... 37 1.1 l.b p53 ...... 3 8 1.11.c 3p ...... 3 9 1.1 l.d Other recessive oncogenes...... 40 1.12. DOMINANT ACTING ONCOGENES...... 40 1.12.a myc fam ily ...... 41 1.12.b ras family: K-, H- and N-ras...... 42 1.12.c Other dominant-acting oncogenes ...... 43 1.12.C i. c-Raf-] ...... 43 1.12.c ii. c-erbB-1 ...... 44 1.12.C Hi. Others: c-myb, c-fms...... 44 1.13. Current status of therapeutic approaches to SCLC- ...... 45 1.13.a Maintenance therapy ...... 45 1.13.b Dose intensity...... 46 1.13.c Alternating chemotherapy ...... 46 1.13.d Biological response modifiers ...... 46 1.13.e Thoracic radiation therapy ...... 47 1.13.f Elective cranial irradiation ...... 47 1.13.g Relapse ...... 47 1.14. Drug resistance mechanisms ...... 48 1.15. GROWTH FACTORS FOR LUNG CANCER...... 49 1.16. Polypeptide hormones ...... 50 1.16.a Epidermal growth factor ...... 50 1.16.b -like growth factor-1 (IGF-I)...... 51 1.16.C Platelet derived growth factor ...... 52 1.16.d C’ k it ...... 3 2 1.16.e Hepatocyte growth factor/Scatter factor ...... 52 1.16.f Transferrin...... 53 1.17. NEUROPEPTIDE GROWTH FACTORS AND SMALL CELL LUNG CANCER...... 53 1.18. Gastrin-releasing peptide ...... 55 1.19. Vasopressin ...... 3 7 1.20. Bradykinin...... 58 1.21. Galanin...... 3 8 1.22. Cholecystokinin...... 59 1.23. Gastrin...... 60 1.24. Neurotensin...... 61 1.25. Other peptides ...... 61 1.25.a Haemopoietic growth factors...... 61 1.25.b Opiods ...... 62 1.25.C Tachykinins ...... 63 1.25.d Vasoactive intestinal peptide ...... 63 1.25.e ...... 63 1.26. BLOCKING GROWTH FACTOR ACTION...... 64 1.27. Specific bombesin antagonists ...... 67 1.28. Testing bombesin antagonists in SCLC ...... 68 1.29. PLAN OF STUDY ...... 68

CHAPTER 2 MATERIALS AND METHODS ...... 70 SCLC-cell lines ...... 70 Cell culture ...... 71 Determination of intracellular Ca^+ concentration:...... 71 Measurement of membrane potential...... 72 Accumulation of inositol phosphates ...... 72 Separation of inositol phosphate by FPLC ...... 73 Liquid Growth A ssay: ...... 73 Clonogenic assay ...... 74 Amplification cloning and sequencing of the human CCKg/gastrin receptor from brain and SCLC: ...... 74 Cloning of the human CCKg/gastrin receptor from human fetal brain library ...... 74 Cloning of the CCKg/gastrin receptor from SCLC cell lines H510 and H 345...... 75 Northern Blot Analysis of CCKg/gastrin receptor mRNA: ...... 76 Materials ...... 76

CHAPTER 3 MULTIPLE NEUROPEPTIDES STIMULATE CLONAL GROWTH OF SMALL CELL LUNG CANCER: EFFECTS OF BRADYKININ, VASOPRESSIN CHOLECYSTOKININ, GALANIN AND NEUROTENSIN...... 78 Mobilization of intracellular Ca2+ in SCLC by multiple neuropeptides...... 78 Multiple neuropeptides stimulate accumulation of inositol phosphates ...... 82 Multiple neuropeptides stimulate clonal growth of SCLC ...... 82 Summary and Discussion...... 89 CHAPTER 4 GALANIN STIMULATES Ca2+ MOBILISATION, INOSITOL PHOSPHATE ACCUMULATION AND CLONAL GROWTH IN SMALL CELL LUNG CANCER CELLS...... SO Galanin increases [Ca2+]j in SCLC...... 92 Effect of EGTA, pertussis toxin and phorbol 12,13-dibutyrate ...... 93 Dissociation of Ca2+ mobilizing effects of galanin from changes in membrane p o ten tial ...... 9 6 Galanin stimulates accumulation of inositol phosphates ...... 96 Galanin stimulates clonal growth in SCLC ...... 99 Summary and Discussion...... 102

CHAPTER 5 Gastrin stimulates Ca2+ mobilization and clonal growth in smali celi lung cancer ceiis ...... 104 Gastrin stimulates Ca2+ mobilization...... 104 Effect of various CCK/Gastrin Antagonists...... 109 Gastrin stimulates clonal growth in SCLC ...... 112 Summary and Discussion ...... 119

CHAPTER 6 MOLECULAR CLONING OF THE CCKg/GASTRIN RECEPTOR ...... 120 Cloning of the human CCKg/gastrin receptor: ...... 120 Expression of the CCKg/gastrin receptor in SCLC: ...... 123 Summary and Discussion...... 127

CHAPTER 7 CCKa RECEPTORS ARE EXPRESSED IN SMALL CELL LUNG CANCER CELLS AND MEDIATE Ca2+ MOBILISATION AND CLONAL GROWTH ...... 129 CCK and Gastrin Ca^+ mobilisation in H510 and GLCl 9 SCLC cell lines ...... 129 CCKa and CCKg/gastrin Receptor Subtypes Stimulate Clonal Growth in SCLC ...... 133 Summary and Discussion...... 136

CHAPTER 8 INTERRUPTION OF AUTOCRINE AND PARACRINE GROWTH STIMULATION BY NEUROPEPTIDE ANTAGONISTS...... 138 Introduction...... 138 Broad spectrum Vs specific neuropeptide antagonists as inhibitors of growth in SCLC ...... 138 R esults...... 140 Summary and Discussion ...... 145

CHAPTER 9 INCREASED RANGE AND POTENCY OF NEUROPEPTIDES ABLE TO RAISE [Ca2+ ]; AND STIMULATE CLONAL GROWTH DURING PROGRESSION OF SCLC...... 147 Summary and Discussion...... 156

CHAPTER 10 GENERAL DISCUSSION ...... 158 Summary and Discussion...... 158 Ca2+ mobilization in SCLC cell lines ...... 158 Multiple neuropeptides stimulate clonal growth in SCLC cells ...... 159 Cholecystokinin receptors in SCLC...... 160 Blocking the action of multiple neuropeptides: broad spectrum antagonists ...... 162 Broad spectrum antagonists block SCLC growth ...... 163 Neuropeptides and progression of SCLC ...... 164 Conclusions...... 165 Future Prospectives...... 166

REFERENCES...... 169 PUBLICATIONS UST OF TABLES.

Table 1.1 Amino-acid sequence of bombesin like peptides ...... 11 Table 1.2 CYTOTOXIC DRUGS FOR SCLC...... 45 Table 1.3 PEPTIDES AND HORMONES SECRETED BY SCLC...... 5 4 Table 1.4. The effect of multiple peptide hormones and neuropeptides on Ca^tnobilization in SCLC cell lines ...... 5 5 Table 1.5 Broad spectrum and specific antagonists of bombesin/GRP...... 65 Table 1.6 Processes blocked by [DArg^DPhe^.DTrp^'^.Leu^ ^ ] substance P in Swiss 3T3 cells...... 67 Table 2.1 Characteristics of SCLC cell lines ...... 70 Table 3.1: Multiple Ca^+ -mobilizing neuropeptides stimulate clonal growth of SCLC cell lines ...... 86 Table 4.1 : Effect of EGTA, pertussis toxin and membrane depolarization on the increase in [Ca^Jj induced by galanin...... 5 3 Table B.liThe effect of multiple peptide hormones and neuropeptides on Ca^tnobilization in the SCLC cell line WX322 ...... 142 Table 9.1 Ca^+ moblising neuropeptides in GLC SCLC cell lines ...... 149 Table 9.2 A[Ca^+]| in response to peptides in GLC 16 and 19 SCLC cell lines 150 Table 9.3: % increase in colony fomation in response to peptides in GLC 16 and GLC 19 SCLC cell lines ...... 152 LIST OF FIGURES

Figure 1.1 Summary of the two major receptor mediated pathways ...... 2 Figure 1.2: Activation of phospholipase C p through G protein-linked receptors ...... 6 Figure 1..3: Summary of the known and suspected routes of metabolism of compounds containing inositol and phosphate...... 9 Figure 1 ..4 : Bombesin-mediated signal transduction ...... 15 Figure 1.5 Structural features of receptor tyrosine kinases...... 24 Figure 3.1 : Dose-dependent effects of bradykinin on [Ca^-Jj in SCLC cells ...... 79 Figure 3.2: Effect of neurotensin, cholecystokinin and vasopressin on [Ca^+jj in H69, H510 and H345 SCLC cell lines ...... 80 Figure 3.3 : Changes in the level of total inositol phosphates in bradykinin stimulated H69, vasopressin stimulated H510 and cholecystokinin stimulated H510 SCLC cells as a function of time...... 81 Figure 3.4: Dose-dependent effects of bradykinin on colony formation in SCLC cells...... B3 Figure 3.5: Effect of the bradykinin antagonist [DArgO, Hyp^, Thi^*®, DPhe^] bradykinin on bradykinin induced Ca^+ mobilization and colony formation in SCLC cells H69 and H345 ...... 83 Figure 3.6: Effect of neurotensin, cholecystokinin and vasopressin on colony formation in H69, H510 and H345 SCLC cell lines ...... 87 Figure 3.7: Effect of mixtures of neuropeptides on colony formation in H69 and H510 SCLC cell lines...... 88 Figure 4.1 : Effect of galanin on [Ca^li in SCLC cells ...... 91 Figure 4.2: Dose-dependent effect of galanin on [C a ^ in SCLC cells...... 92 Figure 4.3: Effect of EGTA, pertussis toxin and PBt 2 on galanin induced Ca^+ mobilization in SCLC cell lines H69 and HSIO ...... 94 Figure 4.4: Effect of galanin on membrane potential and [Ca^Jj in SCLC cells H69 and HSIO ...... 95 Figure 4.5: Effect of galanin on the accumulation of inositol phosphates...... 97 Figure 4.6: Upper: Changes in the level of InsPi, InsPz and InsPg in galanin stimulated H69 and H510 SCLC cells as a function of time ...... 98 Lower: Elution profile of lns(l,4 ,5)P3 andlns(l, 3,4 )P3 in H69 cells stimulated by galanin ...... 98 Figure 4.7: Effect of galanin on colony formation in H69 and H510 SCLC cells...... 108 Figure 4.8: Top: Effect of antagonist [Arg®, D-Trp^»®, MePhe®] substance P on galanin-stimulated Ca^+ mobilization in H69 SCLC cells ...... 101 Bottom: Effect of [Arg^, D-Trp^*^, MePhe®] substance P on galanin induced colony formation ...... 101 Figure 5.1 : Effect of gastrin-l and CCK -8 on [Ca^+ ]j in comparison to other neuropeptides in various SCLC cell lines...... 106 Figure 5.2: Effect of sequential additions of gastrin-l and/or CCK -8 on Ca^+ mobilisation in the HSIO cell line ...... 107 Figure 5.3: Dose dependent effect of gastrin-l, gastrin-ll, CCK -8 and des(S 03)CCK-8 on [Ca2+]j...... 108 Figure 5.4: Effect of gastrin II and CCK -8 and their antagonists and benzotript on [Ca^+Jj in the H510 SCLC cell line ...... 1 10 Figure 5.5: Effect of antagonist L365,260 on gastrin induced Ca^"*mobilization in SCLC cell line H510...... I l l Figure 5.6: Effect of gastrin-l and CCK -8 on colony formation in H510 SCLC cells...... 113 Figure 5.7: Dose-dependent stimulation of gastrin-l on colony number and size in H510 SCLC cells...... 114 Figure 5.8: Gastrin-l, gastrin-ll, CCK -8 and des-(S 03)CCK-8 stimulate colony formation in H510 SCLC cells...... 115 Figure 5.9: Effect of CCK 10-20 on [Ca^+Jj and colony formation in H510 SCLC cells...... 116 Figure 5.10: Effect of L365,260 on gastrin-ll and CCK -8 induced increase in colony formation in H510 SCLC cells...... 117 Figure 5.11: Effect of broad spectrum antagonists on gastrin induced Ca^+ mobilization and colony formation ...... 118 Figure 6 .1 : Human CCKB/gastrin receptor sequence ...... 122 Figure 6.2: Upper panel: Northern blot analyses of different SCLC cell lines Lower panel: Effect of 1 OOnM gastrin on [Ca^+Jj in SCLC cells ...... 124 Figure 6.3: Deduced amino acid sequence of the third cytoplasmic domain of SCLC CCKB/gastrin receptor cDNA ...... 127 Figure 7.1 : Upper panel: Effect of sequential additions of gastrin and CCK -8 on [Ca2+]i in SCLC cells lines H510 and GLC 19. Lower panel: Dose-dependent increase in [Ca^+jj induced by gastrin and CCK-8 in H510 and GLC 19 SCLC cell lines ...... 130 Figure 7.2: Effect of CCKg/gastrin and CCK^receptor antagonists on the increasein [Ca^+ ], induced CCK-8 in the H510 and GLC 19 SCLC cell lines...... 131 Figure 7.3: Upper panel: Effect of sequential additions of gastrin and CCK -8 on [Ca2+]j in the SCLC cell line GLC 28 ...... 132 Lower panel: Effect of CCKg/gastrin and CCKAreceptor antagonists on CCK-8 and gastrin mediated Ca^tnobilisation in the GLC 28 SCLC cell line...... 132 Figure 7.4: Effect of gastrin and CCK -8 on colony formation in GLC 19 SCLC cells...... 134 Figure 7.5: Effect of CCK-8 in the presence or absence of CCK antagonists on colony formation in HSIO and GLC 19 SCLC cell lines ...... 135 Figure 8.1 : Dose-dependent effects of [Leu^Dsi-(CH 2NH)Leu^^ ] bombesin on colony number and size" in H345"SCLC cells...... 139 Figure 8.2: Effects ligand specific and broad-spectrum antagonists on basal, bradykinin, vasopressin and GRP stimulated colony formation in H345 SCLC cells...... 141 Figure 8.3: Effect of agonists and broad spectrum neuropeptide antagonists on [Ca2+]j and growth in the SCLC cell line WX322 ...... 143 Figure 8.4: Effect of agonists and broad spectrum neuropeptide antagonists on [Ca^+]j and growth in the SCLC cell line H69 ...... 144 Figure 9.1 : Panel A: Effects of bombesin, , bradykinin, cholecystokinin and serum on [Ca^+]i in SCLC cells lines GLC 14, GLC 16 and GLC 1 9 ...... 148 Panel B: Dose dependent effects of bombesin, neuromedin B, bradykinin and cholecystokinin on [Ca^+ ]j in SCLC cell lines GLC 16 and GLC 19 ...... 148 Figure 9.2: Dose dependent effects of bombesin, bradykinin,cholecystokinin and neuromedin B on colony formation in GLC 16 and 19 SCLC cells ...... 151 Figure 9.3: Panel A Effect of [Arg®, D-Trp^*^ Me Phe®] substance P (6-11) and [D-Arg\ D-Phe®, Trp^«®, Leu**^ ] substance P antagonists on the increase in [Ca^+Jj induced by bombesin, neuromedin B, bradykinin and cholecystokinin in GLC 19 SCLC cell line ...... 154 Panel B: Effect of [Arg®, D-Trp^*® Me Phe®] substance P (6-11) and [D-Arg\ D-Phe®, Trp^ ®, Leu^^ ] substance P antagonists on growth in liquid culture of GLC 19 and GLC 16 SCLC cell lines ...... 154 Panel C Effect of [Arg®, D-Trp^*® Me Phe^] substance P (6-11) and [D-Arg\ D-Phe®, Trp^*®, Leu^^ ] substance P antagonists on colony growth in agarose semi-solid medium of GLC 19 and GLC 16 SCLC cell lines...... 154 Figure 10.1 :Bombesin-mediated mitogenesis can be blocked at receptor and post-receptor level ...... 168 CHAPTER 1

1.1 INTRODUCTION

The cells of many tissues and organs are maintained in a non-proliferating state (the G q /G i phase of the cell cycle), but can be stimulated to resume DNA synthesis and cell division in response to external stimuli. The regulation of normal cell proliferation is central to many physiological processes, including embryogenesis, growth and development, haemopoeisis, tissue repair and immune responses. Cell proliferation is controlled by positive and negative diffusible factors, as well as cellular interactions with the extracellular matrix proteins. Cancer cells are characterized by unrestrained proliferation, and acquire complete or partial independence from mitogenic control signals in the extracellular environment through different mechanisms (Cross and Dexter 1991; Westermark and Heldin 1991). These include production of growth factors that act on the same cells that produced them (autocrine) or on adjacent cells (paracrine). This hypothesis is supported by the observation that cancer cells require fewer exogenous growth factors than normal cells in serum free medium. Another mechanism by which cancer cells may escape normal regulatory controls is by alterations in the number or structure of cellular receptors and changes in the activity of post-receptor signalling pathways that either stimulate or suppress cell growth (Sager 1989; Bishop 1991). This is supported by the discovery of increasing numbers of oncogenes coding for growth factors, their receptors and post-receptor signalling molecules. Hence, the identification of the extracellular factors and the molecular mechanisms which modulate cell proliferation is crucial to understanding cancer biology and identification of novel therapeutic strategies. Early studies on growth factors were hindered by the use of heterogeneous cell populations and their dependence on serum for maintenance in culture. The development of clonal cell lines, such as the murine Swiss 3T3 fibroblast, greatly facilitated the study of growth factors. These cells are cultured as an adherent cell monolayer and become quiescent in the G i /G q phase of the cell cycle, when they deplete the serum of it's growth promoting activity. The cells remain viable and addition of fresh serum or chemically defined serum free medium containing pharmacological agents or growth factors leads to the reinitiation of DNA synthesis and cell division. Figure 1.1

G PROTEIN-LINKED RECEPTORS

Acetylcholine, histamine NA. 5-HT, ATP. PAF. TXAg. S e c o n d Glutamate. tt. Vasopressin. Bradykinin. Substance P. Bombesin, . Thrombin Cholecystokinin. Neuromedin. TRH. GnRH. PTH Odorants. Light InsP^R

TYROSINE KINASE-LINKED RECEPTORS

Cellutar PDGF. EOF activity

mitogenesis A ntigen I PI-3K I ------► PIP

ra t - 1 M A P -2 k in a s e

Figure 1.1 Summary of the two major receptor mediated pathways.

Many agonists bind to 7-membrane spanning receptors (R) to activate phospholipase C-^l

(PLC-pi), whereas PLC-yl is stimulated by the tyrosine kinase-linked receptors, both cause the formation of inositol trisphosphate (InsP^) and diacylglycerol (DAG). Other effectors activated by tyrosine kinase receptors include phosphatidylinositol 3-OH kinase (PI-3K) which generates the putative lipid messenger phosphatidyl-inositol(3,4,5)-trisphosphate (PIP 3) and ras. GTPase-activating protein

(GAP); InsPgR, InsPg receptor; PKC, protein kinase C; NA, noradrenaline; 5-HT, 5-hydroxytryptamine;

PAF, platelet activating factor; TXA 2 , thromboxane A 2 ; TRH, thyrotrophin-releasing hormone; GnRH, gonadotrophin-releasing hormone; PTH, . ______

This cell system has proved useful in identifying growth factors and signal transduction pathways (Rozengurt 1985). An important feature of mitogenic signalling that has emerged from these studies is that cell proliferation can be stimulated through multiple, independent signal-transduction pathways which act synergistically (Rozengurt 1986). At least two major signal transduction pathways initiate cascades of molecular events leading to proliferation of cultured cells: one involves polypeptide growth factors that^ind to receptors with intrinsic tyrosine kinase activity whiTe thë~otTîër involves receptors coupled to guanine nucleotide binding regulatory proteins (G- proteins). (Fig 1.1)

1.2 CELL PROLIFERATION VIA THE G-PROTEIN PATHWAY

In recent years an increasing number of small regulatory peptides or neuropeptides have been discovered in the neural and neuroendocrine cells of the gastrointestinal tract and central nervous system (Walsh 1987). In the central and peripheral nervous system they are synthesised and stored in pre^naptic neurones and act as fast acting , while in peripheral neuroendocrine cells they act both systemically as hormones circulating through the blood stream and locally in a paracrine or autocrine fashion. Multiple peptides are found in both neuronal and endocrine cells, and can modulate each other's effects, e.g. bombesin/gastrin-releasing peptide (GRP), stimulates the release of other biologically active peptides and galanin inhibits GRP induced insulin release from the pancreas. The role of these peptides as fast-acting neuro-humoral signallers has recently been expanded by the discovery that they also stimulate cell proliferation (Zachary, Woll et al. 1987; Rozengurt 1991a). In addition, these neuropepetides are increasingly implicated as growth factors in human cancer (reviewed in section 1.3) Consequently, it is very important to understand in detail the receptors and signal transduction pathways that mediate the mitogenic action of neuropeptides because they may provide novel targets for therapeutic intervention. Swiss 3T3 cells have been used as a model system to identify the molecular pathways by which neuropeptides elicit mitogenesis. The list of neuropeptides that can act as mitogens in these cells has now grown considerably and includes bombesin, bradykinin, endothelin and vasopressin. Some general points about G-protein linked signal transduction will first be considered, before examininging the evidence linking neuropeptides with growth and malignancy. The early cellular and molecular responses elicited particularly by bombesin, have been studied in detail in Swiss 3T3 cells and will also be reviewed.

1.2.8 G protein-coupled receptors. The molecular cloning of over 75 of these receptors has revealed a superfamily of G-protein coupled receptors (GPRs) (reviewed in (Probst, Snyder et al. 1992)). All of the proteins are single polypeptide chains. The shortest sequence represents the rat mas oncogene (324 amino acids) and the longest sequence represents the human thyroid-stimulating hormone receptor (744 amino acids). The predicted protein structures contain seven stretches of 20-30 hydrophobic amino acids which are believed to form membrane spanning a-helices. These helices are referred to as transmembrane domains 1-7 (TM1-7). The proteins have extracellular amino termini and cytoplasmic carboxyl termini. The areas of greatest homology among the GPRs are in the seven transmembrane regions. Proline residues in TM 4, 5, 6 , and 7 introduce kinks in the a-helices and may be important in the formation of the binding pocket. Overall there is little sequence homology among the receptors in the first extracellular domain. However, GPRs have single conserved cysteine residues in each of the first two extracellular loops that are believed to form a disulphide bond that stabilises the functional protein. The amino termini of these proteins vary greatly in length. They range from as few as 7 residues in the adenosine A 2 receptor to over 300 residues for the glycoprotein hormone receptors. The amino termini of nearly all the GPRs contain consensus sequences for /V-glycosylation. Glycosylation may contribute to the proper expression of the receptor. Phosphorylation and palmitoylation of the carboxy-terminal sites can influence th e signal transduction of som e GPRs. Most GPRs contain potential phosphorylation sites in the third cytoplasmic loop and or carboxyl terminus. For several receptors, phosphorylation by protein kinase A (PKA) and specific receptor kinases mediates receptor desensitisation. The palmitate on carboxy-terminal cysteine(s) would be expected to insert into the membrane, thereby forming an additional cytoplasmic loop which may influence receptor mobility and optimally position the carboxyl terminal residues for G-protein coupling. The transmembrane domains are necessary for ligand binding and confer ligand specificity, while the extracellular and intracellular domains are not directly involved in ligand binding. The ligands for the glycoprotein hormone receptors, thyroid -stimulating hormone (TSH) and follicle-stimulating hormone (FSH), and the lutinizing hormone/chorionic gonadotrophin (LH/CG), are much larger than the ligands for the other GPRs. Presumably because of the large size of the ligands, this receptor subclass has evolved a distinct structure containing an extremely long first cytoplasmic domain encompassing the high-affinity hormone binding site. The large amino terminal extracellular domain of these receptors may be involved in high affinity extracellular binding sites which serve to capture the hormone and present it to the intramembranous binding pocket for signal transduction. All of the intracellular domains are implicated in efficient G-protein coupling. However, amino acids which are proximal to the membrane in third cytoplasmic loop and possibly the carboxyl terminus appear particularly critical in determining the specificity of G-protein coupling for many receptors. The most highly conserved intracellular sequence is the aspartate-arginine-tyrosine triplet adjacent to TM 3. The arginine is invariant, and the aspartate and tyrosine are conservatively replaced in several GPRs. Mutation in this region of the adrenergic-1 p receptor resulted in high affinity ligand binding but reduced or absent G-protein coupling (Dixon, Sigal et al. 1988). Phosphorylation of cytoplasmic residues has been identified as an important mechanism for the regulation of G-protein coupling of some GPRs. The third cytoplasmic loop and the carboxyl terminus are rich in serine and threonine residues that are potential phosphorylation sites. Many GPRs have consensus protein kinase C (PKC) and PKA phosphorylation sites (reviewed in (Probst, Snyder e t al. 1992)).

1.2.b G-proteins and phospholipase C. G proteins constitute a large family of highly homologous proteins. The proteins are heterotrimers consisting of a-, p- and y-subunits. The a-subunit appears to be the most diverse and has traditionally been used to define the purified heterotrimeric proteins. The a-subunit can often account for the primary activity of the G protein. Thus GjO stimulates adenylate cyclase and transducin a (Gta) activates a cyclic GMP-dependent phosphodiesterase. The Gj proteins were identified as inhibitory regulators of adenylate cyclase. More recently, the Gj and Go proteins have been implicated in the regulation of several ion channels. The Gt, Gj and Go proteins are substrates for pertussis toxin induced ADP-ribosylation; attenuation of hormone action by this toxin implicates the participation of one or more of these proteins. In the basal state the a-subunit contains bound GDP, and association of a- and py- subunits is highly favoured. Stimulation of the G protein results when it binds GTP rather than GDP. Receptors interact most efficiently with the heterotrimeric form of the G protein and accelerate activation by increasing the rate of dissociation of GDP and potentially enhancing association of GTP. When activated, the affinity between the a and py-subunits of the G protein is decreased.This increases the likelihood of dissociation of subunits and the generation of two potential pathways (a(GTP) and free py-subunits) for downstream regulation. Finally, the G protein a-subunit has an intrinsic hydrolytic activity that slowly converts GTP to GDP and returns the G protein to it's inactive form. A mutation has been characterised that interrupts this GTP-driven cycie in the a-chain of Gs, the G-protein that stimulates adenyl cyclase. The mutation inhibits GTPase activity and results in a constitutively activated adenyl cyclase mediated adenosine monophosphate production. These mutations are commonly found in growth hormone secreting pituitary adenomas (Landis, Masters et al. 1989). Figure 1.2

Agonist

DAG

OH

ON GTP

GTP GDP

Figure 1.2: Activation of phospholipase C P through G protein-linked receptors. The heterotrimeric G proteins dissociate into Gg and Gp^ subunits both of which can activate different PLC isoenzymes. The figure shows the phospholipase C (PLC) catalysed hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP 2) in the plasma membrane. This reaction produces inositol 1,4,5-trisphosphate (InsPg), and also generates 1,2-diacylglycerol (DAG). ______

The PLC-p subfamily of PLC has been shown to be regulated by G proteins (reviewed in (Sternweis and Smrcka 1992)). The participation of G-proteins in the regulation of this activity was initially suggested by specific requirements for guanine nucleotides for stimulation by hormones and the more direct effects of non- hydrolysable guanine nucleotides and aluminium fluoride (AIF'4, a universal activator of the heterotrimeric G proteins). An important development in delineating this signal transduction pathway has been the purification and molecular cloning of novel heterotrimeric G proteins of the Goq subfamily. When incorporated into phospholipid vesicles together with PLC-pi, the guanine nucleotide regulation of this PLC isoform was reconstituted (reviewed in (Sternweis and Smrcka 1992)). This suggests that Goq activates PLC-p when cells are stimulated by growth promoting neuropeptides.

1.2.0 inositol Phosphates and Calcium. The formation of inositol 1,4,5, tris phosphate (lns(1,4,5)P3) is a focal point for the two major pathways (reviewed (Berridge 1993)). The activation of PLC leads to the hydrolysis of inositol 4,5, bisphosphate leading to the formation of lns(1,4,5)Pg and 1,2 diacylglcerol (DAG). As can be seen from Fig 1.1 while G- protein linked receptors activate PLC-p, receptors with intrinsic tyrosine kinase activity activate PLC-y (see below). These separate receptor mechanisms are coupled to energy requiring (GTP or ATP) transducing mechanisms which activate PLC. Thus there is receptor heterogeneity , in addition to is extensive diversity of down stream elements, e.g. G-proteins, PLC, IP3 receptor (IP3R) and PKC. Despite this molecular heterogeneity, the common theme is that external signals use the lnsP 3/Ca^+ and DAG/PKC pathways to regulate a wide variety of cellular activities (Fig 1.1). The dynamics of these different transducing mechanisms have been compared in Swiss 3T3 cells, which carry both G-protein linked receptors (e.g. bombesin) and tyrosine kinase receptors (e.g. PDGF). When compared to bombesin PDGF-induced formation of lnsP3 was much slower and the resulting Ca^+ response was not only smaller but had a much longer latency. A family of IP3RS has now been identified. The IP3R contains typical membrane-spanning domains in the C-terminal region which anchor the protein in the membrane with four of the subunits combining to form the functional lnsP 3- sensitive calcium channel. The large N-terminal domain lies free in the cytoplasm with the lnsP 3 binding site located at it's end, away from the channel forming C- terminal region. Upon binding lnsP 3 the receptor undergoes a large conformational change which is perhaps related to the coupling process leading to channel opening. The intracellular stores from which Ca^+ is released contain three major components: pumps to sequester Ca^+, binding proteins (such as calsequestrin and calreticulin) to store Ca^+ and the specific IP3R channels to release Ca^+ back into the cytosol. When lnsP 3 binds to it's receptor, Ca2+ contained within intracellular stores is released into the cytosol. The exact mechanism by which this occurs is poorly understood. The response is extremely fast reaching a maximum within 140 ms of adding lnsP 3 to synaptosomes. As the level of lnsP 3 rises, a fixed proportion of the stored Ca^+ is released, with the remainder of the Ca^+ only becoming accessible at higher concentrations of InsPg. The intracellular Ca^+ stores are not uniformly sensitive to InsPg. The IP 3R displays a bell-shaped response to Ca^+ which thus functions as a coagonist with InsPg to release stored Ca^+. In the absence of Ca^+, lnsP 3 has little effect, but becomes increasingly active as the concentration of Ca^+ rises, reaching a maximum at about 300 nM, after which it begins to be inhibitory. There is growing evidence that inositol phosphates ( particularly InsPg and lnsP 4) may have direct effects on Ca^+ channels within the plasma membrane. The influx of Ca^+ seems to be regulated by the Ca^+ content of intracellular Ca^+ stores. In some cells, Ca^+ entry is stimulated when the intracellular Ca^+ stores are artificially emptied by applying the Ca^+ pump inhibitor thapsigargin, or Ca^+ ionophore ionomycin. When the intracellular Ca^+ stores are fully charged, entry is prevented, but as soon as InsPs drains Ca^+ out of these stores, the influx of Ca^tswitches on automatically. Many of the cells that respond to Ca^+-mobilising agonists display a repetitive pattern of Ca^+ spikes whose frequency is sensitive to both agonist concentration and the level of external Ca^+ (reviewed in (Berridge and Irvine 1989; Berridge 1993)). The lnsP 3/Ca^+ signalling pathway is activated by many mitogenic stimuli. Pasturella multicida toxin is a very effective stimulus of the phosphoinositde system and a very potent sole mitogen in Swiss 3T3 cells (Staddon, Barker et al. 1991). Bombesin/GRP is also a sole mitogen that stimulates phospholipid hydrolysis, whereas vasopressin, which also increases intracellular Ca^+ and InsPg, is only mitogenic in synergistic combination (reviewed in (Rozengurt 1991a)). Thus the lnsP 3/Ca^+ signalling pathway is part of a complex array of early signals that interact synergistically causing mitogenesis (Rozengurt 1986).

l.Z.d Inositol phosphate metabolism and lithium. The details of some of the metabolic pathways linking many of the inositol phosphates, that have now been found in eukaryotic cells (reviewed in (Berridge and Irvine 1989)), are summarised in Fig. 1.3. Inositol phosphates fall into two functional groups: those whose levels change in response to agonist stimulation and thus have functions related to intracellular signalling, and the agonist insensitive group concerned with the synthesis of InsPg and InsPg whose levels, if they change, do so comparatively slowly during stimulation. In the agonist sensitive group cyclic inositol phosphates are unlikely to have any physiological purpose, they are unavoidably formed as a consequence of the way phosphoinositidase C works. Much of the remaining proliferation of agonist sensitive inositol phosphate originates from the metabolism of lns(l ,4,5)Ps. It can be sequentially dephosphorylated to free inositol or it can be phosphorylated to lnsP(l, 3,4 ,5)P4 by an lns(l,4 ,5)P3 kinase. The latter is strongly activated in v-src transformed cells, resulting in a seven fold elevation in the level of an lnsP 4. Lithium inhibits the enzymes InsP phosphatase and inositolpolyphosphate phosphatase, resulting in the sustained accumulation of lns(l, 4 )P2, lns(l)P. The initial increase in lns(l,4,5)Ps is independent of lithium. A later rise in the InsPs fraction in the presence of lithium, is due to an accumulation of lns(l,3,4)Ps formed by dephosphorylation of lns(l, 3,4 ,5)P4 (reviewed in (Berridge and Irvine 1989)). Figure 1.3

Ptd OH Diacylglycerol P hs h t d c c d IMP-Phoaphatidata IP hosphatidic acid ICM ICDP-DAGI ^ U pids I 1 P tdlnsl3IP I

<5% P tdtns P td ln sU lP

>95% )

Cyclic

InsP 's

Ins 11:2 cyclP

lns(1.4.5)P,

Déphosphorylation

lnsl1,4)Pj I IniP /InsP I Pathw ay I /@u"

ln slt.3 )P ,

INOSITOL

ln sI3 ie

Glucose-6-Phosphate I

S y n th e sis - "y^ InsP. g o( InsP. ® .T . V

lnsl1,3,4,5.6)P

Figure 1..3: Summary of the known (solid arrows) and suspected (dashed arrows) routes of metabolism of compounds containing inositol and phosphate. myo-Inositol is represented in it's chair configuration. The Lithium sensitive enzymes (-Li+) 13, InsP phosphatase; and 14, inositolpolyphosphate phosphatase are shown and the inositol phosphates that would be expected to accumulate in the presence of lithium are boxed. 10

1.3. NEUROPEPTIDES AND CELL GROWTH

Many studies to identify the molecular pathways by which neuropeptide mitogens elicit cellular growth have exploited cultured murine 3T3 cells as a model system. The list of neuropeptides that can act as mitogens in these cells has now grown considerably and includes bombesin (Rozengurt and Sinnett-Smith 1983), bradykinin (Woll and Rozengurt 1988), endothelin and VIC (Takuwa, Takuwa et al. 1989; Fabregat and Rozengurt 1990a), vasoactive intestinal peptide (Zurier, Kozma et al. 1988) and vasopressin (Rozengurt, Legg et al. 1979). In additon, two receptors of the G-protein superfamily have been shown to have transforming potential: the mas oncogene (Young, Waitches et al. 1986) and the serotonin receptor (5-HT 1c) (Julius, Macdermott et al. 1988). The serotonin receptor is coupled to signal transduction pathways elevating intracellular Ca^+. Introduction of the serotonin receptor into quiescent mouse fibroblast cells results in transformed foci. The injection of cells derived from transformed foci into nude mice results in tumour growth. The greater the number of receptors per cell the higher the transforming potential. The phenotype of the tumour cells differ from that of the parental foci in that the tumour cells have a 2-7 X greater density of receptors on their surface than the parental foci. These tumour cells were able to form colonies in soft agarose in the absence of serotonin (Julius, Livelli et al. 1989). In what follows some fundamental features of the mechanism of action of bombesin as a growth factor in 3T3 cells will be discussed.

1.3.a Bombesin/Gastrin-releasing peptide. Bombesin, the prototype for a large family of naturally occurring bombesin­ like peptides with a highly conserved amino-terminal heptapeptide is a 14-amino acid peptide first isolated from the skin of the frog Bombina bombina. Structurally related peptides (Table 1.1) have been found in many species but the principal mammalian counterpart is GRP. 11

Table 1.1 Amino-acid sequence of bombesin like peptides.

MAMMALIAN

GRP(l-27) Human Pro Leu Pro Ala Gly Gly Gly Thr Val Leu Thr Lys —

Met Tyr Pro Arg Gly Asn His Ala Yal filX His Lsil Mêî NH2

GRP(l-27) Pcffcine Ala Pro Val Ser Val Gly Gly Gly Thr Val Leu Ala Lys —

Met Tyr Pro Arg Gly Asn His la i Ala Yal Gly His Lau Mai NH2

GRP(14-27) Porcine Met Tyr Pro Arg Gly Asn His Im Ala Val Gly His Met NH2

GRPIO (Neuromedin C) Gly Asn His Ala Val Gly His JLau Met NH2

Neuromedin B Gly Asn Leu Im Ala Thr Gly His Leu Met NH2 AMPHIBIAN

BcMnbesin pGlu Gin Arg Leu Gly Asn Gin Ala Yal Gly His Laii Mai NH2

BcMnbesin(8-14) lie Ala Yal Gly His Lan Met NH2

Ranatensin pGIu Val Pro Gin Jot Ala Yal Glv His Lau Mai NH2

Litorin pGlu Gin Im Ala Yal Gly His Leu Met HH2

GRP has 9/10 N-terminai amino acids identical to bombesin. Studies with synthetic bombesin-like peptides have demonstrated that full biological activity requires more than 7 but no more than 9 N-terminal amino acids (Heimbrook, Boyer et al. 1988). Further bombesin-like peptides have been isolated from porcine brain and spinal cord, designated neuromedins. The human GRP gene is located on chromosome 18 at 18q21 ((Naylor, Sakaguchi et al. 1987)), whereas the human neuromedin B gene lies on the long arm of chromosome 15 (Krane, Naylor et al. 1988). Bombesin-like peptides are thought to function both as neurotransmitters and gut hormones (Moody 1984). They are localised to neurones and neuroendocrine cells in the central and peripheral nervous systems (Panula 1986), although autoradiographic studies in gut suggest that their receptors have a wider distribution. In the pancreas, for example, GRP is found in nerve fibres closely associated with exocrine pancreatic tissue, and electrical stimulation of the vagus causes release of GRP (Knutsen, Holst et al. 1987). Infused GRP has a plasma half life of 2.8 minutes (Knigge, Holst et al. 1984). It causes secretion of gastrin, , insulin, glucagon, cholecystokinin and gastric inhibitory peptide, leading to amylase and gastric acid secretion (Lezoche, Basso et al. 1981; Knigge, Holst et al. 1984). GRP and it's receptor are most plentiful in the hypothalamus, and this has lead to speculation that it may regulate pituitary hormone secretion. In support of this, GRP infusion has been shown to stimulate the secretion of ACTH, cortisol and p-endorphin in normal subjects (Knigge, Holst et al. 1987). Behavioural effects of bombesin-like peptides have been studied in several species; in rodents, both peripheral and 12 intraventricular administrations of bombesin suppress food intake and increase grooming and scratching activities.

1.3.b Bombesin/GRP as a growth factor. Bombesin/GRP are potent mitogens for Swiss 3T3 cells (Rozengurt and Sinnett-Smith 1983). Chronic bombesin administration to rodents leads to antral gastrin cell proliferation and pancreatic hypertrophy (Lehy, Accary et al. 1983; Lhoste and Longnecker 1987). The latter effect appears to be partly direct and partly mediated by cholecystokinin (Douglas, Woutersen et al. 1989). Bombesin/GRP is increasingly implicated In many human tumours; e.g. GRP mobilises Ca^+ in breast (Yano, Pinski et al. 1992), thyroid (Abe, Kanamori et al. 1992) and colon (Frucht, Gazdar et al. 1992) cancer cell lines and has been shown to stimulate the growth of a human gastrinoma xenograft (Chung, Evers et al. 1992) and breast cancer cells (Yano, Pinski et al. 1992). Breast cancer cells also express the GRP gene and peptide (Pagani, Papotti et al. 1991; Vangsted, Andersen et al. 1991). Medullary thyroid carcinomas can also secrete GRP, resulting in one case in pituitary dependent Cushing's syndrome (Howlett, Price et al. 1985; Conlon, McGregor et al. 1988). GRP is also secreted by other endocrine tumours (Price, Kruseman et al. 1985). Carcinoid tumours commonly secrete GRP In addition to other peptides (Ghatei, Stratton et al. 1987; Moody, Lee et al. 1990). GRP production has been reported in carcinogen-induced rat hepatocellular tumours (Seglen, Skomedal et al. 1989) and GRP has been implicated in azaserine-induced rat pancreatic tumours (Lhoste and Longnecker 1987). Though bombesin Inhibits the growth of human pancreatic adenocarcinoma in nude mice (Alexander, Upp et ai. 1988b). Bombesin/GRP stimulated growth has been demonstrated In a prostatic cancer cell line, and ''25|.grp binds specifically to these cells, suggesting that GRP could regulate prostatic cancer growth in vivo (Bologna, Festuccia et al. 1989). Autocrine growth regulation has not been postulated for any of these tumours. A role for bombesin In SCLC will be discussed in a later section.

1.3.C Bombesin/GRP In development. Bombesin-like peptides are sparsely present in the neuroendocrine cells lining the bronchi of human adult lung. In contrast, they are abundant in fetal lung, with maximum levels being found around term (Moody 1984). Pro-GRP mRNA and GRP-associated peptides are also present in fetal lung, indicating that the GRP is synthesised there (Spindel, Sunday et al. 1987; Cuttitta, Fedorko et al. 1988). GRP mRNAs are first detectable in fetal lung at 9-10 weeks gestation, becoming maximal (25-fold adult levels) between 16 and 30 weeks, and declining to adult levels by 34 13 weeks (Spindel, Sunday et al. 1987). In neonates with acute respiratory distress syndrome, bombesin levels throughout the lungs are significantly lower than normal (Ghatei, Sheppard et al. 1983).These findings have led to the hypothesis that GRP could act as an important growth factor for fetal lung. Immunoreactive GRP and GRP mRNA are found in human thyroid - containing neuroendocrine cells (C-cells) and are maximally expressed in infants of about 2 months, suggests an analogous role for GRP in the developing thyroid, and it may also have a role in thyroid carcinoma (Sunday, Wolfe et al. 1988), where it increases [Ca^+jj and stimulates secretion of calcitonin (Abe, Kanamori et al. 1992).

1.4. BOMBESIN/GRP SIGNAL TRANSDUCTION.

Bombesin is a model peptide with which to investigate the signal transduction pathways underlying peptidergic mediated cell growth. The early cellular and molecular responses elicited by bombesin and structurally related peptides in Swiss 3T3 cells (Fig. 1.4) have been elucidated in detail. In serum-free medium it stimulates DNA synthesis and cell division in the absence of other growth-promoting agents. The ability of bombesin, like platelet-derived growth factor (PDGF), to act as a sole mitogen for these cells contrasts with other peptide growth factors which are active only in synergistic combinations (Rozengurt 1986). The mitogenic effects of bombesin are markedly potentiated by insulin, which both increases the maximal response and reduces the bombesin concentration required for half-maximal effect (Rozengurt and Sinnett-Smith 1983). Furthermore, receptors for bombesin-like peptides have been well characterized at the molecular level. The cause-effect relationships and temporal organisation of the early signals and molecular events induced by bombesin provide a paradigm for the study of other growth factors and mitogenic neuropeptides and illustrate the activation and interaction of a variety of signalling pathways (Rozengurt 1991a).

1.4.8 Bombesin/GRP Receptors Bombesin and GRP bind to a single class of high affinity receptors (Kd approx. 1 nM) in Swiss 3T3 ceils. The receptors are transmembrane glycoproteins of Mr 75,000-85,000 with a core of Mr 43,000 (reviewed in (Rozengurt and Sinnett- Smith 1990)). The receptor is coupled to one or more G proteins as judged by the modulation of ligand binding in either membrane preparations or in receptor solubilized preparations and of signal transduction in permeabilized cells (Rozengurt, Fabregat et al. 1990). The bombesin/GRP receptor has been cloned and sequenced (Spindel, Giladi e t al. 1990; Battey, Way e t al. 1991). The deduced amino acid 14 sequence predicts a polypeptide core of Mr 43000 and demonstrates that it belongs to the superfamily of G protein coupled receptors.

1.4.b Phosphatidyl inositol turnover, calcium mobilisation and activation of protein kinase C Binding of bombesin/GRP to its receptor initiates a cascade of intracellular signals (summarised in Fig. 1.2) culminating in DNA synthesis 10-15 h later. One of the earliest events to occur after the binding of bombesin to its specific receptor is the rap[d stimulation of PLC (Fig 1.3) catalysed hydrolysis of phosphatidyl inositol 4,5- bisphosphate (PIP 2) in the plasma membrane. This reaction produces lns( 1,4 ,5)P3, which, as a second messenger binds to an intracellular receptor and induces the release of CaZ+ from internal stores (Berridge, 1993). Bombesin causes a rapid increase in lns(1,4,5)Ps, which coincides with a transient increase in the intracellular concentration of Ca^+ ([Ca^+Jj) and with CaZ+ efflux from the ceils ((Mendoza, Schneider et al. 1986; Lopez, Mendoza et al. 1987; Nanberg and Rozengurt 1988).PLC-mediated hydrolysis of PIP 2 also generates DAG. DAG can also be generated from other sources, such as phosphatidylcholine (PC) hydrolysis, and acts as a second messenger in the activation of PKC. PKC is activated by multiple extracellular stimuli (Nishizuka 1988) including bombesin (Erusalimsky, Friedberg et al. 1988). In accord with this, bombesin strikingly increases the phosphorylation of an acidic, myristoylated, alanine rich protein that migrates with an apparent molecular mass of 80 kDa (80K/MARCKS). 80K/MARCKS has been identified as a prominent substrate for PKC in cultured cells and several tissues (Rozengurt, Rodriguez-Pena et al. 1983; Erusalimsky, Brooks et al. 1991). Bombesin phosphorylates 80K/MARCKS within 15 seconds. The cDNAs encoding this substrate from Swiss 3T3 cells has been cloned (Brooks, Herget et al. 1991). PKC activation by phorbol ester causes a dramatic down-regulation of the expression of mRNA and protein of the 80 kDa substrate from these cells through a post-transcriptional mechanism which may involve alterations in mRNA stability (Brooks, Herget et al. 1991; Brooks, Herget et al. 1992). Similar results were observed with physiological activators of PKC e.g. bombesin and PDGF. Expression of 80K/MARCKS mRNA and protein was also down regulated in cells treated with activators of the cAMP-dependent protein kinase and in PDGF treated cells in which PKC was down regulated by prolonged treatment with phorbol ester. 80K/MARCKS appears to be a calmodulin and actin binding protein (reviewed in (Blackshear 1993)). 80K/MARCKS binds calmodulin in a Ca^+ dependent manner, which can be prevented by PKC phosphorylation of 80K/MARCKS (the calmodulin- binding domain turned out to be identical to the phosphorylation site domain). 15

Figure 1.4

Bombesin, GRP Specific Broad Spectrum Antagonists Antagonists

PLA PLC PIR Tyr Kinase Arachidonic lns{1,4,5)P3 Activation Acid Release

Ca mobilization p i25 Tyr - (g ) 80K-® -FAK PGE2 [c -r a f] I I_____ N# /H r - [Cap, MAPKK cAMP

Ca^* efflux

Cell ^ I M o tility Shape T Expression of Immediate Adhesion early genes c-fos c-myc

Figure 1..4 : Bombesin-mediated signal transduction.

Initiation of cell proliferation in Swiss 3T3 cells is stimulated by multiple signal transduction pathways that act in a synergistic and combinatorial fashion. The interactions have been well defined in these cells and provide experimental evidence for a model that involves multiple pathways. The mechanism of action of neuropeptide growth factors are explained within the framework of this model.

The abbreviations used: PLC, phospholipase C; PIP 2 , phosphatidylinositol 4,5-bisphosphate;

Ins(l,4,5)P3, inositol 1,4,5 trisphosphate; [Ca^+Jj, intracellular [Ca^+]; PLA 2 , phospholipase A 2 ; G, guanine nucleotide binding protein; PKC, protein kinase C; DAG, diacylglycerol; PC, phosphatidylcholine; PGE 2 , prostaglandin E 2 ; EGFr, epidermal growth factor receptor; Tyr, tyrosine;

Ser, s e r in e . ______

Activation of PKC and increases in [Ca^+J, could regulate the interaction of 80K with both the cytoskeleton and the plasma membrane, and 80K/MARCKS may play a suppressor role in the control of cell proliferation. Bombesin/GRP also stimulates a rapid exchange of Na+, H+ and K+ ions across the cell membrane, leading to cytoplasmic alkalinization and increased intracellular [K+] and induces a striking PKC-dependent transmodulation of the epidermal growth factor receptor (reviewed in (Rozengurt and Sinnett-Smith 1990)). 16

1.4.C Arachidonic acid reiease and prostagiandin synthesis: While bombesin/GRP stimulate DNA synthesis in the absence of other factors, vasopressin is mitogenic for Swiss 3T3 cells only in synergistic combination with other factors e.g. insulin (Rozengurt, Legg et al. 1979; Rozengurt, Rodriguez-Pena et ai. 1983). Binding of vasopressin to its distinct receptor on quiescent cultures of Swiss 3T3 ceils causes a rapid production of lns( 1,4 ,5)P3, mobilization of Ca2+ from intracellular stores and sustained activation of PKC via a G-protein linked transduction pathway (reviewed in (Rozengurt 1991a)). Independent signal- transduction pathways act synergistically in the initiation of DNA synthesis, hence, the ability of bombesin to act as a sole mitogen could be due to activation of a signalling pathway not stimulated by vasopressin. Bombesin, but not vasopressin, has been shown to induce a marked, biphasic reiease of arachidonic acid into the extracellular medium (Millar and Rozengurt 1990a; Domin and Rozengurt 1992). A first phase involves rapid activation of phospholipase A 2 (PLA2). The major phase of arachidonic acid mobilisation begins 20 min after the addition of ligand. PLA 2 could contribute to the first but not to the second phase, which is dependent upon de novo protein synthesis. These results showed a clear difference in the pattern of early signals induced by the neuropeptides bombesin and vasopressin in Swiss 3T3 ceils. The stimulation of arachidonic acid reiease by bombesin is likely to contribute to bombesin-induced mitogenesis because externally applied arachidonic acid potentiates mitogenesis induced by agents that stimulate polyphosphoinositide breakdown but not arachidonic acid reiease, e.g. vasopressin (Millar and Rozengurt 1990a). Arachidonic acid released by bombesin is converted into E-type prostaglandins which acting in an autocrine and paracrine manner enhance cAMP accumulation in the ceil (Millar and Rozengurt 1988; Millar and Rozengurt 1990a). Since elevated cAMP levels constitute a mitogenic signal for Swiss 3T3 cells (reviewed in (Rozengurt 1991 a)) at least one consequence of arachidonic acid reiease may be the modulation of intracellular cAMP levels. Arachidonic acid has been shown to inhibit the ability of a GTPase activating protein (GAP), resulting in increased pZI'^^in it's activated GTP bound state, however PGE 2 stimulates GAP. Thus arachidonic acid reiease and it's cyciooxygenase metabolites may finely regulate p21'^*(Rozengurt 1991b).

1.4.d Bombesin induction of the proto-oncogenes c-fos and c-myc. Bombesin rapidly and transiently induce the expression of the cellular oncogenes c-fos and c-myc in quiescent fibroblasts (Rozengurt and Sinnett-Smith 1988). Enhanced expression of c-fos occurs within a few minutes of bombesin addition, and is followed by increased expression of c-myc some hours later. Since 17 these cellular oncogenes encode nuclear proteins it is possible that their transient expression may play a role in the transduction of the mitogenic signal in the nucleus (reviewed in (Lewin 1991 )). DNA synthesis does not require both oncogenes to be expressed: agents that activate cAMP but not protein kinase can stimulate mitogenesis in the absence of c-fos induction. Similarly, bombesin can stimulate DNA synthesis with insulin in PKC-down regulated cells despite an almost complete absence of c-fos induction (Mehmet and Rozengurt 1991). The demonstration that the product of the proto-oncogene c~jun, identified as a major component of the transcription factor AP-1, forms a tight complex with fos protein is consistent with a role for c-fos in the regulation of gene transcription (Lewin 1991). There has been considerable interest in elucidating the signal transduction pathways involved in c-fos induction. TbereJs-iricreasing evidence implicating PKC activation in bombesin induction of c-fos (reviewed in (Rozengurt and Sinnett-Smith 1990)). Accordingly, bombesin-induced oncogene expression is markedly reduced by down-regulation of PKC. Interestingly, PKC activation leads to the activation of a cascade of protein kinases (Satoh, Nakafuku et al. 1992) that include c-/?af and the mitogen activated protein (MAP) kinases. The activated MAP kinase directly phosphorylates transcription factor regulators which results in the increased expression of c-fos (Treisman 1992). However, neither direct activation of PKC by phorbol esters nor addition of vasopressin evoke a maximal increase in c-fos mRNA levels. It is likely th a t th e induction of c-fos by bombesin is mediated by the co­ ordinated effects of PKC activation, Ca^+ mobilization and an additional pathway dependent on arachidonic acid release (Rozengurt 1991b).

1.4.8 Neuropeptide stimulation of tyrosine kinase activity The receptor for peptides of the bombesin family is coupled to a G-protein(s) and does not possess intrinsic tyrosine kinase activity. Recently, however, bombesin has been shown to rapidly increase tyrosine phosphorylation of multiple substrates in intact quiescent Swiss 3T3 cells (Zachary, Gil et al. 1991). Vasopressin and endothelin elicit a similar response. Because the increase in tyrosine phosphorylation is not dependent on Ca2+ or PKC signals, it is likely that it represents a distinct pathway (Fig. 1.2). The substrates for neuropeptide tyrosine phosphorylation in these cells appear to be unrelated to known targets for the PDGF receptor. Thus, bombesin and other neuropeptides that act through receptors linked to G-proteins can increase tyrosine phosphorylation of protein substrates in intact cells (Fig. 1.2). The stimulation of tyrosine phosphorylation by neuropeptides is due to activation of a cellular tyrosine kinase (Zachary, Sinnett-Smith et al. 1991). Indeed, a novel cytosolic protein tyrosine kinase, named focal adhesion kinase (pi has 18 been identified as a major substrate for bombesin-stimulated tyrosine phosphorylation in Swiss 3T3 cells (Zachary, Sinnett-Smith et al. 1992). At present, it is not known whether plZS^'^^ is phosphorylated by a proximal kinase or autophosphorylated after its activation; both possibilities are depicted in Fig. 1. Recently, pi has also been shown to be regulated by activation of the adhesive receptors of the integrin family and certain oncogenes (e.g. src). Thus, pi 25^^^ appears to be a point of convergence in the action of neuropeptides, integrins and oncogenes and could be involved in the regulation of cell shape, adhesion and motility (reviewed in (Zachary and Rozengurt 1992)). Given the importance of tyrosine phosphorylation in the action of growth factors and non-receptor oncogenes, it is likely that this novel event plays a role in neuropeptide mitogenic signalling. In fact, recent results have shown that inhibitors of tyrosine kinases can inhibit bombesin stimulation of DNA synthesis in Swiss 3T3 cells by 60% (SeckI and Rozengurt 1993).

1 A .f Regulation of cellular responsiveness to bombesin-stimulated mitogenesis Exposure of cells to many peptide hormones or neurotransmitters decreases the subsequent response of target cells to further challenge with the same ligand (homologous desensitisation) or with a structurally unrelated ligand which elicits responses through a separate receptor (heterologous desensitisation). Desensitisation has been well-documented for hormones that elicit short-term metabolic responses and it is increasingly recognised to play a role in the regulation of cell growth. Bombesin induces at least two distinct desensitisation mechanisms in Swiss 3T3 cells, (i) acute desensitisation which is heterologous, stimulation through the vasopressin and bradykinin receptors are also down-regulated and (ii) chronic desensitisation due to homologous down-regulation of bombesin receptors (Millar and Rozengurt 1990b). Furthermore mitogenic responsiveness to either bombesin alone or vasopressin in the presence of insulin is desensitised in Swiss 3T3 cells deficient in PKC activity, while binding to the vasopressin and bombesin receptors is unaffected (Collins and Rozengurt 1984). Prolonged exposure to vasopressin causes homologous mitogenic desensitisation. Onset of desensitisation is both time- and concentration- dependent, reversed by removal of hormone, however only a minor fraction of the cell surface vasopressin receptors are lost. One of the most interesting features of this process is that the onset of desensitisation occurs at approximately a similar time as the onset of DNA synthesis, suggesting that entry into DNA synthesis and onset of mitogenic desensitisation are co-ordinately regulated (Dicker and Rozengurt 1980). Loss of some of these controls could lead to malignant growth. Prolonged treatment 19

with vasopressin also induces heterologous desensitisation of the mitogenic activity of bombesin and PDGF, this was not due to functional uncoupling of the receptor, this block occurs at a post-receptor locus, namely a complete block of bombesin and PDGF induced release of arachidonic acid and its cyciooxygenase metabolite prostaglandin E (PG E2) into the medium (Millar and Rozengurt 1990a). The existence of homologous and heterologous mitogenic desensitisation suggests that the control of cell proliferation by neuropeptides may result from a delicate interplay between growth- stimulatory and growth-inhibitory signals.

1.5. OTHER NEUROPEPTIDE GROWTH FACTORS.

1.5.3 Vasopressin. I I Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH 2

Vasopressin or antidiuretic hormone (ADH) is a cyclic nonapeptide synthesized in the supra-optic nucleus of the hypothalamus, ADH containg vesicles pass passes down the neural axons to the posterior pituitary before being secreted into the circulation. As an endocrine hormone, it has antidiuretic effects on the kidney, pressor effects mediated through arteriolar smooth muscle and stimulates hepatic glycogenolysis. Two types of vasopressin receptor have _been distinguished functionally and pharmacologically (Huffman, Kinter et al. 1988). The Vi receptor mediates the vascular and hepatic effects of vasopressin, by activating inositol phosphate turnover. The V2 receptors are coupled to adenylate cyclase and mediate antidiuretic responses of the kidney. Subtypes of these receptors have been identified in other tissues including the adenohypophysis (Jard, Gillard et al. 1986). The demonstration that vasopressin was a mitogen for Swiss 3T3 cells provided the first unambiguous evidence that nëurô^ptides can act as growth factors (Rozengurt, Legg et al. 1979). It has been shown to bind to specific high affinity receptors (Collins and Rozengurt 1984) of the Vi type, which are blocked by the specific antagonist {l-(p-mercapto-B, B-cyclopentamethylene proprionic acid), 2- (0-metyl) tyrosine] arginine-vasopressin (Zachary and Rozengurt 1986). Although vasopressin alone does not stimulate cell proliferation in serum free medium, it acts synergistically with insulin, serum, EGF and PDGF at nanomolar concentrations. Vasopressin causes a rapid production of lns(l, 4,5)P3, mobilisation of Ca2+ from intracellular stores and sustained activation of PKC via a G-protein linked transduction pathway (reviewed in (Rozengurt 1991a)). Vasopressin has also been shown to rapidly increase tyrosine phosphorylation of multiple substrates in 20

quiescent Swiss 3T3 cells, and this is due to activation of cellular tyrosine kinases (Zachary, Gil et al. 1991; Zachary, Sinnett-Smith et al. 1991) In vitro, Miller e t al (Miller, Husain e t al. 1977) showed th a t vasopressin stimulated [^H] thymidine incorporation into monolayers of fetal rat chondrocytes. In vivo, homeostatic cell proliferation occurs in the bone marrow in response to haemorrhage and in the liver in response to partial hepatectomy. In both cases, rapid and dramatic increases in mitotic rate are observed, which are maintained for periods of up to a few days, then return to baseline when normality has been restored. Vasopressin has been implicated in both these processes. Following haemorrhage, congenitally deficient vasopressin (Brattleboro) rats, and hypophysectomised rats were unable to mount the early mitotic response of normal animals. Exogenous vasopressin was shown to stimulate marrow proliferation, suggesting that vasopressin is a vital component of the response to haemorrhage (Hunt, Perris et al. 1977). More recent studies have shown that rapid elevations of vasopressin levels occur in the supraoptic nucleus, median eminence and plasma after haemorrhage, in addition to site and time-dependent changes in the distribution of brain and (Feuerstein, Molineaux et al. 1985). Similar experiments have demonstrated that liver regeneration following partial hepatectomy is also dependent on vasopressin secretion (Russell and Bucher 1983). The Brattleboro rat has proved useful for investigating the role of vasopressin in development. These rats show impaired brain development that can be prevented by prenatal vasopressin treatment (Boers 1985), suggesting a possible role for vasopressin in neurogenesis.

I.S.b Bradykinin. Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-NHz

The nonapeptide bradykinin is generated in the plasma or tissues from large molecular weight precursors (kininogens) by the action of kallikreins, which are activated during proteolysis and clotting (Regoli, Drapeau et al. 1986; Steranka, Farmer et al. 1989). Bradykinin is usually present in plasma at very low concentrations, due to it's rapid degradation by carboxypeptidase N and angioconverting enzyme. Bradykinin is one of the most potent pain producing substances and receptors have been localised to the nocioceptive sensory pathways, where it acts as a (Steranka, Farmer et al. 1989). Bradykinin is implicated in smooth muscle contraction, vasodilatation and vascular permeability, the cough caused by angiotensin-converting enzyme inhibitor, captopril, may be due in part to bradykinin-stimulated bronchial inflammation. Synthetic bradykinin 21 analogues, agonists and antagonists, define at least two receptor subtypes: Bi and 62, although there is no clear functional separation between the two receptor subtypes (Steranka, Farmer et al. 1989). Bradykinin in the presence of insulin is mitogenic for Swiss 3T3 cells ( W o 11 and Rozengurt 1988). Bradykinin acting through a B 2 receptor rapidly mobilises [Ca2+]j and stim ulated phosphorylation of 80K/MARCKS. However, in contrast to bombesin and vasopressin the stimulation of 80K/MARCKS phosphoylation by bradykinin is slower, reaching a maximum after 1 min of incubation, and then rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce PKC mediated events such as inhibition of ^25|_£qp binding or enhancement of cAMP accumulation. Bradykinin induced rapid accumulation of total inositol phosphates, but in contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase (Issandou and Rozengurt 1990). Bradykinifv-production^-is-associated-with the flushing caused by carcinoid tumours, whioh-mav secrete large quantities of vasoactive peptides (Balks, Conlon et al. 1988). Bradykinin and [hydroxyprolyP]bradykinin has been detected in the ascitic fluid from patients with carcinomas (Matsumura, Kimura et al. 1989). Because of the short plasma half-life, bradykinin could be acting as a paracrine growth factor without being detected with current assay methods.

1.5.C Tachykinins.

Substance P Arg Pro Lys Pro Gin Gin £h£ Phe Gly L£U Msi NH2

Substance K: His Lys Thr Asp Ser Ehfi Val GlX Lsu Mfil NH2 ()

Neurokinin K: Asp Met His Asp Phe Val Gly L£U Mfil NH2 ()

The three- mammalian — - ' . —— tachykinins illustrated - -- mill--- above . have similar biological activities associated with their shared carboxy-terminal sequence, but bind to distinct receptors. Substance P and substance K are derived from the same gene by alternative splicing. The tachykinins are widely distributed in the brain (hypothalamus, trigeminal ganglion, caudate nucleus and olfactory bulb), the spinal cord and gut neurones. The best studied is the undecapeptide substance P, which is synthesised in the dorsal root ganglia and transported axoplasmically to be stored in the sensory nerve endings. A variety of stimuli lead to it's release, causing local pain, smooth muscle spasm and vasodilatation, in addition to it's systemic effects of inhibition of pancreatic secretion and bile flow, stimulation of salivation and renal natriuresis (Payan 1989). 22

The tachykinin receptors have been characterised pharmacologically using diverse agonists and antagonists (Regoli, Drapeau et al. 1989; Snider, Constantine et al. 1991). The receptors for substance P, substance K and neuromedin K have been designated as NK-1 (substance P), NK-2 (substance K) and NK-3 (neuromedin K). Substance P has been shown to have mitogenic effects on T lymphocytes, at concentrations as low as lOOpM, mediated through it's specific receptor (Payan, Brewster et al. 1984). Tachykinins have been shown to stimulate growth of human skin fibroblasts, arterial smooth muscle cells and kératinocytes (Nilsson, von-Euler et al. 1985). It has been hypothesised that tachykinins could mediate inflammation and wound healing (Payan 1989). in support of this neuropeptides have been observed to become depleted during wound healing in rat skin, and limb regeneration in newts requires innervation or exogenous substance P (Brockes 1984). Substance P receptors are expressed by glia in vivo following neural injury (Mantyth, Johnson et al. 1989). Substance P also stimulates prostaglandin E 2 release and proliferation of synoviocytes (Lotz, Carson et al. 1987), and may be implicated in the pathogenesis of rheumatoid arthritis. Tachykinins are secreted by carcinoid tumours (Bishop, Hamid et al. 1989). Substance P can act as a growth factor for aortic smooth muscle from rat embryos, but not from adult rats (Mitsuhashi and Payan 1987), suggesting a possible developmental role for substance P.

I.S.d Vasoactive intestinal peptide. This 28 amino acid polypeptide is closely related to , glucagon, PHI (porcine histidine, isoleucine amide containing peptide). It is found in large amounts in the mammalian brain and in the gut mucosa and muscle, where it is localised to postganglionic nerves. In addition it is found in the salivary glands, pancreas, respiratory and urogenital tracts (Fahrenkrug and Emson 1982). Neural stimulation causes release of VIP, which binds to specific receptors that can also bind related hormones with lower affinity. VIP causes relaxation of smooth muscle, vasodilatation and enhanced small intestinal and colonic secretion. Pancreatic endocrine tumours secreting VIP (VIPomas) result in the Verner-Morrison syndrome of watery diarrhoea, hypokalaemia and achlorhydria. In Swiss 3T3 cells, VIP stimulates mitogenesis in the presence of insulin and cAMP phosphodiesterase inhibitors. In contrast to bombesin and vasopressin, VIP is a weak mitogen and it's effects are mediated through elevation of cAMP without Ca^+ mobilisation or PKC activation (Zurier, Kozma et al. 1988). VIP also stimulates adenylate cyclase activity and cell proliferation in kératinocytes. VIP has been found in pancreatic, neural and cervical tumours and phaeochromocytomas. 23

1.6 . SIGNAL TRANSDUCTION BY TYROSINE KINASE RECEPTORS

Among the best characterised polypeptide growth factors are PDGF and EGF, and they serve as models for studying the signalling pathways utilised by receptor tyrosine kinases. PDGF is a potent mitogen for connective tissue cells and has been implicated in a wide variety of physiological and pathological processes (Ross, Raines et al. 1986). PDGF is a 32 kDa dimer consisting of two homologous polypeptide chains, A and B, which are encoded by different genes. All three dimeric forms of PDGF have been found to occur naturally. PDGF A and B chains bind differently to two distinct receptor molecules, the PDGFa and p receptors. The a receptor binds both PDGF A and B chains whereas the p receptor binds only the PDGF B chain (W estermark and Heldin 1991).

1.6.3 Structural features of receptor tyrosine kinases. The family of tyrosine kinases share an overall basic structure (Fig 1.5). A binding domain on the outside of the cell where it can specifically recognise it's ligand. The link to the cytoplasm is provided by a stretch of hydrophobic amino acids which cross the plasma membrane once. This part plays more than a passive role in signai transduction; the most striking example is the neu oncogene whose only difference from it's non transforming, receptor like homologue is a single amino acid substitution in the transmembrane region (Weiner, Nordberg et al. 1990). The cytoplasmic domain contains a tyrosine kinase catalytic domain. This is the most conserved domain of these receptors and contain several residues that form a typical ATP-binding site, as found in all protein kinases. The kinase domain catalyses the transfer of the y-phosphate of ATP to tyrosine residues on substrate proteins, as well as onto themselves in an autophosphorylation reaction. Within the family of receptor tyrosine kinases, several subclasses have been defined that display differences in their extracellular and intracellular domains (Üllriüh'âhd 5cHessingerT990). Subclass I has a prototype the EGF receptor and also includes the c-erbBz {neu) and c-erbBi. It is characterised by cysteine-rich regions in the ligand binding domain and a regulatory C-terminal extension that follows the catalytic domain and contains the major sites for autophosphorylation, Subclass II (e.g. the insulin and IGF-I and II receptors) is distinguished by a heterotetrameric tt2p 2 structure with disulphide bonds forming covalent dimers. Subclass III is represented by the two types of PDGF receptors (a and p) the colony stimulating factor-1 (CSF-1) receptor and the stem ceil factor receptor {c-kit). 24

Figure 1.5

Cyxlein* ncA region

• Conserved cysteine 0 IgG-like domain 1 Transmembrane region ■ Kirwse domain 0 Kinase-insed

EGFR IR PDGFR FGFR NGFR HGFR

Figure 1.5 Structural features of receptor tyrosine kinases.

A representative example of each receptor subclass is shown. EGFR, epidermal growth factor receptor; IR, ; PDGFR, platelet derived growth factor receptor; FGFR, fibroblast growth factor receptor; NGFR, nerve growth factor receptor; HGFR, hepatocyte growth factor receptor. ______

These are characterized by five immunoglobulin-like repeats in the extracellular domain and the catalytic (kinase) domain varies in length due to insertion amino acid sequences not involved in catalysis. Autophosphorylation sites have also been found in this region. Similar characteristics are found in subclass IV receptors (e.g. FGF) with the difference of three instead of five repeats. As more receptors are being characterised and sequenced e.g. nerve growth factor receptor (trk), hepatocyte growth factor (HGF) receptor (c-met) more structural variations are observed, increasing the number of receptor sub-classes .

1.6 .b Receptor activation Ligand binding drives dimerization or oligomerization of receptor (with the exception of the subclass II subfamily (insulin-like receptors) where dimers are already formed). This process promotes the interaction of kinase domains, leading to their activation. Dimerization can also be seen in the absence of ligand, as for example 25 th e neu/c-erbBz receptor-like molecule. In this case, the oncogenic mutation in the transmembrane domain results in increased dimerization and constitutive activation (Weiner, Liu et al. 1989). The immediate result of activation is the phosphorylation of the receptors themselves on tyrosine residues, probably in a trans-phosphorylation reaction within dimers (Honegger, Schmidt et al. 1990). Kinase activation appears to be essential for biological response. PDGF triggers a complex array of early events in the membrane, cytosol and nucleus. These include phosphoinositide breakdown, Ca^+ mobilization, PKC activation, 80K/MARCKS phosphorylation, EGF receptor transmodulation, biphasic release of arachidonic acid, prostaglandin synthesis, Na+, K+ and H+ fluxes and increased expression of early response genes. Although this set of responses is similar to that elicited by bombesin, the primary mechanism of signal transduction utilised by PDGF involves the intrinsic tyrosine kinase activity of the a and p PDGF receptors. The tyrosine kinase activity of the receptor is essential for mitogenic signal transduction as shown using selective tyrosine kinase inhibitors and kinase-inactive receptor mutants (reviewed in (Ullrich and Schlessinger 1990)). Thus PDGF as well as other polypeptide growth factors (e.g. EGF, fibroblast growth factors) activate their corresponding receptors by inducing receptor dimerization and subsequent transphosphorylation at specific tyrosine residues (Ullrich and Schlessinger 1990). These liganded receptors physically associate with and phosphorylate a set of cytoplasmic proteins implicated in intracellular signal transduction pathways. These include PLC-yl, phosphatidylinositol 3' kinase (PI3K), Ras GTPase-activating protein (/?asGAP), tyrosine kinases of the Src family and other cellular proteins that have not, as yet, been identified (reviewed in (Cantley, Auger et al. 1991)). In every case, the formation of stable complexes requires the tyrosine kinase activity of the receptor. Thus, these receptors transmit the mitogenic signal through tyrosine phosphorylation of specific cellular targets rather than via activation of heterotrimeric G-proteins.

1.6.C Phosphatidylinositol 3-kinase. The first signalling protein to be identified in receptor complexes was the phosphatidylinositol 3-OH kinase (PI3K). This enzyme phosphorylates the inositol ring of phosphatidylinositol (PI) PI(4)P and Pl(4,5)P 2 at the D3 position to produce PI(3)P, PI(3,4)Pz and PI( 3,4,5)P3 (Stephens, Hughes e t al. 1991). PI3K is composed of two subunits with apparent molecular masses of 85 and 110 kDa (Carpenter, Duckworth, et. al. 1990). The 85 kDa subunit has been cloned in different laboratories and shown to contain SH 2 domains which mediate the association 26 of PI3K to phosphorylated receptors. The 110 kDa subunit encodes the catalytic activity (Escobedo, Navankasattusas et al. 1991). PI3K becomes phosphorylated on tyrosine and activated upon PDGF stimulation of cells. Two phosphorylation sites (Tyr 740 and Tyr 751 in the human PDGF p-receptor) are necessary for a high affinity interaction. Apart from the PDGF receptor, most other receptor tyrosine kinases also interact with the PI3K in a direct way. An exception is the insulin receptor; although insulin clearly stimulates PI3K activity, the enzyme is not found in physical association with the receptor but with one of its substrates, termed IRS-1, which is phosphorylated on tyrosine residues and thus mediates complex formation (Myers, Backer e t al. 1992). PI3K it is thought to play a role in eliciting cell proliferation since deletion mutants in the p PDGF receptors abolished both PI3K association and DNA synthesis (Cantley, Auger et al. 1991; Valius and Kazlauskas 1993).

1.6 .d Phospholipase C-Y PLC-y was first identified as a substrate for tyrosine phosphorylation in PDGF and EGF treated cells. The receptor was found to co-immunoprecipitate with PLC-y, suggesting a tight physical association (Kim, Kim et al. 1991). Recent evidence demonstrates that PLC-y phosphorylation is directly responsible for the activation of this enzyme in living cells. Thus, overexpression of PLC-y increases tyrosine phosphorylation of these enzymes and inositol phosphate formation in response to PDGF. The mechanism of this activation was investigated by substituting phenylalanine for tyrosine at specific phosphorylation sites and expressing the mutant enzymes in recipient 3T3 cells. Mutation of a single tyrosine residue (phenylalanine substitution at Tyr-783) completely blocked the accumulation of inositol phosphates induced by PDGF (Kim, Kim et al. 1991). These results provide direct evidence that the PDGF receptor stimulates the function of an intracellular signal transducing protein by site-specific tyrosine phosphorylation. Nevertheless, a mutational analysis of the PDGF receptor has shown that PLC-y phosphorylation may not be required for mitogenesis (Cantley, Auger et al. 1991). Recent further mutants of the PDGF receptor, showed that permitting association of the PDGF receptor with either PLC-y or PI3K restored Ras activation and a mitogenic response. However even though binding of a 64 kd protein almost fully restored Ras activation, it did not rescue the receptors ability to trigger DNA synthesis. Thus Ras activation is insufficient to trigger PDGF-dependent DNA synthesis, and PLC-y and PI3K are independent downstream mediators of PDGF's mitogenic signal (Valius and Kazlauskas 1993). This 27 further indicates the necessity for interaction between the various signal transduction pathways.

1.6.e GTPase-activating protein The protein encoded by the ras gene (p2K^^) piays an important role in growth control. Its activity is regulated by the GTPase activating protein (GAP) which converts the active GTP-bound Ras to the inactive GDP-bound form (Hall 1990). GAP is therefore considered to occupy a key role in signal transduction mediated through tyrosine kinase receptors. GAP associates with activated tyrosine kinases. GAP becomes phosphorylated on tyrosine residues and translocates to the plasma membrane upon PDGF stimulation, and forms a direct complex with the activated PDGF receptor. A recent further link between growth factor activation and ras signalling has been provided through the characterisation of the GRB 2 protein, which associates with activated EGF and PDGF receptors. This protein lacks any known catalytic activity, it stimulates mitogenesis when micro-injected into quiescent fibroblasts together with ras, whereas these two components have no effect when introduced on their own (Lowenstein, Daly et al. 1992). Oncogenic forms of ras remain permanently active as a ras-GTP complex. A region of the neurofibromatosis gene product (NF-1) that has sequence similarity to the catalytic domain of GAP also stimulates p2K " GTPase activity. NF-1 and GAP are regulated in vitro by lipid mediators, particularly arachidonic and phosphatidic acid (Rozengurt 1991b). The direct downstream targets of Ras are not understood but Increasing evidence implicates several serine/threonine kinases Including Raf~\, MAP kinase kinase and MAP kinase (Satoh, Nakafuku et al. 1992). An attractive aspect of this pathway is that MAP kinase may be able to phosphorylate and activate transcription factors providing a continuous pathway that connects the Initial events In the plasma membrane with the transcriptional response In the nucleus. This pathway can also be stimulated by activation of PKC. It Is Interesting that bombesin does not Increase the GTP-bound form of Ras (Satoh, Endo et al. 1990) and consequently. It Is likely that small regulatory peptides do not utilise the Ras pathway to Initiate intracellular events. In contrast, neuropeptide mitogens utilise PKC to connect with the MAP kinase cascade. This difference emphasises again the existence of multiple signalling pathways lead]ng_to_ cell proliferation. 28

1.6 .f SH2 domains mediate the interaction of signalling molecules to recep to rs Specific phosphotyrosine-containing sequences in activated growth factor receptors form the binding sites for signalling proteins. A conserved domain is shared by these diverse proteins (with the exception of Raf) and has been demonstrated to mediate binding to autophosphorylation sites. This domain has been termed src- homology 2 (SHz), as it was first identified by it's sequence similarity to a region of pp60®"®''‘^. A second conserved domain, termed SH 3, is found in most proteins that contain SH2 domains and may mediate interactions with the cytoskeleton, although it's precise function is unclear. The association of cytoplasmic proteins with the activated receptors is mediated by the src homology (SH 2) region, a non-catalytic domain of approximately 100 amino acids (reviewed in (Pawson 1992)). These domains are sufficient to form stable complexes in vitro with the liganded receptors. It has been proposed that tyrosine phosphorylation of specific residues in the polypeptide chain of the receptor acts as a switch to induce high affinity binding of SH 2-containing cytoplasmic proteins. Differences in the amino acid sequences between SH 2 domains appears to result in distinct binding affinities. Hence, SH 2 domains play a crucial role in the association of cytoplasmic proteins with cellular and receptor tyrosine kinases.

1.7. POLYPEPTIDE GROWTH FACTORS AND CANCER

v-s/s was the first oncogene shown to encode a growth factor. The amino acid sequence of the B subunit of PDGF is homologous to p28s/s, the major transforming protein of simian sarcoma virus suggests that overproduction of PDGF-iike growth factors may underlie the unrestrained proliferation of some cancer cells (reviewed in (Westermark and Heldin 1991)). A functional autocrine loop driven by PDGF has been demonstrated in cultured cells (Fleming, Matsui et ai. 1989). Expression of PDGF and its receptor has also been demonstrated in certain human tumours such as glial-derived neoplasms (Westermark and Heldin 1991). Other oncogenes encoding growth factors include /nt-2, hst and FGF-5, which encode members of the FGF family. Similarly, transforming growth factor a (TGFa), which is a single polypeptide of 50 amino acids that is structurally and functionally related to EGF and binds to the same receptor, is also expressed in carcinomas (Derynck, Goeddel et al. 1987). Although deletions and mutations of the EGF receptor lead to its constitutive activation (Ullrich and Schlessinger 1990), such mutations appear to play a minor role in human cancer. In contrast, enhanced expression of the EGF receptor and/or other 29

members of this receptor family (e.g. erbB-2 and erbB-3) has been frequently implicated in human cancer, particularly in carcinomas of breast, lung, stomach and ovary (reviewed in (Aaronson 1991)). TGF-p may act as a negative growth regulator in breast cancer, reminding us that many growth factors can have positive and negative effects depending on context. A number of other tyrosine kinase systems have been elucidated recently and appear to play a role in cancer. A series of studies revealed a relationship between hepatocyte growth factor (HGF), scatter factor and the c-met proto-oncogene tyrosine kinase receptor. HGF is a potent mitogen for hepatocytes in culture and promotes liver regeneration after partial hepatectomy. HGF also stimulates the proliferation of endothelial and epithelial cells. Independently, an activity that stimulated the motility of certain epithelial cells (e.g. MDCK cell line) had been isolated and termed scatter factor (reviewed in (Gherardi and Stoker 1991)). The sequence of cDNA clones encoding for these polypeptides were virtually identical. Moreover, both purified HGF and scatter factor bind and stimulate tyrosine phosphorylation of c-met (Naldini, Weidner et al. 1991). Thus, scatter factor and HGF are the same polypeptide and are the ligands for the c-met tyrosine kinase receptor. The met gene can be transformed into an oncogene either by gene amplification or overexpression. The HGF/scatter factor receptor has been found in human stomach carcinoma cell lines (reviewed in (Aaronson 1991)). The c-met gene lies on chromosome 7 and monosomy 7 or 7q in breast tumours correlates with significantly shorter metastasis-free survival (Bieche, Champeme et al. 1991) and it has been thus hypothesised that c-met can exert tumour suppressor activity in certain cases. Indeed, the reduced expression of met in tumours could be related to the extent of tumour differentiation (Tsarfaty, Resau et al. 1992). The met ligand HGF has been reported also to inhibit the growth of hepatocellular carcinoma cells (Shiota, Rhoads et al. 1992). A novel putative receptor protein tyrosine kinase of the met family has recently been cloned and localised to human chromosome region 3p21 (Ronsin, Muscatelli et al. 1993), a region frequently deleted in small cell lung cancer and in renal cell carcinoma, a region believed to harbour unidentified tumour suppressor genes. The ras proteins are important components of the signal transduction machinery of tyrosine kinase receptors. Activating mutations of the ras family are frequently detected as oncogenes in lung, colon and pancreatic carcinomas (Boss 1989). The existence of autocrine or paracrine loops involving polypeptide growth factors also provide opportunities for therapeutic intervention. Antibodies directed against receptors have been studied (Kumar, Shepard et al. 1991). The fact that the tyrosine kinase activity of the receptor is central for the transmission of the 30

mitogenic signal has prompted the identification of cell-permeable inhibitors that block unregulated tyrosine kinase activity inside the cell. The tyrphostins are a series of molecules that inhibit tyrosine kinase activity and block cell proliferation (Levitzki 1992). They provide potential antiproliferative agents that could inhibit cell proliferation through a novel mechanism.

1.8. LUNG CANCER.

l.B .a INTRODUCTION

Lung cancer is the commonest fatal malignancy in the developed world. In 1987, 1 in 4 deaths in England and Wales were from cancer. Lung Cancer accounts for 1 in 3 male and 1 in 6.5 female cancer deaths (W.H.O. 1988.). Lung cancer mortality in women now exceeds that from breast cancer. Despite decades of anti-smoking campaigns, the incidence of lung cancer deaths is expected to rise even further over the next twenty years with an increasing number of cases unrelated to smoking (Osann 1991; W.H.O., 1982 a). Lung tum ours are carcinomas originating from respiratory epithelium and are classified on the basis of histological type (W.H.O. 1982b). The two main types are small cell lung cancer (SCLC) with few subtypes and non-small cell lung cancer (NSCLC) with a large number of subtypes. Patients with NSCLC, including squamous cell carcinoma, adenocarcinoma and large cell carcinoma, which collectively accounts for 75% of all new cases, the hope for long term survival relates to resectability of the tumour at the time of presentation. However, this is successful for less than 30% of all patients. Even for those who undergo a curative resection, the 5 year survival rate is less than 35% (reviewed in (Carney and De-Leij 1988)). SCLC constitutes 25% of all primary pulmonary cancers, hence it is the sixth most common malignancy and the fourth leading cause of death from cancer. SCLC distinguishes itself from other forms of lung cancer in that it metastasises early and most patients have disseminated disease at first presentation. Primary resection is therefore rarely possible. Indeed, patients that were surgically resected and died with-in one month of surgery were found at autopsy to have a high incidence of residual cancer, most often distant métastasés (Matthews, Kanhouwa et al. 1973). The predominant influence of systemic cancer on survival in SCLC is confirmed by the trivial effect of surgery and radiotherapy on the life span of patients. The chance for cure/long term survival is directly related to the sensitivity of the tumour to cytotoxic therapy, including combination chemotherapy and radiotherapy. The epidemic proportions of the disease are contrasted sharply by the general failure of 31 conventional treatment. SCLC has a sinister reputation with a median survival time was 2 months. In the mid 70's SCLC was found to be extremely sensitive to both chemotherapy and radiotherapy in the majority of cases. A variety of chemotherapy regimens have been used with response rates of up to 80% in unselected patients, and complete response rates of 50-60% in patients with limited disease (Minna, Higgins et al. 1985). Despite Initial sensitivity to radio and chemotherapy, it almost invariably relapses and is resistant to further treatment, so that the 2 year survival of patients with SCLC remains less than 5% ((Smyth, Fowlie et al. 1986). Moreover, for those patients who survive in excess of 2 years from initial diagnosis of SCLC, there is an increased risk of mortality from other types of lung carcinoma (Minna, Higgins et al. 1985). This dismal prognosis has shifted interest from pharmacology to a more detailed understanding of the fundamental biology of SCLC, which may serve as a basis for the development of novel therapeutic approaches.

1.8.b Classification of Lung Cancers.

By definition, bronchogenic carcinomas arise from the mucosa of the tracheo­ bronchial tree (W.H.O. 1982b; Gazdar and McDowell 1988). Squamous cell carcinomas (SQCC) arise from metaplastic squamous epithelial cells. Cell lines demonstrating ultrastructural features of squamous differentiation include well and poorly differentiated SQCC, adenosquamous and mucoepidermoid carcinomas. All these tumour types demonstrate upregulation of EGF receptor expression and markers characteristic of squamous differentiation (involucrin, transglutaminase activity, higher molecular weight keratins and cornified envelope formation). Adenocarcinomas appear to arise from several cell types including surface epithelium (ciliated and mucus producing cells) and give rise to mucin-containing or mucin-secreting tumours, and the progenitor cells of the peripheral airways (Clara cells and Type II pneum ocytes ) giving rise to peripheral airway cell tum ours (W.H.O. 1982b; Gazdar and McDowell 1988). The latter types often demonstrate morphological features of lepidic and papillary growth, and include the WHO adenocarcinoma subtypes bronchioloalveolar and papillary. Large cell carcinomas are predominantly undifferentiated tumours, which may represent its basal or stem cell origin. SCLC were previously thought to arise from pulmonary neuroendocrine cells (Gazdar, Bunn et al. 1985), however it now seems more likely that they arise from a common stem cell (see below). Bronchial carcinoid are a minor category of lung cancers which have neuroendocrine features. These tumojj^ are thought to arise from submucosal glands 32

_gni arp n?t highly malignont^ Atypical carcinoids lie between the SCLC and the carcinoid tumours. As mentioned above each of the four major types of lung cancer reflects phenotypic features of the cell types that make up the normal bronchial epithelium. A new hypothesis that is becoming increasingly accepted is that the four types of lung cancer are related through a common differentiation pathway in the bronchial epithelium (reviewed in (Mabry, Nelkin e t al. 1991)). This proposal is supported by clinical evidence that demonstrates potential for transitions between the different tumours (i.e. that tumours in patients can be admixtures of the different phenotypes). Neuroendocrine (NE) features can be found in NSCLC tumours. This occurs in about 25% of adenocarcinomas and confers an initial sensitivity to chemotherapy and radiotherapy, but these tumours with NE features are faster growing and have a worse prognosis compared to adenocarcinomas without NE features. Furthermore, individual cells have been observed to manifest features of the SCLC and NSCLC phenotypes simultaneously. It is proposed that these transitions mimic normal cellular transitions in bronchial mucosa and are mediated by the consistent genetic abnormalities now being described for lung cancer. A cell culture model system for one of these transitions, SCLC to the large cell undifferentiated phenotype has been developed (Mabry, Nakagawa et al. 1988). Overexpression of a mutated Harvey (Ha)- ras gene in classic SCLC that do not overexpress the c- or N-myc genes causes no phenotypic changes. In contrastih'senion of a mutated Ha-ras gene into SCLC cells that overexpress either an endogenously amplified c-myc or N-myc gene, or atransTicted~ human c-m yc gene causes transition to the larae-cell undifferentiated cancer phenotvpe (Mabrv^akaaawa et al. 1989). Thus, in an in vitro model, the combination of two genetic abnormalities that can occur in lung cancer results in a transition of phenotype, where there are losses of NE markers, acquisition of drug resistance patterns typical of NSCLC. There is a concomitant fall in cellular production of GRP, and an increase in expression of EGFr on their surface and production of TGF-a. This model has been extended linking differentiation of adult normal bronchial epithelial cells and transitions between the lung cancer phenotypes with normal interactive roles for the chromosome 3p, Rb, and pSS genes and interplay between the myc, ras, and PKC gene products (Mabry, Nelkin e t al. 1991).

1.8.C SMALL CELL LUNG CANCER: Clinical features.

SCLC tends to present centrally and to infiltrate the submucosa. This causes bronchial obstruction with consequent cough, dyspnea, wheezing, chest discomfort, haemoptysis or post-obstructive pneumonia. Two-thirds of patients will have 33 evidence of distant métastasés, most often to the liver, bone, bone marrow, or brain, at the time of original diagnosis. The clinician must also be alert to signs and symptoms which may be manifestations of the many paraneoplastic syndromes associated with SCLC and which may be confused with symptomatic métastasés (Bunn and Ridgway 1989). These syndromes are often due to the synthesis and release by the tumour of various hormones and peptides. Adrenocorticotrophic factor (ACTH) secretion causes ectopic Cushing's syndrome. Arginine vasopressin (AVP) and atrial natriuretic (ANF) (Bliss, Battey et al. 1990; Gross, Steinberg et. al. 1993) release causes hyponatraemia and the syndrome of inappropriate antidiuretic hormone secretion. Immune reactions to tumour antigens, often result in neurological syndromes such as the Eaton-Lambert myaesthenic syndrome, in which the presence of circulating antibodies react with voltage-gated calcium channels (de Aizpurua, Lambert et al. 1988). Visual deficits produced by retinal degeneration and sensory neuropathy are two other syndromes most commonly found in SCLC, in which serum antibodies reacting with both tumour and neuronal tissue are also observed (Bunn and Ridgway 1989). The characteristics of classic SCLC cells are that they have little cytoplasm and indistinct or absent nucleoli. Cells are 2-3 times the size of mature lymphocytes and their nuclei are darkly stained. Small dense-corje-ettaniüîerà fé ^ ig e n in the cytoplasm, and are aumanifestation of the peptide secretion that Is the hall mark of the "neuroendocrine" properties observed in SCLC, but relatively infrequently in other, types of lung cancer. SCLC cell lines grow as tightly clustered spheroids in suspension in culture, form colonies in soft agarose and are tumourigenic in athymic nude mice. They have a doubling time ranging from 32-72 hours. In contrast NSCLC cell lines grow as attached monolayer cultures demonstrating substrate adherence. They are also tumourigenic in nude mice and will form colonies in soft agarose. SCLC has distinct neuroendocrine biochemistry, these include: L-Dopa decarboxylase and Neuron Specific Enolase, the BB isoenzyme of Creatine Kinase, Chromogrannin A and various peptide hormones including Gastrin-releasing peptide, vasopressin, neurotensin, parathormone and many others. 25% of adenocarcinomas express endocrine markers J_-Dopa Decarboxylase, Neuron^ecific enolase. thesecells are faster growing, have a worseprognosis and are chemosensitive in contrast to other adenocarcinomas (Gazdar, Helman e t al. 1988). •*-— — In studies of a large number of SCLC cell lines it is clear that heterogeneity of expression exists, allowing subclassification of SCLC into two distinct types (Carney, Gazdar et al. 1985): Classic cell lines which express high levels of all 4 biomarkers and variant cell lines in which there is selective loss of some of these biomarkers. In vitro studies have shown that variant cell lines have a more rapid growth rate and a 34

higher cloning efficiency in soft agar and nude mice than classic cell lines. Variant cell lines have larger cell morphology^more undifferentiated and are relatively chemo and radio resistant. Variant cell lines have been proposed to represent the in vitro counterpart of the poor prognosis, mixed Small cell/large cell SCLC subtype. 1 0 % ^ 'SCLC cell lines are biochemical variants which lack DDC and other biochemical markers, another 20% are morphological variants which grow as loose aggregates or monolayers with short doubling times and high cloning efficiencies. Although a conversion from classic to variant phenotype might explain the clinical development of resistance to chemotherapy in some cases , other mechanisms are also likely to be operative in the change from responsive to nonresponsive in SCLC, this Is because only 10% of SCLC patients have tumours showing histology in which large cell admixtures are present . At post-mortem this figure has moderately increased to 13- 28%, despite the maiority of these tumours being completely refractory to treatment (Sehested, Hirsch et al. 1986). Seyeral longitudinal studies show that the more extensiye the disease the higher the incidence of positivity of endocrine markers (Bork, Hansen et al. 1988; Johnson, Pandian et. al. 1991 and reviewed in (Carney 1991)). Nejjron Specific Enolase (NSE), is a highly specific m^keJ-iac*,ae,ucQ:Des. Its levels are more^elevateFln extensive disease. Sequential determinations of NSE levels are of value in monitoring the response to chemotherapy and the detection of relapse. In several studies a rise in NSE levels was detected 12-24 weeks prior to the clinical detection of relapse. Levels of Chromogrannin A, BB isoenzyme of Creatine Kinase and Carcinoma embryonic antigen are also of prognostic value and showed a significant correlation with tumour response to cytotoxic therapy and relapse after therapy. Brambilla and his colleagues (Brambilla, Moro et al. 1991) have also showed in a longitudinal study (before and after the acquisition of resistance to chemotherapy and radiotherapy) involving 20 patients, an increase M neurosecretaQLgmQulê^j[L^CLCLcd!§J(n^ cells appear more differentiated after chemotherapy. Large cells in these treated tumours were not anaplastic, but seemed to be differentiated tumour cells showing epithelial and NE signs of differentiation within one tumour, and sometimes within the same tumour cell. This is not surprising, in view of the common stem cell origin of lung cancer is taken into account (Mabry, Nelkin et al. 1991). However, these results though in agreement with those of Bepler et. al. (Bepler, Jaques et al. 1987), are in contradiction with other observations of cell lines and biopsies from treated patients (Berendsen, De-Leij, et al. 1988) and with long-term untreated SCLC cultures, in which it has been shown that l^ge cells exhibited a dedifferentiated phenotype with loss of NE markers. On balance it seem s m ost likely th at in the m ajofil^^flzâëes of ^SCCtr cells are of the classic phenotype and maintain this classic phenotype during chemotherapy and the acquisition of chemoresistance , in a minority of cases there 35 may be conversion from classic to variant phenotype or a maintenance of variant phenotype during the course of the disease.

1.9. Causative agents. All bronchial carcinomas are related to smoking with the exception of bronchial carcinoids. The association with smoking of bronchoalveolar carcinomas, a subgroup of adenocarcinomas which arise from the terminal bronchioles and alveoli, is not as strong as that of the other types. Smoking accounts for 91 % of male lung cancer deaths in the USA in the 1970's (W.H.O. 1982a). Further avoidable causes of lung cancer include exposure to occupational hazards such as asbestos, arsenic, bischloromethyl ether, chromium, mustard gas, nickel, polycyclic hydrocarbons and ionising radiation. These account for 5% of female and 15% of male lung cancer deaths (W.H.O. 1982a). There are synergistic interactions between different carcinogens, e.g. smoking and asbestos or radiation exposure.

1.10. GENETIC CHANGES

The multistep model of carcinogenesis proposes that two or more genetic mutations are required to transform a cell and to enable its monoclonal expansion into a tumour. Inheritance of one or more these mutations allows tumours to develop earlier than if several spontaneous mutations have to accumulate in a cell. Genetic factors thought to influence the incidence of lung cancer include the inducibility and activity of several enzyme systems that metabolically activate components such as polynuclear aromatic hydrocarbons and /V-nitrosamine present in cigarette smoke. Cigarette smoke induces the cytochrome P-450-dependent microsomal mixed function oxygenase aryl, hydrocarbon hydroxylase, and another microsomal monooxygenase associated enzymatic system, dimethlynitrosamine demethylase. The activity nf thMA pnyyme*; are higher in lung tumour tissues from jmokers than from non-smokers (Petruzzelli, Camus et al. 1988). Extensive metabolisers of debrisquine were found to have a significantly elevated risk of developing lung cancer (Ayesh, Idle et al. 1984). Cigarette smoke also increases the levels of microsomal epoxide hydrolase and UDP-glucuronosyltransferase and decreases the level of the enzyme, glutathione S-transferase (Petruzzelli, Camus et al. 1988). The normal levels of some of these enzymes covers wide ranges and the extents to which they can be induced might reflect a genetic component regulating susceptibility to lung cancer among smokers. The long term retention (up to 4 months) of a number of more than 3000 compounds present in cigarette smoke 36

(Petruzzelli, Camus et al. 1988) points to the importance of efficient deactivating systems as a protection against their harmful qualities. These genetic abilities to deactivate carcinogens might play a very important role in preventing the kinds of chromosomal damages that lead to the inactivation of suppressor genes and to the activation of oncogenes that have been documented in lung cancer (see below). Enhanced formation of benzo(a)pyrene:DNA adducts has been detected in the monocytes of individuals predisposed to lung cancer (Rudiger, Nowak et al. 1985). These findings suggest that enzymes systems responsible for DNA-adduct excision and repair might be defective in these individuals.

1.10.8 Hereditary predisposition - lung cancer families. Numerous studies have been conducted to assess the role genetics plays in the development of lung cancer following exposure to long term carcinogens, especially cigarette smoke (Heighway, Thatcher et al. 1986). That susceptibility has a genetic component is indicated by these studies which show that ( 1) not all individuals who smoke develop lung cancer; ( 2) some individuals who do not smoke develop lung cancer; and (3) some high cancer families have an increased incidence of lung cancer. There is a significant excess of deaths due to lung cancer among relatives of lung cancer patients, as well as to non-cancer respiratory diseases compared with controls. A synergistic effect between hereditary predisposition and cigarette smoking has been noted. This is strikingly demonstrated by the report of deaths of 58-year old identical male twins 2 months apart, both having smoking-related alveolar cell carcinoma which was metastatic to the brain. Reports linking inherited mutations in the retinoblastoma gene on chromosome 13ql4 (Sanders, Jay et al. 1989), the p53 gene at chromosome 17p13 (Malkin, Li et al. 1990), and a Ha-ras allele on chromosome lip (Heighway, Thatcher et al. 1986) to an increased risk of NSCLC and SCLC have been published. Inheritance of a major autosomal gene has been postulated to determine susceptibility to lung cancer at ages younger than 50 years (Sellers, Bailey-Wilson et al. 1990; Sellers, Elston et al. 1992).

1.10.b Chromosomal abnormalities. Cytogenetic analysis has been performed on established lung tumour cells as well as fresh lung tumour specimens. Several chromosomes in SCLC cell were found to be abnormal (Whang-Peng, Bunn et al. 1982). Total chromosome distributions ranged from hypodiploid to octaploid, with most structural abnormalities occurring on chromosome 1, 2, 3, and 10 (Whang-Peng, Bunn et al. 1982). Cytogenetic data obtained for 12 SCLC established cell lines and 2 fresh SCLC tumour specimens indicated that the region of chromosome 3p14-p23 was consistently deleted in 100% 37 of the cells analysed (Whang-Peng, Bunn et al. 1982). This loss of material from the short arm of chromosome 3 has also been identified in NSCLC tumours and established cell lines (Whang-Peng, Knutsen et al. 1991). Further studies on 64 fresh lung tumours from 47 patients, showed a 100% loss of heterozygosity occurred on chromosomes 3p, 13q, and 17p in SCLC (Yokota, Wada et al. 1987). These chromosomal losses occurred early in the development of the tumour before N-myc amplification, chromosome 1 1 p deletion, or clinical appearance of m etastasis. Only chromosome 3 lost heterozygosity in adenocarcinoma. A study of 54 NSCLC tumours showed loss of heterozygosity at 17p and 11, in 8 /9 SQCC (W eston, Willey e t al. 1989). Recently, more detailed analyses indicated numerous 'hot spots' for abnormalities. These 'hot spots' could signal the presence of a gene that might be involved in the pathogenesis of lung cancer either through activation or inactivation. Several of the chromosomal regions pinpointed by these studies e.g. 17p, 13q, and 1 Ip have been shown to be sites of known dominant and recessive oncogenes (Whang-Peng, Knutsen et al. 1991). Suppressor activity of chromosome 11 is demonstrated by experiments in which a single isolated chromosome 11 from a normal human fibroblast was able to suppress tumourigenesis of cells from a uterine carcinoma and a Wilm's tumour following microcell fusion (Saxon, Srivatsan et al. 1986).

1.11. RECESSIVE ONCOGENES

1.11.a Retinoblastoma gene. The existence of recessive oncogenes or tumour suppressor genes, in tumours, was put forward in a model by Knudson to explain the early appearance and familial pattern of retinoblastomas in young children. This model proposed that two genetic lesions are required for tumour development. In the hereditary form of the disease, one lesion is transmitted through the germ line, whereas the second lesion is acquired postnatally as a somatic mutation. In the sporadic forms of the disease, both lesions have to occur postnatally, thus explaining the relative rarity of this form of the disease. Later cytogenetic analyses on retinoblastoma cells demonstrated a deletion of chromosome 13 material at band ql4, as well as loss of heterozygosity (Cavenee, Dryja et al. 1983; Dryja, Cavenee et al. 1984). The cloning and characterisation of the retinoblastoma gene (RBI) showed that loss of the gene or its protein product by homologous deletion or mutation was the event that resulted in tumour development. There is evidence that survivors of hereditary retinoblastoma are at a higher risk of developing lung cancers as adults and develop them at an earlier age than the general population (Leonard, MacKay et al. 1988). Relatives of retinoblastoma 38

patients who are carriers of an RBI mutation have a 15-fold increased risk of lung cancer than the general population (Sanders, Jay et al. 1989). Cytogenetic analyses of SCLC tumours and established cell lines indicate an increased number of deletions involving chromosome 13 as well as a more frequent loss of the entire chromosome 13, loss of heterozygosity at 13ql 2-13q33 in over 75% of cases (Whang-Peng, Bunn et al. 1982; Yokota, Wada et al. 1987; Johnson, Sakaguchi et al. 1988). Hence loss of the RBI gene may play some role in the development of lung tumours. In fact, analysis of 50 established lung tumour cell lines and 8 primary SCLC lung tumour tissues revealed structural abnormalities at the DNA level and abnormalities in size and expression of the 4.7 kb RBI mRNA in 65% of SCLC and 75% of carcinoid lines (Harbour, Lai et al. 1988); no abnormalities were detected in NSCLC cell lines. Rbl protein product, pRB, is approximately 105 kDa and is located in the nucleus. This protein is expressed in all cell types and is phosphorylated at multiple sites just prior to DNA replication at the Gl/S boundary (Buchkovich, Duffy et al. 1989). Both phosphorylated and unphosphorylated forms of pRB complex with DNA viral proteins involved in cellular transformation e.g. SV40 large T antigen, adenovirus El a protein and the human papillomavirus E7 protein (Whyte, Buchkovich et al. 1988; Dyson, Howley et al. 1989). The picture that emerges is that each of these viral proteins is specifically inactivatirig the RBI gene product as a requisite to transforming the infected cell, a view consistent with the proposed role of pRB as a tumour suppressor. This is further supported by the demonstration that réintroduction of a cloned RBI cDNA into retinoblastoma and osteosarcoma cells that lack active RBI genes results in a decreased growth rate of these cells and loss of the ability to grow in soft agar (Huang, Yee et al. 1988).

1.1 l.b p53. p53 was originally cloned from chemically transformed mouse cells. The human p53 recessive oncogene maps to chromosome 17pl 3 (Isobe, Emanuel et al. 1986), a region that has been found to suffer loss of heterozygosity in colon, breast, brain tumours and also in NSCLC and SCLC cell lines and primary tumours. Wild type p53 is a nuclear protein which is thought to be active in the Go to G i transition in cell division and efficiently and specifically binds DNA virus-derived transforming proteins: the SV40 large T antigen, the adenovirus Elb protein and the papillomavirus E6 protein. p53 acquires oncogenenic potential at the DNA level primarily as a result of a point mutations in the wild-type gene whose normal function appears to be suppression of cellular transformation (Lane 1992). This results in abnormal mRNAs encoding m utant p53 proteins which have an increased half life and which bind to heat shock protein hsc70. A study of 30 lung cancer cell lines of a variety of 39

histological types detected mRNA abnormalities, including changes in size, reduced or absent expression of message as well as point or small mutations within the open reading frame, in 74% of cases. Gross abnormalities at the DNA level, rearrangements and two homozygous deletions, were found in 7% of cases. Point mutations in p53 mRNA and the resulting proteins of increased stability have been detected in primary lung tumour specimens and p53 is a very common mutation in SCLC (Takahashi, Nau et al. 1989; Takahashi, Takahashi et al. 1991). It is becoming increasingly clear that inactivation of normal p53 protein in lung cells is involved at some stage in lung tumour development. A recent report demonstrates germ-line p53 mutations in all 5 Li-Fraumeni syndrome families studied; lung cancer is one of the tumours that members of these families develop (Malkin, Li et al. 1990). There are sev ^l mechanisms to remove wild-type p53 which include homozygous deletions at the DNA level and point mutations in exons or introns that result in no or in mutated mRNA encoding mutant or no p53 protein. Mutated p53 protein can sequester wild-type p53 in heteroduplexes rendering the cells functionally homozygous for the mutant. The importance of p53 inactivation to the development of lung cancers has been shown by the high incidence of lung adenocarcinomas that develop in transgenic mice carrying a mutant p53 gene (Lavigueur, Maltby et al. 1989). Transfection of wild-type p53 suppressed the growth of a human lung cancer cell line expressing p53 (Cajot, Anderson et al. 1992). Alterations of the p53 gene are common and critical events for the maintenance of malignant phenotypes in small­ cell lung carcinoma (Sameshima, Matsuno et al. 1992). Transfection with wild-type p53 cDNA caused a significant decrease in the cloning efficiency and reduced tumourigenic potential in nude mice compared to transfection with mutant p53 and control. Transfection of wild-type p53 also induced apoptosis in a colon cancer cell line (Shaw, Bovey e t al. 1992). The induction of apoptosis by p53 following genotoxic insult may act as a defence mechanism to protect the organism from propagation of cells that have sustained mutation. Abrogation of this p53 pathway is the most common specific alteration in human cancer, and may be central to the progression of the disease and to it's response to treatment by radiation and chemotherapeutic drugs (Lane 1993).

1.11.C 3p. The consistent loss of the region of chromosome 3 between bands p14-p23 in SCLC has prompted speculation that this region encodes a tumour suppressor gene. Loss of heterozygosity at chromosome 3p is seen in SCLC (Naylor, Johnson et al. 1987), NSCLC (Weston, Willey et al. 1989), cervical cell cancer, sporadic renal cell 40 carcinoma, hereditary renal cell carcinoma and in breast cancer. Studies of aminoacylase-1 (ACY-1), which maps to 3p21.1, indicate a consistent decrease of this enzym e in SCLC (Miller, Minna e t al. 1989). The c-erM ^ proto-oncogene encodes a 55 kDa thyroid hormone receptor (THR-p) and is found on human chromosome 3p21-25. This region of chromosome 3 lies within the portion of the short arm found to be deleted in SCLC, and loss of heterozygosity for c~erbAp w as detected in 6/6 SCLC cell lines examined (Dobrovic, Houle et al. 1988). The gene encoding the retinoic acid receptor-p is also within the 3p deleted region at 3p24 (Gebert, Moghal et al. 1991). Some of the effects of retinoic acid on lung tumours might be related to alterations in this gene. Deletion of the protein tyrosine phosphatase-gamma gene on the short arm of chromosome 3 has also been described (LaForgia, Morse et al. 1991). This gene may be important in regulating the level of protein phosphorylation on tyrosine residues. As has been described earlier tyrosine phosphorylation is a major pathway in signal transduction, which is activated by polypeptide growth factors as well as neuropeptides. Hence, deletions of the protein tyrosine phosphatase-gamma gene could increase the level of protein phosphorylation on tyrosine residues with-in the cell resulting in transformation. A new putative receptor protein tyrosine kinase of the m et family with structural similarity to the product of the C-MET proto-oncogene, the receptor for hepatocyte growth factor and scatter factor, has been localised to 3p21 (Ronsin, Muscatelli et al. 1993).

1.11 .d Other recessive oncogenes. Several additional putative tumour suppressor genes have recently been reported. These include the Wilm’s tumour gene at chromosome 11pl4 (Call, Glaser et al. 1990), the DCC gene (cell-adhesion molecule) first detected as a frequent deletion in colon cancer at chromosome 8q22 (Fearon, Cho et al. 1990), the neurofibromin-1 gene (GTPase-activating protein) at 17ql 1.2 (Wallace, Marchuk et al. 1990) and the interferon a and p genes at 9p22 (Einhom, Grander et al. 1990). All of these genes lie at chromosomal locations which are involved in deletions and non-reciprocal translocations in SCLC and NSCLC

1.12. DOMINANT ACTING ONCOGENES

Transforming genes are present and often expressed as proto-oncogenes In normal tissues and are highly conserved during evolution suggesting that they are important cellular components. The identification or these proto-oncogenes as constituents of normal cells and the elucidation of the mechanisms of genetic mutation, translocation, amplification and deletion by which they are activated to play dominant 41 roles in oncogenesis represent a major advance in understanding the molecular basis of cancer. At least 11 of the known dominant-acting oncogenes have been shown to be activated in a variety of lung cancers. The precise stages at which these oncogenes become activated in the lung tumours and the exact role they play in the development and progression of these lung tumours has not been delineated.

1. 12.8 m yc family c-myc: The c-myc proto-oncogene is located on human chromosome 8. Two phosphoproteins, p64'^‘myc and p67'^’myc, are translated from the c-myc mRNA (Little, Nau et al. 1983). The nuclear localisation and in vitro DNA binding properties of these proteins suggest that they play an important role in regulating cell growth, c- myc was the first proto-oncogene found to have altered expression in SCLC established lines (Seifter, Sausville et al. 1986) and in lung tumours (Johnson, Makuch et al. 1988) and is the only member of the myc family to have correlation with clinical outcome in patients with these tumours. The DNA of this proto-oncogene was found to be amplified 20-76-fold in 8/18 SCLC cell lines examined (Little, Nau et al. 1983). In addition c-myc expression as 2.7kb mRNA was found to be over-expressed 15-35 times in these cells even in the absence of DNA amplification, this occurred by increased rates of transcription from the myc prom oter (Seifter, Sausville e t al. 1986; Krystal, Birrer et al. 1988), the cytoplasmic half-life of the c-myc mRNA was unchanged. A block to normal c-myc transcription elongation is located in the first intron of the c-m yc gene. This block is absent in some of those cell lines expressing elevated levels of c-m yc mRNA without gene amplification (Krystal, Birrer et al. 1988). Over-expression of c-myc was found predominantly in variant SCLC cell lines and rarely in classic SCLC cell lines (Johnson, Ihde et al. 1987). Thus amplification/overexpression of c-m yc is associated with a more virulent clinical course and shorter survival, suggesting a role for c-myc in tumour growth which may be clinically significant. Over-expression of c-myc in NSCLC tumours has also been reported (Cline and Battifora 1987).

N-myc: The N-myc proto-oncogene was first detected as amplified in cells of advanced neuroblastomas. The N-myc proto-oncogene is located on human chromosome 2. Though different genes, c-myc and N-myc, encode proteins with deduced amino acid sequence with significant similarities in the second and third exons (Schwab, Varmus et al. 1985). N-myc demonstrates the same uncoupling of DNA and mRNA amplification as was noted for c-myc. N-myc was amplified 5-fold in primary lung tumours prior to metastasis but not in normal tissue. N-myc amplification and over­ expression were found in both classic and variant SCLC cell lines. 42

L-myc: L-myc proto-oncogene resides on human chromosome lp32 and was found to be amplified 10-20-fold in 4/8 SCLC cell line DMAs and to a similar level in a SCLC tumour sample but not in adjacent normal lung tissue DNA. Again discordant over-expression of L-myc RNA was noted. Subsequent experiments showed that a family of L-myc mRNAs are generated by alternative splicing of the primary transcript and by use of alternative polyadenylation signals (Kaye, Battey et al. 1988). L-myc expression is limited to SCLC tumour cells (Nau, Brooks et al. 1985). The c-myc, N-myc and L-myc genes are highly homologous in their second and third exons which are translated into protein (Kaye, Battey et al. 1988). The first exon of each gene is untranslated and diverges most widely among the three genes. The c-myc protein is known to be involved in cell division and its mRNA is amplified following stimulation by growth factors or fetal calf serum (Greenberg and Ziff 1984). This proto-oncogene resides in the nucleus and is involved in the immortalisation of primary cells in culture (Land, Parada et al. 1983). The c-, N-, L-myc proteins cooperate with the Ha-ras oncogene in transforming primary rat fibroblasts with L-myc producing foci at 1-10% the rate observed for c- and N-myc (Land, Parada et al. 1983; Schwab, Varmus et al. 1985). Although the myc family members demonstrate a high degree of conservation at the nucleic acid and amino acid levels, suggesting common intracellular functions, the differential tissue expression of these genes during embryogenesis and adult life and their differing abilities to complement ras suggest that they each play important and unique roles (Zimmerman, Yancopoulos et al. 1986).

1 .12.b ras family: K-, H- and N-ras. Members of the ras family of proto-oncogenes, comprised of K- ,H- and N- ras , are inner plasma membrane-associated GTPases that bind GTP to cleave it to GDP; they are involved in signal transduction and act as a link between tyrosine kinases and serine/threonine kinases (Barbacid 1987). Activated ras oncogenes have been found in a variety of human tumours, ras can cooperate with myc to transform fibroblasts (Land, Parada et al. 1983). The involvement of ras proto-oncogenes in human lung tumours was first detected by transfection of tumour DNAs into NIH3T3 cells. K-ras restriction fragments were found in cells transformed by DNAs from SCLC, adenocarcinoma, squamous cell carcinoma and undifferentiated lung carcinoma cell lines; the K-ras -2 gene 12pl 2 was found to be mutated in some of the original tumour tissue (Land, Parada et al. 1983). Activating point mutations were localised predominantly in codons 12, 13 and 61. 14/77 NSCLC tumour samples were found to have K-ras mutations, all of which were single point mutations in codon 12 from 43 adenocarcinomas (Santos, Martin-Zanca et al. 1984). H-ras mutated at codon 61 was detected in SQCC cell line. Loss of heterozygosity of the c-H-ras gene on chromosome 11p15 has been associated with more aggressive NSCLC tumours. N-ras was found in one undifferentiated lung cancer. The preponderance of ras mutations in lung tumours were K-ras (Suzuki, Orita et al. 1990). The K-ras activation in these lung tumours was thought to represent an early event which was the direct consequence of exposure to tobacco smoke. All of the K-ras lesions were detected in tumours from smokers and all the tumours were small and non-metastatic. These findings, together with the ability of mutated H-ras to transform normal human bronchial epithelium following transfection in vitro (Yoakum, Lechner et al. 1985) and to induce lung adenomas in mice made transgenic for H-ras (Suda, Aizawa et al. 1987), reinforce the suggestion that mutations in ras proto-oncogenes are involved at an early stage in lung tumourigenesis. Conversely, involvement of ras proto-oncogenes in later stages of lung carcinogenesis is indicated by several studies noting the association of activated ras proto-oncogenes with metastatic potential of transformed human bronchial epithelial cells (Bonfil, Reddel et al. 1989) and with differentiation of established SCLC lines to increased endocrine production (Mabry, Nakagawa et al. 1988) or to a large cell lung cancer phenotype (Mabry, Nakagawa et al. 1989).

1.12.C Other dominant-acting oncogenes.

1.12.c l. c-Raf-} The c-/?af-1 proto-oncogene maps to chromosome 3p25, a region of chromosome 3 frequently deleted in SCLC and NSCLC lung tumours. Cytogenetic analyses of SCLC cell lines show th at one c-/?a7-1 allele is deleted in approximately 80% of the lines (Sithanandam, Dean et al. 1989). However, in another study, c-Raf~ 1 mRNA was expressed at high levels in 12/12 SCLC cell lines (Kiefer, Bepler et al. 1987). Transcriptional activation of c~Raf-^, along with other proto-oncogenes, was considered to be a common feature of NSCLC (Kiefer, Wegmann et al. 1990) and co­ transfection of c-/?a7-1 and c-myc into human bronchial epithelial cells immortalised with SV40 large T antigen resulted in the transformation and growth of these cells as large cell carcinomas in nude mice (Pfeifer, Mark et al. 1989). These large cell tumours expressed high levels of neuron-specifc enolase. The c-Raf protein, p74^*C is a serine/threonine-specific kinase which is membrane localised. Phosphorylation at serine and threonine residues activates the kinase activity of p74^®^ which may participate in signal transduction from the membrane to the cytoplasm (Morrison, Kaplan et al. 1988; Morrison, Kaplan et al. 1989). It has been demonstrated that loss 44 of the amino-terminal one-third of the protein results in a constitutively-active transforming phosphoprotein fragment p48^^^ (Schultz, Copeland et al. 1988).

l.lZ.cii. c-erbB-1 The c-erbB-] proto-oncogene encodes the epidermal growth factor receptor (EGF-R), a tyrosine specific protein kinase capable of autophosphorylation; the c- erbS-1 gene is on chromosome 7 (Downward, Yarden et al. 1984). Expression of the 170kDa EGF-R on lung tumours was detected in 8/8 NSCLC (Haeder, Rotsch et al. 1988). The presence of EGF-R in SCLC cell lines is controversial (Cemy, Barnes et al. 1986; Haeder, Rotsch et al. 1988; Damstrup, Rygaard et al. 1992). The highest receptor levels were on SQCC cells, increased protein levels were accompanied by amplification of the c-erbB-1 gene (Hendler, Shum-Siu et al. 1989). High levels of the EGF-R were detected in the more proliferative, less differentiated cells an SQCC specimens . Recent phase I clinical trials demonstrated that ^ ^ ^In-labelled anti-EGF- R antibody localised predominately to the lung tumour and liver, suggesting that this molecule might serve as a useful target in tumour treatment (Divgi, Welt et al. 1989). EGF at a concentration of lOng/ml has been found to inhibit growth and colony formation in SQCC cell lines in vitro (Kamata, Chida et al. 1986). c-erbB-2 (neu) proto-oncogene found on chromosome 17q21 encodes an EGF- R-related receptor which is also a tyrosine kinase, this was amplified in one adenocarcinoma (Cline and Battifora 1987).

1.12.c III. Others: c-myb, c-fms. c-m yb has been localised to human chromosome 6q24. Examination of a number of established lung cancer cell lines indicated 4/4 classic SCLC and 3/4 variant SCLC and 0/5 NSCLC expressed a 3.5 kb c-myb mRNA. It is speculated th a t c- myb expression reflects an early stage in bronchial epithelium differentiation and might be a feature of a common bronchial mucosal stem cell proposed to give rise to all the differentiated cells in the bronchial mucosa (Kiefer, Wegmann et al. 1990). The protein kinase product of c-fms is th e CSF-1 receptor for m onocyte growth factor. High levels of the c-fms 8.5 kb mRNA was detected in variant SCLC cell lines (Kiefer, Bepler et al. 1987). However, the c-fms mRNA expressed in these cells lacked homology to the 5' portion of human c-fms, leading to the suggestion that the SCLC mRNA might represent a c-fms-related gene product and not the CSF-1 receptor. 45

1.13. Current status of therapeutic approaches to SCLC.

Before the 1970s, surgery and radiotherapy were the most common forms of treatment. Subsequent ciinical studies showed that SCLC metastasises early and widely; moreover, this tumour was found to be sensitive to various cytotoxic treatment extending the median survival time from 3 to 15 months. Unfortunately , the past decade has seen no change in overall results of treatment (Smyth, Fowlie et al. 1986). No standard treatment exists for SCLC. Less than 5% of properly staged patients will be candidates for primary surgical resection. The most frequent chosen agents for combination regimens are shown Table 1.2:

Table 1.2 CYTOTOXIC DRUGS FOR SCLC.

Alkylating agents Cyclophosphamide Ifosfamide Hexamethylmelamine Lomustine Vinca alkaloids Vincristine Vindesin Podophyllotoxin derivatives Etoposide Teniposide Platinum analogues Cisplatinum Carboplatin Miscellaneous Doxorubicin Metotrexate Mitomycin C

These agents are active singly against SCLC. Current combination chemotherapy regimens centre around CAV (cyciophosophamide, doxorubicin, vincristine) and PE (cisplatinum, etoposide) (reviewed in (Hansen and Kristjansen 1991)). The main areas of debate will now be discussed.

1.13.a Maintenance therapy Most trials including two recent large British trials show a small survival advantage with maintenance treatment. Six to eight cycles of the most active regimens are today thought of as an acceptable standard, corresponding with a total treatment duration of 5-8 months (Bleehen, Fayers et al. 1989; Spiro, Souhami et al. 1989). 46

1.13 .b Dose intensity. The intensity of chemotherapy can be increased by the use of autoiogous bone- marrow infusion, toxic effects are severe and no therapeutic advantages have emerged (Leonard, Duncan et al. 1990). Continuous etoposide therapy produces a better outcome than intermittent treatment (Clark, Cottier et ai. 1990). Anticoagulants may also find a place in therapy. Response rates among 328 patients with extensive SCLC treated with warfarin were superior to those in controls, as was overall survival (Chahinian, Propert et ai. 1989). However, these differences did not reach conventional levels of statistical significance. Warfarin is still regarded as an experimental drug in SCLC.

1.13.C Alternating chemotherapy. Resistant clones of SCLC ceils, which develop either at the time of diagnosis or during chemotherapy, are the reason for treatment failure. However, attempts to intensify treatment by alternating supposedly non-cross resistant drug combinations have not improved outcome (Klastersky and Sculier 1989).

1.13.d Biological response modifiers. Interferons-SCLC ceils have reduced expression of specific ceil surface antigens, namely the class I major histocompatabiiity antigens. Since these molecules play a crucial role in immune recognition, their lack of expression may be relevant to the escape of SCLC from immune surveillance and contribute to It's metastatic potential. Doyle et. al. (Doyle, Gazdar et al. 1984; Doyle, Martin et al. 1985) found that SCLC cell lines, in contrast to other lung cancer lines, are deficient in the expression of HLA-A, B and C antigens as well as of p 2-mlcroglobulin, but that treatment with leukocyte-A interferon as well as y-interferon could induce the expression of these antigens. However, In a different panel of SCLC cell lines. Ball et al (Ball, Sorenson e t al. 1986) were able to d etect expression of class I and II major histocompatabiiity antigens but did note increase expression after treatment with y- interferon. Decreased proliferation of SCLC has been noted In response to y-lnterferon (Ruff, Farrar et al. 1986). y-interferon Induced modulation of class I major histocompatabiiity antigen expression is associated with increased lysis by cytolytic T lymphocytes (Weynants, Wauters et al. 1988) but decreased sensitivity to natural killer cells in vitro (Stam, Kast et al. 1989). Preliminary results show greater survival for patients receiving interferon maintenance therapy than those receiving no such therapy (Mattson, Niiranen et al. 1992). In addition the SCLC cell line WX322 (see chapter 8) is also sensitive to interferon resulting in decreased growth (Langdon, Rabiasz et al. 1991). 47

The colony stimulating factors, Granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor, given to counter the haematological effects of combination chemotherapy, can lessen the severity of neutropenic episodes; treatment delays can be eliminated, reduction of cytotoxic drug dose can be avoided in some patients, and the number and duration of hospital admissions may be reduced (Crawford, Ozer et al. 1991; Green, Trillet et al. 1991). Improvement in survival has not yet been reported. Growth factors-Clinical trials of an anti gastrin-releasing peptide monoclonal antibody are ongoing, however the results so far seem to be inconclusive (Mulshine, Avis e t al. 1990).

1.13.e Thoracic radiation therapy. in patients with limited disease, a small but definitive benefit of 5-15% in disease-free survival at two years is seen when thoracic irradiation is added to combination chemotherapy. Chest irradiation also significantly decreases the rate of local recurrence. Acute and late toxicity is increased when thoracic radiation and chemotherapy are given together (Arriagada, Pignon et al. 1989).

1.13.f Elective cranial irradiation. Cranial métastasés are common in patients with SCLC; 10% of patients in complete remission develop brain métastasés as the sole site of recurrence. Cranial irradiation iengthens the time before brain métastasés become symptomatic, but does not prevent their occurrence. Patients receiving cranial irradiation are at a greater risk of late neurological complications, e.g. leukoencephalopathy and neuropsychological impairment.

1.13.g Relapse. When patients relapse, and most do in the first two years, the results of second-line treatment with combination chemotherapy are frequently disappointing (Andersen, Kristjansen et al. 1990). Multifocal relapse normally leads to a change in chemotherapy to include agents not given in the original treatment. Responses after such non-cross-resistant regimens are seen in 20-25% with a median survival time of 3-4 months. Provided that there is a chemotherapy free interval before relapse, the drug combination that produced a response earlier in the course of treatment seems to be more effective, with response rates exceeding 50%. if the patient with a local relapse has not received previous irradiation, then radiotherapy is the treatment of choice (Bergman, Sullivan e t al. 1991). 48

1.14. Drug resistance mechanisms.

The earliest data were obtained on the mechanisms of resistance to the antimetabolite methotrexate. Before reaching its major intracellular target, the enzyme dihydrofolate reductase, methotrexate must enter the cell through an energy- dependent specific carrier, after which its intracellular accumulation is facilitated by polyglutamation. Sensitivity to dihydrofolate reductase inhibition depends in part on the continuous consumption of reduced folates by thymidylate synthesis. As a consequence of these complex events, resistance to methotrexate can result from alterations in many different aspects of the drug’s interactions (Ozols and Cowan 1986). In SCLC the relative resistance to methotrexate was correlated with reduced intracellular drug accumulation due to decreased polyglutamation, as well as decreased thymidylate synthetase activity (Curt, Jolivet et al. 1984). In addition, unstable methotrexate resistance in cultured SCLC cell lines was occasionally found to be due to increased levels of dihydrofolate reductase caused by gene amplification associated with the presence of double-minute chromosomes (Curt, Jolivet et al. 1985). Although these early observations emphasised the ability of neoplastic cells to develop mechanisms to escape the toxic effects of specific chemotherapeutic agents, they did not address the broader question of concomitant resistance to diverse classes of agents. Variants of the "multi-drug resistance" phenotype have been attributed to the high-level expression of a drug efflux pump, the pi 70 glycoprotein encoded by the ^multi-drug resistance gene MDR-1 (Gerlach, Endicott et al. 1986), the expression of efficient drug-detoxifying enzymes, such as the placental form of glutathione transferase (Batist, Tulpule et al. 1986), or an enhanced ability to repair DNA damage (Ozols and Cowan 1986). Studies with panels of SCLC cell lines derived from previously treated and untreated patients, should allow the eventual correlation of drug sensitivity patterns with cellular mechanisms of drug resistance. The role of the MDR-1 gene In lung cancer was explored by quantitating the level of expression of MDR-1 specific message in mRNA extracted from a panel of lung cancer cell lines and tumours. The level of expression was found to be elevated in only 4/23 SCLC cell lines and 3/6 SCLC primary tumours studied. In no instances were elevations of MDR-1 expression to the very high levels sometimes observed In colon cancer. In addition there was no correlation between MDR-1 expression and whether tumour material was derived from previously treated or untreated patients who did or did not respond to chemotherapy. There was no correlation between mRNA levels and in vitro chemosensitivity (Lai, Goldstein et al. 1989). Hence the MDR-1 gene expression Is not a major mechanism in the clinical drug resistance of SCLC. The discovery that the high-level expression of the placental form of the drug metabolising enzyme glutathione-S-transferase correlated with a phenotype of 49 pleiotropic drug resistance distinct from drug accumulation defects induced in a breast cancer cell line in vitro has several important implications (Batist, Tulpule et al. 1986). This enzyme can not only detoxify a wide range of xenobiotics but also has it's expression induced by exposure to these compounds. Many drugs in common use are derivatives of natural products which can be detoxified by this mechanism. This therefore provides a general framework within which many therapeutic failures could be explained. In fact it has been shown that the level of glutathione-S-transferase enzymatic activity was increased in the lung tissue of smokers and that this was proportional to the cumulative tobacco exposure measured in pack-years (Petruzzelli, Camus et al. 1988). There are several reports indicating the frequent high levels of this enzyme in NSCLC tumours and cell lines (Carmichael, Mitchell et al. 1988). In contrast, SCLC tissues and cell lines express lower levels of glutathione-S-transferase mRNA, immunoreactivity, and enzymatic activity (Carmichael, Mitchell e t al. 1988; Eimoto, Tsutsumi e t al. 1988). There is also no correlation of glutathione-S-transferase activity and sensitivity to chemotherapeutic agents. A SCLC cell line made resistant in vitro by graded exposure to doxorubicin was found to show collateral resistance to vincristine, etoposide, cisplatin and X- irradiation, but not to melphalan, colchicine and actinomycin D. This drug resistance phenotype was explained on the basis of a combination of decreased intracellular levels of doxorubicin, increased DNA repair, and reduced DNA topoisomerase II activity (Zijistra, de et al. 1987; de Jong, Zijistra et al. 1990). In summary the drug resistance mechanisms used by SCLC remain largely unknown.

1.15 GROWTH FACTORS FOR LUNG CANCER.

Initial attempts to establish lung cancer cell lines in vitro yielded poor results. The non-selective medium containing fetal calf serum had a success rate of only 10% for clinical specimens of SCLC. The need to selectively grow the tumour cells and eliminate normal fibroblasts led to the development of a defined medium (HITES = hydrocortisone (lOnM), bovine insulin (Spg/ml), human transferrin (lOng/ml), 1 Zp-estradiol (lOnM) and sodium selenite (30nM) in RPMI-1640). This medium increased the success rate to about 45% (Simms, Gazdar et al. 1980.). Successful establishment and maintenance of cell lines derived from NSCLC and carcinoid tumours through the use of other defined medium have been reported (Gazdar and Minna 1986). 50

Conditioned serum free medium from SCLC cell lines is able to promote the growth of colonies of normal rat fibroblasts and established human cancer cell lines in soft agar (Carney and De 1988). More than 20 peptide hormones have been detected in SCLC and NSCLC culture fluids (Sorenson, Pettengill et al. 1981; Gazdar, Carney et al. T8857'Wlth“up to ten différent hormones being produced by a single cell line (Sorenson, Pettengill et al. 1981; Gazdar, Carney et al. 1985).

1.16. Polypeptide hormones.

1.1 S.a Epidermal growth factor The epidermal growth factor (EGF) is a polypeptide hormone which has both growth stimulatory and growth inhibitory effects on normal and tumour cells in vitro (Kamata, Chida et al. 1986). Examination of EGF receptors on lung cancer cells indicated that they were present on both NSCLC (1,300-2,700 fmol/mg protein) and SCLC (10-120 fmol/mg protein) from binding studies and northern blot analysis of mRNA (Damstrup, Rygaard et al. 1992). Although EGF receptor expression is generally elevated in human lung squamous carcinoma , the biological significance of this phenomenon and the role of EGF and TGF-a in this disease are poorly understood. Three human squamous carcinoma cell lines have been shown to express EGF receptors using an antibody against the receptor. Ligand binding studies indicated high concentrations (1,300-2,700 fmol/mg protein) of a single low affinity binding site (Kd = 3-5 nM). Addition of EGF or TGF-a at concentrations greater than 0.1 nM resulted in growth inhibition of all three cell lines and was associated with an accumulation of cells in the G2/M phase of the cell cycle. mRNA for TGF-a was detected in all three cell lines (Rabiasz, Langdon et al. 1992). However, several other studies have reported that EGF and TGF-a increase [^H] thymidine incorporation in squamous and adenocarcinoma cell lines at 10 ng/ml (Haeder, Rotsch et al. 1988; Soderdahl, Betsholtz et al. 1988; Veale, Kerr et al. 1989; Fang, Li et al. 1991). This stimulating effect on DNA synthesis could be blocked by suramin, an anti-helminthic, which competitively blocked the binding of [^25|]7Qp.oj to the EGF receptor (Putnam, Yen et al. 1992). EGF and TGF-a also stimulated NSCLC cell clonal growth in soft agarose and growth in nude mice. A monoclonal antibody (Ab 108) to the EGF receptor inhibited this growth stimulation in vitro and in vivo (Lee, Draoui e t al. 1992). Interestingly, in one study where the parental cell line growth was stimulated by TGF-a, brain métastasés were non-responsive to TGF-a and though [T^^I]EGF binding assays indicated EGF receptors antibodies failed to recognise them in an immunoprécipitation assay. These EGF receptors were not intrinsically phosphorylated. Hence the brain métastasés had an altered autocrine growth 51

mechanism compared to the parental cell (Fang, Li et al. 1991). These results suggest that EGF and related peptides may have a growth regulatory role in lung cancer cells.

1.1 6.b insulin-like grow th factor-1 (IGF-I). iGF-l (also known as somatostatin C) is a 70 amino acid closely related to insulin (Clemmons 1989), but with distinct high affinity receptors. IGF-i binds to a receptor with intrinsic tyrosine kinase activity (Czech 1989). Although insulin was shown to be necessary for serum-free culture of SCLC (Simms, Gazdar et al. 1980.), supraphysiological concentrations were required for optimal growth, suggesting that insulin was binding with low affinity to the IGF-I receptor. IGF-I is secreted by SCLC and NSCLC cell lines and tumours (Macauly, Teale et al. 1988; Minuto, Del et al. 1988; Jaques, Kiefer et al. 1989). It has been shown to be mitogenic in a variety of cell types including fibroblast and erythroid progenitor cells, breast and thyroid cells (Clemmons 1989; Williams, Williams e t al. 1989; Rosen, Yee e t al. 1991). High affinity binding sites have been shown for IGF-I on SCLC cell lines (Jaques, Rotsch et al. 1988; Macauly, Teale et al. 1988). The growth of SCLC cell lines was also stimulated by exogenous IGF-I (Nakanishi, Mulshine et al. 1988). Furthermore, a monoclonal antibody to the IGF-I receptor inhibited IGF-I and insulin-stimulated cell growth in 4 SCLC cell lines (Nakanishi, Mulshine et al. 1988). Hence, IGF-I acts as an autocrine growth factor in SCLC. Since bombesin and insulin act synergistically to stimulate mitogenesis in Swiss 3T3 cells (Rozengurt and Sinnett-Smith 1983), this may also occur in SCLC. IGF-I receptor mRNA was detected by northern blot analysis in all SCLC and NSCLC cell lines tested and insulin-like growth factors were able to stimulate clonal growth in soft agarose in all of these lines (Havemann, Rotsch et al. 1990; Macaulay, Everard et al. 1990). Preliminary clinical data showed that serum IGF-I levels were significantly higher in patients with lung cancer than controls, and patients with métastasés showed significantly higher levels of IGF-I than patients without. However, no significant difference in IGF-I mean values were seen before and after surgical removal of tumours. IGF-II receptors have also been characterised on small cell lung cancer cell lines (Schardt, Rotsch et al. 1993) The production of IGF-binding proteins which are also produced by lung cancer cell lines modifies the autocrine/paracrine model for IGFs since these proteins can either enhance or inhibit the effects of IGFs on tumour growth (Jaques, Kiefer et al. 1989; Kiefer, Jaques et al. 1991). 52

1.16.c Platelet derived growth factor PDG F normally circulates in blood stored in the alpha granules of platelets. These cells have an affinity for injured sites, aggregating there and releasing their contents. In the lung the processes of inflammation, fibrosis and carcinogenesis appear to be closely intertwined. The proto-oncogene c-s/s (PDGF B-chain) are upregulated in activated alveolar macrophages from fibrotic lungs; these and possibly others may play an important role in asbestosis (Antoniades, Galanopoulos et al. 1992). Elevated levels of PDGF have been found in adenocarcinoma pleural effusions. Significant stromal development is a specific feature of adenocarcinoma of the lung, this may be due to PDGF acting as a chemotactic and growth factor for mesenchymal cells (Safi, Sadmi et al. 1992).

1.16.d c-kit Stem cell factor (SCF) is a pluripotent growth factor which is suggested to play an important role in proliferation and differentiation in various types of fetal and adult tissues as the ligand for a transmembrane tyrosine kinase receptor encoded by the c-kit oncogene. Expression of the ligand and the receptor is seen in SCLC. The human SCF gene is transcribed into two major forms of alternatively spliced mRNAs with different molar ratio in fetal, adult and malignant tissues (Hibi, Takahashi et al. 1991). This aberrant expression is seen almost exclusively in SCLC. c -k it is autophosphorylated in response to SCF, resulting in significant chemotaxis and moderate proliferation of SCLC cells in vitro. Molecular analysis of the c-kit gene also revealed an amino acid substitution within the transmembrane domain in a SCLC cell line (Sekido, Takahashi et al. 1993).

1.16.e Hepatocyte growth factor/Scatter factor Hepatocyte growth factor/Scatter factor (HGF/SF) is a cytokine which is produced by mesenchymal cells and stimulates the motility of some epithelial cells. Small cell lung cancer cells have been shown to produce a protein indistinguishable from HGF/SF. These cell lines showed no apparent response to exogenous HGF/SF. In addition, c-m et mRNA was detectable. However, these did not occur together in the same lines, hence no autocrine loop has been demonstrated as yet (Rygaard, Nakamura e t al. 1993). 53

1.16.f Transferrin. Transferrin is an 80kDa (3-globulin that is synthesised in the iiver and transports iron in the plasma. It is required for serum-free growth of SCLC (Simms, Gazdar et al. 1980.). The SCLC cell lines NCI-H345 and NCi-HSIO secrete immunoreactive transferrin and have a transferrin requirement for growth (Nakanishi, C uttitta e t al. 1988). Gallium salts, which blocked iron uptake, inhibited SCLC growth in vitro (Vostrejs, Moran et al. 1988). Thus there is preliminary evidence for an autocrine growth loop involving transferrin in SCLC. Transferrin has also been identified as a lung-derived growth factor that stimulates the growth of iung-metastasizing tumor cells (Cavanaugh and Nicolson 1991).

1.17. NEUROPEPTIDE GROWTH FACTORS AND SMALL CELL LUNG CANCER

The neuroendocrine nature of SCLC has been long recognised on morphological grounds (presence of neurosecretory granules), as well as biochemical grounds (expression of chromogrannin A and the key biogenic amine synthetic enzyme L-DOPA decarboxlase) (Gazdar, Helman et al. 1988). The most striking manifestation of this phenotype is the elaboration of a large number of peptide hormones and growth factors, several of which were first identified as neuropeptides. The first peptide to be specifically documented to be produced by SCLC was GRP. This list continues to expand, the most recent additions being atrial naturetic peptide (Bliss, Battey et al. 1990; Gross, Steinberg et al. 1993), multiple opiod peptides (Roth and Barchas 1986) and neuromedin B (Giaccone, Battey et al. 1992; Moody, Staley et al. 1992) (see table1.3). These peptides and hormones have been detected by immunohistochemistry of tumour samples, measuring plasma levels and examining cell lines. Most of these products are present in only a minority of tumours, although some SCLC appear capable of synthesising many ectopic hormones. Some have also been found in NSCLC (Luster, Gropp et al. 1985). 54

Table 1.3 PEPTIDES AND HORMONES SECRETED BY SCLC ACTH (Becker, Silva et al. 1984) Atrial natriuretic peptide (Bliss, Battey et al. 1990; Gross, Steinberg et al. 1993) Calcitonin gene related product (Bepler, Rotsch et al. 1988) CCK (Moody 1988; Rehfeld, Bardram et al. 1989) Chorionic gonadotrophin (Sorenson, Pettengill et al. 1981; Gazdar and Carney 1984) FSH (Sorenson, Pettengill et al. 1981) GRP (Moody, Pert et al. 1981; Erisman, Linnoila et al. 1982) Gastrin (Gazdar and Carney 1984; Rehfeld, Bardram et al. 1989) GM CSF (Abe, Kameya et al. 1984) Growth hormone (Sorenson, Pettengill et al. 1981) Glucagon (Sorenson, Pettengill et al. 1981; Bepler, Rotsch et al. 1988) IGF-I (Macaulay, Everard et al. 1990; Reeve 1991) Lipotrophin (Sorenson, Pettengill et al. 1981; Abe, Kameya et al. 1984) Neuromedin B (Cardona, Rabbitts et al. 1991; Giaccone, Battey et al. 1992) Neurotensin (Moody, Carney et al. 1985b; Bepler, Rotsch et al. 1988) Opioid peptides (Roth and Barchas 1986) (Sorenson, Pettengill et al. 1981; Maurer 1985; Sausville, Carney et al. 1985) Parathyroid hormone (Sorenson, Pettengill et al. 1981; Yoshimoto, Yamasaki et al. 1989) (Lazarus and Hernandez 1985) (Sorenson, Pettengill et al. 1981) Serotonin (Sorenson, Pettengill et al. 1981) Somatostatin (Wood, Wood et al. 1981) Vasopressin (North, Maurer et al. 1980; Gross, Steinberg et al. 1993)

In addition many of these bioactive peptides can be produced by normal lung (Becker and Gazdar 1985). Many of these peptides are synthesised as prohormones that acquire biological activity only after specific post-translational modifications, one example of this is carboxy-terminal alpha-amidation (Quinn, Treston et al. 1991). The effect of these peptides were investigated in detail by (Woll and Rozengurt 1990a). The effect of 32 neuropeptides and hormones on Ca2+-sensitive fluorescence in fura-2/AME loaded SCLC cells was examined. 55

TABLE 1.4. The effect of multiple peptide hormones and neuropeptides on [Ca2+ ]j mobilization in SCLC cell lines Effective Non Effective Bradykinin ACTH Cholecystokinin Angiotensin I, H, IB Galanin Atrial natriuretic peptide Bombesin/GRP Calcitonin Neurotensin Chorionic gonadotrophin Vasopressin a-endorphin Endothelin Epinephrine Follicle stimulating hormone GHRH GIF Glucagon 5-hydroxytryptamine Leu- Neuropeptide-Y Parathyroid hormone Substance K Substance P TRH

Intracellular Ca^+ was measured in SCLC cell lines NCI H69, H510, H345, H209, H128 with the indicator fura-2/AME described in Materials and Methods. Effective peptides resulted in consistant large restx)nses at 1 uM , the responses in the various cell lines were heterogeneous. ______

Thus expression of the corresponding receptors has been found for some of these hormones (Sorenson, Pettengill et al. 1981; Gazdar, Carney et al. 1985; Woll and Rozengurt 1989a; Bunn, Dienhart et al. 1990). Some of these substances are known to act as mitogens in other systems (see above) and if even a few are also mitogenic for SCLC, a potentially complex network of autocrine and paracrine interactions could be envisaged. A novel growth factor derived from normal porcine and rat lung that differentially promotes the growth of lung métastasés (Cavanaugh and Nicolson 1991) substantiates this proposal. Some of these hormones and peptides will now be discussed in relation to small cell lung cancer

1.18. Gastrin-releasing peptide. Willey et al (Willey, Lechner e t al. 1984) reported th a t bombesin and GRP acted as growth factors for cells derived from explants of normal human bronchial epithelium cells, in serum-free medium, colony-forming assays. The maximum effect (about 150% X control) was obtained with 0.1 pM bombesin/GRP, which is almost 100-fold greater than required for maximal effect on DNA synthesis in Swiss 3T3 cells (Rozengurt and Sinnett-Smith 1983). 56

In the normal lung, secretion of GRP by pulmonary neuroendocrine cells occurs in response to alterations in pulmonary oxygenation, such as those associated with birth (increased oxygenation) or with chronic obstructive airways disease (decreased oxygenation) (Schuller 1991). Elevated levels of GRP have been found in broncho-alveolar lavages of normal smokers compared to non smokers (Aguayo, King et al. 1990). The high incidence of SCLC in smokers with a history of chronic obstructive lung disease, and the successful induction of bombesin-producing atypical carcinoids in hamsters by simultaneous exposure to hyperoxyia and carcinogenic nitrosamines (Schuller 1991), may indicate a role for GRP and abnormal oxygenation in the initiation of smoking related tumours. Bombesin-like peptides were first detected in SCLC in the early 1980s (Moody, Pert et al. 1981). Although present in greatest amounts in SCLC, smaller quantities were detected in some lung adenocarcinomas. Plasma GRP has been suggested as a tumour marker for patients undergoing treatment for SCLC, but is not suitable for routine use unless a highly sensitive radioimmunoassay is used (Maruno, Yamaguchi et al. 1989). Demonstration of pre-pro GRP mRNA, pro-bombesin C-terminal peptide and multiple GRP gene associated peptides in SCLC confirm that the GRP and related peptides originate in these tumours (Sausville, Lebacq-Verheyden et al. 1986; Cuttitta, Fedorko et al. 1988; Sunday, Choi et al. 1991). GRP secretion by SCLC can be stimulated by treatment with secretin or vasoactive intestinal peptide (Korman, Carney e t al. 1986). In addition to secreting bombesin-like peptides, SCLC also exhibit receptors for them, as demonstrated by specific binding of [^25|.Tyr4] bombesin to SCLC (Moody, Carney et al. 1985a). Estimates of receptor number are in the range of 1-3 X 10^ per cell. This suggests that GRP could act as an autocrine growth factor. GRP/bombesin have been shown to act as growth factors in SCLC in vitro and in vivo : Weber et al (Weber, Zuckerman et al. 1985) reported that GRP enhanced DNA synthesis in 2 SCLC cell lines, but not in 2 NSCLC cell lines, grown in liquid culture in the presence of 10% serum. Maximal effects were seen at concentrations of 5pg/ml (1.8pM) which is 1000 times the maximum required for Swiss 3T3 cells. This observation has not been repeated by others. In another study, colony formation in 9/10 SCLC cell lines was stimulated up to 150-fold by GRP, with maximal effects at 50nM (Carney, Cuttitta et al. 1987). However, there was no correlation between amounts of GRP secreted, response to exogenous GRP, and the number of binding sites in individual cell lines. Growth of SCLC xenografts (NCI-H69) was reported to be increased 77% above control in nude mice treated thrice daily with intraperitoneal injections of bombesin 20ng/kg (Alexander, Upp et al. 1988a). The paucity of GRP receptors in H69 SCLC cells suggests that this effect may be partly direct and partly 57 mediated by some other mechanism e.g. bombesin stimulating the release of other peptides: insulin, glucagon, gastrin and cholecystokinin etc. which in turn stimulate the growth of the SCLC xenograft. The hypothesis of autocrine growth stimulation by GRP in SCLC was tested by Cuttita et al. (Cuttitta, Carney et al. 1985), using a monoclonal antibody to [Lys^] bombesin (2A11). It inhibited the clonal growth of 2 SCLC cell lines in serum free medium, and retarded the growth of one growing as xenografts in nude mice, these results strengthened the hypothesis that GRP is an autocrine growth factor for SCLC. Because fewer cell lines appear to express receptors for GRP than to secrete it, autocrine stimulation probably only occurs in a subset of SCLC (Kado-Fong and Malfroy 1989). GRP has been shown to activate PLC in SCLC, both in metabolically labeled whole cell and membranous preparations (Trepel, Moyer et al. 1988b). This activation is accompanied by an elevation of [Ca^+Jj (Heikkila, Trepel et al. 1987), but PKC response has not yet been characterised. However, GRP stimulation of InsPg and increase in [Ca^+Jj were inhibited by prior treatment with PKC activator phorbol 12-myristate 13-acetate, suggesting that PKC might exert negative feedback regulation on this response. Because non-hydrolysable GTP analogues could modulate PLC activation in response to GRP (Trepel, Moyer et al. 1988b; Sharoni, Viallet et al. 1990), it was concluded that the GRP receptor in SCLC was coupled to PLC by a G- protein. The GRP receptor and neuromedin B receptors have been cloned from SCLC cells, two consensus PKC phosphorylation sites are conserved in these receptors, and there were no structural changes in the GRP-receptor and neuromedin B-receptor (Corjay, Dobrzanski et al. 1991). It seems likely that SCLC may be stimulated to grow by the normal intracellular signals evoked by ligand-dependent activation of bombesin peptide receptors. In the study by Woll and Rozengurt (Woll and Rozengurt 1989a) GRP stimulated an increase in [Ca^+Jj in 3/5 SCLC cell lines, which suggets that in 2/5 SCLC cell lines, unrestrained growth is due to other factors and not a GRP mediated autocrine growth loop.

1.19. Vasopressin. Vasopressin at nanomolar concentrations increases [Ca^+]| in all SCLC cell lines tested through Vi receptors (Woll and Rozengurt 1989a). Vasopressin (with other ) is secreted by up to 65% of SCLC (North, Maurer et al. 1980; Sorenson, Pettengill et al. 1981; Sausville, Carney et al. 1985). The expression of the vasopressin gene has been identified in SCLC. Vasopressin mRNA has been 58 identified in 3/26 (2 classic and 1 variant) SCLC cell lines, including the H345 cell line (Verbeeck, Elands et al. 1992). Vasopressin has not previously been shown to be a growth factor for SCLC

1.20. Bradykinin. Bradykinin is generated in the plasma or tissues from large molecular weight precursors (kininogens) by the action of kallikreins, which are activated during proteolysis and clotting (Regoli, Drapeau et al. 1986; Steranka, Farmer et al. 1989). Bradykinin is usually present in plasma at very low concentrations, due to it's rapid degradation by carboxypeptidase N and angioconverting enzyme. However, lung tumours are frequently surrounded by areas of tissue and tumour necrosis and local concentrations of bradykinin may be significantly higher. Bradykinin at nanomolar concentrations increases [Ca^+jj in all SCLC cell lines tested through 82 receptors (Woll and Rozengurt 1989a). Bradykinin was not so far shown to stimulate growth of SCLC cells.

1.21. Galanin. Galanin, a 29 amino acid peptide initially isolated from porcine intestine (Tatemoto, Rokaeus et al. 1983), has widespread distribution occurring in central and peripheral neurones (Rokaeus 1987). It elicits a variety of rapid biological responses including modulation of the release of several hormones (Fisone, Wu et al. 1987), stimulation of smooth muscle contractility and inhibition of neuronal excitability (Ekblad, Hakanson et al. 1985). Since galanin may play an important role in the regulation of endocrine, neuronal and smooth muscle function, its mechanism of action is attracting considerable attention. In the endocrine pancreas and in pancreatic p cell models in vitro, galanin inhibits the release of insulin (for review see (Ahren, Rorsman et al. 1988)). Galanin activates an ATP-sensitive K+ channel, hyperpolarizes the plasma membrane (de-Weille, Schmid-Antomarchi et al. 1988.; Dunne, Bullett et al. 1989) and thereby inhibits the activity of voltage-dependent Ca^+ channels (Nilsson, Arkhammar et al. 1989; Sharp, Le-Marchand-Brustel et al. 1989). In this manner, galanin reduces Ca^+ influx and blocks the activity of various agents that increase the intracellular concentration of Ca^+ ([Ca^+Jj) in the pancreatic p cell. These effects are induced via a pertussis toxin-sensitive G protein (Dunne, Bullett et al. 1989). In a rat insulinoma cell line, it was shown that galanin receptors are negatively coupled to adenylate cyclase through a pertussis toxin sensitive inhibitory guanine nucleotide binding regulatory protein (Ahren, Rorsman et al. 1988). In myenteric neurones, galanin also hyperpolarizes the plasma membrane and blocks Ca^+ influx via voltage 59 gated Ca2+channels (Rokaeus 1987; Tamura, Palmer et al. 1988). Furthermore, galanin inhibits muscarinic agonist stimulated breakdown of inositol phospholipids in tissue slices of ventral hippocampus (Palazzi, Fisone et al. 1988) and galanin inhibits noradrenaline-induced accumulation of cAMP in the rat cortex. Elevated levels of galanin were found in 5/11 pheochromocytomas (Bauer, Hacker et al. 1986). Galanin co-exists with other neuropeptides, and a moderate supply of galanin containing nerve fibres have been described in the respiratory tract of humans (Uddman and Sundler 1987). To date, galanin has not been found to stimulate inositol phosphate production or Ca2+ mobilisation from internal stores in target cells nor to act as a direct regulator of proliferation in any cell type. In view of the fact that galanin opposes the Ca2+ signals and modulates the action of other neuropeptides in various cell systems, it was important to determine weither galanin could reduce [Ca^+]| and antagonise the Ca^+-mobilising effects of other neuropeptides. The preliminary finding that galanin (IpM) increased [Ca^+jj rather than decreasing it in SCLC cell lines H69 and HSIO (Woll and Rozengurt 1989a) was a surprising result that warranted further more detailed experimental work to elucidate the signal transduction pathways activated by galanin in SCLC cells..

1.22. Cholecystokinin Cholecystokinin (CCK) mobilised intracellular [Ca^+]| in 4/5 cell lines tested (Woll and Rozengurt 1989a) however, the nature of the receptor initiating these events was not determined. Gastrin and CCK share a common C-terminal pentapeptide and bind to at least two different receptor subtypes (Jensen, Wank et al. 1989; Lin, Holladay et al. 1989). The CCKg/gastrin receptors, which are found mainly in the central nervous system and in the gastrointestinal tract, bind either CCK or gastrin with approximately equal affinities whereas the CCKa receptors exhibit a 500-fold higher affinity for CCK than for gastrin (Jensen, Wank et al. 1989). These receptor subtypes can be also distinguished by their sensitivity to specific antagonists (Bock, DiPardo et al. 1989; Lotti and Chang 1989). CCK has been reported to exert trophic effects on normal pancreas and human pancreatic cancer cells as shown by weight measurements and DNA synthesis, to stimulate the growth of rat stomach and mouse pancreas in vivo (Heald, Kramer e t al. 1992). CCK elevates [Ca^+]j in human pancreatic cells (Smith, Kramer et al. 1991) and has also been implicated in the growth of gut tumours (Lamers and Jansen 1988; Douglas, Woutersen et al. 1989). CCK has been demonstrated in a liver metastasis from an islet cell tumour (Madsen, Larsson et al. 1986). While these observations 60

suggest that CCK can act as a growth factor, it is difficult to obtain unambiguous evidence, that CCK acting through CCKa receptors stimulate growth. Other laboratories have also reported receptors for CCK in some SCLC cell lines (Yoder and Moody 1987). Interestingly, SCLC cells have also been shown to express gastrin and CCK peptides (Rehfeld, Bardram et al. 1989; Geijer, Folkesson et al. 1990). The effect of CCK on SCLC cell growth has not been determined. The type of receptor through which CCK increases [Ca^+]| in SCLC is also unknown.

1.23. Gastrin The effects of gastrin have not been determined in SCLC. Gastrin exists in multiple molecular forms with carboxy-terminal homology, including (G14), big gastrin (G34) and the most common gastrin-l (G17). are found in the proximal duodenum and gastric antrum, where they are secreted by the neuroendocrine G-cells. Gastrin release stimulates gastric acid secretion, gastric motility and contraction of the lower oesophageal sphincter. In addition, gastrin is found in the hypothalamus and pituitary, where it may act as a neurotransmitter (Walsh 1987). Gastrinomas, a component of multiple endocrine neoplasia type 1 syndrome, cause the Zollinger-Ellison syndrome of peptic ulcers and diarrhoea. The possibility that the gastrointestinal peptide gastrin could act as a hormonal growth factor has attracted considerable interest. A considerable body of evidence has demonstrated that the administration of gastrin induces growth promoting effects in the digestive tract and exocrine pancreas (Johnson 1984). Exogenous gastrin stimulates DNA synthesis in the fundic antral mucosa. In particular, an increase in the circulating levels of gastrin has been related to hyperplasia of the gastric enterochromaffin-like cells (Ryberg, Axelson et al. 1990; Brenna and Waldum 1992). Furthermore, a decrease in circulating gastrin induced by antrectomy resulted in reduced DNA synthesis in the pancreas, oxyntic glands, duodenal and colonic mucosa and atrophy of colonic mucosa in the rat, an effect reversed by administration of pentagastrin (Johnson and Guthrie 1984). Gastrin also appears to be a growth promoting hormone for malignant cells (gastric and colonic) grown as xenografts in nude mice (Singh, Walker et al. 1986; Watson, Durrant et al. 1989). While these observations strongly suggest that gastrin acts as a growth factor, it is difficult to obtain unambiguous evidence for a direct growth-promoting effect of gastrin in vivo because the administration of this peptide could stimulate the release of other biologically active peptides or growth factors which could act as the proximal effectors of the action of gastrin. Cultured cells have provided useful experimental systems for elucidating the extracellular factors that promote cell growth without the many complexities of whole 61 animal experimentation. Nevertheless, compelling evidence in favour that gastrin acts as a cellular growth factor or as an autocrine factor in tumours has been difficult to document using clonal cell populations. Indeed, studies using receptor antagonists and colon carcinoma cell lines resulted in controversial results (Hoosein, Kiener et al. 1988; Hoosein, Kiener et al. 1990; Thumwood, Hong e t al. 1991; Yapp, Modlin e t al. 1992). The lack of a convenient model system has impeded the elucidation of whether gastrin can act as direct growth factor in vitro. SCLC cells are an excellent system In which to test the early signalling events and growth potential of gastrin and also to evaluate novel gastrin receptor antagonists

1.24. Neurotensin. Neurotensin a 13 amino acid polypeptide, is present in the hypothalamus and in the mucosal endocrine cells of the ileum. It's biological activity resides in amino acid residues 1-8. Neurotensin in the presence of EGF stimulated a 5-10 fold incorporation of [^H] thymidine in a dose dependent manner 10'^-10"^° M in hepatocytes; this effect was inhibited by TGF-p 1 ng/ml (Carr, Hasegawa et al. 1992). Neurotensin may function as an autocrine growth factor in colon cancers. Neurotensin release and expression has been identified in human colon cancer cell lines. mRNA for neurotensin was detected by northern blot analysis in 4/4 cell lines and neurotensin was identified by radio-immunoassay in 2/4 cell lines and elevates [Ca^+]j in 2/4 cell lines (Evers, Ishizuka et al. 1992). Neurotensin was also able to stimulate the growth of normal small bowel and colonic mucosa. Neurotensin also stimulated the growth of a mouse colon cancer and human colon cancer cell line in vivo (Yoshinaga, Evers et al. 1992). Neurotensin mRNA expression has also been detected in a human pancreatic carcinoid tumour. Neurotensin raises [Ca^+]j in SCLC (Woll and Rozengurt 1989a). Neurotensin is also secreted by some SCLC cell lines (Goedert, Reeve et al. 1984; Moody, Carney et al. 1985b; Staley, Fiskum e t al. 1989).

1.25. Other peptides

1.25.a Haemopoietic growth factors Haemopoietic growth factors are increasingly given to lessen the haematological effects of chemotherapy. It is therefore important to be sure that these growth factors do not stimulate the growth of SCLC cells as well. Granulocyte- macrophage colony-stimulating factor (GM-CSF) 1-1000 U/ml had no effect what so ever on the growth of 10 SCLC, 1 adenocarcinoma and 1 large cell lines (Nemunaitis and Singer 1989). In another study 5 haemopoietic growth factors, GM-CSF, granulocyte colony-stimulating factor, IL-4, IL-6 and IL-3 were tested against a 62 panel of 11 SCLC cell lines. All had absolutely no effect on growth, except IL-3, which stimulated the growth of one of the SCLC cell lines in three different growth assays (Vellenga, Biesma et al. 1991). GM-CSF and IL-3 were found to have growth stimulating effects on three solid tumours (breast cancer, NSCLC, and hypernephroma cell lines) (Nachbaur, Denz et al. 1990). These results suggest that the application of haematopoetic growth factors in cancer patients undergoing chemotherapy should be viewed in the light of possible co-stimulation of the malignant cells.

1.25.b Opiods. Endogenous opiate peptides including the enkephalins, and dynorphins are widely distributed in the central nervous system. Multiple subtypes of receptors have been identified using a variety of agonists and antagonists. Because of their central role in pain transmission, opiate pharmacology has been studied in detail (Snyder 1980). p-endorphins has been shown to stimulate lymphocyte proliferation in vitro (Davis, Burgess et al. 1989), although this effect may not be mediated directly through opiate receptors, as it was not blocked by naloxone . Dynorphins and enkephalins appear to be involved with vasopressin in the proliferative response of the marrow to haemorrhage (Feuerstein, Molineaux et al. 1985). p-endorphin stimulates clonal growth in SCLC (Davis, Burgess et al. 1989) . Some NSCLC secrete the pentapeptide neo-kyotorphin (Zhu, Hsi et al. 1986), but it is not known whether this can stimulate tumour or stromal growth. Neuroblastoma xenograft growth has been inhibited by naltrexone, an opiate antagonist (Zagon and McLaughlin 1987), suggesting a possible role for these drugs in treating some cancers. Opiod peptides, p-endorphin, enkephalin and dynorphin are secreted by SCLC cells. The p-endorphin levels detected were 0.2 and 1.1 pmol/mg total protein. The detection of 50 and 100 fmol opiod receptors/mg protein on the same cells postulated the existence of an opiod autocrine loop (Roth and Barchas 1986). High affinity membrane receptors for the p, 6 and k opiod agonists are present on SCLC and NSCLC cell lines (Maneckjee and Minna 1990). It appears that opiods have an inhibitory effect on SCLC growth through atypical opiate receptors (Maneckjee and Minna 1992). This suggests that the opiod autocrine loops act in a negative fashion to control lung cancer cell growth. The addition of nicotine together with an opiod abrogated the growth suppression although nicotine alone had no effect on cell growth. Methadone has been found to significantly inhibit the in vitro and in vivo growth of human lung cancer cells. The in vitro growth inhibition (occurring at 1- lOOnM methadone) was associated with changes in cell morphology and viability 63

detected with-in 1 hr and was irreversible after 24-hr exposure to the drug. These effects of methadone could be reversed in the first 6 hr by naltrexone, actinomycin D and cycloheximide, suggesting involvement of opioid-like receptors and the requirement for de novo mRNA protein synthesis. The inhibitory effects of methadone could also be achieved with the less addictive (+) isomer of methadone. The binding sites were high affinity (nM), but the binding characteristics appeared to be different from methadone sites present in rat brain. Methadone decreases cAMP levels in lung cancer cells but the receptors are not coupled to a pertussis sensitive guanine nucleotide-binding regulatory protein (Maneckjee and Minna 1992). These receptors have been further characterised and are found to be non-conventional opioid receptors which are not antagonised by naloxone.

1.25.C Tachykinins A recent study reported that tachykinins were able to mobilise Ca^+ in SCLC cells but not stimulate growth in liquid culture (Takuwa, Takuwa et al. 1990). Physalaemin, an amphibian tachykinin, is detectable in some SCLC (Lazarus and Hernandez 1985) and has been shown to inhibit clonal and mass culture of SCLC in vitro at picomolar concentrations (Bepler, Rotsch et al. 1988).

I.ZS.d Vasoactive intestinal peptide. VIP elevates cAMP in SCLC, an effect which is abolished by somatostatin (Taylor, Moreau et al. 1991), and recently VIP analogues have been shown to inhibit the growth of SCLC cell lines (Moody, Zia et al. 1992), however VIP has been shown to stimulate the growth of a human lung adenocarcinoma (Scholar and Paul 1991).

1.25.0 Somatostatin Somatostatin and it's analogues inhibit the endogenous production of IGF-I and insulin (Nilsson, Arkhammar et al. 1989), and have been shown to inhibit breast cancer growth in vitro (Scambia, Panici et al. 1988). They also inhibit the growth of experimental prostatic tumours (Murphy, Lance et al. 1987; Schally 1988). Somatostatin receptors are present on many tumours (Reubi, Waser et al. 1990; Reubi, Laissue et al. 1992) and somatostatin analogues are useful in the treatment of hormone-secreting tumours including apudomas and carcinoids (Gorden, Comi et al. 1989). It is therefore interesting to note that a somatostatin analogue when administered twice daily as a perilesional infusion, was able to inhibit the growth of 4 SCLC cell lines including NCI-H69 and H345 in vitro and in xenografts. When perilesional treatment was terminated some tumours regrew, suggesting cytostatic activity (Taylor, Bogden et al. 1988). This inhibition could be mediated by 64

suppressing the effects of GRP or IGF-I. Two active octapeptide analogues of somatostatin inhibited clonal growth of SCLC cells and the VIP-stimulated cAMP formation (Taylor, Bogden et al, 1988; Taylor, Moreau et al. 1991). However more recent studies showed that somatostatin receptors were present on 3/4 SCLC cell lines but not in two NSCLC cell lines. The growth of 1/3 somatostatin receptor positive SCLC cell lines was significantly inhibited by the long acting somatostatin analogue octreotide (10 9 M). 20 SCLC patients were treated with octreotide 250 pg three times per day, before chemotherapy (6 patients) and after chemotherapy (14 patients). Octreotide was well tolerated, and serum levels of IGF-I were suppressed to 62 ± 7% of pretreatment levels, however there was no evidence of anti-tumour activity. Hence there was no correlation between somatostatin receptor expression and growth inhibition by octreotide, either in vitro or clinically (Macaulay, Smith et al. 1991).

1.26. BLOCKING GROWTH FACTOR ACTION

As understanding of the effects of growth factors in cancer increases, it has become possible to plan rational therapeutic interventions. If an autocrine growth loop is considered, in which cells synthesise, secrete, bind and respond to the same growth factor, it is evident that interruption of this cycle at any point will block mitogenesis. Paracrine growth could be blocked in the same way. Secreted factors can be cleared by antibodies, such as the bombesin monoclonal antibody 2A1 1 used to retard the growth of SCLC xenografts in nude mice (Cuttitta, Carney et al. 1985). This antibody has entered Phase I clinical trial (see Mulshine, Avis e t al. 1990). Efforts have been directed to developing peptide antagonists which are not antigenic and should have higher tissue penetration than antibody proteins. Two classes of neuropeptide antagonist have been characterized in the model Swiss 3T3 fibroblast system and tested for their effects on SCLC in vitro. These results are summarised as an example of growth factor antagonist development. The first bombesin antagonist to be described was an analogue of substance P,

[DArg\DPro^,DTrp^'9 Leuii Jsubstance P (antagonist A, Table 1.5). 65

TABLE 1.5 Broad spectrum and specific antagonists of

Broad spectrum antagonists (substance P analogues)

Substance P: Arg-Pro-Lys-Pro-Gln-GIn-Phe-Phe-Gly-Leu-Met-NH 2 Antagonist A: [ DArgL DPro^, DTrp^>^, Leu^^ ] substance P Antagonist D; [ DArgL DPhe^, DTrp^-®, Leu^^] substance P Antagonist G: [ Arg^, DTrp^»^, MePhe^ 1 substance P (6-11) ______Specific antagonists (bombesin analogues) Bcnnbesin;

pGIu-GIn-Arg-Leu-Gly-Asn-Gln-Trp-Ala-VaI-Gly-His-Leu-Met-NH2

Antagonist L: [ Leu^^-psi(CH 2NH)Leu^^ ] bombesin Antagonist N: N-acetyl-GRP (20-26) ______

Substance P is structurally unrelated to the bombesin-like peptides, but antagonist A. which is a substance P antagonist, was found to block the secretary effectsjoL^JTlbesin on a pancreatic preparation (Jensen. Jones et al. 1984). It was subsequently found to block ’25|_grp binding and bombesin-stimulated mitogenesis in Swiss 3T3 cells with half-maximal effect at 118 pM (Zachary and Rozengurt 1985). It did not affect mitogenesis stimulated by polypeptide growth factors, such as EGF and platelet-derived growth factor (PDGF), but was found to block vasopressin- stimulated mitogenesis (Zachary and Rozengurt 1986). Further substance P analogues were therefore studied in order to identify more potent bombesin antagonists that could be tested in SCLC (Woll and Rozengurt 1988b; Woll and Rozengurt 1990b). Two interesting compounds were identified in this study. They were [DArg\ DPheS, DTrp^'9, Leu"" ^ ] substance P (antagonist D, Table 1.4) and [ArgG, DTrp^'S, MePheS] substance P(6-11) (antagonist G, Table 1.5) Both antagonists reversibly inhibited GRP-stimulated mitogenesis in Swiss 3T3 cells, and antagonist D was 5-fold more potent than antagonist A, although antagonist G was more potent than A (half- maximal inhibitory concentrations with 3.6 nM GRP and 1 pg/ml insulin: 118 pM A, 22 pM D, 85 pM G). In contrast, when tested as competitive Inhibitors of vasopressin-stimulated mitogenesis, antagonists D and G were equipotent, with a half- maximal effect at 1 pM in the presence of 14 nM vasopressin and 1 pg/ml Insulin. In addition, the antagonists were found to block mitogenesis stimulated by the neuropeptides bradykinin and endothelin (Woll and Rozengurt 1988; Fabregat and Rozengurt 1990b), but not that stimulated by the polypeptide growth factors EGF and PDGF, phorbol esters, prostaglandins or the cAMP activators 8-bromo-cAMP and vasoactive intestinal peptide. Thus, the substance P analogue antagonists showed broad 66 spectrum specificity against several neuropeptide mitogens: bombesin/GRP, vasopressin, bradykinin and endothelin. These findings prompted the question: is this growth inhibition receptor- mediated? Binding of radioisotope-labelled ligand to receptors provides evidence for receptor-mediated action. Consequently, [^HJvasopressin and ^^5; _ endothelin binding has been measured in Swiss 3T3 cells. Antagonists D and G competed with these radio-labelled ligands for binding in a dose-dependent fashion (Woll and Rozengurt 1988b; Fabregat and Rozengurt 1990b; Woll and Rozengurt 1990b). Further evidence that these neuropeptides utilise independent receptors, which are all recognised by this class of antagonists was obtained using specific antagonists for bombesin, vasopressin and bradykinin. These had no cross-reactivity against the other ligands (Woll and Rozengurt 1988). The neuropeptide mitogens bombesin/GRP, vasopressin, bradykinin and endothelin thus act through distinct receptors in Swiss 3T3 cells. A common feature in their signal transduction pathways is the rapid and transient mobilization of intracellular Ca^+. Antagonists D and G inhibited Ca^+ mobilization stimulated by each of these peptides, in addition to other early intracellular signals triggered by them (Woll and Rozengurt 1988a; Fabregat and Rozengurt 1990b). This has led to speculation that these antagonists might recognise a common domain on these neuropeptide receptors, each of which is a member of the G protein linked, Ca2+ mobilising receptor family, with seven helical transmembrane domains (Probst, Snyder et al. 1992). Alternatively, the antagonists might bind to a separate protein that interacts with the receptor and regulates its activity. An obvious candidate could be the G proteins themselves, which are capable of binding basic and hydrophobic peptides (e.g.. (Gil, Higgins et al. 1991)). To distinguish between these models, it will be necessary to determine whether the broad spectrum antagonists inhibit ligand binding to purified receptors. Recent studies using the substance P analogue [DPro^, DTrp^'^'TO] substance P (4-11), purified G protein and purified receptors showed that this novel truncated substance P-related peptide inhibited the activation of Gj or Go by M2 muscarinic cholinergic receptor or of Gg by p-adrenergic receptor in reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis (Mukai, Munekata et al. 1992). This inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G proteins. Changes in concentration of muscarinic agonist did not alter th e inhibitory effects of [DPro^, DTrp^*®TO] substance P (4-11) on M2 muscarinic cholinergic receptor promoted GTPase by Go (Mukai, Munekata et al. 1 9 9 2 ). 67

Table 1.6 Processes blocked by [DArg^DPhe5,DTrp^»9,Leu^ substance P in Swiss 313 cells

^^I-GRP binding (Zachary and Rozengurt 1986) ^^I-GRP cross-linking to GRP receptor (Zachary and Rozengurt 1987) [^H]vasopressin and ^^I-endothelin binding (Woll. 1988; Fabregat and Rozengurt 1990b) Activation of Na+/K+ pump (Mendoza, Schneider et al. 1986) Ca^+ mobilisation (Mendoza, Schneider et al. 1986) 80K phosphorylation (Zachary and Rozengurt 1986) EGF receptor transmodulation (Zachary and Rozengurt 1986) Activation of c-fos and -myc (Rozengurt and Sinnett- Smith 1987) Bradykinin stimulated mitogenesis (Woll and Rozengurt 1988) Bombesin stimulated mitogenesis (Zachary and Rozengurt 1985) Vasopressin stimulated mitogenesis (Corps, et al. 1985; Zachary and Rozengurt 1986)

1.27. Specific bombesin antagonists

The study of substance P analogues yielded several broad spectrum antagonists more potent than antagonist A, but no more specific antagonist. [LeuT 3-psi(C H 2NH) Leu^4] bombesin (antagonist L, Table 1.4) is a pseudopeptide bombesin analogue that was shown to inhibit bombesin-stimulated amylase release from guinea pig pancreatic acinar cells (Coy, Heinz-Erian et ai. 1988). its actions in Swiss 3T3 cells were characterized and found it to be a potent and specific, competitive antagonist of bombesin-stimulated mitogenesis (Woll and Rozengurt 1988a). Half-maximal effect was obtained with 240 nM antagonist L in the presence of 2.7 nM GRP and 1 pg/mi insulin. It had no effect on mitogenesis stimulated by vasopressin, bradykinin, EGF, PDGF, phorbol 12,13-dibutyrate, cholera toxin, 8-bromo-cAMP, prostaglandins or vasoactive intestinal peptide. Antagonist L was shown to inhibit specific ^^^l-GRP binding in a dose-dependent fashion, indicating that its effects were receptor mediated. In addition, early intracellular signals stimulated by bombesin, including Ca^+ mobilization and trans-modulation of EGF receptor binding (mediated by protein kinase C activation) were inhibited by this antagonist. A further specific bombesin antagonist was tested in Swiss 3T3 cells. N- acetyl-GRP(20-26) (antagonist N, Table 1.4) was found, like antagonist L, to act at the bombesin receptor to block bombesin/GRP-stlmulated mitogenesis. It was about 4-fold less potent than antagonist L. 68

1.28. Testing bombesin antagonists in SCLC The compounds characterized as broad spectrum and specific antagonists In Swiss 3T3 ceils were tested as inhibitors of bombesin mediated signals and growth in SCLC cell lines. Because SCLC is a heterogeneous group of tumours, each compound was tested in several cell lines. All five antagonists reversibly inhibited GRP- stimulated Ca^+ mobilization in cells loaded with the fluorescent indicator fura- 2/AME, confirming that they could act as competitive antagonists of the bombesin receptor on SCLC. As expected, the specific antagonists L and N had no effect on Ca^+ mobilization stimulated by other ligands, but the broad spectrum antagonists A, D and G inhibited Ca^+ mobilization stimulated by GRP, vasopressin, bradykinin, cholecystokinin and galanin in diverse cell lines (Trepel, Moyer et al. 1988a; Woll and Rozengurt 1990b).

1.29. PLAN OF STUDY

Human small cell lung cancer (SCLC) constitutes 25% of lung cancers and follows an aggressive clinical course. SCLC is characterised by the presence of intracytoplasmic neurosecretory granules and by its ability to secrete many hormones and neuropeptides. Only bombesin-like peptides, which Include gastrin-releasing peptide (GRP), have been shown to act as autocrine growth factors for certain SCLC cell lines. This thesis will focus on other neuropeptides and particularly their ability to mediate SCLC growth. An initial screen of neuropeptides showed that In addition to GRP, bradykinin, cholecystokinin (CCK), galanin, neurotensin and vasopressin were also able to stimulate an increase in [Ca^+Jj in responsive SCLC cell lines (Woll and Rozengurt 1990a). To extend these observations, the following will be investigated: 1 ) The precise dose-response relationships between these peptides and an Increase in [Ca2+], 2) To determine weither these Ca^+.mobilislng neuropeptides increase inositol phosphates in SCLC cells. 3) Most importantly, to investigate if Ca2+-mobilising neuropeptides are able to stimulate SCLC growth. This will test the hypothesis that SCLC cell growth Is mediated by an extensive network of autocrine and paracrine interactions. 4) Galanin a 29 amino-acid peptide opposes Ca2+ signals and modulates the action of other neuropeptides in various cellular systems. Thus the effect of galanin In SCLC cell lines will be Investigated. The surprising preliminary result that galanin (IpM) Increased rather than decreased [Ca^+Jj, warrents further detailed experimental work to elucidate signal transduction pathways activated by galanin In SCLC cells. Galanin 69 has not previously been shown to evoke inositol phosphate, Ca^+ mobilisation and growth responses in any cell type. 5) It became increasingly apparent that virtually all SCLC cell lines tested were able to respond to cholecystokinin ( 7 out of 8 cell lines) (Bunn, Dienhart e t al. 1990; Woll and Rozengurt 1990a; Bunn, Chan et al. 1992). In addition gastrin and cholecystokinin are circulating hormones. Their serum post-prandial levels can reach 100-500pmol, though local levels may be much higher than systemic levels. Also the possibility that the gastrointestinal peptides gastrin and cholecystokinin could act as a hormonal growth factors has attracted considerable interest. Therefore gastrin and cholecystokinin responses in SCLC cell lines will be studied in greater detail. The effect of gastrin has never been studied in SCLC cells. Are gastrin and CCK growth factors in SCLC cells and what is the nature of the receptor(s) through which they signal? 6 ) The broad-spectrum neuropeptide antagonists [D-Arg^ ,D-Phe5,D-Trp7»9 Lg^l 1 ] substance P and [Arg6,D-Trp^»9,MePhe^] substance P (6-11) block growth of SCLC cell lines in liquid culture (Woll and Rozengurt 1990b). These findings will be extended to determine if these broad spectrum antagonists can inhibit bradykinin, CCK, galanin, gastrin, GRP and vasopressin stimulated clonal growth in addition to inhibiting basal clonal growth. Further supporting the idea that broad spectrum neuropeptide antagonists constitute potential anticancer agents . 7) The advantages of responsiveness to multiple Ca^+-mobilising neuropeptides^ correlation with tumour progression and the potential role for broad spectrum antagonists in the clinical setting will also be addressed. 70

CHAPTER 2

MATERIALS AND METHODS

SCLC-ceii lines

SCLC cell lines H510, H69 and H345 (Carney, Gazdar et al. 1985) were generously donated by Dr. A. Gazdar (Bethesda, USA.) and purchased from the American Type Culture Collection. GLC 19 GLC 16 and GLC 14; and GLC 28 were the kind gift of Dr. de Leij (Groningen, Holland) (Berendsen, de-Leij et al. 1989; Damstrup, Rygaard et al. 1992). WX322 SCLC cell line was provided by Prof. J. Smyth (Edinburgh) (Langdon, Rabiasz et al. 1991). All of these lines have been well characterized.

Table 2.1 Characteristics of SCLC cell lines. (Carney, Gazdar et al. 1985; Berendsen, De et al. 1988; Langdon, Rabiasz et al. 1991; Damstrup,

Cell line H69 H345 H510 WX322 GLC 19 GLC 16 GLC14 Sex M MMMFFF Prior Rx Yes Yes Yes No Yes Yes No Source PE adrenal BM Sub. Lung Lung L node Cut Class C C CC CCC DDC u/m g 2 40 98 2 1 4 180 57.2 72.5 50.8 CK-BB pg/mg 2.2 5.8 2.7 0.7 0 .4 6 0 .2 4 0.22 NSE ng/mg 8 1 7 4 0 7 5 491 BLLpmol/m^ 1.7 4.7 7.4 + +

Abréviations: Prior Rx, Prior treatment; PE, pleural effusion; BM, bone marrow; Sub. Cut, subcutaneous, L. node, supraclavicular lymph node; C, classic; DDC, L-dopa decarboxylase (elevated > 1.0 u/mg); CK-BB, creatine kinase brain isoenzyme (elevated > 0.4 pg/mg); NSE, neuron specific enolase (elevated > lOOng/mg); BLI, bombesin-like immunoreactivity (elevated > 0.1 pmol/mg). ______71

Cytogenetic studies established that these cell lines were all of human origin and they had the following chromosome numbers: H69 63-73 chromosomes/cell and double minutes in some. H345 53-69 chromosomes/cell H510 37-49 chromosomes/cell WX322 GLC 19} 78 chromosomes/cell and double minutes in some. GLC 16} 77 chromosomes/cell GLC 14} and double minutes in some. For experimental purposes all cell lines were used at the lowest possible passage number and regularly screened for mycoplasma infection. All lines were consistantly negative.

Cell culture Stocks were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal " bovine serum (heat-inactivated at 57°C for 1 h) in a humidified atmosphere of 10% CÛ2:90% air at 37°C. They were passaged every 7 days. For experimental purposes, the cells were grown in HITESA which consists of RPMI 1640 medium supplemented with 10 nM hydrocortisone, 5 pg/ml insulin, 10 pg/ml transferrin, 10 nM estradiol, 30 nM selenium and 0.25% bovine serum albumin (essentially fatty-acid and globulin free. Sigma A-7030) (Simms, Gazdar et al. 1980.). Stock solutions: Hydrocortisone hemisuccinate 100 pM in water; Bovine insulin 1 mg/ml in 6 mM MCI. Transferrin 10 mg/ml in PBS. Estradiol 100 pM in PBS. Sodium selenite 100 pM in PBS. All solutions were stored at -20° C.

Determination of intracellular calcium concentration:

Aliquots of 4-5 x 10® SCLC cells cultured in HITESA for 3-5 days, were washed and incubated for 2 h at 37°C in 10 ml fresh HITESA medium. Then, IpM fura-2-tetraacetoxymethylester AME from a stock of ImM in dimethyl-sulphoxide was added and the cells were incubated for a further 5 min. The cell suspension was centrifuged at 2,000 rpm for 15 s, and the cells were resuspended in 2 ml of electrolyte solution (140 mM NaCI, 5 mM KCI, 0.9 mM MgCl 2, 1.8 mM CaCI, 25 mM glucose, 16 mM Hepes, 16 mM Tris and a mixture of amino acids at pH 7.2), transferred to a quartz cuvette, and stirred continuously at 37°C. Fluorescence was 72

recorded continuously in a Perkin-Elmer LS5 luminescence spectrometer with an excitation wavelength of 336nm and an emission wavelength of SlOnm. [Ca2+]j was calculated using the formula (Tsien, Pozzan et al. 1982; Mendoza, Schneider et al. 1986):

[Ca^li nM = where F is the fluorescence at the unknown [Ca^+]j, F,^ax's the fluorescence after the trapped fluorescence is released by the addition of 0.02% Triton-X-100 and F^j^ is the fluorescence remaining after the Ca2+ in the solution is chelated with 10 mM EGTA. The value of K was 220 for fura-2 (Tsien, Pozzan et al. 1982).

Measurement of membrane potential

Membrane pr^toptiai with thn lipnphilic fluorescent dye bis( 1,3 diethyltiobarbiturate)-trimethineoxonol (bis-oxonol) (Tsien, Pozzan et al. 1982). Cells cultured in HITESA were washed and incubated for 2 h in fresh HITESA. The cells were then resuspended in 2 ml electrolyte solution (see above), placed in a quartz cuvette and stirred continuously. Bis-oxonol was added at a final concentration of 100 nM from a stock solution of 1 pM in DMSO to the cell suspension in electrolyte solution at 37°C for 5 min before starting the experiment.Fluorescence was monitored in a Perkin-Elmer Ls5 luminescence spectrometer at 37®C. Excitation and emission wavelenghts were 540 and 580 nM respectively (Tsien, Pozzan et al. 1982).

Accumulation of inositol phosphates

The SCLC cell lines were maintained in culture as previously described. 2 x 10^ cells were labelled in 20 ml HITESA with 10 pCi/ml of myo-[3H]-inositol for 24 h. For determination of the production of total inositol phosphates, cells were washed twice in HITESA 0.02 M-Hepes/Na pH 7.2 at 37®C. Approximately 1.5 x 10® cells were resuspended in 1 ml HITESA plus Hepes and incubated with 20 mM LiCI for 20 min prior to the addition of galanin at the concentrations and times as indicated. Following the incubation at 37®C the cells were then lysed using 200 pi 18% perchloric acid and left at 4®C for 30 min. The supernatant was collected after centrifugation and neutralised with 0.5 M KOH, 25 mM Hepes, 10 mM EDTA using 0.01% phenol red as an indicator. The precipitated salts were removed by centrifugation and the supernatant was diluted in water and loaded on Dowex columns which were subsequently washed four times with water and the total inositol phosphates were eluted using 5 ml 0.1M formic acid + 1M ammonium formate 73

(Nanberg and Rozengurt 1988). Aliquots (1 mi) of eiuates were transferred to scintillation vials containing TO ml P^cbfluor and radioactivity determined in a Beckman p-Counter.

Separation of Inositol phosphate by FPLC

Cells were maintained in culture as previously described, washed at 3-5 days post-passage and labelled in HITESA with 50 pCi/ml myo[3H]-inositol for 24 h. Cells were then washed twice in HITESA at 37°C pH 7.2 and 3-5 x 10® cells resuspended in 1 ml electrolyte solution and 20 mM LiCI for 20 min before addition of neuropeptide (lOOnM). The cells were then incubated at 37°C for various times as indicated. The cells were then lysed with 250 pi trichloroacetic acid (TCA), cooled rapidly on ice and left at 4°C for 30 min. The extract was centrifuged and the TCA in the supernatant was removed by six extractions with water saturated ether. Excess ether was blown off under nitrogen and the sample was diluted to 10 ml with Buffer A (lOmM Hepes, 100 pM EDTA, pH 7.4). The inositol phosphates were separated by anion-exchange chromatography using a Mono Q column fitted in a Pharmacia FPLC system (Nanberg and Rozengurt 1988). The inositol phosphates were eluted with a gradient of sodium sulphate at a flow rate of 1 ml/min at pH 7.4. The gradient used was from 0 (Buffer A) to 0.5 M sodium sulphate (Buffer B) as follows. 25 min Buffer A 100%: a 20 min gradient to 20% Buffer B, a 25 min gradient to 32.5% Buffer B, a 15 min gradient to 50% Buffer B and a 25 min elution at 100% Buffer B. Fractions (1 ml) were collected and counted in 4.5 ml Picoflour in a Beckman p-Counter. Separation of [3H]lns(1 ,4,5)P3 from [3H]lns(1,3,4)Pg was carried out by the same method except that the gradient used was 25 min Buffer A (100%), a 17 min gradient to 15% Buffer B and then an isocratic elution at 15% for 40 min followed by 25 min elution at 100% Buffer B. Radioactivity peaks were identified by use of ^H inositol standards added to controls not pretreated with myo-[3 H] inositol which were then lysed and treated as previously described, or by co-elution of ^ 2p labelled inositol standards with samples. All samples were run with the addition of 10Ong ATP.

Liquid Growth Assay:

SCLC cells, 3-5 days post-passage, were spun down at 2,000 rpm for 30s,washed and resuspended in HITESA. Cells were resuspended at a density of 5 x 10^ cells in 1 ml HITESA in the presence or absence of antagonists in triplicate.^ various times, cell number was determined using a Coulter Counter, after cell clumps were 74 disaggregated by passing the cell suspension through 19 and 21 gauge needles (modified from (Wolî and RozengurTl'SSS)).

Cionogenic assay

SCLC cells, 3-5 days post-passage, were washed and resuspended in HITESA. Cells were then disaggregated into an essentially single cell suspension by two passes through a 19 g needle and then through a 20 pm pore size nylon gauze. Viability was judged by trypan blue exclusion on a standard haemocytometer. Cell number was determined using a Coulter Counter. 10^ viable cells were mixed with HITESA containing 0.3% agarose and agonist/antagonist at the concentrations indicated, and layered over a solid base of 0.5% agarose in HITESA with agonist/antagonist at the same concentration, in 35 mm plastic dishes. The cultures were incubated in humidified 10% C0£:90% air at 37°C for 21 days, then stained with the vital stain ; ) nitro-blue tétrazolium. Colonies of >120 pm diameter (16 cells) were counted using a microscope and using a X4 lens, the image is relayed via a TV camera and analysed on a Macintosh Ilex computer running the digital image processing and analysis program Image (modified from (Carney, Gazdar et al. 1980)). ^

Amplification cloning and sequencing of the human CCKg/gastrin receptor from brain and SCLC:

Cloning of the human CCKg/gastrin receptor from human fetal brain library

In order to generate a DMA probe by PCR for isolating cDNAs encoding the human CCKg/gastrin receptor two oligonucleotides were synthesised. Primer No.2 (5'-GAG C/AGA TAC/T A/GGC GCC ATC TGC -3') was deduced from a consensus sequence of the second cytoplasmic domain of the dog CCKg/gastrin (Kopin, Lee et al. 1992) and the rat CCKy^ receptor (Wank, Pisegna et al. 1992). Primer No.4 (5'-CGC TTC TTG GCC/HT AA/TC AGG/C TTG G-3') was reverse complement to a consensus sequence of the cDNAs coding for the third cytoplasmic domain. PCR experiments were performed under the following conditions. 3 x 10^ plaque-forming units of a human fetal brain cDNA library in XZAP II weTeljsetTas templates and incubated with 50 pmol of each of the appropriate primers (No.2 an d T ^ " for the cDNA library) at 100°C for 8 min, then quickly chilled on ice^ECR was car^d^puTwith 2.5 DNA pq[ymerase iji^finarhVdnjmFof 50 jJ^reœTnrnended 75 by the manufacturer (Boehringer Mannheim). The reaction was allowed to proceed on a thermal cycler (Perkin-Elmer) for 30 cycles, each cycle consisting of a 1-min denaturing step at 94°C, a 1-min annealing step at 60®C and a 3-min polymerisation step at 72°C. ((Sambrook, Fritsch et al. 1989)). In this way a product of about 550 nucleotides was amplified. This PCR product was trimmed by treatment with the Klenow-fragment (Amersham), isolated from an agarose gel by the GeneClean (Bio 101) method and cloned directly by blunt-end ligation into the H/ndll-site of the pBluescript(SK-) vector (Stratagene). Plasmids containing the PCR fragment were sequenced by the dideoxy chain termination method (Sanger, Nicklen et al. 1977) using a modified T7 DNA polymerase (Sequenase Version 2.0, USB). Double stranded plasmid DNA was sequenced from both strands using reverse and forward vector primers and PCR primers that corresponded to the CCKg/gastrin receptor. Using this PCR fragment two cDNA libraries were screened under standard conditions (Sambrook, Fritsch et al. 1989) with the cloned 550bp PCR fragment as probe: firstly, a cDNA library synthesised from mRNA of a 17-18 week old human fetal brain, cloned in the XZAPII vector (Stratagene); secondly, a 5'-stretch cDNA library from a 26 week old human fetal brain cloned in the Xgtl 1 vector (Clontech). Both libraries were oligo(dT) and random primed. Inserts of positive bacteriophages were cloned either by in vivo excision (XZAPII) or after gel-purification of the insert (Xgtll) into the EcoR\ site of the pBluescript(SK-) plasmid. A full length CCKg/gastrin receptor clone was isolated the complete sequence of which was obtained (as described for the PCR fragment) by custom primer-directed sequencing using specific primers complementary to internal cDNA sequences.

Cloning of the CCKg/gastrin receptor from SCLC cell lines H510 and H345.

The following primers were used for PCR analysis of the coding region of SCLC CCKg/gastrin receptor cDNA: 24185 (5’-CGG GCG CTG CAG TGC GTG CAT CGT TGG CCC -3'), 3F (5'- GTC GCC TAC GGG TTG ATC TC -3'), 6F(5'- AAC GGG CGT TGC CGG CCT GAG -3'), 6R(5'-CTC AGG CCG GCA ACG CCC GTT-3’), 7R(5'- GGC ACT ATA AAC TGG CAA CC 3') and 10R(5'- CTA CTC CTC AGC CAG GGC CCA GTG TGC -3'). These primers were deduced from the sequence of the cloned human CCKg/gastrin receptor cDNA obtained in this study (see above). Primers were ethanol precipitated and adjusted to a concentration of 50 pmol/pl in H 2O and were used directly for PCR. First strand cDNA was synthesised from total RNA from each SCLC cell line (H510 and H345). The reverse transcription mixture consisted of lOpg of cellular RNA, lOOng of oligonucleotide primer ICR, ImM of each dNTP, 4mM DTT, 50mM 76

Tris pH 8 3, 75mM KCI and 3 mM MgCl 2 and 40 units of RNase inhibitor. The reaction was initiated by adding 200 units of Moloney murine leukemia virus reverse transcriptase (Superscript kit, BRL) and carried out at 42° C for one hour. Using PCR with appropriate primers and 1 ng SCLC first strand cDNA as a template, the amplified DNA fragments were cloned and sequenced as described above.

Northern Blot Analysis of CCKg/gastrin receptor mRNA:

For performing Northern blot analys«*«= fhf^ start codon to the third cytoplasmic domairL.JUsiag^h.e. upstream primer No.38 (5'- GCC AT G GAG CTG CTA AAG CTG AAC-3') in combination with primer No.4 and 1 ng of the full length CCKg/gastrin receptor cDNA (digested by fcoR I), as a template resulted in the amplification of a region of about 1 kb as expected. RNA was isolated from different SCLC lines according to the method described by Chirgwin (Chirgwin, Przybyla et al. 1979). 30 pg of total RNA were separated on a 1% agarose / 2.2M formaldehyde gel, transferred onto Hybond N+ membrane (Amersham) and hybridised with this Ikb human CCKg/gastrin receptor probe, radiolabeled by random-priming (Feinberg and Vogelstein 1983) using both [32p]adATP and [32p]adCTP. Membranes were washed at high stringency (final washes: 3 x 0.5 h with O.lxSSC / 0.2% SOS at 60°C) and exposed to pre-flashed Kodak XAR-5 films for three days at -70°C with intensifier screens. The blots were de-hybridised and probed with a human 28S rRNA probe (Herget, Brooks et al. 1992).

Materials

Bradykinin, vasopressin, neurotensin, cholecystokinin, GRP, gastrin-17-1 (unsulfated), gastrin-17-11 (sulphated on tyrosine residue 12, position 6 from the COON terminus), cholecystokinin (CCK) residues 26-33 sulphated on tyrosine residue 27, the 7th position from the COOH terminus (CCK- 8), desulfated CCK-8 [des(S03)CCK-8], CCK 10-27, and galanin were purchased from Sigma Chemical Co., St. Louis, MO; [D-Arg\ D-Phe^, D-Trp^.s, Leu^i] substance P and [Arg®, D-Trp7,@, MePhe®] substance P were purchased from Peninsula Laboratries, Belmont, CA; Antagonists L364,718 and L365,260 were a kind gift from Prof. John Walsh, University of California. The gastrin/CCKg and CCKy^ antagonists ( CAM 2200 and CAM 1481 respectively ) were the generous gift of Dr. J. Hughes, Parke Davis, Cambridge, England. Fura-2-AME was from Calbiochem Corporation, LaJolla, CA; Agarose was obtained from Seakem, Rockland, ME; Dowex (mesh size 200-400) from Bio-Rad 77

Laboratories, Richmond, CA; ^Pertussis toxin from List Biological Laboratories, Campbell, CA. Bis-oxonol was obtained from Molecular Probes, Eugene, OR97402. ic e i a s e e b a n d r mta l bovine^s e ^ m was from Gibco Europe (UK). Jiochemicals were obtained from^t Amersham International (UK). 32pjnositol phosphate standards were the kind gift of Prof. R.H. Mitchell. 32p labelled nucleotides adATP and adCTP, and Hybond N+ filters were purchased from Amersham, Aylesbury, Bucks, England. Human fetal brain library and pBluescript vector were purchased from Strategene, Cambridge, England. were purcha§siLfenx.BQfibücm^ East Sussex, England. Sequenase 2.0 was purchased from USB, Cambridge, England. All other reagents used were of the highest grade commercially available. 78

CHAPTER 3

MULTIPLE NEUROPEPTIDES STIMULATE CLONAL GROWTH OF SMALL CELL LUNG CANCER: EFFECTS OF BRADYKININ, VASOPRESSIN CHOLECYSTOKININ, GALANIN AND NEUROTENSIN.

SCLC is characterized by the presence of intracytoplasmic neurosecretory granules and by its ability to secrete many hormones and neuropeptides (Sorenson, Pettengill et al. 1981; Maurer 1985) including bombesin, neurotensin, cholecystokinin and vasopressin (North, Maurer et al. 1980; Sorenson, Pettengill et al. 1981; Wood, Wood et al. 1981; Gazdar and Carney 1984; Goedert, Reeve et al. 1984; Sausville, Carney et ai. 1985; Bepler, Rotsch et al. 1988). Among these, only bombesin-like peptides, which include gastrin-releasing peptide (GRP),have been shown to stimulate mobilization of intracellular Ca^+ and inositol phosphate turnover in SCLC cells (10,51,52) and act as autocrine growth factors for certain SCLC cell lines (Cuttitta, Carney e t al. 1985; Heikkila, Trepel e t al. 1987; Bepler, Rotsch e t al. 1988; Moyer, Trepel et al. 1988; Trepel, Moyer et al. 1988b). In contrast, the role of other neuropeptides in the proliferation of SCLC cells remains poorly understood, as emphasized in sections 1.19-1.24, in the intrr>Hnrtinn Multiple were screened for their ability to induce a rapid increase in [Ca^+Jj in different SCLC cell lines fWoll and irt..l$&aa)..This assay should be regarded as an indicator ofT ^oductive ligand-receptor interaction. Ca^t^mobili^gi^iQn Is one of the early components in a complex signalling cascade leading to mitogenesis, rather than the signal that promotes cell growth. In the light of this initial screen, a more.jletaile.d analysis of the Ca^+ reoonse to neuropetides was undertaken, the mechanisms underlying this response and the ability of these neuropeptides to stimulate SCLC cell

^growth was investigated------

Mobilization of intracellular calcium in SCLC by multiple neuropeptides. Bradvkinin wasable to mobilise J iiUacellular in, alt ^CLC cell lines screened (Woll and Rozengurt 1989a). This peptide was therefore investigated in detail. Addition of bradykinin to H69, H510 or H345 cells loaded with the Ca^+- sensitive indicator fura-2, increased [Ca^+Jj without any measureable delav fFia. 3.1). Peak [Ca2+]. was reached 20-30 s after addition of the peptide. 79

Figure 3.1

H69 H510 H345 ^ 200 120 c

225- 231- 150 100 144-

125- 144-/ 105- 1 Min 1 Min 1 Min 100 100 Bradykinin nM

Figure 3.1: Dose-dependent effects of bradykinin on [Ca2+]i in SCLC cells.

SCLC cell lines H69 (left), H510 (middle) and H345 (right) were cultured in HITESA for 3-5 days. [Ca^'*’]j was determined at the concentrations of peptide indicated as described in Materials and

Methods. The insert shows the fluorescence tracing obtained when 100 nM bradykinin was added to cells loaded with fura- 2 . Typical [Ca^+]; dose response curves are shown for each cell line. The basal [Ca^+], is the mean value for that experiment ± SEM. The increases in [Ca^+]; induced by 100 nM bradykinin were repeated in several independent experiments and the data is given in the text. 80

Figure 3 .2

H69 H510 H345

208- +

1 Mm 1 Mm 0 10 100 1000 0 10 100 ""0 10 100 Neurotensin nM Cholecystokinin nM Vasopressin nM

Figure 3.2; Effect of neurotensin, cholecystokinin and vasopressin on [Ca^+]j in H69, H510 and

H345 SCLC cell lines. [Ca^+]| was determined as described in Materials and Methods. The insert shows the fluorescence tracing obtained when 100 nM of the peptide indicated was added to cells loaded with fura- 2. Typical [Ca^+]j dose response relationships are shown. The basal [Ca^'^Jj is the mean value for that experiment ± SEM.______

Bradykinin, at 100 nM, increased [Ca^+]j from 100 ± 8 (n = 6 ) to 192 ± 9 (n = 6 ) nM in H69 cells, from 134 ± 17 (n = 5) to 206 ± 17 (n = 5) nM in H510 cells, and from 89 ± 7 (n = 4) to 126 ± 9 (n = 4) nM in H345 cells. In each cell line bradykinin increased [Ca^+]| in a dose-dependent fashion in the nanomolar range; typical dose-response relationships are depicted in Fig. 3.1. Other neuropeptides were also tested for their effects on Ca^+ mobilization. The peptides neurotensin, cholecystokinin and vasopressin increase [Ca^+ji in responsive SCLC cell lines through distinct receptors (Woll and Rozengurt 1 989a). Fig. 3.2 shows that these peptides increased [Ca^+Jj in H69, FI510 and H345 cells respectively, in a dose-dependent fashion in the nanomolar and physiological range. The expression of these receptors is heterogeneous among these lines. 81

Figure 3.3

Bradykinin 80 CCK ^ 60 60

40

20

0 10 min30 sec 10 min30 30 sec 10 min 80 Vasopressin

30 sec 10 min

Figure 3.3 : Changes in the level of total inositol phosphates in bradykinin stimulated H69, vasopressin stimulated H510 and cholecystokinin stimulated H510 SCLC cells as a function of time.

H69 and H510 cells were prelabelled with myo-[^H]inositol and incubated in HITESA containing 20

mM LiCI for 20 min. Then, neuropeptide (100 nM) was added either for 30 sec or for 10 min as

indicated, before the termination of the experiment. Parallel cultures were incubated in the presence of

LiCI but without neuropeptide (controls). Incubations were stopped by addition of 250 p,l ice cold TCA.

The samples were analysed for their composition of inositol phosphates by anion exchange

chromatography on a Mono Q column. All other experimental conditions were as described in Materials

and Methods. Total inositol phosphate was calculated and the results are shown as a percentage increase

of control. Each bar represents the mean of 2 experiments, error bar indicates the range. 82

The Ca2+-mobilizing effects are mediated by distinct receptors as shown by the use of specific antagonists and by the induction of homologous desensitization (Woll and Rozengurt 1989a). Studies carried out by other laboratories are in agreement with these findings (Bunn, Dienhart et al, 1990).

Multiple neuropeptides stimulate accumulation of inositol phosphates.

The binding of a variety of iigands to their specific receptors causes breakdown of phosphatidyiinositoi 4,5-bisphosphate by a phospholipase C, yieiding lns(1,4,5)P3 which is released into the cytosol and mobilizes Ca^+ from intracellular stores (for review see (Berridge and Irvine 1989; Berridge 1993)). Consequently, the ability of neuropetides to stimulate the accumulation of inositol phosphates in SCLC cell lines was studied. In order to characterize the inositol response in more detail, the major inositol phosphate fractions were separated by FPLC after various times of neuropeptide treatment. The inositol phosphate response was amplified by adding LiCI for 20 min prior to the termination of the incubation (Berridge and Irvine 1989; Berridge 1993). As shown in Fig. 3.3 addition of bradykinin to H69 cells, and CCK or vasopressin to H510 cells, labelled with myo-[3|H]inositol stimulated the accumulation of total inositol phosphates in a time-dependent manner. An increase in total inositol phosphate was detectable within seconds of neuropeptide addition. Vasopressin and CCK caused a sustained accumulation of inositol phosphates over 10 min in the presence of lithium. In contrast bradykinin stimulation resulted in a small increase in total inositol phosphates at 30s, however at 10 min no further increase in inositol phosphates were deteceted despite lithium. The results shown in Figs. 3.3 A and 3.3.B demonstrate that neuropeptides stimulate an inositol phosphate response in SCLC cell lines, though the intensity and duration of the response may vary depending on the neuropeptide. A similar situation has been observed in Swiss 3T3 cells (Issandou and Rozengurt 1990).

Multiple neuropeptides stimulate clonal growth of SCLC.

It has been hypothesized that SCLC growth is regulated by multiple autocrine and/or paracrine circuits involving Ca^+-mobilizing neuropeptides (Woll and Rozengurt 1989a). A crucial test of this hypothesis is to determine whether Ca^+- mobilizing neuropeptides can act as growth factors for SCLC cell lines. Consequently,the effect of multiple Ca^+-mobilizing neuropeptides to promote clonal growth in semi-solid medium in different SCLC cell lines was studied. 83

Figure 3 .4

H69 H510 H345

100 200 û 100

o "2 100 o o

O ■ 1 20 Bradykinin nM

Figure 3.4: Dose-dependent effects of bradykinin on colony formation in SCLC cells.

SCLC cell lines H69 (left), H510 (middle) and H345 (right) were cultured in HITESA for 3-5 days.

Colony formation were determined at the concentrations of peptide indicated as described in Materials

and Methods.

Each point in the colony formation assay represents the mean ± SEM of 3-4 independent experiments

(each with 5 replicates). 84

Figure 3.5

H69 H345 175

O 110 X

— BKA — BKA — BKA — BKA BK

« 200 100

(/) 0) c o 50 o o

— BKA — BKA — BKA — BKA BK BK

Figure 3.5: Effect of the bradykinin antagonist [DArg®, Hyp^, Thi^’^, DPhe^] bradykinin on bradykinin induced Ca^+ mobilization and colonyformation in SCLC cells H 69 (left) and

H 345 (right).

Bradykinin (BK) and [DArg®,Hyp^,Thi^’^,DPhe^]bradykinin (BKA) were added at 10 nM and 10 |iM respectively. Top; [Ca^+Jj was determined as described in Materials and Methods. Basal [Ca^+]| is represented by the open bar. Each bar represents the mean ± SEM of 3-6 experiments. Bottom: 10"^ cells in 0.3% agarose were layered onto 0.5% agarose containing bradykinin either in the absence (—) or presence (BKA) of the bradykinin antagonist. After 21 days colonies > 16 cells were counted under a microscope. The open bar represents spontaneous colony formation. Each bar represents the mean ± SD of 5 replicates. 85

Tumour and transformed cells including SCLC are able to form colonies in agarose medium. Indeed, there is a positive correlation between cloning efficiency of the cells and the histological involvement and invasiveness of the tumour in specimens taken from SCLC (Carney, Gazdar et al. 1980). The effect of bradykinin on the ability of H69, H510 and H345 cells to form colonies in semi-solid medium was investigated. Fig. 3.4 (lower panels) shows that bradykinin markedly increased colony growth of these SCLC cell lines in a steeply dose-dependent manner. Optimal colony stimulation in H69 and H345 cell lines was achieved at 10 nM bradykinin and in HSIO cells at 5- 10 nM bradykinin. At higher concentrations the stimulatory effect decreased, presumably due to homologous desensitization in this long-term assay (Fig. 3.4). Time-dependent mitogenic desensitization has been reported in other cellular systems as discussed in section 1.4.f of the introduction ( Millar and Rozengurt 1989; Millar and Rozengurt 1990b). The role of bradykinin receptors in mediating Ca^+ mobilization and cell growth was tested using [DArgO, Hyp^, Thi^»®, DPhe^] bradykinin, a specific competitive antagonist of the 82 receptor (Steranka, Farmer et al. 1989). The antagonist, at 10 jiM, completely blocked the increase in both [Ca^+Jj and colony formation induced by 10 nM bradykinin in either H69 cells or H345 cells. [DArgO, Hyp®, Thi®'®, DPhe^] bradykinin at 10 pM had no effect on the basal [Ca^+jj or on spontaneous colony formation in the absence of bradykinin (Fig. 3.5). Crucially, neurotensin, cholecystokinin and vasopressin at nanomolar concentrations stimulated clonal growth in semi-solid medium (Fig. 3.6). Cholecystokinin, vasopressin and GRP in H69 cells or galanin in H345 cells caused little or no rise in [Ca^+jj and did not stimulate colony formation in these cell lines (Table 3.1). The ability of multiple Ca^+-mobilizing neuropeptides to promote clonal growth in semi-solid medium in different SCLC cell lines is shown in Table 3.1. These neuropeptides stimulate inositol phosphate accumulation, Ca^+ mobilization and colony formation in SCLC cells, and GRP was also included in parallel experiments for comparison. The results demonstrated that, at optimal concentrations, bradykinin, vasopressin, cholecystokinin, neurotensin and GRP induce comparable increases of SCLC clonal growth in responsive cell lines (Table 3.1). Thus, multiple Ca2+- mobilizing neuropeptides, via distinct receptors, can act directly as growth factors for SCLC. 86

TABLE 3.1: Multiple calcium mobilizing neuropeptides stimulate

Cell line Peptide FnMl [CaZ+li %Colony formation

H69 —— 100 ( 8 0 )

Bradykinin 10 4- 360 ± 19 (24) Galanin 50 252 ± 8 ( 3 4 )

Neurotensin SO 4- 455 ± 37 ( 9 )

H 510 — 100 ( 7 0 )

Bradykinin 5 - 1 0 4- 359 ± 23 ( 1 9 )

Vasopressin 100 4- 344 ± 12 ( 1 8 )

CCK 25 4- 291 ± 8 ( 9 )

H345 — — 100 ( 5 0 )

Bradykinin 10 4- 321 ± 37 ( 2 0 )

Vasopressin 150 4- 257 ± 19 ( 9 )

GRP 5 -1 0 4- 232 ± 7 ( 1 0 )

[Ca^+]i was measured with the fluorescent indicator fura-2 asdescribed in Materials and Methods. The positivity of the [Ca^+]j response reflects a productive ligand-receptor complex. Colony formation was determined using the cionogenic assay as described in Materials and Methods. Spontaneous colony formation, i.e. in theabsence of any exogenously added peptide (—), 98 ± 4, 57 ± 4 and 56 ± 6 in H69,

H510 and H345 respectively is normalized to 100% colony formation is expressed as the mean ± SEM,

The number of 35 mm dishes counted are indicated in brackets at the concentration or range of concentrations indicated. In H69 cells, GRP, vasopressin and cholecystokinin bad little or no effect on both [Ca^+]j and colony formation. 87

Figure 3 .6

H69 H510 H345

(0 b w û) "c o o ü 10 100 0 10 100 0 100 200 Neurotensin nM Cholecystokinin nM Vasopressin nM

Figure 3.6; Effect of neurotensin, cholecystokinin and vasopressin on colony formation in H69, H510 and H345 SCLC cell lines.

Colony formation was determined as described in Materials and Methods. Each point in the colony assay represents the mean ± SEM of 2-3 independent experiments (each with 5 replicates). Figure 3.7

200 H510 500 JO c/)

(/) 300 100 Q) 'c O o o Con Gai BK NT Mix Con Gai BK VP CCK Mix

300H69 JO U) 200 (/) Q) C O 100 o O

Control Gal BK NT Mixture

Figure 3.7: Effect of mixtures of neuropeptides on colony formation in H69 and H510 SCLC cell lines. UpperrEffect of neuropeptides at maximal colony stimulating concentrations individualy and in combination in H69 and H510 cells. Colony formation was determined as described in Materials and

Methods. Abbreviations and final concentrations used: Gal, galanin 50nM; BK, bradykinin lOnM; NT, neurotensin 50nM, VP vasopressin lOOnM; CCK, cholecystokinin 50nM. Mix contained a mixture of the preceding peptides at the final concetrations as indicated. Each bar in the colony assay represents the mean ± SEM of one independent experiment (each with 5 replicates). Lower: Time dependent effects of neuropeptides at sub-maximal colony stimulating concentrations individualy and in combination in H69 cells. Abbreviations and final concentrations used: Gal, galanin lOnM; BK, bradykinin 5nM; NT, neurotensin lOnM, Mix contained a mixture of the preceding peptides at the final concetrations as indicated. Each bar in the colony assay represents the mean ± SEM of one independent experiment (each with 5 replicates). Closed bars indicate colony growth after 7 days, open bars indicate colony growth after 21 days. 89

The effect of mixtures of neuropeptides was investigated (Fig 3.7). At maximal colony stimulating concentrations, only a marginal increase in colony stimulation was seen with mixtures of neuropeptides compared with the stimulation seen for individual neuropeptides (though in the case of galanin and bradykinin in H69 cells an increase of 60% and 65% was seen which was significant p<0.05 using an unpaired student t test). This lack ofjsvnergy or additivity suggests that these neuropeptides are all signalling-through-the-samw-pathways and once maximaJ!y,sMmu!«e±no further... increase is possible. Fjow ever,^^ siihnptimal c»leny- ^fimnimting" rnnrrntrations

marked in the early stages of clonal growth. This suggests that in the early stages of clonal growth (single cells to smalLxolomesT-theFe-"is-a--growth-advantag&-to expressing multiple receptors for neuropeptides espeç[9lly„in._the^-presence.oifjQW concentrations of these peptides. This may be particularly relevant in the clinical setting with regard to tumour progression and the formation of metastasis (this will be developed further in chapter 9)

Summary and Discussion It is known that GRP, vasopressin, cholecystokinin and neurotensin are - I ------—ITTI I |- - r -ri,,W d - |# I Y T |- 1 11' 111* * n i tTiaWS ----- secreted by some SCLC tumours (North, Maurer et al. 1980; Sorenson, Pettengill et al. 1981; Wood, Wood et al. 1981; Gazdar and Carney 1984; Goedert, Reeve et al. 1984; Sausville, Carney et al. 1985; Bepler, Rotsch et al. 1988). Other peptides may be released by a variety of normal cells in the lung or, like bradykinin, produced extracellularly as a result of the proteolytic cleavage of plasma precursors in the damaged tissue surrounding tumours (Steranka, Farmer et al. 1989). Collectively, these findings support the hypothecs that SCLC growth is sustained by an extremely -varied ■ajidL_c_omplex network of autocrine and paracrine i^»^n7tinP*= 'ruiAWwri jnultiple neuropeptides. In view of this, it would appear unlikely that a therapeutic jtraitëqy aimedjat.aiiv.sinale qrowth^^ctor^ much clinical impact"ancTtKat any serious attempt at interupting these trophic effects must take into account this heterogeneity and mitogenic complexity. 90

CHAPTER 4

GALANIN STIMULATES CALCIUM MOBILISATION, INOSITOL PHOSPHATE ACCUMULATION AND CLONAL GROWTH IN SMALL CELL LUNG CANCER CELLS.

Galanin, a 29 amino acid peptide (Tatemoto, Rokaeus et al. 1983), has widespread distribution occurring in central and peripheral neurones (Rokaeus 1987). It elicits a variety of rapid biological responses including modulation of the release of several hormones (Fisone, Wu et al. 1987), stimulation of smooth muscle contractility and inhibition of neuronal excitability (Ekblad, Hakanson et al. 1985). Since galanin may play an important role in the regulation of endocrine, neuronal and smooth muscle function, its mechanism of action is attracting considerable attention. In the endocrine pancreas and in pancreatic p cell models in vitro, galanin inhibits the release of insulin (for review see (Ahren, Rorsman et al. 1988)). Galanin activates an ATP-sensitive K+ channel, hyperpolarizes the plasma membrane (de-Weille, Schmid-Antomarchi et al. 1988.; Dunne, Bullett et al. 1989) and thereby inhibits the activity of voltage-dependent Ca^+ channels (Nilsson, Arkhammar et al. 1989; Sharp, Le-Marchand-Brustel et al. 1989). In this manner, galanin reduces Ca^+ influx and blocks the activity of various agents that increase the intracellular concentration of Ca2+ ([Ca^+jj) in the pancreatic p cell. These effects are induced via a pertussis toxin-sensitive G protein (Dunne, Bullett et al. 1989). In myenteric neurons, galanin also hyperpolarizes the plasma membrane and blocks Ca^+ influx via voltage gated Ca^+ channels (Rokaeus 1987; Tamura, Palmer e t al. 1988). Furthermore, galanin inhibits muscarinic agonist stimulated breakdown of inositol phospholipids in tissue slices of ventral hippocampus (Palazzi, Fisone et al. 1988). To date, galanin has not been found to stimulate inositol phosphate production or Ca^+ mobilization from internal stores in target cells nor to act as a direct regulator of proliferation in any cell type. It has been shown in chapter 3 , that multiple neuropeptides stimulate Ca^+ mobilizatioiu jid clonal growth in a variety of SCLC cell lines. In view of the fact that galanin opposes Ca^+ signals and rnpdulates the action of other neuropeptides in various cellular svstems (see above), it was important to determine whether galanin could reduce [Ca^+Jj and antagonize the Ca^+ mobilizing effects of other neuropeptides in SCLC cell lines. Surprisingly, a preliminary result indicated that galanin increased rather than decreased [Ca^-^j in certain SCLC cell lines (Woll and Rozengurt 1989a). 91

Figure 4.1

H510 200

120 150

80 L -"100

alloonM BK BK+ GallOOnM VP VP+ 121 150

130 100

VP 95 en 76 'Gai 'BK Gai Gai Gai

150 150

130 130

106 BK BK + VP+ J VP BKA °®' 1 Min

Figure 4.1: Effect of galanin on [Ca^+]; in SCLC cells.

SCLC cell lines H69 (Left) and H510 (Right) were cultured in HITESA for 3-5 d. Aliquots of 4-5 x 10^

cells were washed and incubated in 10 ml fresh HITESA medium for 2 h at 37°C. Then, 1 jiM fura-2-

AME was added for 5 min. The cells were washed and resuspended in 2 ml of electrolyte solution. This cell suspension was placed in a quartz cuvette. Fluorescence was monitored and [Ca^"*"]! was calculated

as described in Materials and Methods. Agonists and antagonists were added either independently

(upper) or sequentially (middle and lower) at the following final concentrations: BK, 10 nM bradykinin;

BK+, 100 nM bradykinin; VP, 10 nM vasopressin; VP+, 100 nM vasopressin; BKA, 10 |iM [D-Arg®,

Hyp^, Thi^'^, D-Phe] bradykinin; PVP, lOOnM [ Pmp^ ,OMe, Tyr^,Arg^i vasopressin; galanin (Gal) was

added either at 100 nM (upper) or 25 nM (middle and lower). 92

Figure 4 .2

H69 H510 20 1 5 0

1 3 0 00 +

8 0 1 00 1 00

Galanin (nM)

Figure 4.2; Dose-dependent effect of galanin on [Ca^+]; in SCLC cells.

H69 (left panel) and H510 (right panel). Cells (4-5 x 10^) were loaded with fura-2-AME (1 p,M) and

resuspended in 2 ml electrolyte solution. Galanin was added at the concentrations indicated. Fluorescence was monitored continuously as described in Materials and Methods. Basal [Ca^"'']j and

peak [Ca^+]| were calculated at the concentrations indicated. The results represent peak [Ca^+]; values

obtained at the given concentrations of 3-5 independent experiments expressed as mean ± S.E.M. ______

Hence, the elucidation of the signal transduction pathways activated by galanin in

SCLC cell lines warranted further experimental work.

Galanin increases the intraceilular concentration of calcium in SCLC

Addition of 100 nM galanin to either H69 or H510 cells loaded with the fluorescent Ca^+ indicator fura-2 AME increased [Ca^+Jj without any measurable delay (Fig. 4.1). At this concentration, galanin increased [Ca^+jj from 81 ± 4.5 (n=35) to

115 ± 6.3 (n=10) nM in H69 cells and from 107 ± 5.9 (n=27) to 152 ± 9 (n=10) nM in H510 cells. [Ca^+Jj reached peak values at 20-30 s and subsequently declined towards the basal level. The magnitude and kinetics of the [Ca^+]j response induced by 93 galanin were comparable to those induced by other Ca^+-mobilizing neuropeptides like bradykinin (H69) or vasopressin (H510). Repeated additions of galanin (at 25 nM) caused homologous desensitization of Ca2+ mobilization but did not prevent the increase in [Ca^+jj induced through other neuropeptide receptors such as bradykinin and vasopressin (Fig. 4.1). Accordingly, addition of specific bradykinin and vasopressin antagonists blocked the eiffect of the corresponding neuropeptides but did not interfere with the rapid h ci^se in [Ca^+]; induced by galanin (Fig. 4.1 ). Galanin increased the peak level of [Ca^+jj in a concentration-dependent manner in both H69 and HSIO cells (Fig. 4.2). The concentrations of galanin required to induce half-maximum stimulation (EC50) of [Ca^+Jj increase were 22 and 20 nM in H69 and H510 cells, respectively. Maximum stimulation was achieved at 100 nM galanin in both SCLC cell lines.

Effect of EGTA, pertussis toxin and phorbol 12,13-dibutyrate

Since the effect of galanin on [Ca^+Jj in the SCLC cell lines was entirely different from that observed in other cellular systems, the Ca^+ response to galanin was characterized in more detail (Fig. 4.3 and Table 4.1).

Table 4.1:Effect of EGTA, pertussis toxin and membrane depolarization on the increase in [Ca^+Jj induced by galanin ______Increase in [Ca2+]j nM______

Addition H69 ceils H510 cells

- 31 ± 3.5 37 ± 3.8 EGTA 24 ± 1.8 26 ± 2 Pertussis toxin 30 ± 2.8 37 ± 3.6 K+, 145 mM 31 ± 3.9 41 ± 3.8

Experimental conditions are identical to those described in the legend to Figures 4.3 and 4.4. The increase in [Ca^+]j caused by 25 nM galanin in the absence or presence of various additions was calculated by subtracting the basal [Ca^+jj from the [Ca^+I^ peak. The values shown are the means ± S.E.M. of 3-6 independent determinations. ______94

Figure 4 .3

H69 Control EGTA P.Tx PBt 20 20 20 20 1 (0 4—" c D 0 0 Q> Gai O c Gai Gai Gai 0) I 1 o 1 Min Q) 20 20 20 1— O 3 LL 0 0 0 Gai Gai Gai

Figure 4.3: Effect of EGTA, pertussis toxin and PBt 2 on galanin induced Ca^+ m obilization in

SCLC cell lines H69 (Top) and H510 (Bottom).

Cells were preloaded with fura-2-AME and fluorescence was monitored continuously as previously

described.

EGTA: The Ca^"*" chelator EGTA was added to a final concentration of 1.8 mM 1-2 min prior to the

addition of galanin.

Petussis toxin: Cells cultured in HITESA for 3-5 d were washed and incubated in 10 ml fresh HITESA.

Pertussis toxin was added to a final concentration of 200 ng/ml and incubated for 4 h at 37°C. Fura-2-

AME (1 |iM) was then added for 5 min and the cells were then washed and fluoresence monitored as

previously described.

PBt2 : Cells were pretreated with PBt 2 at a final concentration of 500 nM for 3 min prior to the addition

of galanin. In all cases, the final concentration of galanin (Gal) was 25 nM. The results obtained in 3-6

independent experiments are shown in Table 1. 95

Figure 4.4

H69 Ext.K'*' 145mM 0

c o 0 4-^ f cc Gai N Gai i5 iH510 o a . 0 o Q t 0 Gai Gai 1 Min

Figure 4.4: Effect of galanin on membrane potential and [Ca^+]; in SCLC cells. H69 (Top)

and H510 (Bottom).

Left: Cells cultured in HITESA were washed and incubated for 2 h in fresh HITESA. The cells were

then resuspended in 2 ml electrolyte solution and placed in a quartz cuvette. Bis-oxonol at a final

concentration of 100 nM was then added to the cell suspension which was continuously stirred for 5 min

prior to the sequential addition of galanin 25 nM (Gal) and 80 mM KCl (K+). Fluorescence was

monitored as described in Materials and Methods.

Right: Cells preloaded with fura-2-AME were resuspended in electrolyte solution (140 mM, Na'*’) or in

a modified electrolyte solution in which Na"*" was replaced by K+, giving a concentration of 145 mM K+. [Ca^+]j was calculated as described in Materials and Methods.

The increase of [Ca^+Jj resulted from Ca^+ mobilization from internal stores since it still occurred after the addition of 1.8 mM EGTA to chelate extracellular Ca^+ just prior to the addition of galanin (Fig. 4.3). In pancreatic p cells, galanin receptors are coupled to the ATP-sensitive K+ channel via a pertussis toxin sensitive G protein (Dunne, Bullett et al. 1989). Treatment with pertussis toxin (200ng/ml for 4 h) did not prevent galanin-induced Ca^+ mobilization in SCLC cells (Fig. 4.3). fn_^ther cellular systems activation of protein kinase C (PKC) attenuates Ca^+ mobilization 96

from intracellular stores (Nanberg and Rozengurt 1988). Similarly, direct activation of PKC with phorbol 12,1 3-dibutyrate (PBt2) inhibited the Ca2+response to galanin in both H69 and H510 cells (Fig. 4.3).

Dissociation of caicium mobiiizing effects of gaianin from changes in membrane potentiai It has been suggested that the inhibitory effects of galanin on pancreatic secretion and neuronal excitability are mediated by increases in membrane potential (Rokaeus 1987; Tamura, Palmer et al. 1988).. Therefore, the effect that galanin exerts on membrane potential of SCLC cell lines was examined using cells loaded with the membrane potential sensitive dye bis-oxonol. Fig. 4.3. shows that 25 nM galaaitv did not cause any detectable change in membrane potential, as judged by bis-oxonol

^ells. tiL was next determined whether galanin can induce Ca^+ mobilization in depolarized cells. Galanin increased [Ca^+Jj in either H69 or HSIO cells suspended in medium in which the extracellular Na+ was substituted by K+ (Fig. 4.4; Table 4.1). Thus, the effects of galanin on [Ca^+Jj in SCLC cells can be dissociated from changes in membrane potential.

Gaianin stimulates accumulation of inositol phosphates

The binding of a variety of ligands to their specific receptors causes breakdown of phosphatidyiinositol 4,5-bisphosphate by a phosphoiipase C, yielding ins(1,4,5)Pg which is release into cytosol and mobilizes Ca^+ from intracellular stores (for review see (Berridge and Irvine 1989)). Consequently, we determined whether galanin stimulates the formation of inositol phosphates in SCLC cell lines. The inositol phosphate response was amplified by adding LiCI for 20 min prior to the termination of the incubation (Berridge and Irvine 1989). As shown in Fig. 4.5, addition of galanin to H69 cells labelled with myo-[3h]inositol stimulated the accumulation of total inositol phosphates in a time and dose-dependent manner. The response can be detected at a concentration of 5 nM and the EC 50 value was 10 nM. Similar results were obtained when HSIO cells were used instead of H69 cells. 97

Figure 4 .5

8

0 ) CO CO CD o 4 c

(Min) 0

Galanin (nM)

Figure 4.5: Effect of galanin on the accumulation of inositol phosphates.

H69 SCLC cells incubated in HITESA for 3-5 d were washed and labelled in HITESA with 10 |iCi/ml myo-[^H]-inositol for 24 h. Cells were then washed twice in HITESA at 37°C. Approximately 1.5 xlO^ cells were resuspended in 1 ml HITESA containing 0.02 M Hepes and incubated at 37°C with 20 mM

LiCl for 20 min before the addition of galanin at the concentrations indicated. Cells were incubated with galanin for 20 min. The accumulation of total inositol phosphates was determined as described in

Materials and Methods. The increase in inositol phosphates at a particular galanin concentration is expressed as a percentage increase above the control (i.e. cultures incubated in the presence of LiCl for

40 min) and represent the mean of 4 independent experiments ± SEM. Average control value was 1405 cpm, n=10. Average 100 nM galanin value was 2141 cpm, n=10. Insert: Time course of the accumulation of inositol phosphates. 100 nM galanin (closed circles) was added for the times indicated in the presence of LiCl. Percentage increase in total inositol phosphates from control is shown. The control (open circles) (LiCl only for 40 min) value was 2194 cpm; 100 nM galanin for 20 min value was

3330. All other details were as described in Materials and Methods. 98

150 H69 H510 Figure 4.6 100 InsP

(D (0 (Q (D O InsP C

InsP

Time (Min) 2000 H69 lns(1,4,5)1%

lns(1,3,4)^

Q , 1000 ü

0 25 30 Fraction Number

Figure 4.6: Upper: Changes in the level of InsP^, InsP 2 and InsPg in galanin stimulated H69

and H510 SCLC cells as a function of time.

H69 and H510 cells were prelabelled with myo-[^H]inositol and incubated in HITESA containing 20 mM LiCl for 20 min. Then, 100 nM galanin was added for various times. Parallel cultures were incubated in the presence of LiCl but without galanin (controls). Incubations were stopped by addition of 250 |il ice cold TCA. The samples were analysed for their composition of inositol phosphates by anion exchange chromatography on a Mono Q column. All other experimental conditions were as described in Materials and Methods. Each point has an appropriate control and the results are expressed as a percentage change from the control. Each point represents the mean of 3-5 experiments ± SEM. Lower: Elution profile of Ins(l,4,5)P3 andIns(l^,4)P3 in H69 cells stimulated by galanin.

H69 cells were prelabelled with myo-[^H]inositol as described above and incubated in HITESA containing 20 mM LiCl for 40 min. Galanin (100 nM) was added either for 30 sec (circles) or for 20 99 min (triangles) before the termination of the experiment Parallel cultures were incubated for 40 min in the presence of LiCl without galanin (squares). [^H]Ins(l,4,5)?3 was separated from [3H]Ins(l,3,4)?3 as described under Materials and Methods. The peak of radioactivity corresponding to Ins(l,4,5)?3 ^ Üie sample was assigned on the basis of co-elution with standards [3H]Ins(l,4,5)?3 and [^^P]Ins(l,4,5)P3. In this system, the peak eluting immediately prior to Ins(l,4,5)P3 is ascribed to Ins(l,3,4)P3 (25). A similar profile was obtained in 3 independent experiments.

In order to characterize the inositol response in more detail, the major inositol phosphate fractions were separated by FPLC after various times of galanin treatment. As shown in Fig.4.6, an increase in InsPg and Insp 2 was detectable within seconds of galanin addition, this was followed by a marked and slower increase in the InsPi fraction. A marked increase in [^H]lns(l,4,5)P3 was observed after 30 sec of galanin treatment whereas [3H]lns(1,3,4)Pg was predominant after 20 min of incubation (Fig. 6, lower panel). In both H69 and H510 there was a rapid rise in InsP^, which was detectable at 30 sec (increase of 40-60% above control) and maintained for up to 20 min (increase 50-190% above control) . The results shown in Figs. 4.5 and 4.6 demonstrate that galanin stimulates an inositol phosphate response in SCLC cell lines.

Galanin stimulates clonal growth In SCLC

The rapid stimulation of Ca^+ mobilization and inositol phosphate production induced by galanin in SCLC cells prompted the question : what is the effect of this neuropeptide on the growth of these cells? Transformed or tumour cells including SCLC are able to form colonies in semisolid media (Carney, Gazdar et al. 1980; Carney, Cuttitta et al. 1987). Consequently, the ability of H69 and H510 cells to form colonies in this assay was tested in the presence of increasing concentrations of galanin. Galanin caused a marked stimulation of colony-formation in a concentration- dependent fashion (Fig. 4.7). The concentrations required to promote half-maximum stimulation were approximately 20 nM for H69 and H510 cells. The maximum effect was achieved within a narrow range of galanin concentration (about 50 nM). At higher concentrations, the growth promoting effect of galanin was sharply reduced, presumably due to homologous desensitization. 100

Figure 4 .7

H 69 H 510A s: 300 300

- O ^ 200 0) 200 C O o 1 00 1 00 ü

-w 1 0 1 0 0 0 1 0 1 0 0 Galanin (nM)

Figure 4.7: Effect of galanin on colony formation in H69 (Left panel)and H510 (Right panel) SCLC cells.

Cells 3-5 d post passage were washed, resuspended in HITESA and then disaggregated into an

essentially single cell suspension. Cell number was determined using a Coulter Counter and 10^ cells in

0.3% agarose were layered on top of 0.5% agarose, both layers containing galanin at the same

concentration in 33 mm plastic dishes. Colonies represent aggregates of cells >16 counted under a

microscope after 21 d. H69: Each point represents the mean of 7 experiments (each with 5 replicates ) ±

SEM. H510: Each point represents the mean of 5 experiments (each with 5 replicates) ±SEM. 101

Figure 4.8

300 .C co b O) 200 0) 'c o O 1 00 O

10 25 50 100 200 Galanin (nM)

Figure 4.8:

Top: Effect of antagonist [Arg^, D-Trp^’^, MePhe*] substance P on galanin-stimulated Ca^+

mobilization in H69 SCLC cells.

Additions: Galanin, 25 nM (Gal); Galanin 1 |iM (Gal+); [Arg^, D-Trp^'^, MePhe^]substance P, 20 fiM

(Ant).

Bottom: Effect of [Arg^, D-Trp^»^, MePhe*] substance P on galanin induced colony formation.

10"^ cells in 0.3% agarose containing galanin at concentrations indicated either in the absence (closed bars) or in the presence (striped bars) of 20 pM [Arg^, D-Trp^’^, MePhe^j substance P Colonies >16 cells were counted after 21 d under a microscope. Columns represent the mean of 2 experiments (each with 5 replicates), bars indicate ± S.D. 102

The half-maximum concentrations required to induce clonal growth were similar to those required to stimulate Ca^+ mobilization (Fig. 4.2) or inositol phosphate accumulation (Fig.4.5). Galanin did not increase [Ca^+jj in H345, a SCLC cell line responsive to GRP. In this cell line, galanin failed to promote clonal growth whereas GRP, added to parallel assays, caused a marked stimulation (Table 3.1). Thus, the growth-promoting effects of galanin are clearly associated with the ability of this neuropeptide to induce early signalling events. ^cently, [Arg6,D-Trp^.9.MePhe8]substanç,e^CjSd..lLMs_beenJdentjfM as„ a broad spectrum neuropeptide an ^ q o n ist (Woll and RozeDcmcL.19J9jQbL.. Fig. 4.8 shows that addition ofThisT^rrtigonist, at 20 pM, prevented the increase in [Ca2+]j caused by a subsequent addition of galanin in H69 cells. This prompted us to determine whether this antagonist could also prevent galanin from stimulating clonal growth in these cells. As shown in Fig. 4.8 (lower), [Arg®,D-Trp^*9,MePhe®]substance P, added at 20 pM, caused a profound inhibition of colony-formation. This inhibitory effect was reversed by high concentrations of galanin. The results presented here demonstrate that the neuropeptide galanin induces a rapid and translentJjKxej^fcJixJCa^,^ of inositol phdspHates ahci’ stimulates clonal growth of SCLC cell lines. The findings demonstrate that galanin can act as a direct growth factor for cultured human cells. _

Summary and Discussion Galanin is widely distributed and elicits a multiplicity of physiological responses (Rokaeus 1987). However, the only model system in which the signal transduction pathways activated by galanin have been studied in detail is the pancreatic p cell (Ahren, Rorsman et al. 1988). In these cells, galanin stimulates an ATP-sensitive K+ channel which increases the plasma membrane potential, blocks the influx of Ca2+ through voltage-gated Ca 2+ channels and thereby decreases [Ca 2+]j (Nilsson, Arkhammar et al. 1989). These effects are mediated by a pertussis toxin- sensitive G protein (Dunne, Bullett et al. 1989). The results presented here demonstrate that galanin initiates an entirely different set of early events in SCLC cell lines. Galanin stimulates a rapid increase in [Ca^+Jj from internal stores through a pertussis toxin-insensitive pathway. This Ca^+-mobilizing action of galanin can be completely dissociated from changes in membrane potential. Furthermore, galanin stimulates the production of inositol phosphates, consistent with the hypothesis that galanin-induced Ca2+ mobilization is mediated by lns(l, 4,5)P3. This is the first time that galanin is shown to evoke inositol phosphate and Ca^+ mobilization responses in any cell type. 103

Pharmacological and molecular cloning studies provided evidence that neuropeptide receptors are frequently expressed in multiple molecular forms (Woll and Rozengurt 1989b). This suggests that exists in at least two different molecular subtypes. One couples to K+ channels via a pertussis toxin- sensitive G protein e.g. in the pancreatic p cell. A second subtype, present in certain SCLC cells, is coupled to phosphoiipase C which generates lns( 1,4,5)P3 and thereby leads to Ca2+ mobilization from internal stores. In this context, H69 and H510 SCLC cells may provide a useful model to study these novel effects of galanin. In view of the Ca^+.mobilizing actim sJ 3iLWaaiajËie!im.ia^^ important to determine whether this neuropeptide influenceaTbg.^^ of responsive SCLC. Consequently, the effect of galanin on the ability of H69 and H510 cells to form colonies in semisolid medium was determined. This study demonstrated that galanin markedly stimulates the clonal growth of either H69 or H510 cells in semisolid medium. The EC50 values for promoting colony formation are in excellent agreement with the EC50 values for Ca^+ mobilization and inositol phosphate accumulation. Furtherm ore, [Arg®,D-Trp^-®,MePhe®] substance P (6-11), recently identified as a broad spectrum neuropeptide antagonist (Woll and Rozengurt 1990b) prevented Ca^+ mobilization induced by galanin and strikingly inhibited basal and galanin-stimulated colony formation. This is thefirst time that galanin is shown to act as a growth factor for any cell type. G^nin^s widely distributed (Rokaeus 1987) and in human lung is associated with other peptides in neuroendocrine cells (Uddman and Sundler 1987) from which it is presumed that SCLC derive (Gazdar and McDowell 1988). In view of its widespread distribution, it is likely that galanin regulates the proliferation of other cell types, a possibility that warrants further experimental work. The finding that galanin can act as a direct growth factor for SCLC cells further supports the proposition that the growth of these tumours may be regulated in a complex manner by multiple autocrine/paracrine interactions involving neuropeptides. 104

CHAPTER 5

GASTRIN STIMULATES CALCIUM MOBILIZATION AND CLONAL GROWTH IN SMALL CELL LUNG CANCER CELLS.

The possibility that the gastrointestinal peptide gastrin could act as a hormonal growth factor has attracted considerable interest. A considerable body of evidence has demonstrated that the administration of gastrin induces growth promoting effects in the digestive tract and exocrine pancreas (Johnson 1984; Ryberg, Axelson et al. 1990). In particular, an increase in the circulating levels of gastrin has been related to hyperplasia of the gastric enterochromaffin-like cells (Brenna and Waldum 1992). Gastrin also appears to be a growth promoting hormone for malignant cells grown as xenografts in nude mice (Singh, Walker et al. 1986; Hoosein, Kiener et al. 1988; Watson, Durrant et al. 1989). While these observations strongly suggest that gastrin acts as a growth factor, it is difficult to obtain unambiguous evidence for a direct growth-promoting effect of gastrin in vivo because the administration of this peptide could stimulate the release of other biologically active peptides or growth factors which could act as the proximal effectors of the action of gastrin. Cultured cells have provided useful experimental systems for elucidating the extracellular factors that promote cell growth without the many complexities of whole animal experimentation. Nevertheless, compelling evidence in favour that gastrin acts as a cellular growth factor or as an autocrine factor in tumours has been difficult to document using clonal cell populations. Indeed, studies using receptor antagonists and colon carcinoma cell lines resulted in controversial results (Hoosein, Kiener et al. 1988; Hoosein, Kiener et al. 1990; Thumwood, Hong et al. 1991; Yapp, Modlin et al. 1992). The lack of a convenient model system has impeded the elucidation of whether gastrin can act as direct growth factor in vitro. Cell lines established from SCLC provide a useful model system to study the effects of hormonal peptide agonists and antagonists on early signalling events and on cell proliferation (as shown in the previous chapters). The SCLC cell line H345 has been shown to express receptors for gastrin (Staley, Jensen et al. 1990; Bunn, Chan et al. 1992) but the effect of this peptide on H345 growth was not determined.

Gastrin stimulates calcium mobilization

Addition of gastrin-l or CCK -8 to either H69 or H345 SCLC lines loaded with the fluorescent Ca^+ indicator fura-2 AME caused a rapid and transient increase in 105

[Ca2+]j. These findings (Fig.5.1) are in agreement with other reports that demonstrated that gastrin induces Ca^+ mobilization in the SCLC cell line H345 (Staley, Jensen et al. 1990; Bunn, Chan et al. 1992). However, the increase of [Ca2+]j by gastrin or CCK -8 (A[Ca2+]j20-30 nM) was considerably smaller than that induced by other peptides including bradykinin (H69) or GRP (H345) (A[Ca2+]j80- 100 and 85-120 nM respectively). The salient feature shown in Fig. 5.1 is that gastrin-l and CCK -8 induced a prominent Ca^+ mobilization in the SCLC cell line H510. The magnitude of the [Ca^+]j response induced by gastrin-l in this cell line (A[Ca^+];150 nM) was greater than the responses induced by other Ca2+-mobilizing neuropeptides including bradykinin, vasopressin or galanin (A[Ca^+]|100, 70 and SO nM respectively). Identical results were obtained when CCK -8 was added instead of gastrin-l (Fig. 5.1). The increase in [Ca^+]j induced by gastrin-l in H510 cells results from Ca^+ mobilization from internal stores since it still occurred after the addition of 1.8mM EGTA to chelate extracellular Ca^+, just prior to the addition of gastrin-l (results not shown). In view of the findings depicted in Fig. 5.1, the H510 SCLC line has been used in additional experiments to characterize the Ca^+ mobilizing effects of gastrin-l and CCK-8. Repeated additions of gastrin-l caused homologous desensitization of Ca2+ mobilization. Furthermore, addition of gastrin-l attenuated the increase in [Ca^+]j induced by CCK-8 and reciprocally, brief exposure to CCK -8 prevented the [Ca^+]| response induced by gastrin-l. Neither gastrin-l nor CCK -8 prevented the increase in [Ca2+]i induced through a distinct such as bradykinin (Figure 5.2). These results suggest that gastrin-l and CCK -8 induced Ca^+ mobilization in H510 through a common receptor. Gastrin-l, gastrin-ll, des (S 03)CCK-8 and CCK-8 increased the peak level of [Ca^+]j in a concentration-dependent fashion (Fig.5.3). The concentrations required to induce half-maximum stimulation (ECgg) by these agonists were 7, 2.5, 5 and 2.5 nM, respectively. Thus, the receptors expressed by H510 cells recognize gastrin and CCK agonists with approximately equal apparent affinities. 106

Figure 5.1

H69 -1 8 0 H345

-1 7 0 206 —

145 - -1 3 0 1 2 7 - -1 3 0

110 - -1 1 0 I 120 - - 1 0 0 BK CCK Gastrin - H510 267 -

275 - 200 -

208 -

-1 5 6

10- - 1 1 0 100- -1 0 0

Gastrin

Figure 5.1: Effect of gastrin-I and CCK-8 on [Ca^+]; in comparison to other neuropeptides in various SCLC cell lines.

SCLC cell lines H69 (top left), H345 (top right) and H510 (lower) were cultured in HITESA for

3-5 days. Aliquots of 4-5 x 10^ cells were washed and incubated in 10 ml fresh HITESA medium for 2 h at 37°C. Then, 1 p.M fura-2/AME was added for 5 min. The cells were washed and resuspended in 2 ml of electrolyte solution whose composition was described in Materials and Methods. This cell suspension was placed in a quartz cuvette and was continuously stirred. Fluorescence was monitored in a Perkin- Elmer Ls5 luminescence spectrophotometer and basal and peak [Ca^+]j values were determined.

Gastrin-I, CCK-R. cholecystokinin 23-26. BK^Jbradykinin; GRP, gastrin releasing peptide; VP, vasopressin; Gal, galanin. All peptides were used at a final concentration of 100 nM. 107

H510 Figure 5.2:

1 9 0 - - 1 5 3

-1 5 6

112 -1 1 2 100 -1 0 0 CCK BK Gastrin Gastrin ^K Gastrin

+ CM (Ü 205 - Ü 1 9 0 - —163 —160

-121 85 - — 85 1 0 0 - Gastrin BK 1 Min des CCK Gastrin BK

Figure 5.2: Effect of sequential additi , is of gastrin-I and/or CCK-8 on C a^ mobilisation in

the H510 cell line.

H510 cells were washed and loaded with fura-2/AME and resuspended in electrolyte solution and placed in a quatz cuvette. Fluorescence was monitored and basal and peak [Ca^+]i calculated as described in

Materials and Methods. The following abbreviations and final concentrations were used: Gastrin-I,

gastrin-I (5nM); CCK-8, (5nM); des CCK-8, desulphated CCK-8 (5nM) and BK, bradykinin (lOnM). 108

Figure 5.3

Gastrin-l Gastrin-ll

150

100

+ CM CO des-CCK-8 CCK-8 ü 150 c

100

100 1 10 100 [Peptide] nM

Figure 5.3: Dose dependent effect of gastrin-I, gastrin U, CCK-8 and des(S 0 3 )CCK-8 on [Ca2+]|.

H510 cells were preloaded with fura 2/AME and fluorescence was monitored as previously described. desCCK-8, des(S0 3 )CCK-8. A[Ca^+]j i.e. Peak [Ca^^lj - Basal [Ca^'*']} was calculated at each concentration. Each point represents the mean ± S.E.M. from 3-5 independent experiments. 109

Effect of various CCK/Gastrin Antagonists

In order to gain insight into the receptor that mediates the [Ca^+jj response to gastrin and CCK -8 in H510 cells the-^ e c t of various receptor antagonists were tested. Proglumide, a derivative of glutamic acid, and benzotript, a tryptophan derivative, block the binding and biological effects of both gastrin and CCK analogues (reviewed in (Jensen, Wank et al. 1989)). Accordingly, the Ca^+-mobilizing effect of either gastrin-ll or CCK -8 were prevented by addition of either proglumide or benzotript (Fig. 5.4). These antagonists, however, do not discriminate between different types of CCK/gastrin receptors. Recently, a CCK receptor antagonist has been identified that is selective for gastrin/CCKg receptors as compared to pancreatic CCK^ receptors (Bock, DiPardo et al. 1989; Huang, Zhang et al. 1989; Lotti and Chang 1989). Fig. 5.5 shows that addition of the specific gastrin/CCKg receptor antagonist L- 365260 (15 nM) completely blocked the increase in [Ca^+Jj induced by 5 nM gastrin-ll. The antagonist inhibited the [Ca^+Jj response by either gastrin-ll or CCK- 8 in a concentration dependent manner; IC 50 values were 4 and 1 nM, respectively. In contrast, the CCK;^ preferring antagonist L-364,718 (15 nM) had little effect on the increase in [Ca^+Jj induced by 5 nM gastrin-ll (results not shown). The fact that gastrin and CCK agonists were able to induce Ca^+ mobilization with comparable potencies (Fig.5.3) and that L-365260 was a_|îOtç_ntjnhMtor of these responses (Fig.4) indicates that H510 cells express a gastrin/CCKp type receptor. 110

Figure 5 .4

216-

-154

1 3 1 - Proglu Gastrin .nGastrin .n+ BK p^ogiy BK

+ 1 9 1 - CNJ 03 180- O

117 — 120- Benz^~ I ^BK ^ ^ ^pri/'CCK-8+ o . 'BK Gastrin -II CCK-8

1 Min

100'K

G3 40

100 1000 1 10 [Benzotript] |iM [Proglumide] mM

Figure 5.4: Effect of gastrin II and CCK-8 and their antagonists proglumide and benzotript on [Ca^+1; in the H510 SCLC cell line.

Upper: H510 cells, cultured, washed and loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^'^Jj calculated as described previously. The following abbreviations and final concentrations were used:

Gastrin-II, gastrin-II (5 nM); CCK-8, CCK-8 (5 nM); Proglu, progumide (10 mM); gastrin-Il4-, gastrin-II

(100 nM); BK, bradykinin (10 nM); Benz, benzotript (1 mM); Gal, galanin (25 nM); CCK-84-, cholecystokinin-8 (100 nM), Lower: Dose-dependent inhibition of Ca^^ mobilization induced by 5 nM gastrin-II by benzotript and proglumide. AlCa^+Jj i,e. Peak [Ca^+]j - basal [Ca^+]^ was calculated at each antagonist concentration, A[Ca^+li induced by 5 nM gastrin-II is taken as 100%, Ill 216 Figure 5.5 141 -

81 - 108 — % 1 Min CCK Gastrin -135

120 - _120 105 - “ 105

- 90

L365,260 CCK+ BK Gal

• Gastrin CCK-8

CNJ

J I t i#i ml L—\ \i I 1 i i j J 1 10 0.1 [L365,260] nM

Figure 5.5: Effect of antagonist L365,260 on gastrin induced Ca^^ mobilization in SCLC cell line H510.

Upper: H510 cells were preloaded with fura-2/AME and fluorescence was monitored as previously described. The following abbreviations and final concentrations were used: Gastrin-II, gastrin-II (5 nM);

BK, bradykinin (10 nM); L365,260 (15 nM); gastrin-II+, gastrin-II (100 nM). Lower: Dose-dependent

inhibition of Ca^+ mobilization induced by 5 nM gastrin-II and 5 nM CCK-8 by L365,260. 112

Gastrin stimulates clonal growth in SCLC

The preceeding findings indicate that the H510 SCLC ceil line can provide a useful model system to determine whether gastrin can act as a direct growth factor in vitro. Consequently, whether gastrin can stimulate colony formation in agarose- containing medium was examined in this SCLC cell line . Rg. 5.6 shows that gastrin-l and CCK-8 cause(j_a^trikiDg_iDcrease,jii.th&abü%.of.tl:%e^c.eiS3o]PbnTrc^ semi-solid medium. Gastrin increased both the number and the size of colonies, In a dose-dependent manner (Fig. 5.7). This increase In clonal growth was comparable to that Induced by other growth factors. Gastrln-I, gastrln-ll, des-(S 03)CCK-8 and CCK-8 stimulated colony formation at comparable concentrations (Fig. 5.8). In contrast, CCK-10-20 which lacks the COOH-terminal sequence critical for receptor binding, neither increased [Ca^+Jj nor stimulated colony formation Fig 5.9. The ability of gastrin to induce colony growth was attenuated by the addition of the specific gastrin/CCKB receptor antagonist L-365260 (Figure 5.10). The peptides [D- Arg\D-Phe5,D-Trp^'^,LeuT ^jsubstance P and [Arg®,D-Trp^'^,MePhe®]substance P (6-11) have been shown to inhibit signal transduction and cell proliferation by multiple neuropeptides that induce Ca^+ mobilization In SCLC cells (Woll and Rozengurt 1990b). These broad-spectrum neuropeptide antagonists inhibited both the [Ca2+]j response and the stimulation of colony formation induced by gastrin (Figure 5.11). 113

Figure 5.6

Gastrin-l

CCK-8

10 50 200 [Peptide] nM

Figure 5.6: Effect of gastrin-I and CCK-8 on colony formation in H510 SCLC cells.

Cultures, 3-5 days post-passage in HITESA, were washed, and lO'^ viable cells/ml were plated in

HITESA medium containing 0.3% agarose on top of a base of 0.5% agarose in culture medium as described in Materials and Methods. Both layers contained either gastrin-I or CCK-8 at various concentrations, as indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% C02/90% air for 21 days and then stained with nitrotetrazolium blue. Colonies on a control dish and from dishes with 10, 50 and 200 nM gastrin-I and CCK-8 are shown. 114

Figure 5.7

0 ) n E 3 z > , C o o Ü

0.41 Control Area Sq. n m Gastrin nM

Figure 5.7: Dose-dependent stimulation of gastrin-I on colony number and size in H510 SCLC cells.

Cultures, 3-5 days post-passage, were washed and resuspended in HITES A. Single cells (104) in 0.3% agarose were layered on top of 0.5% agarose, both layers containing gastrin-I at the same concentration as indicated. Colonies were incubated and stained as described. Colonies represent aggregates of cells

>0.01 mm^ visualised by a TV camera and analyzed using tbe Macintosh Image program. 115

Figure 5.8

Gastrin Gastrin-ll

_C (/)

(/) Q) desCCK-8 CCK-8 C o o O

0 10 50 100 0 10 50 100 [Peptide] nM

Figure 5.8: Gastrin-I, gastrin-II, CCK-8 and des-(SOg)CCK-8 stimulate colony formation in

H510 SCLC cells.

Cultures, 3-5 days post-passage, were washed and resuspended in HITESA. Single cells (lO^) in 0.3% agarose were layered on top of 0.5% agarose, both layers containing peptide at the same concentration as indicated. Colonies were incubated and stained as described in Materials and Methods. - Colonies represent aggregates of cells >16 cells (120 pm diameter) counted under a microscope. Each bar represents the mean of 3-4 independentexperiments (each with 5 replicates ± SEM). 116

Figure 5.9

300

+ CN (Q CJ

140 —

CCK-8 ^CCK 10-20^8 K 1 min 600 CCK-8 CCK 10-20

Q 400

O 200

0 10 50 100 0 10 50 100 [Peptide] nM

Figure 5.9: Effect of CCK 10-20 on [Ca^+]; and colony formation in H510 SCLC cells.

Upper: H510 cells, loaded with fura-2/AME, were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^+]j calculated as described in

Materials and Methods. CCK 10-20, and CCK-8 were used at a final concentration oflOOnM.

Lower: Single H510 cells (10^) in 0.3% agarose were layered on top of 0.5% agarose, both layers containing peptide at the same concentration as indicated. Colonies were incubated and stained as described in Materials and Methods. CCK 10-20, and CCK-8 were used at the final concentrations indicated. 117

Figure 5.10

— 184

:T 160 _ “ 154

100 — 116

Gastrin+ Gastrin

1 Min

400 -

-C co 300 -

(/) 200 - ' c _o o CJ 100

Gll CCK' L ' L+ ' L+ Gli CCK

Figure 5.10: Effect of L365,260 on gastrin-II and CCK-8 induced increase in colony formation

in H510 SCLC ceils.

L ow er: Single H510 cells (10^) in 0.3% agarose were layered on top of 0.5% agarose, both layers containing peptide at the same concentration as indicated. Colonies were incubated and stained as described in Materials and Methods. The following abbreviations and final concentrations were used: spontaneous colony growth (control); GII, gastrin-II at 50 nM; CCK-8 at 50nM; L,L365,260 (50nM). -3 1 0 118

Figure 5.11

+ 160 CJ <ü o

100 136

Gastrinii Gastrimi l' Min

400

-C (/) 300

(/) .22 200 c o o o 100

Figure 5.11: Effect of broad spectrum antagonists on gastrin induced Ca^^—mobilization and

colony formation.

Upper: H510 cells, loaded with fura-2/AME, were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^"^]! calculated as described in

Materials and Methods.

The following abbreviations and final concentrations were used: Gastrin-II, gastrin-II (5 nM); D, [D-

A rg’, D-Phe^, D-Trp^'^, Leu'^] substance P (20 |iM); gastrin-II+, gastrin-II (100 nM).

Lower: Single H510 cells (10^) in 0.3% agarose were layered on top of 0.5% agarose, both layers

containing peptide at the same concentration as indicated. Colonies were incubated and stained as

described in Materials and Methods. The following abbreviations and final concentrations were used:

spontaneous colony growth (control); GII, gastrin-II at 50 nM; D, [D-Arg^, D-Phe^, D-Trp^’^, Leu"]

substance P at 20 )iM; G, [Arg^, D-Trp^’^,MePhe^] substance P (6-11) at 20 pM. 1

i> A- V] 119

Summary and Discussion

Hence, this study demonstrates that addition of gastrin at nanomolar concentrations to the SCLC line H510 causes a rapid mobilization of Ca%+ from intracellular stores. The magnitude of the [Ca^+Jj signal evoked by gastrin was more pronounced than that elicited by other Ca^+-mobilizing peptides in this cell line. Thus, the SCLC cell line MS 10 has been identified as a useful model system to study effects of gastrin in vitro. Gastrin and CCK share a common C-terminal pentapeptide and consequently bind to common cell surface receptors. The CCK* receptors have a 500-fold higher affinity for CCK-8 than for gastrin. In contrast, CCKg receptors bind either CCK-8 or gastrin with approximately equal apparent affinities (Jensen, Wank et al. 1989; Lin, Holladay et al. 1989). This study shows that gastrin-I, gastrin-II, CCK-8 and des-(S 03)CCK-8 induce rapid Ca%+ mobilization in SCLC H510, at comparable concentrations. Furthermore, the selective gastrin/CCKg antagonist L360265 (Bock, DiPardo et al. 1989; Huang, Zhang et al. 1989; Lotti and Chang 1989) blocked the increase in [Ca^+Jj induced by either gastrin or CCK-8. Thus, the effects of gastrin and CCK in SCLC H510 are mediated by gastrin/CCKg-eceptors. Gastrin has been postulated to act as a cellular growth factor but compelling evidence in vitro has been difficult to obtain. SCLC cell lines including H510 are able to form colonies in semi-solid medium and their clonogenic ability is markedly increased by multiple neuropeptides (chapter 3). The SCLC cell lineJjSIO provide^ an excellent model system in which to study whether gastrin can act as a direct growth factor^Consequently, we determined the effëCT bf gastrin on the ability of these cells to form colonies in agarose-containing medium. Gastrin markedly stimulates the clonal growth of H510 cells. Gastrin-I, gastrin-II, des-(S 03)CCK-8 and CCK-8 induce colony formation at comparable concentrations, inagreement with the effects obtained on Ca^+ mobilization. [D-Arg**, D-Phe^, D-Trp^-^, Leu^ ^ ] substance P and [ArgG, D-Trp^-^, MePhe® ] substance P (6-11), which have been identified as broad spectrum neuropeptide antagonists (Woll and Rozengurt 1990b), prevented Ca^+ mobilization induced by gastrin and strikingly inibited basal and gastrin-stimulated colony formation. These results demonstrate that gastrin acts as a direct growth j factor in vitro and show, for the first time, that this hormonal peptide can stimulate the proliferation of cells outside the gastrointestinal tract. Interestingly, SCLC cells have been shown to express gastrin and CCK peptides (Rehfeld, Bardram et al. 1989; Geijer, Folkesson et al. 1990). Thus, the findings presented here demonstrating that gastrin and CCK-8 can act as direct growth factors for the SCLC cell line H510 further extend the hypothesis that SCLC growth may be regulated by multiple autocrine and paracrine loops involving neuropeptides. 120

CHAPTER 6

MOLECULAR CLONING OF THE CCKg/GASTRIN RECEPTOR.

The hormonal peptides gastrin and cholecystokinin (CCK) share a common C- terminal pentapeptide and bind to at least two different subtypes of receptors (Jensen, Wank et al. 19^9; Lin, Hoiiaday eTaTTsSS). The CCKg receptors, found mainly in the central nervous system and also in the gastrointestinal tract, bind either CCK or gastrin with approximately equal affinities whereas the CCK^ receptors exhibit a 500-fold higher affinity for CCK than for gastrin (Jensen, Wank et al. 1989). These receptor subtypes can be also be distinguished by their sensitivity to specific antagonists (Bock, DiPardo et al. 1989; Huang, Zhang et al. 1989; Lotti and Chang 1989). Recently, cDNAs encoding the rat and canine CCKg/gastrin receptors have been cloned and sequenced (Kopin, Lee et al. 1992; Wank, Pisegna et al. 1992).. Small cell lung carcinoma (SCLC) cell lines provide a useful model system to study the effects of hormonal peptide agonists and antagonists on early signalling events and on cell proliferation (chapters 3,4 and 5). As shown in chapter 5 Gastrin and CCK cause a rapid mobilization of Ca^+ from internal stores and act as direct growth factors for the SCLC cell lines H 510 through receptors with a CCKg/gastrin pharmacology. Gastrin and CCK also induce a rapid mobilization of Ca^+ in other SCLC cell lines (e.g. H 69 and H 345) but the magnitude of the Ca^+ response was markedly lower than in the H 510 SCLC cell lines. To extend these fingings, the expression of the CCKg/gastrin receptor mRNA in different SCLC cell lines was determined. This study, employed polymerase chain reaction (PCR) methodology with oligonucleotide primers designed according to the sequence of rat CCK^ and canine CCKg/gastrin receptors (Kopin, Lee et al. 1992; Wank, Pisegna et al. 1992) and a human brain cDNA library as a template.

Cloning of the human CCKg/gastrin receptor:

Initially part of the coding region of the human CCKg/gastrin receptor was cloned in order to analyse its expression at the mRNA level in SCLC. Primers were designed.tp^mplify the region between the second and third intracellular loops. The primer sequences were deduced according to the nucleotide sequences of the recently cloned cDNAs encoding the canine CCKg/gastrin (Kopin, Lee et al. 1992) and the rat CCK^ receptor (Wank, Pisegna et al. “[992). PCR using primers No.2 and 4 (see Materials and Methods) and a human fetal brain cDNA library cloned in XZAPII as a 121 template resulted in the amplification of a single DNA fragment of the expected about 550 bp. The PCR product was cloned and the nucleotide sequence was determined. The corresponding peptide sequence showed homology with the rat and canine receptor sequence but also revealed a surprising diversity. Specifically, the pentapeptide

Ala/Thr-Ala/Gly-Pro-Gly-Pro (residues 272-276) of the canine/rat gastrin receptor corresponding to the third cytosolic loop, a region thought to play a critical role in signal transduction, was absent in the amplified human sequence. This observation prompted us to confirm the difference in sequence by isolating cDNA clones encoding the human CCKg/gastrin receptor. Therefore, we screened two independent cDNA libraries; one constructed in the AZapll vector (2x10® plaques) and the other in Xgtl 1 (10® plaques) using the cloned PCR fragment as a probe. We isolated in total ten independent clones from the A.Zapll library all of which were subcloned into the pBluescript phagemid by in vivo excision. Their sequences revealed that these clones were derived from three independent clones with insert sizes of 1.0, 1.8 and 2.1 kb. The largest clone was isolated four times and contained an intron of 210 bases (Fig. 6.1 A) including an Ecc^l restriction site and all splice-signals (Sambrook, Fritsch et al. 1989), but showed no significant homology to any other known sequence. The intron was confirmed by analysis of human genomic DNA (Wu, V. and Walsh, J., unpublished data). Its position in the complete CCKg receptor cDNA is denoted by an asterix in Fig.6.IB. All clones from the cDNA library confirmed the divergencies to the dog and rat receptors noted previously by PCR but did not provide a complete sequence. The screening of the A.gt11 library yielded four clones, one of which with an insert-size of 1.7 kb contained the complete coding region. The final cDNA sequence (Fig.IB) of overlapping clones is 2152 bp in length including a poly (A) tail of 45 bases. At the 5' end, 185 nucleotides precede an ATG codon, which is present in the consensus sequence for translation initiation (Sambrook, Fritsch, et. al. 1989). After the translation start there is a single open reading frame, which predicts a protein of 447 amino acid residues (calculated 48.5 kDa) including seven putative transmembrane spanning regions. At the 3' end the termination codon (TGA) is followed by 578 bp of untranslated sequence containing a typical polyadenylation signal. The complete nucleotide and the deduced amino acid sequence are shown in Fig. 6.18. ACC TG flL.gaûct.tgcccataaaggctatcctaggaatCoctttctcacccctattagatg 122 T W

cttacgaccattgcccagaatcttcctccagcttcccggagaattaccacgccaactcctat

tctgcatccaccaccctggagttccagt ttggggcccctccccagttctctctcccttccca F ig u re 6.1

gcggcacccccaaatcctactcctactt£afl G TCC GTA S V 2 3 0

1 CCAGOCGGGOCGAGCCGCGOGAGAGTOGACXJOCAaGCGCCTOGOCTCGOGCSCGOGGACCA 61 OGCXJOGGCAOGGOGCAGOGAGAGGAGGGCOGCGOGAOCCTGAGCCOGAATCGCAGCGTCA 121 GCAGGTGGAOCCOCOGTCGGAOCCCSCCGGGTCGAGCrcAGTAAOGCGGCGOGCTCGGCGG 181 GGOCCATOGAGCT3CTAAAGCTCAACCGGAGCGTGCAGOGAACCGGACCCOGGCCOGOGG MELLKLNRSVQGTGPGPGA

241 CrrrCCCTGTOCCO«XS3GGOGCGCCIXrrCCTCAACAOCAGCAGTGTGGOCAACCTCAOCT SLCRPGAPLLNSSSVGNLSC

301 OCGAOCCœCTCOCATTCGCGGAGCCOGGACACGAGAATTGGAOCTGGCCATTAGAATCA EPPRIRGAGTRELELAIRIT ______I ______361 CTCrrTACGCAGTGATCTTCCTCATXyiGCGTTGGAGGAAATATOCTCATCATCGrOGTCC lyaviflmsvggnmliivvl

TVTNAPLLS 99

481 OCGACCTCCTGCTGOCTGT>3GCTTOCATGœCITCACCCTCCTOCCCAATCTCATOGOCA DLLLAVACMPFTLLPNLMGT 119

541 CJkTrCATCTTTGœACCGTCATrTOCJJlGœGGTrrCCTACCTCATGGGGGTCTGTGTGA FIFGTVICKAVSYLMGVSVS 139 ______III ______60 1 GTG7GTCCACGCTAAGCCTCGTGGCCATCOCACTGGAGCGATATAOCGCCATCTGCCGAC VSTLSLVAIALERYSAICRP 159

ACGCGOCTCGCGTCATTGTAOCCACGT lAARVIVATW 179

CCGTGTACACTGTCGTGCXACCAGTGG >VYTVVQPVG 199 A _ CCAGTOCGCGOGTCCGCCAGACCTOGT >SARVRQTWS 319

CAGGTGTGGTTATGGCCGTGOCCTACG ’GVVMAVAYG 339

:OCTTTGACOGCGACAGTGACAGCGACA IFDGDSDSDS 359

:CAGOGOCTGnCACCAGAACOGOCGTT PGAVHQNGRC 379

lOCGATOGCTGCTACGTGCAACTTCCAC 5DGCYVQLPR 399

iCGGCTCCTaGOCCGGGATCCaGCTCCC TAPGPGSGSR 319

.'OCGTGGTGCGAATGTTGCTGGTGATCG RVVRMLLVIV 339

rATAGTOCCAACACGTOGCGCOCCTTTG YSANTWRAFD 359

XrrCCTATCTCCTTCATTCACTTOCTGA APISFIHLLS 379

rACTOCTrCATOCACCGTCGCTITCOCC YCFMHRRFRQ 399

rCCCOGCCTCCACGAGCTCGCOCCAOGG PRPPRARPRA 419

kTTGCTTCGCTGTCCAGGCTTAGCTACA lASLSRLSYT 439

TTAGAGOGGCCGrOOGOGTrGAGOCAGG 447

3AAAACACAAACCACAACTGACACAOGA rACAOGAAAAGGTAGCTTACCTGACTCA 3AGGCATGCCTCTGATAT00GACTCAGC 3AGACACAGCGTCCCTA0CAGTGAACTA ACCTOCCTCTCACACACATAGATTAATG GACAOGACTGACTCTGOGATGCTOCTAG PGAAAATACCX3TCAGGCCTAATCTCATA GTTCTTCATCCCrTTCCAGTTAAOGACC GTTCAAGAAATAATAAATTGTTTQGCTT AAAAAAAAAAAAAAAAAAAA 2 1 5 2

NQGGLPGA...... VHQNGR CRPETGAVGE NQGGLPGGA APGPVHQNGG CRPVTSVAGE NQGGLPGGT APGPVHQNGG CRHVT-VAGE SQGGLRGGA GPGPAPPNGS CRPEGGLAGE

33 3 TAPGPG--S GSRPTQAKLL AKKRWR TTPTPGPVP GPRPNQAKLL AKKRWR TTPTPGPGL ASA-NQAKLL AKKRWR TAPTPGPGG GPRPYQAKLL AKKRWR SEREVENT 123

Figure 6.1: Human CCKB/gastrin receptor sequence (A) HuCCKg receptor intron sequence. The sequence of the intron found in the HuCCKg receptor cDNAs is printed in small letters, of the coding region in capital letters. Tbe bold numbers indicate the position of the amino acids of the coding region, the small numbers mark the start and the end of the intron. Bases corresponding to splice signals are underlined, the £coRl site is written in italic. (B) Nucleotide and deduced protein sequences of the human fetal brain CCKg receptor cDNA. The lines above the nucleotide sequence indicate the seven putative transmembrane spanning regions (I - VII). The AATAAA RNA cleavage and polyadenylation signal is underlined. The star marks the location of the intron. (Fig.l). Position for the nucleotide sequence are given on the left side, for the amino acid sequence on the right side. (C) Alignment of the third cytoplasmic domain of the human (this study), rat (Wank, Pisegna et al. 1992), Mastomys (Nakata, Matsui et al. 1992) and dog (Kopin, Lee et al. 1992) CCKg receptor.

Alignment of the human CCKg receptor (this study) shows a high degree of over-all similarity of about 89% with the one from canine (Kopin, Lee et al. 1992), rat (Wank, Pisegna et al. 1992) and Mastomys (Nakata, Matsui et al. 1992). However, the sequence comparison also emphazises the important difference in the region corresponding to the less conserved third cytosolic domain (Fig.6.1C). Interestingly, two potential sites for protein kinase C phosphorylation on serines (S 82 and S 300) and the two potential sites for protein kinase A phosphorylation (S 154 and S 437) are conserved among the CCKg receptors of all four species. The role of these potential phosphorylation sites in receptor function and signal transduction remains to be elucidated. While a manuscript of this study was in preparation, Pisegna e t al. (Pisegna, deWeerth et al. 1992) published the cloning and sequencing of the human CCKg receptor from brain and stomach. This sequence is in good agreement with the human fetal braûn sequence presented he^re^^ However, residue 288, which in the PCR fragment and in one of our cDNAs is a lysine (K) residue, is a glutamate (E) in the three other cDNAs and in the sequence published by Pisegna et al. (1992). Furthermore, the clone isolated in this study covers the complete coding region as cDNA and provides additional 5’ untranslated sequences (Fig. 6.IB).

Expression of the CCKg/gastrin receptor in SCLC:

CCK and gastrin induce a rapid mobilization of Ca^+ from intracellular stores and promote clonal growth of certain SCLC lines (see chapter 5). A[Ca ^+]; nM

H 5 1 0

H 3 4 5

H 6 9 5 1 0 GLC28 3 4 5 GLC 19

H 5 1 0 GLC 28 H 3 4 5 GLC 19 H 69 ro (Q GLC 28 CO 00 C (/) 0) CD GLC 19

% Scanning Units 125

Figure 6.2: Upper Panel: Northern blot analyses of different human SCLC lines. 30 mg of total RNA from the SCLC lines H 510, H 345, H 69, GLC 28 and GLC 19 were separated on a 1% agarose / 2.2M formaldehyde gel, blotted, then hybridised with a radiolabelled 1 kb human CCKg

receptor cDNA probe and washed under high sfringent conditions (upper panel). Exposure time was three days. The migration positions of the 28S and 18S rRNA are labelled on the right. The blot was re-probed with a 288 rRNA probe to demonstrate equal loading of all lanes of the gel. The density of each band was determined by scanning densitometry, the level of H510 expression of CCKg/gastrin receptor mRNA was taken as 100%, each bar represents the mean +/- range of 3

independent experiments. Lower panel: Effect of lOOnM gastrin on [Ca^]j in SCLC cells.

SCLC cell lines H510, H345, H69, GLC 28 and GLC 19 were cultured in HITESA for 3-5 d. Aliquots of 4-5 X 10^ cells were washed and incubated in 10 ml fresh HITESA medium for 2 b at 37°C. Then, 1 pM fura-2-AME was added for 5 min. The cells were washed and resuspended in 2 ml of electrolyte solution. This cell suspension was placed in a quartz cuvette. lOOnM gastrin was added and fluorescence monitored, [(Za^+]j was calculated as described in Materials and Methods. Each bar represents the mean

4-/- S.E.M. o f 4-8 independent experiments. ______

Therefore, to determine whether the responsiveness of SCLC lines to CCK and gastrin is correlated with the expression of the CCKg/gastrin receptor, specific probes were designed, using the sequence of the humajn CCKg/gastrin receptor determined in this study (Fig. 6.IB), to analyse the expression of it's corresponding mRNA in SCLC by Northern blot hybridisation. Six different SCLC lines were grown in serum-free HITESA medium (Simms, Gazdar et al. 1980.) for three days, their total RNA isolated, gel-fractionated and hybridized with a radiolabelled human CCKg receptor probe (see Materials and Methods). The autoradiogram.(Fig. 6.2) shows a single hybridizing transcript of about 2.4 kb. The intensity varies greatly according to the cell line used. These bands were analysed by scanning densitometry (Fig 6.2 lower panel). The strongest signal was detected in H 510. Intermediate signals were observed in H^345 and H 69, whereas only a faint band was dëtêcteFïh i^ ^ had no detectable expression of CCKg/gastrin receptor mRNA. " " ^ — Parallel cultures to those used to prepare RNA were used to determine the ability of gastrin to induce Ca^+ mobilisation. Addition of gastrin (lOOnM) to either H510, H69 or H345 and GLC28 SCLC lines, loaded with the fluorescent Ca^+ indicator fura-2 AME caused a rapid and transient increase in [Ca^+Jj. Gastrin induced a prominent Ca^+ mobilisation in the SCLC cell line H510. The magnitude of the [Ca^+jj response induced by gastrin in this cell line was greater than the responses induced in the cell lines H345 H69 and GLC28 (A[Ca^+]j were 155, 30 25 and lOnM 126 respectively in response to lOOnM gastrin ). in the GLC 19 SCLC line gastrin did not cause any increase in [Ca^+]j (Fig 6.2 lower panel) . In order to provide direct evidence that SCLC cell lines express CCKB/gastrin receptors, mRNAs isolated from H510 and H345 cells were reverse transcribed and the resulting cDNAs were used for PCR analysis. Amplification of these cDNAs using primers 24185 and 6R; 3F and 7R; 6F and 7R; and 6F and 10R, (see Materials and Methods) resulted in PCR DNA fragments of 243bp, 140 bp, 231 bp and 528 bp respectively, exactly as predicted from the human brain CCKg/gastrin receptor sequence (this study and 14). These overlapping PCR fragments from each cell line were subcloned and sequenced. The deduced amino acid sequence of the CCKg/gastrin receptor from SCLC cells was identical to that of the CCKg/gastrin receptor from human brain. In particular, the nucleotide sequence of the third cytoplasmic loop (the most variable region between species) of the CCKg/gastrin receptors in both SCLC cell lines lack residues 272-276 present in the canine/rat CCKg/gastrin receptors (Fig 6.3). Hence, we provide direct evidence that CCKg/gastrin receptors are .expressed Jn SCLC cells and these receptors are identical to those in human brain. 127

F ig u r e 6.3

244

SCLC CCKB: ELYLGLRFDG DSDSDSQSRVRN QG G LPG A- VHQNGR CRPETGAVGE HumCCKB: ELYLGLRFDGDSDSDSQSRV RN QG G LPG A- VHQNGR CRPETGAVGE RatCCKB: ELYLGLHFDG ENDSETQSRA RNQGGLPGGA APGPVHQNGG CRPVTSVAGE MasCCKB: ELYLGLRFDGDNDSDTQSRV RNQGGLPGGT APGPVHQNGG CRHVT-VAGE DogCCKB: ELYLGLRFDE DSDSE--SRV RSQGGLRGGA GPGPAPPNGS CRPEGGLAGE

323 SCLC CCKB DSDGCYVQLP RSRPA LELTA LTAPG PG --S GSRPTQAKLL AKKR'/'v/R HumCCKB DSDGCYVQLP RSRPA LELTA LTAPG PG --S GSRPTQAKLL AKKRWR. RatCCKB DSDGCCVQLP RSR--LEM TT LTTPTPGPVP GPRPNQAKLL AKKRW'R MasCCKB DNDGCYVQLP R S R -- LEMTTLTTPTPGPGL ASA-NQAKLL AKKRWR DogCCKB DGDGCYVQLP RSRQT LELSA LTAPTPGPGG GPRPYQAKLL AKKRWR

Figure 6.3: Deduced amino acid sequence of the third cytoplasmic domain of SCLC CCKg/gastrin receptor cDNA.

mRNA from SCLC cell lines H510 and H345 was prepared, cDNAs were synthesized by reverse transcriptase with primer lOR. Amplification by PCR with appropriate internal primers produced

DNA fragments which were cloned and sequenced (see Materials and Methods). The amino acid

sequence was deduced. Alignment of the third cytoplasmic domain of the SCLC, human (this study), rat

(Wank, Pisegna et al. 1992), Mastomys (Nakata, Matsui et al. 1992) and dog (Kopin, Lee et al. 1992) CCKg/gastrin receptor. Amino acid position is indicated nos. 244-323 according to the human sequence.

Summary and Discussion The results presented here have several important implications. The initial observation that a region of the human CCKg/gastrin receptor amplified by PCR revealed significant differences to the known canine and rat gastrin receptors prompted the cloning and sequencing of cDNAs encoding the complete human receptor. In five independent clones from two different human fetal brain cDNA libraries interesting divergence between sequences encoding the human, canine and rat receptor was substantiated. The difference is located in the third cytoplasmic loop, a region generally thought to provide the specificity for the interaction with trimeric G proteins. Further studies will be necessary to elucidate whether the differences have any impact in the coupling of these receptors to signal transduction pathways. This present study also establishes that the expression of the mRNA coding for CCKg/gastrin receptors correlates extremely well with the responsiveness of SCLC 128 for these hormones. The results provide direct evidence that the growth promoting effects of CCK and gastrin in the H510 SCLC cell line are mediated by the CCKg/gastrin receptor whose complete sequence is presented in Fig. 6.1. Nucleotide sequencing of PCR derived fragments from SCLC lines H510 and H345 cDNAs were identical to the brain CCKg/gastrin receptor. In particular, the predicted amino acid sequence of the third cytoplasmic loop of the CCKg/gastrin receptors in both SCLC cell lines lack residues 272-276 present in the canine/rat CCKg/gastrin receptors. Since SCLC growth is stimulated by multiple autocrine and paracrine circuits involving multiple neuropeptides including CCK and gastrin, the elucidation of the structure of the human CCKg/gastrin receptor may facilitate the design of novel antagonists that may be useful in the treatment of SCLC as well as in other areas of clinical pharmacology. Previous studies have shown that multiple neuropeptides induce Ca^+ mobilization in a variety of SCLC cell lines. These studies also identified a considerable heterogeneity in the response of individual SCLC cell lines. In theory, this variability could be due to changes in the expression or function of various constituents of neuropeptide signal transduction pathway leading to Ca^+ mobilization, namely surface receptors, transducing G-proteins and effector phospholipase C. Our results indicate that the variability in the responsiveness to gastrin in various SCLC cell lines can be accounted for by th expression of the mRNA encoding the CCKg/gastrin receptor. Similarly, variability in the response to bombesin/GRP (Corjay, Dobrzanski et al. 1991) and neuromedin B (Giaccone, Battey et al. 1992; Moody, Staley et al. 1992) among SCLC cell lines can also be explained in terms of differential expression of the corresponding mRNAs. We conclude that a major mechanism leading to the heterogeneity of neuropeptide responsiveness amnog SCLC cell lines is the differential expression of the genes encoding for the neuropeptide receptors. 129

CHAPTER 7

ccka receptors are expressed in small cell lung cancer cells AND MEDIATE CALCIUM MOBILISATION AND CLONAL GROWTH

None of the previous studies provided evidence indicating that SCLC cell lines also express CCKy^ receptors.

CCK and Gastrin calcium mobilisation in H510 and GLC 19 SCLC cell lines

The expression of the CCKg/gastrin receptor in SCLC cell lines H510, H345 and H69 can account for the responsiveness of these cell lines to both gastrin and CCK- 8. Thus addition of gastrin to H510 cells attenuated the increase in [Ca^+]j induced by CCK-8 and reciprocally, brief exposure to CCK -8 prevented the [Ca^+Jj response induced by gastrin (Figure 7.1 A). Neither gastrin nor CCK -8 prevented the increase in [Ca^+îi induced through a distinct neuropeptide receptor such as bradykinin. Similar results were obtained in the SCLC cell lines H345 and H69 (results not shown). In contrast to the results obtained with H510, H345 and H69, addition of gastrin (lOOnM) to the SCLC cell line GLC 19 did not stimulate Ca^+ mobilization w hereas addition of lOOnM CCK -8 caused a rapid and transient increase in [Ca2+]j. Furthermore, prior addition of gastrin did not attenuate the increase in [Ca2+]j induced by CCK-8 in this cell line (Fig 7.1 upper panel). These results suggest that GLC 19 SCLC cell line express CCK/^ rather than CCKg/gastrin receptors . This conclusion was further substantiated by the dose-responses of CCK -8 and gastrin on [Ca2+]j in H510 and GLC 19 cells (Fig 7.IB). CCK -8 and gastrin increased [Ca2+]j in H510 cells at identical concentrations (ECgQ=5nM). In contrast, CCK -8 caused a dose dependent increase in [Ca^+jj in the GLC19 cell line (ECgQ= 1 SnM), while gastrin, over the same concentration range (1-IOOnM) had no effect. 130

Figure 7.1

H510 GLC 19 200 210

110 170 -

Gastrin CCK BK CCK BKGastrin

205 A 195 - (ü ü

1 5 0 ------1

CCK Gastrin BK CCK Gastrin BK

1 Min B H510 GLC19

150

40

20

0 1 10 100 1000 10 100 1000 [Agonist] nM

Figure 7.1: Upper panel: Effect of sequential additions of gastrin and CCK-8 on [Ca^+]; in SCLC cells lines H510 and GLC 19.

Cells loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^+]j calculated as descrit)ed previously. Gastrin,

CCK-8 (CCK) and bradykinin (BK) were added sequentially at the following final concentrations: lOOnM, lOOnM and 5 nM respectively. Lower panel: Dose-dependent increase in [Ca^+]| induced by gastrin and CCK-8 in H510 and

GLC 19 SCLC cell ünes. Peak [C!a^+]j was measured and calculated as descrit>ed in Materials and Metliods. A representative dose response curve to gastrin (open circles) and CCK-8 (closed circles) is shown for each cell line. 131

Figure 7 .2

GLC 19 H510 _ 100

CM (Q 60 - O

H510 GLC 19 0.01 0.1 1 1 10 CAM-2200 CAM-1481 Antagonist [nM]

Figure 7.2: Effect of CCKg/gastrin and CCKy^ receptor antagonists on the increase in [Ca^]j

induced CCK-8 in the H510 and GLC 19 SCLC cell lines.

H510 (closed circles) and GLC 19 (open circles) cells loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^+]j calculated as described previously. Dose-dependent inhibition of mobilisation induced by

lOnM CCK-8 in H510 and GLC 19 SCLC cell lines, by CCKg/gastrin receptor antagonist (CAM-2200)

(left panel) and CCK^ receptor antagonist (CAM-1481) (right panel) was measured. A[Ca^+]j i.e. Peak

[Ca^+]| - basal [Ca^+], was calculated at each antagonist concentration. A[Ca^+]j induced by lOnM

CCK-8 is taken as 100%. 132

Figure 7 .3

GLC 28 5 0 -

1 6 0 -

1 5 0 -

1 4 0 - 20 - S C Gastrin CCK BK CCK Gastrin BK r CM (Q ü 1 6 0 -

140 -

1 3 0 - 140 -

Ant A Gastrin C C K BK Ant B Gastrin CCK BK

1 Min

Figure 7.3: Upper panel: Effect of sequential additions of gastrin and CCK-8 on [Ca^+]j in the SCLC cell line GLC 28.

Cells loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^+]j calculated as described previously. Lower panel: Effect of CCKg/gastrin and CCK^^ receptor antagonists on CCK-8 and gastrin mediated [Ca^+]; mobilisation in the GLC 28 SCLC cell line.

GLC 28 cells loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and [Ca^+]| calculated as described previously. Agonists and antagonists were added sequentially at the following final concentrations: Gastrin, lOOnM; CCK, lOOnM ; BK, 20 nM bradykinin; Ant A, 25nM CCK^ antagonist CAM-1481; Ant B, 3.5nM CCKg/gastrin antagonist CAM-2200. 133

Effect of specific CCK^ and CCKg/gastrin antagonists.

Next, we attempted to distinguish the CCK receptors expressed by the SCLC ceil lines H510 and GLC19 by using selective antagonists. Fig 7.2 shows that a specific CCKg/gastrin antagonist (CAM-2200) (Higginbottom, Horweii et ai. 1993) profoundly inhibited the increase in [Ca^+j: stimulated by lOnM CCK -8 in H510, % ÏC^= BOpM). n contrast, this antagonist had little effect on the Ca^+ mobilisation mediated by 1 OnM CCK -8 in GLC 19. Conversely, the CCK/^ antagonist CAM-1481 (Boden, Higginbottom et al. 1993) profoundly inhibited the increase in [Ca^+jj Induced by lOnM CCK -8 In GLC 19 (ICgQ = 3nM), but had little effect on the Ca^+ mobilisation stimulated by lOnM CCK -8 in H510.

As shown in Fig 6.2, the GLC 28 SCLC ceil line shows detectable expression of CCKg/gastrin receptor mRNA. in agreement with this , addition of gastrin caused a rapid and transient increase in [Ca^+jj (Fig 7.3).Addition of CCK -8 also stimulated Ca2+ mobilization in this cell line, but the magnitude of the response (A[Ca2+]j=30nM) was consistently higher than that induced by gastrin (A[Ca2+]j=l0nM). Repeated additions of gastrin caused homologous desensitisation of Ca^+ mobilisation (results not shown). However, addition of lOOnM gastrin did not prevent CCK-8 increasing [Ca^+Jj. Conversely, prior exposure to lOOnM CCK -8 prevented the [Ca^+Jj response induced by gastrin. Fig 7.3 also shows th a t prior treatment with CAM 1481 did not block the gastrin induced [Ca2+]j response but markedly depressed the Ca^+ mobilization in response to CCK- 8. Conversely, addition of CAM 2200 prevented the [Ca^+]j response to gastrin, without affecting the Increase in [Ca^+]j induced by CCK-8. These results strongly suggest that the SCLC cell line GLC28 express both CCKy^and CCKg/gastrin receptors.

CCK;^ and CCKg/gastrin Receptor Subtypes Stimulate Clonal Growth in SCLC

Fig 7.4 shows th a t in th e GLC 19 SCLC cell line, over the concentration range lO-IOOnM, CCK-8 greatly stimulated clonal growth, while over the same concentration range, gastrin had no effect. 134

Figure 7.4

GLC 19

300

b V) 200 g ) 'c o o 100 X X

— 10 100 10 25 50 100

[Gastrin] nM [CCK] nM

Figure 7.4: Effect of gastrin and CCK-8 on colony formation in GLC 19 SCLC cells. GLC 19 cell cultures, 3-5 days post-passage in HITESA, were washed, and lO'^ viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of 0.5% agarose in culture medium as described in Materials and Methods. Both layers contained no additions ( open bar), gastrin (hatched bars) or CCK-8 (closed bars) at the concentrations indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% CO2:90% air for 21 d and then stained with nitro-tetrazolium blue. After

21 d colonies were counted. Each point represents the mean ± SD of 2 experiments each of 5 replicates.

In the H510 and GLC19 SCLC cell lines CCK-8 can stimulate clonal growth. In the SCLC cell line H510, the CCKg/gastrin antagonist (CAM-2200) at 1 nM inhibits the colony stimulating effect of 50nM CCK-8, however, the CCK^ antagonist( CAM- 1481) at 10nM had no effect. In the GLC 19 SCLC cell line, the CCK^ antagonist CAM-

1481 at 10nM abolished the colony stimulating effect of SOnM CCK-8. The CCKg/gastrin antagonist (CAM-2200) at 1 nM had no effect on CCK-8 mediated growth (Fig 7.5). These results show th at the occupancy of the CCK^ receptor can also stimulate clonal growth in SCLC. 135

Figure 7.5

H510 GLC 19

400 -

o 200

»

C C K -8 CCK-8

Figure 7.5: Effect of CCK-8 in the presence or absence of CCK antagonists on colony formation in H510 and GLC 19 SCLC cell lines.

H510 (left panel) and GLC 19 (right panel) cultures, 3-5 days post-passage in HITESA, were washed, and lO'^ viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of

0.5% agarose in culture medium as described in Materials and Metliods. Both layers contained no addition, lOnM CCK^ antagonist CAM-1481 (A), or InM CCKg/gastrin antagonist CAM-2200 (B), in the presence or absence of 50nM CCK-8, as indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% CO2:90% air for 21 days and tlien stained with nitro-tetrazolium blue. Each bar represents the mean -h/- SD of 5 replicates. ______136

Summary and Discussion The GLC 19 SCLC cell line had no detectable expression of CCKo/gastrin ^ - - - ' ... ^ ^ ...... receptor mRNA. Accordingly gastrin, at nanomolar concentrations, did not cause any increase in [Ca^+]j in this cell line. In contrast, CCK -8 caused a rapid and transient increase in [Ca^+]j and pretreatment with gastrin did not attenuate the increase in [Ca2+]j induced by CCK-8. In GLC 19 cells, CCK -8 mobilised [Ca^+jj in a dose dependent manner in the nanomolar range, whereas over the same concentration range gastrin had no measurable effect. Furthermore, the selective CCK;^ antagonist CAM-1481 blocked the increase in [Ca2+]j induced by CCK -8 in GLC19 but not in H510 cells. Conversly, the selective CCKg/gastrin antagonist CAM-2200 blocked the increase in [Ca2+]j induced by CCK-8 in H510 but not in GLC19 cells. Taken together, these results indicate that the Ca^+-mobilizing effects of CCK -8 are mediated through a CCKy^ receptor in GLCl 9 cells and via a CCKg/gastrin receptor in H510 cells. In addition to cell lines that express either CCKg/gastrin or CCK^^ receptors, it was also found th a t the SCLC cell line GLC28 expresses both CCK^ and CCKg/gastrin receptors. Several lines of evidence support this conclusion: a) GLC28 cells show a low level of expression of the CCKg/gastrin receptor mRNA; b)CCK -8 induced an increase in [Ca^+]j of higher magnitude than that stimulated by gastrin in these cells; c) prior exposure to gastrin did not prevent CCK -8 increasing [Ca2+]j, whereas CCK- 8 completely prevented the response to a subsequent addition of gastrin and d) CCK^ and CCKg/gastrin seiective antagonists were effective in preventing the increase in [Ca2+]j induced by CCK-8 and gastrin, respectively. The results presented here show, that SCLC cell lines can express the two distinct CCK receptor subtypes, CCK;^ and CCKg/gastrin, either independently or coexisting in the same cell. CCK has been reported to exert trophic effects on normal pancreas and to stimulate the growth of rat stomach in vivo and has also been implicated in the growth of gut tumours (Lamers and Jansen 1988; Douglas, Woutersen et al. 1989; Heald, Kramer et al. 1992). While these observations suggest that CCK can act as a growth factor, it is difficult to obtain unambiguous evidence, that CCK acting through CCK^ receptors stimulate growth. Consequently, we determined the effect of CCK;^ receptor occupancy on the abilitity of GLC 19 cells to form colonies in agarose-containing medium. This study shows that CCK -8 markedly stimulates colony formation in GLCl 9 cells in a dose dependent manner in the nanomolar range. The selective CCK a antagonist CAM-1481 inhibited the CCK-stimulated colony formation in GLCl9 but not in H510 celis, whereas the selective CCKg/gastrin antagonist CAM-2200 inhibited the CCK-stimulated colony formation in H510 but not in GLCl 9 cells. These results demonstrate, for the first time, that CCK -8 acting through CCK^ receptors can stim ulate Ca^+ mobilisation and clonal growth in a SCLC cell line. 137

SCLC is characterised by its ability to secrete many hormones and neuropeptides including gastrin-releasing peptide, neurotensin, and vasopressin (Goedert, Reeve et al. 1984; Cuttitta, Carney et al. 1985; Sausville, Carney et al. 1985)). Results from chapter 3 show that at optimal concentrations, neurotensin, vasopressin, gastrin-releasing peptide, galanin and bradykinin induce comparable increases of SCLC clonal growth in responsive cell lines. This suggests that SCLC growth is regulated by multiple autocrine and/or paracrine circuits involving Ca^+- mobilizing neuropeptides. Interestingly, SCLC cells have also been shown to express gastrin and CCK peptides (Rehfeld, Bardram et al. 1989; Geijer, Folkesson et al. 1990). Thus, the findings presented here, demonstrating that SCLC express two distinct functional CCK receptors both of which can mediate growth for SCLC cell lines, further extends the hypothesis that SCLC growth may be regulated by multiple autocrine and paracrine loops involving neuropeptides, including CCK and gastrin. Indeed, an autocrine loop involving CCK constitutes a unique case in which a single peptide can induce signal transduction and clonal growth through two different receptor subtypes (i.e. CCKy^ and CCKg) with equal potency. Broad spectrum neuro­ peptide antagonists (Woll and Rozengurt 1990b) provide a strategy to block SCLC growth which takes into account the mitogenic complexity of these tumours. 138

CHAPTER 8

INTERRUPTION OF AUTOCRINE AND PARACRINE GROWTH STIMULATION BY NEUROPEPTIDE ANTAGONISTS.

Introduction

As has been demonstrated in chapter 3, GRP is only one of a multitude of peptides with potential growth regulatory roles in SCLC. Also insulin-like growth facto r 1 and transferrin-like growth factor participate in autocrine growth regulatory loops (Nakanishi, Cuttitta et al. 1988; Vostrejs, Moran et al. 1988; Havemann, Rotsch et al. 1990; Macaulay, Everard et al. 1990). In addition, individual SCLC cell lines can be shown to be responsive to a number of distinct hormones. By monitoring the response of [Ca^+jj the SCLC cell line H345 can respond to eight different individual hormones (acetylcholine, ACTH, bradykinin, cholecystokinin, galanin, GRP, neurotensin and vasopressin) (Woll and Rozengurt 1990a). Thus antagonists capable of blocking the biological effects of neuropeptides could provide an effective approach in the treatment of SCLC.

Broad spectrum Vs specific neuropeptide antagonists as inhibitors of grow th in SCLC.

Many neuropeptide receptors in SCLC have been well characterised pharmacologically and now increasingly at a molecular level, and specific antagonists are available. Specific antagonists include the bradykinin 82 receptor antagonist: [DArgO,Hyp3,Thi5.8,DPhe^]bradykinin (BKA) (Regoli, Drapeau et al. 1986; Steranka, Farmer et al. 1989); the vasopressin receptor antagonist [1-(p- mercapto-p, p-cyclopentamethylene proprionic acid), 2 -(0 -methyl) tyrosine] arginine-vasopressin ([Pm p\ OMeTyr^, ArgS] vasopressin) (Zachary and Rozengurt 1986), the bombesin/GRP antagonist [LeuT 3-psi-(CH 2NH)LeuT 4] bombesin (LiyLB) (Coy, Heinz-Erian et al. 1988; Woll and Rozengurt 1988c; Coy, Taylor et al. 1989) and the CCK^ and CCKg/gastrin receptor antagonists (described in chapters 5 and 7 (Bock, DiPardo et al. 1989; Lotti and Chang 1989; Boden, Higginbottom et al. 1993; Higginbottom, Horwell et al. 1993)). 139

Figure 8.1

CO Q) ■H C O

0 p = 0.346

p = 0.016

p=0.582

Figure 8.1: Dose-dependent effects of [Leu^^-psi-(CH 2NH)Leu''*j bombesin on colony number

and size in H345 SCLC cells.

Cultures, 3-5 days post-passage, were washed and resuspended in HITESA. Single cells (IO'*) in 0.3% agarose were layered on top of 0.5% agarose, both layers containing [Leu*3-psi-(CH2NH)Leu*'*] bombesin at the same concentration as indicated. Colonies were incubated and stained as described.

Colonies represent aggregates of cells >0.01 mm^ visualised by a TV camera and analyzed using the

Macintosh Image program. ______

The compounds [D-Arg\ D-Phe^, D-Trp7,9, Leu^^jsubstance P and [Arg^, D- Trp^'9, MePhe^Jsubstance P (6-11) (broad spectrum antagonists) have been shown to inhibit signal transduction and DNA synthesis stimulated by bombesin, GRP, bradykinin and vasopressin by preventing agonist receptor binding in a reversible fashion in swiss 3T3 cells (Woll and Rozengurt 1988; Woll and Rozengurt 1990b). There have been reports recently that the specific bombesin antagonist LyLB was able to inhibit the clonal growth of H345 SCLC cells (Mahmoud, Staley et al. 1991). LyLB appears to cause some degree of inhibition of basal colony growth in agarose semi-solid medium in H345 SCLC cells. 140

Results This was studied in greater detail in H345 cells. L\}/LB is partial agonist as well as antagonist (due to impurities as a result of manufacture) (Fig 8.1). In 5 dose response experiments, the concentration of L\|/LB causing the the maximum inhibition of basal clonal growth varied between IGOnM - IpM. From the maximum inhibition seen in each experiment and using a paired student t test, it was determined that LyLB caused a significant 13% inhibition of basal growth (in the absence of exogenous stimulation) p < 0.01, (Control mean nos. of colonies * 190 ± S.E.M. = 28, LyLB [1 OOnM-1 pM] mean nos. of colonies = 165 ± S.E.M. - 20, (n = 22 )). A similar type of result was obtained using the CCKg/gastrin antagonist CAM-2200 in H510 cells (Fig 7.5), which also caused a 48% inhibition of basal growth p < 0.01 using an unpaired student t test (Control mean nos. of colonies = 178 ± S.E.M. = 5, CAM-2200 [InM] mean nos. of colonies = 92 ± S.E.M. = 6 , (n = 5 ). As might be expected LyLB had no effect on basal colony formation of H69 SCLC cells which have very few GRP receptors (results not shown). Therefore the effects of specific and broad spectrum antagonists on colony formation in H345 cells were compared. Specific antagonists were able to inhibit Ca^+ mobilisation induced by the relevant neuropeptide, while Ca^+ mobilisation induced by other neuropeptides was unaffected (Woll and Rozengurt 1988; Woll and Rozengurt 1990a), in contrast broad spectrum antagonists inhibited Ca^+ mobilisation induced by wide variety of neuropeptides in SCLC (Woll and Rozengurt 1988; Woll and Rozengurt 1990a). Fig 8.2 shows that while BKA was able to block the ability of bradykinin, ([Pmp^, OMeTyr^, ArgS] vasopressin) was able to block the ability of vasopressin and (LyLB) was able to block the ability of GRP, to increase clonal growth, these antagonists are highly ligand specific. In contrast [D-Arg\ D-Phe^, 0- Trp^»5, Leu^ ijsubstance P and [Arg®, D-Trp^»®, MePhe®]substance P (6-11) were able to decrease clonal growth in the presence of a wide range of neuropeptide stimulation ( in additon to galanin chapter 4, gastrin and CCK chapter 5 and 7). Crucially these broad spectrum antagonists also caused a much more dramatic decrease Ig the cloning efficiency (i.e. basal growth in the absence exogenous stimulation) of H345 cells than specific antagonists. Indeed in nine different cell lines tested (H69, H345, H510, WX322, GLC 14, 16 and 19 in previous chapters, and H209, HI 28 (Woll and Rozengurt 1990b)) broad spectrum antagonists inhibited the growth of SCLC cells in both liquid and semi-solid media. 141

Figure 8.2

1 0 H 345

200- 1 00 c o 5 Ô 5 0 U 1

■•5—-—o

0 1 0 200 100 200 Bradykinin nM Vasopressin nM GRP

I I G+Agonist D-i-Agonist (3 Specific antagonist+ Agonist • Agonist only

Figure 8.2: Effects ligand specific and broad-spectrum antagonists on basal, bradykinin, vasopressin and GRP stimulated colony formation in H345 SCLC cells.

Cultures, 3-5 days post-passage, were washed and resuspended in HITESA. Single cells (lO'^) in 0.3%

agarose were layered on top of 0.5% agarose, both layers containing additions as indicated.

Abbreviations used: BKA, [DArg®, Hyp^, Thi^-^, DPhe^] bradykinin (ImM ); PVP, ([Pmp^ OMeTyr^,

Arg*] vasopressin (lOOnM); LLB,[Leu^^-psi-(CH 2 NH)Leu^bombesin (IpM); D, [D-Arg\ D-Phe^, D-

Trp^*^, Leu^^]substance P (20|iM); G, [Arg^, D-Trp^*^, MePhe^Jsubstance P (6-11) (20pM). Colonies

were incubated and stained as described. Colonies represent aggregates of cells >120)jm. 142

Of particular interest is the SCLC cell line WX322, which readily forms tumors in nude mice (Langdon, Rabiasz et al. 1991)) This cell line expresses receptors for multiple neuropeptides. As shown in Figure 8.3A, sequential addition of substance P, vasopressin, neurotensin and bradykinin caused rapid and transient increases in [Ca^+Jj in WX322 cells loaded with the fluorescent Ca^+ indicator fura-2. Table 1 shows that WX322 cells respond in this assay to a surprisingly large number of neuropeptides. TABLE 8.1 The effect of multiple peptide hormones and neuropeptides on calcium mobilization in the SCLC cell line WX322. Effective Non-Effective

Angiotensin 1 -k + ACTH Bradykinin + + Atrial natriuretic peptide Cholecystokinin + Calcitonin Dynorphin + Chorionic gonadotropin Endothelin -k a-endorphin GHRH + Epinephrine Bombesin/GRP -k Galanin Neurotensin -k -k GIP Neuromedin B + Glucagon Oxytocin -k -k 5-hydroxytryptamine Substance P -k -k Leu-enkephalin Vasopressin + -k Neuropeptide-Y Parathyroid hormone Substance K TRH [Ca^+]j was measured as described in Materials and Methods. All peptides were tested at a final concentration of 100 nM. Ca^’*’—mobilization: + indicates an increase in [Ca^+]j of 20-30 nM; ++ indicates an increase [Ca^+], of 60-100 nM. GHRH, growth hormone releasing hormone; ACTH, adrenocorticotrophin hormone; GIP, gastric inhibitory peptide; TRH, thyrotropin releasing hormone.

Hence it becomes obvious that the stimulatory effects on the growth of on the growth of SCLC cells by themselves (autocrine) or by neighboring cells (paracine) may be extremely varied and complex. 143

Figure 8 .3

-152

121 -

97 - 'NT O -210

100 - BK ro O -100 8 0 o NT+ >< 6 0 NT - 2 8 0

4 0 - 2 5 0

20 O -1 4 0 -1 3 0 0 5 1 0 1 5 Day 1 Min Figure 8.3: Effect of agonists and broad spectrum neuropeptide antagon ts on [Ca^]j and

growth in the SCLC cell line WX322. [Ca^+], values were determined as described in Materials and Methods.

Panel A: SP, substance P; VP, vasopressin; NT, Neurotensin; BK, Bradykinin. All peptides were added at a final concentration of 100 nM.

Panel B: SP, substance P (25 nM); SP+, substance P (1(X) nM); NT, Neurotensin (5 nM); NT+, neurotensin (100 nM); D, [D-Arg^ D-Phe^, D-Trp^’^, Leu^^Jsubstance P (20 pM); G, [Arg^, D-

Trp^’^JdePhe^] substance P (6-11) (20 pM).

Panel C: Effect of broad spectrum antagonists on growth of WX322 SCLC cells.

Cells were incubated at a density of 5 x lO'^ cells in 1 ml HITESA substance P (6-11) (open square) or

[D-Arg\ D-Phe^, D-Trp^’^ Leusubstance P (open circle) each at 20 pM. Each point represents mean

+ S.D. of 3 determinations. 144

H69 Figure 8.4 AÎ^

127 -

99 -

-157 20 40 60 80 100 + C\J Antagonist uM ü Galanin Bradykinin t t t t D BK BK+ 'Gai 'Gal+ -1 7 5 —166

-1 4 0 -1 3 0 V, ^BK ^BKt ^Sal ^Galt — 10 25 — 5 10 1 Min Peptide nM + 20 ,uM G

Figure 8.4: Effect of agonists and broad spectrum neurope tide antagonists on [Ca^+J. and

growth in the SCLC cell line H69. Panel A: [Ca^+]- values were deiennined as described in Materials and Meiliods. BK, Bradykinin

(lOnM); Gal, Galanin (25nM); BK+, Bradykinin (lOOnM); Gal+, Galanin (lOOnM). D, [D-Arg^ D-Phe^,

D-Trp^>^, Leu^^jsubstance P (20 pM); G, [Arg^, D-Trp^*^,MePhe^] substance P (6-11) (20 pM). Panel B: Dose-dependent effect of broad spectrum antagonists on basal clonal growth of H69 SCLC cells.

SCLC cell line H69 was cultured in HITESA for 3-5 days. Colony fonnation was detennined in tlie

presence of [Arg^, D-Trp^*^,MePhe^] substance P (6-11) (closed circle) or [D-Arg*, D-Phe^, D-Trp^-^

Leu^ ^Jsubstance P (open square) at tlie concentrations indicated, as described in Materials and Methods.

Each point in the colony formation assay represents the mean ± SEM of 2 independent experiments

(each with 5 replicates). Panel C:Effect of broad spectrum antagonists on galanin and bradykinin stimulated clonal growth growth in H69 SCLC cells.

SCLC cell lines H69 were cultured in HITESA for 3-5 days. Colony formation were detennined at the

concentrations of peptide indicated as described in Materials and Metliods, in the presence of 20pM G,

[Ajg^, D-Trp^’^,MePhe^] substance P (6-11). Each point in the colony formation assay represents tlie

mean ± SEM of one experiment (each witli 5 replicates). 145

In this setting, it would appear unlikely that a therapeutic strategy aimed at any one growth factor would have much clinical impact and that any serious attempt at interrupting these trophic effects must take into account this heterogeneity and complexity. The compounds [D -Arg\ D-Phe^, D-Trp^»^, Leu^^]substance P and [Arg®, D- Trp^'®^ MePhe®]substance P (6-11) prevent neuropeptide signal transduction in . WX322 cells. Addition of either [D-Arg\ D-Phe^, D-Trp^'^, Leu"* ^jsubstance P or [Arg®, D-Trp^'®,MePhe®]substance P (6-11) prevented the increase in [Ca^+]i induced either by substance P or by neurotensin, which acts through a distinct receptor (Figure 8.3B). The antagonists (20 pM) also blocked the increase in [Ca2+]j induced by bradykinin, vasopressin, cholecystokinin and bombesin in this cell line (results not shown) though the relative affinities of these antagonists for the neuropeptide receptors are different (Woll and Rozengurt 1988). Furthermore, both antagonists added at 20 pM caused a profound inhibition of the proliferation of WX322 cells (Figure 8.3C). The SCLC cell line H69 is also known to express multiple neuropeptide receptors (10-12). Addition of bradykinin, galanin or neurotensin induced a marked increase in [Ca^+Jj in H69 cells whereas GRP caused only a slight effect ((Woll and Rozengurt 1989a; Bunn, Dienhart et al. 1990) and chapter 3). Signal transduction and colony formation of this cell line in response to neuropeptides are markedly inhibited by broad spectrum neuropeptide antagonists (Figure 8.4).

Summary and Discussion Thus in SCLC cell lines, these neuropeptide antagonists blocked Ca^+ mobilization by multiple neuropeptides, inhibited cell proliferation in liquid culture and markedly reduced colony formation in semi-solid medium either in the absence or in the presence of exogenously added stimulating neuropeptides Consequently, broad-spectrum neuropeptide antagonists can block multiple autocrine and paracrine growth loops in SCLC. in order to test whether these neuropeptide antagonists could be useful anticancer agents in SCLC, Dr. Lagdon in Professor Smyth's group in Edinburgh has evaluated the effects of [Arg®, D-Trp^ ®, M ePhe®]substance P (6-11) and [D-Arg^, D-Phe®, D-Trp^ ®, Leu^ substance P on the growth of xenografts of the SCLC cell lines WX322 and H69 in nude mice (Langdon, Sethi e t al. 1992). Fragments of the xenograft were implanted subcutaneously in the flanks of nude mice and allowed to grow to a measurable size. Then, a group of animals were treated with the antagonist given peritumorally (45 pg/g) once per day for a week. In other experiments, they found that this was the maximum tolerated dose that could be 146 administered intraperitoneally to non-tumor bearing nude mice for 14 days without lethality. The antagonist profoundly inhibited the growth of the tumor, as compared with the control group. The inhibitory effect was clearly maintained beyond the duration of administration. [A rg6, D-Trp^*^ ,MePhe® ] substance P (6-11) given peritumorally a t 45 pg/g/day for 7 days also produced a marked inhibition of the growth of the H69 xenograft which was maintained beyond the duration of antagonist treatment. To determine whether systemic (rather than peritumoral) administration of [A rg 6 , D-Trp^'9^ MePhe®]substance P (6-11) was also effective, they tested the effects of intraperitoneal injection of this antagonist on the growth of WX322 and H69 xenografts. Although the inhibitory effect of the antagonist administered through this route was smaller, the effect was statistically significant against WX322 xenografts . Next they determined whether the broad spectrum antagonist [D-Arg^ ,D-Phe5, Trp^'9, Leu^ ^jsubstance P (Woll and Rozengurt 1988b) could also inhibit SCLC growth in vivo. An initial experiment demonstrated that this antagonist could be administered for 7 days by continuous infusion using Alzet osmotic minipumps at doses equivalent to 100 pg/g/day without any indication of toxicity while a dose of 20 pg/g/day given as intraperitoneal injections produced lethalities in non-tumor bearing nude mice. Administration of this antagonist at the time of tumor implantation produced a pronounced inhibition of the growth of the WX322 xenograft. Furthermore, continuous infusion of [D-Arg**, D-Phe^, D-Trp^'^, Leu^^Jsubstance P for 7 days also inhibited the growth of previously implanted WX322 xenografts. In other experiments, administration of [D-Arg®, D-Trp^*^, MePhe®] substance P (6-11) by Alzet osmotic minipumps also inhibited the growth of previously implanted H69 xenografts In all of the above described antitumor experiments, there was no evidence of toxicity as indicated by lethalities or body weight loss after injection of these antagonists. There is currently great interest in developing new treatment strategies for SCLC. It has been proposed that the broad-spectrum neuropeptide antagonists [D- A r g \ D-Phe®, D-Trp^*®, Leu^ ^ substance P and [Arg®, D-Trp^*®, MePhe®]substance P (6-11), provide a novel approach to the treatment of these complex tumors, in which multiple neuropeptides interact to stimulate growth. These broad-spectrum neuropeptide antagonists inhibited the growth of the WX322 and H69 SCLC xenografts in nude mice (Langdon, Sethi et al. 1992). The results support the hypothesis that these compounds could constitute useful antiproliferative agents against SCLC . 147

CHAPTER 9

INCREASED RANGE AND POTENCY OF NEUROPEPTIDES ABLE TO RAISE [Ca2+ ]j AND STIMULATE CLONAL GROWTH DURING PROGRESSION OF SCLC.

SCLC is initially sensitive to chemotherapy and radiotherapy and response rates of up to 80% have been achieved. However it almost invariably relapses and is resistant to further treatment, so that the 2 year survival of patients with SCLC remains less than 5% at 2 years (Smyth, Fowlie et al. 1986). One possible approach to breach this impasse is to try and understand the cell biology of SCLC as this may provide novel areas for therapeutic intervention. The identification of changes in the tumor cell, considered in conjunction with the development of clinical resistance, might contribute to an understanding of the mechanisms involved. Particularly useful in this respect is the development of the three classic-type SCLC cell lines (GLC-14. G 1^16 and GLC-19). The GLC lines were derived from a 55 year old female with SCLC and before treatment the GLC 14 cell line was established from a supraclavicular lymph node. After chemotherapy, the patient was in complete remission. After 4 months she relapsed, further chemotherapy resulted in a partial response and the GLC 16 cell lines was established from a recurrence in the lung. After radiotherapy the lung appeared free from tumor. However 3 months later, tumour recurred in the lung, from which the GLC 19 cellJjneLWasjderived, this was resistant to any further treatment and the patient died two months later (Berendsen, de-LeiJ, et al. 1989). Phenotypic and functional characterization of the different cell lines showed that there was a good match between the morphological, biochemical and immunohistological findings in the cell lines as compared to those obtained in the biopsies. Crucially also, the in vitro sensitivity to chemotherapeutic agents reflected the clinically observed development of resistance to treatm ent. Thus the cell lines represent a well-characterised in vitro model system in which to study the development of SCLC (Berendsen, de-LeiJ, et al. 1988; de Vries, Meijer et al. 1989). Initially neuropeptides ( at concentrations of 100 nM), in each of the three cell lines, were screened for their ability to increase [Ca 2+ ]j. Table 9.1 shows that the GLC 14 cell line responded to bradykinin and serum resulting in a large increase in [Ca^+jj and smaller responses were seen to carbamylcholine, CCK, GRP and neuromedin B, no Ca2+ mobilization was observed in response to neurotensin, substance P and vasopressin. 148

Figure 9.1

A G LC19 2 5 0 - 100 1 75- 100

115 j CCK* GLC 16 2 2 5 - 10 100 0 1 10 100 CM [Bombesin] [Neuromedin B] O < 100

120 - CCK NmB Serum GLC 14 2 1 0 -

I 10 25 100 0 10 100 110 — [Bradykinin] [Choiecystokinin] CCK NmB Serur i Min

Figure 9.1: Effects of bombesin, neuromedin B, bradykinin, cholecystokinin and serum on [Ca^liin SCLC ceUs Unes GLC 14, GLC 16 and GLC 19 . Cells loaded with fura-2/AME were resuspended in electrolyte solution and placed in a quartz cuvette. Fluorescence was monitored and basal and peak [Ca^+]^ calculated as described in Materials and

Methods. Panel A: All peptides were added at a final concentration of lOOnM, abbreviations used BN, bonbesin; MNB, neuromedin B, BK, bradykinin; CCK, cholecystokinin. Serum was added at a final concentration of 1%. Panel B: Dose dependent effects of bombesin, neuromedin B, bradykinin and cholecystokinin in SCLC cell lines GLC 16 and GLC 19. ^ Peptides were added at the concentrations indicated. Typical doe response relationships are shown. f \ — 149

However the GLC 19 cell line addition of carbamylcholine, bradykinin, CCK, GRP, neuromedin B, and serum caused large increases in [Ca^+Jj, vasopressin and neurotensin caused smaller but consistent increases in [Ca^+jj and addition of substance P also resulted in a large increase in [Ca^+Jj but this was less consistent. The GLC 19 was the only cell line in which neurotensin caused a measurable increase In [Ca2+]j .

Table 9.1 Calcium mobllsing neuropeptides In GLC SCLC cell lines GLC 14 GLC 16 GLC 19 Carbamylcholine + ++ ++ Bradykinin ++ ++ ++ Cholecystokinin + + ++ GRP ± + ++ Neuromedin B ± + +4- Neurotensin -- 4- Substance P - ± 4-4- Vasopressin - -k 4- Serum ++ +4- 4-4-

The GLC 19,16 and 14 SCLC cell lines were loaded with l|iM Fura2/AME wased and resuspended in

2ml electrolyte solution and the cell suspension placed in a quarzt cuvette and stirred contiuously. Fluorescence was recorded continuously as indicated in Materials and Methods. Ca^+'- mobilization: ± indicates an increase [Ca^+]j of less than 30 nM, + indicates an increase in [Ca^+]j of 30-50 nM; ++ indicates an increase [Ca^+li of greater than 50 nM. The following peptides were also tested at lOOnM-

IpM concentrations and no increase in [Ca^'^lj was ever observed: Adrenocorticotrophin hormone.

Angiotensin 1, Atrial natriuretic peptide. Calcitonin, Chorionic gonadotropin, Dynoiphin, a-endorphin,

Endothelin, Epinephrine, Galanin, Growth hormone releasing hormone. Gastric inhibitory peptide.

Glucagon, 5-hydroxytryptamine, Leu-enkephalin, Neuropeptide-Y, Parathyroid hormone. Substance K,

Thyrotropin releasing hormone.

Thus, not only is there an increase in responsiveness to an individual neuropeptide but also the number of responsive neuropeptides increase during progression of the tumour. Bombesin-like peptides have been shown to be autocrine growth factors for SCLC (Cuttitta, Carney et al. 1985). Bradykinin is produced extracellularly as a result of the proteolytic cleavage of plasma precursors in the damaged tissue surrounding tumours (Steranka, Farmer et al. 1989), and all SCLC cell lines tested have bradykinin receptors (Woll and Rozengurt 1989a; Bunn, Dienhart et al. 1990; Bunn, Chan e t al. 1992). CCK too has been found to mobilize Ca^+ in virtually all SCLC cell lines tested (Woll and Rozengurt 1989a; Bunn, Dienhart e t al. 1990; Bunn, Chan et al. 1992), and in addition may constitute a unique case in SCLC of being able to 150 stimulate growth through two different receptors with equal potency (chapter 7). In view of this we investigated these peptides in greater detail. Cells loaded with the fluorescent Ca^+ indicator Fura 2/AME show that these peptides increase [Ca^+Jj with-out any measurable delay. The CaZ+ mobilisation induced by these peptides at a concentration of IGOnM in the GLC 14 cell line was always consistently lower than that seen in the GLC 16 cell line (Fig 9.1 A). The increase in responsiveness to an individual neuropeptide and also the number of responsive neuropeptides increase during progression of the tumour is clearly seen. 1% Serum caused a rapid increase in [Ca^+Jj in all three GLC SCLC cell lines of 150 - 100 nM (Fig 9.1 A), suggesting that the mobilizable Ca^+ pools were equivalent in each of the three cell lines. Addition of bombesin, neuromedin B, bradykinin and cholecystokinin causes a dose-dependent increase in the nanomolar range in [Ca2+]j in each of the GLC SCLC cell lines (Table 9.2 and Fig 9.1). Typical dose response curves for GLC 16 and 19 (Fig 9. IB)

Table 9.2 A[Ca2 + ]j in response to peptides in GLC 16 and 19 SCLC cell lines GLC 16 GLC 19 Bombesin 25 nM ± 5 (n = 7) 100 ± 15 (n = 7) Neuromedin B 25 nM ± 3 (n = 3) 1 50 ± 20 (n = 4) Bradykinin 105 nM ± 7 (n = 8) 98 nM ±12 (n * 10) Cholecystokinin 32 nM ±4 (n = 3) 56 nM ± 5 (n = 4) All peptides were used at a concentration of 100 nM. Cells were loaded with Fura 2/AME and [Ca^+]j was measured as indicated in Materials and Methods. Values shown are the mean ± SJE.M. Number of observations is indicated by ( n ).

Thys as SCLC progresses from sensitive to resistant to therapy, there is an increase in the number of neuropeptides able to induce a measurable increase in [Ca^+]; and also an increase in the potency of individual neuropeptides. 151

Figure 9.2

Bombesin Neuromedin B I O 15 c o G Cm O - 5 10 15 50 5 10 15 50 - 5 10 25 100 5 10 25 100 c o *■3 CCK 3 E *3 en

- ' 5 10 1 5 ' 2 0 5 10 15 20 — 10 25 50 100 10 25 50 100

Figure 9.2: Dose dependent effects of bombesin, bradykinin,cholecystokinin and neuromedin

B on colony formation in G L C 16 and 19 S C L C cells.

GLC 19 and GLC 16 cell cultures, 3-5 days post-passage in HITESA, were washed, and lO'^ viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of 0.5% agarose in culture medium as described in Materials and Methods. Both layers contained additions at the nanomolar concentrations indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% CO2:90% air for 21 days and then stained with nitro-tetrazolium blue. After 21 days colonies were counted. Each point represents the mean ± SEM of 2-4 experiments each of 5 replicates. 152

Tumour and transformed cells, including SCLC, are able to form colonies in agarose medium, and there is a positive co-ordination between cloning efficiency of the cells and the histological involvement and invasiveness of the tumor in specimens taken from SCLC (Carney, Gazdar et al. 1980). Consequently, the ability of these Ca^+-mobilizing neuropeptides to promote clonal growth in the semi-solid medium was determined in these three GLC SCLC cell lines. Cholecystokinin, bombesin, neuromedin B and bradykinin at nanomolar concentrations stimulate a dose-dependent increase in clonal growth in the GLC 16 and 19 cell lines (Fig 9.2 and Table 9.3). There was a significant increase in the ability of bombesin, neuromedin B and CCK to stimulate clonal growth during tumour progression from GLC 16 to GLC 19 SCLC cell lines. Bradykinin was equally effective in stimulating clonal growth in both GLC 16 and 19 cell lines. The ability of these neuropeptides to stimulate clonal growth in the GLC 14 cell line was consistently less than that seen in the GLC 16 cell line. Serum stimulated clonal growth in the GLC 19, 16 and 14 cell lines (increase 405 - 370%). The cloning efficiency of all three GLC SCLC lines was approximately 1.5%. Hence as the tumor progresses the neuropeptides cholecystokinin, bombesin, neuromedin B and bradykinin have an increased ability to stimulate clonal growth.

Table 9.3: % increase in colony fomation in response to peptides In GLC 16 and GLC 19 SCLC cell lines.

GLC 16 GLC 19 p value Bombesin 202% ± 25 (n = 15) 296% ± 31 (n = 10) p< 0.01 Neuromedin B 192% ± 17 (n = 5) 331% ± 16 (n = 5) p< 0.01 Bradykinin 362% ± 42 (n = 20) 341% ± 38 (n = 20) p> 0.6 Cholecystokinin 232% ± 45 (n = 10) 438% ± 47 (n = 15)s p< 0.01

Colony formation was determined as described in Materials and Methods. The maximum % increase in colony formation above basal growth induced by each peptide was determined in the GLC 16 and GLC

19 SCLC cell lines. The mean values are shown ± S.E.M. and n, the number of observations are shown in parenthesis. Statistical significance was determined using the unpaired student t test ______

[Arg®, D-Trp^'® Me Phe®] substance P (6 - 11) (Antagonist G) and [D-Arg\ D-Phe®, Trp^'9, Leu^ substance P (Antagonist D) are compounds which have been characterised as broad spectrum neuropeptide antagonists (Woll and Rozengurt 1990b) which in SCLC cells inhibit Ca^+ mobilization (Woll and Rozengurt 1990b) and clonal growth stimulated by multiple neuropeptides (see chapter 8). They also cause a dramatic decrease in cloning efficiency of SCLC in the absence of exogenously added peptide (see chapter 8). Broad spectrum antagonists also inhibited the growth of SCLC xenografts in nude mice (Langdon, Sethi et al. 1992). The results shown in 153 chapter 3 suggest that SCLC growth is driven by multiple autocrine and paracrine loops. The increased potency of neuropeptides not only to stimulate a rise in [Ca^+]i, (one of the early signals associated with mitogenesis (Rozengurt 1986)), but also to stimulate clonal growth, suggests that these autocrine/paracrine loops are increasingl\Mmportant as the t prepresses. Hence, to further test the dependence of these cells on neuropeptide mediated growth loops, the ability of broad- spectrum neuropeptide antagonists to inhibit SCLC cell growth was tested. Both antagonist D and G inhibited Ca^+ mobilization induced by cholecystokinin, bombesin, neuromedin B and bradykinin in the GLC 19 SCLC cell line (Fig 9.3A). In addition D and G inhibited the cloning efficiency of the GLC 14, 16 and GLC 11 SCLC cell lines (Antagonist D ICgg 25, 50, 1 pM and antagonist G IC50 25, 25, 5 pM respectively) and the growth of GLC 14, 16 and 19 in liquid culture (Antagonist D IC50 25, 50, and 10 pM and antagonist G IC 50 25, 25 and 15 pM respectively) (Fig 9.3C). 154

Figure 9.3

A GLC 19

220-

r 190 - Ant. D ^NmB + C3 U 145- 1 5 0 - . 1 4 0 - mB Ant BK CCK Bn ^ CCK mB B a o 100 100" S 80 80 o 60 60

oa 40 40 o u 20 20

] 10 100 0 10 100 [Antagonist D] |iM [Antagonist G] |oM C 100"

I 60 ^ 40

0 100 ] 10 100 [Antagonist D] [iM [Antagonist G] |iM 155

Figure 9.3; Panel A Effect of [Arg*, D-Trp"^»^ Me Phe*] substance P (6-11) and [D-Arg^, D-Phe^, Trp"^»^, Leu^^] substance P antagonists on the increase in [Ca^+]; induced by bombesin, neuromedin

B, bradykinin and cholecystokinin in GLC 19 SCLC cell line. GLC 19 cells were loaded with Fura 2/AME. [Ca^+], was determined as described in Materials and

Methods. Abbreviations usediBK, bradykinin (25nM); CCK, cholecystokinin (25nM); BN, bombesin

(25nM); NMB, neuromedin B (25nM); D, [D-Arg\ D-Phe^, Trp^*^, Leu*^] substance P (20 pM); 0,

[Arg^, D-Tip^’^ Me Phe®] substance P (6-11) (20 pM) Panel B: Effect of [Arg^, D-Trp^*^ Me Phe*] substance P (6-11) and [D-Arg^, D-Phe^, Trp^***, Leu^^] substance P antagonists on growth in liquid culture of GLC 19 and GLC 16 SCLC cell lines.

GLC 16 (open) and GLC 19 (closed) SCLC cells, 3-5 days post-passage, were spun down at

2,000 rpm for 30s,washed and resuspended in HITESA. Cells were resuspended at a density of 5 x 10"* cells in 1 ml HITESA in the presence or absence of antagonists in triplicate. At various times, cell number was determined using a Coulter Counter, after cell clumps were disaggregated by passing the cell suspension through 19 and 21 gauge needles Panel C Effect of [Arg®, D-Trp^*^ Me Phe*] substance P (6-11) and [D-Arg', D-Phe^, Trp^»^, Leu^^] substance P antagonists on colony growth in agarose semi-solid medium of GLC 19 and GLC 16 SCLC cell lines.

GLC 16 (open) and GLC 19 (closed) cultures, 3-5 days post-passage in HITESA, were washed, and 10^ viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of 0.5% agarose in culture medium as described in Materials and Methods. Both layers contained additions as indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% CO2:90% air for 21 days and then stained with nitro-tetrazolium blue. Each point represents the mean ^ SEM of 2 experiments each of 5 replicates. ______

Interestingly the GLC 19 seem to be more sensitive to these broad spectrum antagonists both in liquid culture and in semi-solid medium, than the earlier cell lines. This reinforces the Ca2+ and colony results indicating greater neuropeptide dependence as the tumour progresses. In addition, in the GLC 19 cells antagonist D is a more potent gro^h anHgohist than antagonist G (EC 50 1 and 5 respectively) probably reflecting the different specificity's of the two antagonists (e.g. antagonist D is a more potent bombesin/GRP antagonist than antagonist G) (Woll and Rozengurt 1990a). In view of the increasing ability of both bombesin and neuromedin B to mobilize [Ca^+Jj and stimulate colony formation as the tumour progressed, the ability of the bombesin antagonist [Leu^ 3-^ (CH 2NH)-LeuT bom besin (LLB) to inhibit basal growth in the clonogenic assay in the GLC 19 SCLC cell line was determined. LLB was able to inhibit basal colony growth at 1 GOnM and 1 mM by 156

Figure 9.4

100 y)

yj

C O o u

0.1 1 [LLB] |liM

Figure 9.4 Effect of [Leu^^-V|/(CH 2NH)-Lenl'^l bombesin on clonal growth in agarose semi-solid medium of GLC 19 SCLC cell line.

GLC 19 cell cultures, 3-5 days post-passage in HITESA, were washed, and 10"^ viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of 0.5% agarose in culture medium as described in Materials and Methods. Both layers contained additions as indicated. Cultures were incubated at 37°C in a humidified atmosphere at 10% CO2:90% air for 21 days and then stained with nitro-tetrazolium blue. Each point represents the mean ± SEM of 1 experiments each of 5 replicates.

Abbreviations LLB, [Leu*^-\|/(CH 2 NH)-Len*'^l bombesin.

34% (FIG 9.4). Thus, it would appear that in the GLC 19 cell line, clonal growth in semi-solid agarose medium was in part driven by bombesin/neuromedin B autocrine loop.

Summary and Discussion Collectively these findings suggest that as SCLC progresses it is able to respond to a greater range of neuropeptides and with increasing potency. Pulmonary neuroendocrine (NE) cell hyperplasia has been postulated to be an early event in the pathogenesis of both SCLC and NSCLC (Mabry, Nelkin et al. 1991 ). 157

In response to injury, hypoxia and carcinogenic exposure, NE cells produce numerous ^ptides with diverse physiological roles (Schuller 1991). GRP has been identified as an important marker of NE cell hyperplasia. Interestingly, the GLC 16 has been shown to contain abundant GRP mRNA (Verbeeck, Elands et al. 1992). In the context of the multistage evolution of cancer, neuropeptide autocrine and paracrine mitogenic loops may play a role at an early stage in SCLC as a tumor promoter in initiated cells and also later in the unrestrained growth of the fully developed SCLC. In this context broad spectrum antagonists provide a logical adjunct to conventional chemotherapeutic agents and may, in addition, be particularly useful when chemoresistance sets in when the cells appear to be more neuropeptide dependent and may have increased sensitivity to broad spectrum antagonists. 158

CHAPTER 10

GENERAL DISCUSSION

Summary and Discussion

Lung cancer is the commonest fatal malignancy in the developed world. SCLC constitutes 25% of the total and follows an aggressive clinical course, despite initial chemosensitivity (Smyth, Fowlie et al. 1986). Identification of the factors that stimulate the proliferation of SCLC cells will be important in the design of alternative and more effective therapeutic strategies. SCLC is characterized by the presence of intracytoplasmic neurosecretory granules and by its ability to secrete many hormones and neuropeptides (Sorenson, Pettengill et al. 1981; Maurer 1985) including bombesin, neurotensin, cholecystokinin and vasopressin (North, Maurer et al. 1980; Sorenson, Pettengill et al. 1981; Wood, Wood et al. 1981; Gazdar and Carney 1984; Goedert, Reeve et al. 1984; Sausville, Carney et al. 1985; Bepler, Rotsch et al. 1988). Among these, only bombesin-like peptides, which include gastrin-releasing peptide (GRP), have previously been shown to act as autocrine growth factors for certain SCLC cell lines (Cuttitta, Carney et al. 1985; Carney, Cuttitta et al. 1987). In contrast, the role of other neuropeptides in the proliferation of SCLC cells was poorly understood. Consequently, it was important to understand in detail the receptor and signal transduction pathways that mediate the mitogenic action of bombesin and GRP as well as to elucidate the role played by other neuropeptides in SCLC growth.

Calcium mobilization in SCLC cell lines

Studies with SCLC have demonstrated a similar set of early events to those previously elucidated in murine 313 cells. Specifically, GRP stimulates mobilization of intracellular Ca^+and inositol phosphate turnover in SCLC cells (Heikkila, Trepel et al. 1987; Trepel, Moyer et al. 1988b). In a subsequent study, multiple neuropeptides were screened for their ability to induce a rapid increase in [Ca2+]j in different SCLC cell lines (Woll and Rozengurt 1989a). This assay should be regarded as an indicator of a productive ligand-receptor interaction. Ca^+ mobilization is one of the components of a complex array of signalling events rather than the signal that promotes cell growth. Bradykinin, cholecystokinin, galanin, neurotensin and vasopressin mduce^rrapida^ increase jn [Ca^+T in SCLC cell lines. The expression of these receptors is heterogeneous among these lines. These neuropeptides 159

Increased [Ca^+]| in a dose-dependent fashion in the nanomolar range. The Ca^+- mobilising effects are mediated by distinct receptors as shown by the use of specific antagonists and by the induction of homologous desensitisation (chapter 3). Studies carried out in other laboratories are in agreement with these findings (Bunn, Dienhart et al. 1990; Bunn, Chan et al. 1992). The observation that galanin, a 29 amino acid neuropeptide, causes Ca^+ mobilization in SCLC is of special interest. In pancreatic cells galanin activates an ATP-sensitive K+ channel, hyperpolarizes the plasma membrane and inhibits the activity of voltage dependent Ca^+ channels (Ahren, Rorsman e t al. 1988). in this manner it reduces Ca^+ influx and blocks th e activity of various agents that increase the intracellular concentration of Ca^t Surprisingly, in SCLC cell lines galanin caused a rapid and transient increase in [Ca^+jj from internal stores and stimulated early production of inositol phosphates, particularly lns(1,4,5)P3 (chapter 4). In contrast to the pancreatic cells addition of galanin to SCLC cell lines did not alter membrane potential. Thus, these studies suggest that SCLC express a novel type of galanin receptors that are coupled to Ca^+ mobilization. Collectively, these studies indicate that SCLC exhibit receptors for multiple neuropeptides and that the expression of these receptors is heterogeneous among SCLC cell lines.

Multiple neuropeptides stimulate clonal growth in SCLC cells

In view of the findings discussed, it has been hypothesised that SCLC growth is regulated by multiple autocrine and/or paracrine circuits involving Ca2+-mobilising neuropeptides. A crucial test of this hypothesis is to determine whether Ca^+- mobilising neuropeptides can act as growth factors for SCLC cell lines. Consequently, the effect of multiple Ca2+-mobilising neuropeptides to promote clonal growth in semi-solid medium in different SCLC cell lines was studied (chapter 3). The results demonstrate that, at optimal concentrations, bradykinin, neurotensin, vasopressin, cholecystokinin, galanin, and GRP induce comparable increases of SCLC clonal growth in responsive cell lines. Thus, multiple Ca2+-mobilising neuropeptides, via distinct receptors, can act directly as growth factors for SCLC. It is known that GRP, vasopressin, cholecystokinin and neurotensin are secreted by some SCLC tumours (North, Maurer e t al. 1980; Sorenson, Pettengill e t al. 1981; Wood, Wood e t al. 1981; Gazdar and Carney 1984; Goedert, Reeve et al. 1984; Sausville, Carney et al. 1985; Bepler, Rotsch et al. 1988). Other peptides may be released by a variety of normal cells in the lung or, like bradykinin, produced extracellularly as a result of the proteolytic cleavage of plasma precursors in the damaged tissue surrounding tumours (Steranka, Farmer et al. 1989). Collectively, these findings support the 160 hypothesis that SCLC growth is sustained by an extensive network of autocrine and paracrine interactions involving multiple neuropeptides. Approaches designed to block SCLC growth must take into account this mitogenic complexity.

Cholecystokinin receptors in SCLC.

Despite the phenotypic heterogeneity of SCLC, manifested in different growth patterns (Carney, Gazdar et al. 1985), oncogene expression (Johnson, Makuch et ai. 1988; Takahashi, Nau et al. 1989; Takahashi, Takahashi et ai. 1991), secretion of peptides and expression of their receptors, it became increasingly apparent that virtually all SCLC cell lines tested were able to respond to cholecystokinin ( 7 out of 8 cell lines) (Bunn, Dienhart et al. 1990; Woll and Rozengurt 1990a; Bunn, Chan et al. 1992). In addition gastrin and cholecystokinin are circulating hormones. Their serum post-prandial levels can reach lOO-SOOpmol, though local levels may be much higher than systemic levels. Also the possibility that the gastrointestinal peptides gastrin and cholecystokinin could act as a hormonal growth factors has attracted considerable interest. Therefore gastrin and cholecystokinin responses in SCLC cell lines were studied in greater detail (chapters 5,6 and 7) Gastrin has been postulated to act as a cellular growth factor but compelling evidence in vitro has been difficult to obtain. Gastrin markedly stimulates the clonal growth of H510 cells. Gastrin-I, gastrin-ll, des-(S 0 3 )CCK-8 and CCK-8 induce colony formation at comparable concentrations, in agreement with the effects obtained on Ca2+ mobilization. The increase in [Ca^+jj and stimulation of clonal growth induced by gastrin was blocked by the specific CCKB/gastrin antagonist L365,260. These results demonstrate that gastrin acts as a direct growth factor in vitro through CCKB/gastrin receptors and show, for the first time, that this hormonal peptide can stimulate the proliferation of cells outside the gastrointestinal tract. To extend these findings, the expression of the CCKg/gastrin receptor mRNA in different SCLC cell lines was determined using PCR methodology (chapter 6). Initially part of the coding region of the human CCKg/gastrin receptor was cloned and the nucleotide sequence was determined. The corresponding peptide sequence showed homology with the rat and canine receptor sequence but also revealed a surprising diversity. Specifically, the pentapeptide Ala/Thr-Ala/Gly-Pro-Gly-Pro (residues 272- 276) of the canine/rat gastrin receptor corresponding to the third cytosolic loop, a region thought to play a critical role in signal transduction, was absent in the amplified human sequence. This observation was confirmed by isolating cDNA clones encoding the human CCKg/gastrin receptor, one of which with an insert-size of 1.7 kb contained the complete coding region. After the translation start there is a single open 161

reading frame, which predicts a protein of 447 amino acid residues (calculated Mr 48.5 kDa) including seven putative transmembrane spanning regions. Interestingly, two potential sites for PKC phosphorylation on serines (S 82 and S 300) and the two potential sites for PKA phosphorylation (S 154 and S 437) are conserved among the CCKg receptors of all four species. The role of these potential phosphorylation sites in receptor function and signal transduction remains to be elucidated. Northern blot analysis (using a CCKg/gastrin receptor probe), established that the expression of the mRNA coding for CCKg/gastrin receptor correlates extremely well with the responsiveness of SCLC cells to gastrin and provide direct evidence for the expression of CCKg/gastrin receptors in SCLC cells. Nucleotide sequencing of PCR derived fragments from SCLC lines H510 and H345 cDNAs were identical to the brain CCKg/gastrin receptor. In particular, the predicted amino acid sequence of the third cytoplasmic loop of the CCKg/gastrin receptors in both SCLC cell lines lack residues 272-276 present in the canine/rat CCKg/gastrin receptors. The results indicate that the heterogeneity in responsiveness to gastrin among SCLC cell lines can be accounted for by the expression of the mRNA encoding the CCKg/gastrin receptor. Similarly, variability in the response to gastrin releasing peptide (GRP) (Corjay, Dobrzanski et al. 1991) and neuromedin B (Moody, Staley et al. 1992) among SCLC cell lines can also be explained in terms of differential expression of the corresponding receptor mRNAs. Hence, it was concluded that a major mechanism leading to the heterogeneity of neuropeptide responsiveness among SCLC cell lines is the differential expression of the genes encoding for the neuropeptide receptors. The GLC 19 SCLC cell line had no detectable expression of CCKg/gastrin receptor mRNA. Accordingly gastrin, at nanomolar concentrations, did not cause any increase in [Ca^+jj in this cell line. In contrast, CCK-8 caused a rapid and transient increase in [Ca^+jj and pretreatment with gastrin did not attenuate the increase in [Ca2+]j induced by CCK-8. In GLC 19 cells, CCK-8 mobilised [Ca^+Jj in a dose dependent manner in the nanomolar range, whereas over the same concentration range gastrin had no measurable effect. Selective CCKy^ antagonist CAM-1481 and CCKg/gastrin antagonist CAM-2200 indicate that the Ca2+-mobilising effects of CCK-8 are mediated through a CCK^ receptor in GLC 19 cells and via a CCKg/gastrin receptor in MS 10 cells. In addition to cell lines that express either CCKg/gastrin or CCK^ receptors, we also found that the SCLC cell line GLC28 expresses both CCK^ and CCKg/gastrin receptors. 162

These results showed, that SCLC cell lines can express the two distinct CCK receptor subtypes, CCK^ and CCKg/gastrin, either independently or coexisting in the same cell. CCK has been reported to exert trophic effects on normal pancreas and to stimulate the growth of rat stomach in vivo and has also been implicated in the growth of gut tumours (Lamers and Jansen 1988; Douglas, Woutersen et al. 1989). While these observations suggest that CCK can act as a growth factor, it is difficult to obtain unambiguous evidence that CCK acting through CCKy^ receptors stimulate growth. Consequently, we determined the effect of CCK;^ receptor occupancy on the ability of GLC 19 cells to form colonies in agarose-containing medium. We found that CCK-8 markedly stimulates colony formation in GLC 19 cells in a dose dependent manner in the nanomolar range. The selective CCK^ antagonist CAM-1481 inhibited the CCK- stimulated colony formation in GLC 19 but not in H510 cells, whereas the selective CCKg/gastrin antagonist CAM-2200 inhibited the CCK-stimulated colony formation in H510 but not in GLC 19 cells. These results demonstrate, for the first time, that CCK- 8 acting through CCK;^ receptors can stimulate Ca^+ mobilisation and clonal growth in a SCLC cell line. Interestingly, certain SCLC cell lines have also been shown to express gastrin and CCK peptides (Rehfeld, Bardram et al. 1989; Geijer, Folkesson et al. 1990). Thus, the findings presented here, demonstrating that SCLC express two distinct functional CCK receptors both of which can mediate growth for SCLC cell lines, further extends the hypothesis that SCLC growth may be regulated by multiple autocrine and paracrine loops involving neuropeptides, including CCK and gastrin. Indeed, CCK may constitute a unique case in which a single peptide can induce signal transduction and clonal growth through two different receptor subtypes (i.e. CCK;^ and CCKg) with equal potency.

Blocking the action of multiple neuropeptides: broad spectrum antagonists.

As understanding of the effects of growth factors in cancer increases, it has become possible to plan rational therapeutic interventions. If an autocrine growth loop is considered, in which cells synthesise, secrete, bind and respond to the same growth factor, it is evident that interruption of this cycle at any point will block mitogenesis. Paracrine growth could be blocked in the same way. SCLC constitutes a special case in which unrestrained proliferation appears driven, at least in part, by multiple autocrine and paracrine circuits involving Ca^+-mobilising neuropeptides. Secreted factors can be cleared by antibodies, such as the bombesin monoclonal antibody 2A11 163 used to retard the growth of SCLC xenografts in nude mice (Cuttitta, Carney et al. 1985). Peptide antagonists are not antigenic and should have higher tissue penetration than antibody proteins. Neuropeptide antagonists have been characterized in the model Swiss 3T3 fibroblast system and their effects have then been tested on SCLC in vitro and in vivo.

Two interesting compounds were [DArg\ DPhe^, DTrp^*9, Leu^T] substance P (antagonist D) and [Arg®, Dlrp^-^, MePhe®] substance P(6-11) (antagonist G). Both antagonists reversibly inhibited GRP-stimulated mitogenesis in Swiss 3T3 cells, and antagonist D was 5-fold more potent than antagonist G (Woll and Rozengurt 1988; Woll and Rozengurt 1988a). In contrast, when tested as competitive inhibitors of vasopressin-stimulated mitogenesis, antagonists D and G were equipotent, with half- maximal effect at 1 pM (Woll and Rozengurt 1988; Woll and Rozengurt 1988a). In addition, the antagonists were found to block mitogenesis stimulated by the neuropeptides bradykinin and endothelin (Woll and Rozengurt 1988; Fabregat and Rozengurt 1990b). It is important to note that the antagonists neither block DMA synthesis by PDGF which stimulates Ca^+-mobilization through a different mechanism from neuropeptides (i.e. mediated by tyrosine phosphorylation rather than by a G protein) nor inhibit mitogenesis stimulated by vasoactive intestinal peptide which induces cAMP accumulation without Ca^+ mobilization (Woll and Rozengurt 1988; Woll and Rozengurt 1990b). Thus, the substance P analogue antagonists show broad spectrum specificity against the neuropeptide mitogens bombesin/GRP, vasopressin, bradykinin and endothelin, which act through distinct receptors in Swiss 3T3 cells, but activate common signal transduction pathways.

Broad spectrum antagonists block SCLC growth

The compounds characterized as broad spectrum antagonists in Swiss 3T3 cells were tested as inhibitors of neuropeptide mediated signals and growth in SCLC cell lines. Because SCLC is a heterogeneous group of tumours, each compound was tested in several cell lines. The broad spectrum antagonists inhibited Ca%+ mobilization stimulated by GRP, vasopressin, bradykinin, cholecystokinin and galanin in diverse cell lines and inhibited the growth of SCLC cell lines, in liquid and semi-solid media (chapter 8). Antagonist D and G were equipotent, with half-maximal effect at about 20 pM. The broad spectrum antagonists (D and G) caused a dramatic decrease of the cloning efficiency of these cells in the absence of any exogenously added peptide (i.e. basal colony formation). Broad spectrum antagonists also decrease clonal growth in the presence of neuropeptide stimulation (chapter 8). The striking finding that 164

antagonists D and G inhibit the basal and stimulated clonal growth of so many cell lines, regardless of positivity for bombesin receptors, contrasts with smaller and more selective inhibition of specific bombesin and CCKg/gastrin receptor antagonists. This suggests that broad spectrum antagonists could be more useful anti cancer drugs than ligand specific growth factor antagonists. As a first step to test this possibility, the effect of antagonist D and G on the growth of a H69 and WX322 SCLC xenografts in nude mice was examined. The antagonists administered either once daily by peritumoural injection or by continuous infusion via an alzet mini-osmotic pump, profoundly inhibited the growth of the tumours, as compared with the control group. The inhibitory effect was clearly maintained beyond the duration of administration. A lesser but still significant effect was seen with daily intraperitoneal administration (Langdon, Sethi et al. 1992). These results demonstrate that antagonist G can inhibit SCLC growth in vivo as well as in vitro.

Neuropeptides and progression of SCLC.

The identification of changes in the tumour cell, considered in conjunction with the development of clinical resistance, might contribute to an understanding of the mechanisms involved. Particularly useful in this respect is the development of the three classic-type SCLC cell lines (GLC-14, GLC-16 and GLC-19) established from one patient during longitudinal follow-up (Berendsen, de-Leij, et al. 1988). During this period the tumour changed from sensitive to completely resistant to chemotherapy. Phenotypic and functional characterisation of the different cell lines showed a good match as compared to those obtained in the biopsies. Crucially also, the in vitro sensitivity to chemotherapeutic agents reflected the clinically observed development of resistance to treatment. Thus these cell lines represent a well- characterised in vitro model system in which to study the development of SCLC (Berendsen, de-Leij, e t al. 1988; de Vries, Meijer e t al. 1989). During the transition from being sensitive to chemotherapy and radiotherapy(GLC 14 and GLC 16), to complete resistance to further therapy (GLC 19) there was a progressive increase in responsiveness to neuropeptides both in terms of their ability to mobilise Ca%+ and to stimulate clonal growth. in addition the growth of all three GLC SCLC cell lines (sensitive and resistant to chemotherapy) are inhibited by broad spectrum neuropeptide antagonists, and particularly the GLC 19 cell line (chemoresistant cell line) has increased sensitivity to broad spectrum antagonists. Further confirming increasing neuropeptide dependence during tumour progression. Antagonist D and G having half-maximal effect 165 at about 1-5 ^.M respectively. Thus broad spectrum neuropeptide antagonists are a logical adjunct to conventional chemotherapeutic agents and may be particularly useful when the tumour becomes resistant to further conventional therapy. It is also interesting to note that the basal colony formation of the SCLC cell line GLC 19 was inhibited (34% p< 0.01) by the specific bombesin antagonist [Leu‘'3-Y(CH2NH)-Leu^^] bombesin. This cell line was more sensitive in my hands than any other cell line to [Leu^ 3-y (CH 2NH)-LeuT bombesin. This result suggests that under the special circumstance of this particular cell line in a serum-free clonogenic assay in agarose semi-solid medium, that a bombesin/GRP mediated autocrine growth loop may be more important. However, in vivo, the cells would be exposed to a greater variety of peptides capable of stimulating growth. Hence, it would still seem unlikely that ligand specific antagonists would be effective in all but exceptional cases, particularly as bombesin antagonists would only be useful in tumours with GRP receptors (Thomas, Arvelo et al. 1992). The effect of mixtures of neuropeptides on clonal growth,(chapter 3, Fig 3.7). suggests that there is a growth advantage in expressing multiple receptors for neuropeptides especially when these peptides are present at low concentrations. Neuropeptides at submaximal colony stimulating concentrations were able to interact in an synergistic fashion this effect was more marked in the early stages of clonal growth.This may be particularly relevant in the clinical setting with regard to tumour progression and the formation of metastasis. At maximal colony stimulating concentrations, only a marginal increase in colony stimulation was seen with mixtures of neuropeptides compared with the stimulation seen for individual neuropeptides. This lack of synergy or additivity suggests that these neuropeptides are all signalling through the same pathways and once maximally stimulated no further increase is possible.

Conclusions

Neuropeptides are increasingly implicated in the control of cell proliferation and their mechanisms of action are attracting intense interest. The peptides of the bombesin family including gastrin-releasing peptide (GRP) bind to specific surface receptors and initiate a complex cascade of signalling events that culminates in the stimulation of DNA synthesis and cell division in Swiss 3T3 cells in the absence of other growth promoting factors. These peptides may also act as autocrine growth factors for certain SCLC cells. The results in this thesis strongly suggest that the autocrine growth loop of bombesin-like peptides may be only a part of an extensive network of autocrine and paracrine interactions involving a variety of Ca^+- 166 mobilising neuropeptides in SCLC including bradykinin, cholecystokinin, galanin, neurotensin and vasopressin, and as SCLC progresses it is able to respond to a greater range of neuropeptides and with increasing potency. Pulmonary neuroendocrine (NE) cell hyperplasia has been postulated to be an early event in the pathogenesis of both SCLC and NSCLC (Mabry, Nelkin e t al. 1991). In response to injury, hypoxia and carcinogenic exposure, NE cells produce numerous peptides with diverse physiological roles (Schuller 1991). GRP has been identified as an important marker of NE cell hyperplasia. Branchoalveolar lavage specimens from smokers contain higher levels of this peptide compared to non-smokers, and this is thought to indicate an increased risk of cancer (Aguayo, King et al. 1990). GRP stimulates the growth and differentiation of fetal lung cells (Spindel, Sunday et al. 1987; Cuttitta, Fedorko et al. 1988) and the growth of normal bronchial epithelial cells (Willey, Lechner e t al. 1984). It has also been shown to function as an autocrine growth factor in SCLC (Cuttitta, Carney et al. 1985). In the context of the multistage evolution of cancer, neuropeptide autocrine and paracrine mitogenic loops may play a role at an early stage in SCLC as a tumour promoter in initiated cells and also later in the unrestrained growth of the fully developed SCLC. A detailed understanding of the receptors and signal transduction pathways that mediate the mitogenic action of neuropeptides may identify novel targets for therapeutic intervention. In this context, broad spectrum antagonists that prevent the function of multiple Ca^+-mobilising receptors are of special interest. These antagonists block neuropeptide mediated signals in the 3T3 and SCLC cells and inhibit SCLC growth in vitro and in vivo. Thus, broad spectrum neuropeptide antagonists constitute potential anti cancer agents and provide a logical adjunct to conventional chemotherapeutic agents.

Future Prospectives

Broad spectrum antagonists will soon enter clinical trials. The present generation of substance P antagonists are likely to have fairly short half-lives in vivo despite the protection from proteolysis conferred by D-amino acid. Furthermore, they are unlikely to be orally effective. The main problem is how to increase the bio­ availability of these compounds. One possibility is to give continuous high dose infusion for a prolonged period, particularly at diagnosis, with conventional chemotherapeutic agents in the hope that this combination in addition to host defences will eradicate the disease. Future developments could increase the bio-availability of peptide drugs by enclosing them in lipid mycelles. Future research might also focus on non-peptide antagonists, a non-peptide substance P antagonist has recently been developed (Snider, Constantine et al. 1991). This compound is a very potent 167 antagonist but is also highly specific. Further design of these types of compound require a clear and detailed understanding of their exact mechanism of action and this warrants further experimental work. Using the information gained from Swiss 3T3 cells on the mechanisms of neuropeptide mediated signal transduction and mitogenesis, it is possible to use this system as a basis for investigating signal transduction and areas for therapeutic intervention in SCLC cells (see Fig 10.1). This complex network of signals (see (Rozengurt 1991a) for review) involves a degree of redundancy, suggesting the importance of the mitogenic pathway and ensuring the amplification of the stimulus. Strategies to block growth factor action must embrace this complexity. Importantly, studies with SCLC have demonstrated a similar set of early events. GRP stimulates mobilization of intracellular Ca^+ and inositol phosphate turnover in SCLC cells (Heikkila, Trepel e t al. 1987; Moody, Murphy e t al. 1987; Trepel, Moyer e t al. 1988b). Although caution is prudent when considering interventions with such general effects on cell metabolism, this should not exclude these approaches from consideration. Drugs such as calcium blockers (e.g. nifedipine, verapamil) and lithium have been found to have specific therapeutic applications despite their general effects. Thus we must not be timid in exploring the new possibilities identified by our rapidly expanding knowledge of the basic biology of SCLC. 168

Figure 10.1

Neuropeptide ^ 7 O ©

© M

PLC P|P / Arachidonic acid * Diacylgiycerol lns(1,4,5)R,

© ■ ■ ■ ■ - 1 1 Protein Kinase C [Ca^+] Prostaglandin Eg 1 Transcriptional ^Na+/H+ A ctivation

Figure 10.1: Bombesin-mediated mitogenesis can be blocked at receptor and post-receptor levels.

Growth factor-initiated cell proliferation in 3T3 cells is mediated by multiple signal transduction pathways that act in a synergistic fashion. The interactions have been well defined in these cells. The actions of the neuropeptide growth factors are demonstrated here and means of blocking the various pathways are indicated by broken lines as follows:

1) Specific and broad spectrum neuropeptide antagonists; antibodies to the growth factors or receptors.

2) Down-regulation of the receptor.

3) GDPPS, a G protein antagonist that blocks signal transduction.

4) Phorbol dibutyrate down-regulation of protein kinase C.

5) Heterologous desensitisation of arachidonic acid release.

6) Cyclooxygenase inhibitors, e.g.. indomethacin.

7) Selective transcriptional block by anti-sense RNA.

8) Na+ZK'*' pump inhibitors, e.g.. ouabain.

9) Blockers o f antiport. The abbreviations used: PLC, phospholipase C; PIP], phosphatidyl-inositol 4,5-bisphosphate;

Ins(l,4,5)P3, inositol 1,4,5 trisphosphate; [Ca^+k, intracellular PLA], phospholipase A]: G, guanine nucleotide binding protein. 169

REFERENCES

Aaronson, S. A. (1991). Growth factors and cancer. Science. 254: 1146-1153.

Abe, K., Kameya, T., Yamaguchi, K., Kikuchi, K., Adachi, I., Tanaka, M., Kimura, S., Kodama, T., Shimosato, Y. and Ishikawa, S. (1984). Hormone-producing lung cancers, endocrinologie and morphologic studies. The Endocrine Lung in Health and Disease. Becker KL, Gazdar AF, eds. Philadelphia, W.B. Saunders.

Abe, Y., Kanamori, A., Yajima, Y. and Kameya, T. (1992). Increase in cytoplasmic Ca2+ and stimulation of calcitonin secretion from human medullary thyroid carcinoma cells by gastrin-releasing peptide. Biochem. Biophys. Res. Comm. 185: 833-838.

Aguayo, S. M., King, T. J., Waldron, J. J., Sherritt, K. M., Kane, M. A. and Miller, Y. E. (1990). Increased pulmonary neuroendocrine cells with bombesin-like immunoreactivity in adult patients with eosinophilic granuloma. J Clin Invest. 86: 838-44.

Ahren, B., Rorsman, P. and Berggren, P.-O. (1988). Galanin and the endocrine pancreas. FEBS Lett. 229: 233-237.

Alexander, R. W., Upp, J. J., Poston, G. J., Gupta, V., Townsend, C. J. and Thompson, J. C. (1988a). Effects of bombesin on growth of human small cell lung carcinoma in vivo. Cancer Res. 48: 1439-41.

Alexander, R. W., Upp, J. J., Poston, G. J., Townsend, C. J., Singh, P. and Thompson, J. C. (1988b). Bombesin inhibits growth of human pancreatic adenocarcinoma in nude mice. Pancreas. 3: 297-302.

Andersen, M., Kristjansen, P. E. and Hansen, H. H. (1990). Second-line chemotherapy in small cell lung cancer. Cancer Treat Rev. 17: 427-36.

Antoniades, H. N., Galanopoulos, T., Neville, G. J. and O’Hara, C. J. (1992). Malignant epithelial cells in primary human lung carcinomas coexpress in vivo platelet-derived growth factor (PDGF) and PDGF receptor mRNAs and their protein products. Proc Natl Acad Sci USA. 89: 3942-6. 170

Arriagada, R., Pignon, J. P. and Le, C. T. (1989). Thoracic radiotherapy in small cell lung cancer: Rationale for timing and fractionation. Lung Cancer The Netherlands. 5: 4-6.

Ayesh, R., Idle, J. R., Ritchie, J. C., Crothers, M. J. and Hetzel, M. R. (1984). Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature. 312: 169-70.

Balks, H. J., Conlon, J. M., Creutzfeldt, W. and Stockmann, F. (1988). Circulating bradykinin-like immunoreactivity and the pentagastrin-induced carcinoid flush. Clin Endocrinol Oxf. 29: 141-51.

Ball, E. D., Sorenson, G. D. and Pettengill, 0. S. (1986). Expression of myeloid and major histocompatibility antigens on small cell carcinoma of the lung cell lines analyzed by cytofluorography: Modulation by gamma-interferon. Cancer Res. 46: 2335-9.

Barbacid, M. (1987). RAS GENES (270 Refs). Annu Rev Biochem. 56: 779-827.

Batist, G., Tulpule, A., Sinha, B. K., Katki, A. G., Myers, C. E. and Cowan, K. H. (1986). Overexpression of a novel anionic glutathione transferase in multidrug- resistent human breast cancer cells. J Biol Chem. 261: 15544-9.

Battey, J. P., Way, J. M., Corjay, M. H., Shapira, H., Kusano, K., Harkins, R., Wu, J. M., Slattery, T., Mann, E. and Feldman, R. I. (1991). Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci USA. 88: 395-9.

Bauer, F. E., Hacker, G. W., Terenghi, G., Adrian, T. E., Polak, J. M. and Bloom, S. R. (1986). Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas. J. Clin. Endocrinol. Metab. 63: 1372-1378.

Becker, K. L. and Gazdar, A. F. (1985). What can the biology of small cell cancer of the lung teach us about the endocrine lung? Biochem Pharmacol. 34: 155-9.

Becker, K. L., Silva, 0. L., Gazdar, A. F., Snider, R. H. and Moore, C. F. (1984). Calcitonin and small cell cancer of the lung. The Endocrine Lung in Health and Disease. Becker KL, Gazdar AF, eds. Philadelphia, W.B. Saunders. 171

Bepler, G., Jaques, G., Havemann, K., Koehler, A., Johnson, B. E. and Gazdar, A. F. (1987). Characterization of two cell lines with distinct phenotypes established from a patient with small cell lung cancer. Cancer Res. 47: 1883-91.

Bepler, G., Rotsch, M., Jaques, G., Haeder, M., Heymanns, J., Hartogh, G., Kiefer, P. and Havemann, K. (1988). Peptides and growth factors in small cell lung cancer production, binding sites, and growth effects. J Cancer Res Clin Oncol. 114: 235-44.

Berendsen, H. H., de-Leij, L., De-Vreis. E., Mesander, G., Mulder, N. H., De-Jong. B., Buys, C., Postmus, P. E., Poppema, S., Sluiter, H. J. and The, H. T. (1988). Characterization of three small cell lung cancer cell lines established from one patient during longitudinal follow-up. Cancer Res. 48: 6891-9.

Berendsen, H. H., de-Leij, L, Poppema, S., Postmus, P. E., Boes, A., Sluiter, H. J. and The, H. (1989). Clinical characterization of non-small-cell lung cancer tumors showing neuroendocrine differentiation features. J Clin Oncol. 7: 1614-20.

Bergman, B., Sullivan, M. and Sorenson, S. (1991). Quality of life during chemotherapy for small cell lung cancer I. An evaluation with generic health measures. Acta Oncol. 30: 947-57.

Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature. 316: 315-325.

Berridge, M. J. and Irvine, R. F. (1989). Inositol phosphates, and cell signalling. Nature. 341: 197-204.

Bieche, I., Champeme, M. H., Matifas, F., Hacene, K., Callahan, R. and Lidereau, R. (1991). Loss of heterozygosity on chromosome 7q and aggressive primary breast cancer. Lancet. 339: 139-143.

Bishop, A. E., Hamid, Q. A., Adams, C., Bretherton, W. D., Jones, P. M., Denny, P., Stamp, G., Hurt, R. L, Grimelius, L., Harmar, A. J., Valentino, K., Cedermark, B., Legon, S., Ghatei, M. A., Bloom, S. R. and Polak, J. M. (1989). Expression of tachykinins by ileal and lung carcinoid tumors assessed by combined in situ hybridization, immunocytochemistry, and radioimmunoassay. Cancer. 63: 1129-37.

Bishop, J. M. (1991). Molecular themes in oncogenesis. Cell. 64: 235-248. 172

Blackshear, P. J. (1993). The MARCKS family of cellular protein kinase C substrates. J. Biol. Chem. 268: 1501-1504.

Bleehen, N. M., Payers, P. M., Girling, D. J. and Stephens, R. J. (1989). Controlled trial of twelve versus six courses of chemotherapy in the treatment of small-cell lung cancer. Br J Cancer. 59: 584-590.

Bliss, D., Jr., Battey, J. P., Linnoila, R. I., Birrer, M. J., Gazdar, A. P. and Johnson, B. E. (1990). Expression of the atrial natriuretic factor gene in small cell lung cancer tum ors and tum or cell lines. J Natl Cancer Inst. 82: 305-10.

Bock, M. G., DiPardo, R. M., Evans, B. E. et. al. (1989). Benzodiazepine gastrin and brain ligands: L-365,260. J. Med. Chem. 32: 13-16.

Boden, P., Higginbottom, M., Hill, D. R., Horwell, D. C., Hughes, J., Rees, D. C., Roberts, E., Singh, L, Suman-Chauhan, N. and Woodruff, G. N. (1993). Cholecystokinin dipeptoid antagonists: Synthesis and anxiolytic profile of some novel CCK-A and CCK-B selective and “mixed” CCK-A/CCK-B antagonists. J. Med Chem. 36: 552-565.

Boers, G. J. (1985). Vasopressin and brain development: studies using the Brattleboro rat. Peptides. 6: 49-62.

Bologna, M., Pestuccia, C., Muzi, P., Biordi, L and Ciomei, M. (1 9 8 9 ). Bombesin stimulates growth of human prostatic cancer cells in vitro. Cancer. 63: 1714-20.

Bonfil, R. D., Reddel, R. R., Ura, H., Reich, R., Pridman, R., Harris, C. C. and Klein- Szanto, J. P. (1989). Invasive and metastatic potential of a v-Ha-ras transformed human bronchial epithelial cell line. J Natl Cancer Inst. 81: 587-94.

Bork, E., Hansen, M., Urdal, P., Paus, E., Holst, J. J., Schifter, S., Penger, M. and Engbaek, P. (1988). Early detection of response in small cell bronchogenic carcinoma by changes in serum concentrations of creatine kinase, neuron specific enolase, calcitonin, acth, serotonin and gastrin releasing peptide. Eur J Cancer Clin Oncol. 24: 1033-8.

Boss, J. L. (1989). ras Oncogenes in human cancer: A review. Cancer Res. 49: 4682-4689. 173

Brambilla, E., Moro, D., Gazzeri, S., Brichon, P. Y., Nagy-Mignotte, H., Morel, F., Jarcot, M. and Brambilla, C. (1991). Cytotoxic chemotherapy induces cell differetiation in small cell lung carcinoma. J. Clin. Oncol. 9: 50-61.

Brenna, E. and Waldum, H. L. (1992). Trophic effect of gastrin on the enterochromaffin like cells of the rat stomach: establishment of a dose response relationship. Gut. 33: 1303-6.

Brockes, J. P. (1984). Mitogenic growth factors and nerve dependence of limb regeneration. Science. 225: 1280-1287.

Brooks, S., Herget, T., Broad, S. and Rozengurt, E. (1992). The expression of 80K/MARCKS, a major substrate of PKC, is down-regulated through both PKC- dependent and -independent pathways. J. Biol. Chem. 267: 14212-14218.

Brooks, S. P., Herget, T., Erusalimsky, J. D. and Rozengurt, E. (1991). Protein kinase c activation potently down-regulates the expression of its major substrate, 80k, in Swiss 3T3 cells. Embo J. 10: 2497-505.

Buchkovich, K., Duffy, L. A. and Harlow, E. (1989). The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 58: 1097-105.

Bunn, P. A. and Ridgway, E. C. (1989). Paraneoplastic svndromes. Philadelphia, Uppincott.

Bunn, P. J., Chan, D., Dienhart, D. G., Tolley, R., Tagawa, M. and Jewett, P. B. (1992). Neuropeptide signal transduction in lung cancer: Clinical implications of bradykinin sensitivity and overall heterogeneity. Cancer Res. 52: 24-31.

Bunn, P. J., Dienhart, D. G., Chan, D., Puck, T. T., Tagawa, M., Jewett, P. B. and Braunschweiger, E. (1990). Neuropeptide stimulation of calcium flux in human lung cancer cells: Delineation of alternative pathways. Proc Natl Acad Sci USA. 8 7 : 2162-6.

Cajot, J. P., Anderson, M. J., Lehman, T. A., Shapiro, H., Briggs, A. A. and Stanbridge, E. J. (1992). Growth suppression mediated by transfection of p53 in Hut292DM human lung cancer cells expressing endogenous wild-type p53 protein. Cancer Res. 52: 6956-60. 174

Call, K. M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D. A., Rose, E. A., Kral, A., Yeger, H., Lewis, W. H., Jones, C. and Housman, D. E. (1990). Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell. 60: 509-20.

Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R. and Soltoff, S. (1991). Oncogenes and signal transduction. Cell. 64: 281-302.

Cardona, C., Rabbitts, P. H., Spindel, E. R., Ghatei, M. A., Bleehen, N. M., Bloom, S. R. and Reeve, J. G. (1991). Production of neuromedin B and neuromedin B gene expression in human lung tum or cell lines. Cancer Res. 51: 5205-11.

Carmichael, J., Mitchell, J. B., Friedman, N., Gazdar, A. F. and Russo, A. (1988). Glutathione and related enzyme activity in human lung cancer cell lines. Br J Cancer. 58: 437-40.

Carney, D. (1991). Clinical implications of the biochemistry of lung cancer. J. Irish Colleges of Phys. and Surg. 20: 20-24.

Carney, D. N., Cuttitta, F., Moody, T. W. and Minna, J. D. (1987). Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide. Cancer Res. 47: 821-5.

Carney, D. N. and De-Leij. L. (1988). Lung cancer biology. Semin Oncol. 15: 199- 214.

Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, T. W., Zweig, M. H. and Minna, J. D. (1985). Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 45: 2913-23.

Carney, D. N., Gazdar, A. F. and Minna, J. D. (1980). Positive correlation between histological tumor involvement and generation of tumor cell colonies in agarose in specimens taken directly from patients with small cell carcinoma of the lung. Cancer Res. 40: 1820-1823.

Carpenter, C.L. Duckworth, B.C. Auger, K.R.Cohen, B. Schaffhausen, B.S. Cantley, LC. (1990). Purification and characterization of phosphoinositide 3-kinase from rat liver. J. Biol. Chem. 32: 19704-19711. 175

Carr, B. I., Hasegawa, K. and Kar, S. (1992). Novel hepatocyte growth factors: neurotensin, neuropeptide gamma and vasoactive intestinal polypeptide (VIP) (meeting abstract). Proc Annu Meet Am Assoc Cancer Res. 33: A479

Cavanaugh, P. G. and Nicolson, G. L. (1991). Lung-derived growth factor that stimulates the growth of lung-metastasizing tumor cells: Identification as transferrin. J Cell Biochem. 47: 261-71.

Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. F., God bout, R., Gallie, B. L, Murphree, A. L., Strong, L. C. and White, R. L. (1983). Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature. 305: 779-84.

Cerny, T., Barnes, D. and Hasleton, P. S. (1986). Expression of epidermal growth factor receptor in human lung tumours. Br. J. Cancer. 54: 265-269.

Chahinian, A. P., Propert, K. J., Ware, J. H., Zimmer, B., Perry, M. C., Hirsh, V., Skarin, A., Kopel, S., Holland, J. P., Comis, R. L. and Green, M. R. (1989). A randomized trial of anticoagulation with warfarin and of alternating chemotherapy in extensive small-cell lung cancer by the Cancer and Leukemia Group B. J Clin Oncol. 7: 993-1002.

Chirgwin, J. N., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J. ( 1979). Isolation of biologically active ribonucleic acid from scources enriched in riboneuclease. Biochemistry. 18: 5294-5299.

Chung, D. H., Evers, B. M., Beauchamp, R. D., Upp, J. R., Rajaraman, S., Townsend, C. M. and Thompson, J. C. (1992). Bombesin stimulates growth of human gastrinoma. Surgery. 112: 1059-1065.

Clark, P. I., Cottier, B., Joel, S. P., Thompson, P. and Slevin, M. L (1990). Prolonged administration of single-agent oral etoposide in small-cell lung cancer (SCLC) (meeting abstract). Br J Cancer. 62: 498

Clemmons, D. R. (1989). Structural and functional analysis of insulin-like growth factors. Br. Med. Bull. 45: 465-480.

Cline, M. J. and Battifora, H. (1987). Abnormalities of protooncogenes in non-small cell lung cancer. Correlations with tumor type and clinical characteristics. Cancer. 60: 2669-74. 176

Collins, M. K. L and Rozengurt, E. (1984). Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and post-receptor steps. J Cell Physiol. 118: 133-142.

Conlon, J. M., McGregor, G. P., Wallin, G., Grimelius, L and Thim, L (1988). Molecular forms of katacalcin, calcitonin gene-related peptide and gastrin-releasing peptide, in a human medullary thyroid carcinoma. Cancer Res. 48: 2412-6.

Corjay, M. H., Dobrzanski, D. J., Way, J. M., Viallet, J., Shapira, H., Worland, P., Sausville, E. A. and Battey, J. F. (1991). Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J Biol Chem. 266: 18771- 9.

Corps, A. N., Rees, L. H. and Brown, K. D. (1985). A peptide that inhibits the mitogenic stimulation of Swiss 3T3 cells by bombesin or vasopressin. Biochem. J. 231: 781-784.

Coy, D. H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W. T., Gardner, J. D. and Jensen, R. T. (1988). Probing peptide backbone function in bombesin: a reduced peptide bond analogue with potent and specific receptor antagonist activity. Journal of Biological Chemistry. 263: 5056-5060.

Coy, D. H., Taylor, J. E., Jiang, N. Y., Kim, S. H., Wang, L. H., Huang, S. C., Moreau, J. P., Gardner, J. D. and Jensen, R. T. (1989). Short-chain pseudopeptide bombesin receptor antagonists with enhanced binding affinities for pancreatic acinar and Swiss 3T3 cells display strong antimitotic activity. J Biol Chem. 264: 14691-7.

Crawford, J., Ozer, H., Stoller, R., Johnson, D., Lyman, G., Tabbara, I., Kris, M., Grous, J., Picozzi, V., Rausch, G., Smith, R., Gradishar, W., Yahanda, A., Vincent, M., Stewart, M. and Glaspy, J. (1991). Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with small-cell lung cancer. N Engl J Med. 325: 164-70.

Cross, M. and Dexter, T. M. (1991). Growth factors in development, transformation, and tumorigenesis. Cell. 64: 271-280. 177

Curt, G. A., Jolivet, J., Bailey, B. D., Carney, D. N. and Chabner, B. A. (1984). Synthesis and retention of methotrexate polyglutamates by human small cell lung cancer. Biochem Pharmacol. 33: 1682-5.

Curt, G. A., Jolivet, J., Carney, D. N., Bailey, B. D., Drake, J. C., Clendeninn, N. J. and Chabner, B. A. (1985). Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate. J Clin Invest. 76: 1323-9.

Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler, A. and Minna, J. D. (1985). Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature. 316: 823-6.

C uttitta, F., Fedorko, J., Gu, J. A., Lebacq, V. A., Linnoila, R. I. and Battey, J. F. (1988). Gastrin-releasing peptide gene-associated peptides are expressed in normal human fetal lung and small cell lung cancer: a novel peptide family found in man. J Clin Endocrinol Metab. 67: 576-83.

Czech, M. P. (1989). Signal transmission by the insulin-like growth factors. Cell. 59: 235-238.

Damstrup, L, Rygaard, K., Spang, T. M. and Poulsen, H. S. (1992). Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines. Cancer Res. 52: 3089-93.

Davis, T. P., Burgess, H. S., Crowell, S., Moody, T. W., Culling, B. A. and Liu, R. H. (1989). beta-Endorphin and neurotensin stimulate in vitro clonal growth of human SCLC cells. Eur J Pharmacol. 161: 283-5. de Aizpurua, H., Lambert, E. H., Griesmann, G. E., Olivera, B. M. and Lennon, V. A. (1988). Antagonism of voltage-gated calcium channels in small cell carcinomas of patients with and without lambert-eaton myasthenic syndrome by autoantibodies omega-conotoxin and adenosine. Cancer Res. 48: 4719-24. de-Jong, S., Zijistra, J. G., Eg, d.e-Vries. and Mulder, N. H. (1990). Reduced DNA topoisomerase II activity and drug-induced DNA cleavage activity in an Adriamycin- resistant human cell lung carcinoma cell line. Cancer Res. 50: 304-9. de Vries, E. G., Meijer, C., Timmer, B. H., Berendsen, H. H., de-Leij, L , Scheper, R. J. and Mulder, N. H. (1989). Resistance mechanisms in three human small cell lung 178 cancer cell lines established from one patient during clinical follow-up. Cancer Res. 49: 4175-8. de-Weille, J., Schmid-Antomarchi, H., Fosset, M. and Lazdunski, M. (1988.). ATP- sensitive K"^ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone. Proc. Natl. Acad. Sci. USA. 85: 1312-1316.

Derynck, R., Goeddel, D. V., Ullrich, A., Gutterm an, J. U., Williams, R. D., Bringman, T. S. and Berger, W. H. (1987). Synthesis of the epidermal growth factor receptor by human tumors. Cancer Res. 47: 707-712.

Dicker, P. and Rozengurt, E. (1980). Phorbol esters and vasopressin stimulate DNA synthesis by a common mechanism. Nature. 287: 607-612.

Divgi, C. R., Welt, S., Yeh, S. D., Real, F. X., Gralla, R. J., Kris, M. G., Masui, H. and Mendelsohn, J. (1989). Phase 1 and imaging trail with radiolabeled anti-EGF receptor monoclonal antibody in sqaumous cell lung cancer. Proc. Annu. Meet. Am. Soc. Clin. Oncol. 8: A713.

Dixon, R. A. F., Sigal, I. S. and Strader, C. D. (1988). Structure-function analysis of the p-adrenergic receptor. Cold Spring Harbor Symp. Quant. Biol. S3: 487-498.

Dobrovic, A., Houle, B., Belouchi, A. and Bradley, W. E. (1988). erbA-related sequence coding for DNA-binding hormone receptor localized to chromosome 3p21- 3p25 and deleted in small cell lung carcinoma. Cancer Res. 48: 682-5.

Domin, J. and Rozengurt, E. (1992). Heterologous desensitization of platelet-derived growth factor-mediated arachidonic acid release and prostaglandin synthesis. J. Biol. Chem. 267: 15217-15223.

Douglas, B. R., Woutersen, R. A., Jansen, J. B., Rovati, L. C. and Lamers, C. B. (1989). Study into the role of cholecystokinin in bombesin-stimulated pancreatic growth in rats and hamsters. Eur J Pharmacol. 161: 209-14.

Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlessinger, J. and Waterfield, M. D. (1984). Close similarity of epidermal growth factor receptor and w-erb-B oncogene protein sequences. Nature. 307: 521-7. 179

Doyle, A., Gazdar, A., Martin, J., Carney, D., Bunn, P. and Minna, J. (1984). The deficit in HLA expression in small cell lung cancer (SCLC) resides at the transcriptional level and is reversed by interferon (meeting abstract). Proc Annu Meet Am Soc Clin Oncol. 3: A53

Doyle, A., Martin, W. J., Funa, K., Gazdar, A., Carney, D., Martin, S. E., Linnoila, I., Cuttitta, F., Mulshine, J., Bunn, P. and et, al. (1985). Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer. J Exp Med. 161: 1135-51.

Dryja, T. P., Cavenee, W., White, R., Rapa port, J. M., Petersen, R., Albert, D. M. and Bruns, G. A. (1984). Homozygosity of chromosome 13 in retinoblastoma. N Engl J Med. 310: 550-3.

Dunne, M. J., Bullett, M. J., Li, G. D., Wollheim, C. B. and Petersen, 0. H. (1989). Galanin activates nucleotide-dependent K'*’ channels in insulin-secreting cells via a pertussis toxin-sensitive G-protein. EMBO. J. 8: 413-420.

Dyson, N., Howley, P. M., Munger, K. and Harlow, E. (1989). The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 243: 934-7.

Eimoto, H., Tsutsumi, M., Nakajima, A. et. al. (1988). Expression of glutathione-S- transferase placental form in human lung carcimomas. Carcinogenesis. 9: 2325.

Einhorn, S., Grander, D., Bjork, 0., Brondum-Nielsen, K. and Soderhall, S. (1990). Deletion of alpha-, beta-, and omega-interferon genes in malignant cells from children with acute lymphocytic leukemia. Cancer Res. 50: 7781-5.

Ekblad, E., Hakanson, R., Sundler, F. and Wahlestedt, C. (1985). Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br. J. Pharmacol. 86: 241-246.

Erisman, M. D., Linnoila, R. I., Hernandez, 0., DiAugustine, R. P. and Lazarus, L. H. (1982). Human lung small-cell carcinoma contains bombesin. Proceedings of the National Academy of Science USA. 79: 2379-2383. 180

Erusalimsky, J. D., Brooks, S. F., Herget, T., Morris, C. and Rozengurt, E. (1991). Molecular cloning and characterization of the acidic 80-kda protein kinase C substrate from rat brain, identification as a glycoprotein. J Biol Chem. 266: 7073-80.

Erusalimsky, J. D., Friedberg, I. and Rozengurt, E. (1988). Bombesin, diacylglycerols and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells: effect of guanine nucleotides. J. Biol. Chem. 263: 19188-19194.

Escobedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A. and Williams, L. T. (1991). cDNA cloning of a novel 85 kd protein th a t has SH 2 domains andregulates binding of PI3-kinase to the pdgf beta-receptor. Cell. 65: 75-82.

Evers, B. M., Ishizuka, J., Chung, D. H., Townsend, C. J., Thompson, J. C., Debas, B., Moore, T. C. and Andersen, D. K. (1992). Neurotensin expression and release in human colon cancers. Ann Surg. 216: 423-30.

Fabregat, I. and Rozengurt, E. (1990a). Vasoactive intestinal contractor, a novel peptide, shares a common receptor with endothelin-1 and stimulates Ca^ + mobilization and DNA synthesis in Swiss 3T3 cells. Biochem Biophys Res Commun. 167: 161-7.

Fabregat, I. and Rozengurt, E. (1990b). [DArg\DPhe^,DTrp^»^,Leu^ substance P, a neuropeptide antagonist, blocks binding, Ca^'^-mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells. J. Cell. Physiol. 145: 88-94.

Fahrenkrug, J. and Emson, P. C. (1982). Vasoactive intestinal peptide: functional aspects. Br. Med. Bull. 45: 265-270.

Fang, K., Li, L., Jansen, J., Fidler, I. and Roth, J. A. (1991). Brain metastatic variants of human lung cancer cells aquire independence from autocrine growth stimulation by TGF alpha. Proc. Annu. Meet. Am. Soc. Clin. Oncol. 32: A302.

Fearon, E. R., Cho, K. R., Nigro, J. M., Kern, S. E., Simons, J. W., Ruppert, J. M., Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W. and Vogelstein, B. (1990). Identification of a chromosome 18q gene that is altered in colorectal cancers. Science. 247: 49-56. 181

Feinberg, A., P. and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction fragments of high specific activity. Anal. Biochem. 132: 6-13.

Feuerstein, 6., Molineaux, C. J., Rosenberger, J. G., Zerbe, R. L, Cox, B. M. and Faden, A. I. (1985). Hemorrhagic shock and the central vasopressin and system of rats. Am. J. Physiol. 249: E244-E250.

Fisone, G., Wu, C. F., Consolo, S., Nordstrom, 0., Brynne, N., Bartfai, T., Melander, T. and Hokfelt, T. (1987). Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies. Proc. Natl. Acad. Sci. USA. 84: 7339-7343.

Fleming, T. P., Matsui, T., Molloy, C. J., Robbins, K. C. and Aaronson, S. A. (1989). Autocrine mechanism for v-s/s transformation requires cell surface localization of internally activated growth factor receptors. Proc. Natl. Acad. Sci. USA. 86: 8063- 8067.

Frucht, H., Gazdar, A. F., Park, J. A., Oie, H. and Jensen, R. T. (1992). Characterization of functional receptors for gastrointestinal hormones on human colon cancer cells. Cancer Res. 52: 1114-1122.

Gazdar, A. F., Bunn, P., Jr., Minna, J. D. and Baylin, S. B. (1985). Origin of human small cell lung cancer. Science. 229: 679-80.

Gazdar, A. F. and Carney, D. N. (1984). Endocrine properties of small cell carcinoma of the lung. Philadelphia, W.B. Saunders.

Gazdar, A. F., Carney, D. N., Becker, K. L, Deftos, L J., Liang, V., Go, W., Marangos, P. J., Moody, T. W., Wolfsen, A. R. and Zweig, M. H. (1985). Expression of peptide and other markers in lung cancer cell lines. Recent Results Cancer Res. 99: 167-74.

Gazdar, A. F., Helman, L. J., Israel, M. A., Russell, E. K., Linnoila, R. I., Mulshine, J. L., Schuller, H. M. and Park, J. G. (1988). Expression of neuroendocrine cell markers l-dopa decarboxylase, chromogranin a, and dense core granules in human tumors of endocrine and nonendocrine origin. Cancer Res. 48: 4078-82.

Gazdar, A. F. and McDowell, E. M. (1988). Pathobioloav of lung cancers. New York, Marcel Dekker Inc.. 182

Gazdar, A. F. and Minna, J. D. (1986). Cell lines as an investigational tool for the study of biology of small cell lung cancer. Eur J Cancer Clin Oncol. 22: 909-11.

Gebert, J. F., Moghal, N., Frangioni, J. V., Sugarbaker, D. J. and Neel, B. G. (1991). High frequency of retinoic acid receptor beta abnormalities inhuman lung cancer. Oncogene. 6: 1859-68.

Geijer, T., Folkesson, R., Rehfeld, J. F. and Monstein, H. J. (1990). Expression of the cholecystokinin gene in a human (small-cell) lung carcinoma cell-line. Febs Lett. 270: 30-2.

Gerlach, J. H., Endicott, J. A., Juranka, P. F., Henderson, G., Sarangi, F., Deuchars, K. L and Ling, V. (1986). Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance. Nature. 324: 485-9.

Ghatei, M. A., Sheppard, M. N., Henzen-Logman, S., Blank, M. A., Polak, J. M. and Bloom, S. R. (1983). Bombesin and vasoactive intestinal peptide in the developing lung: marked changes in the respiratory distress syndrome. J. Clin. Endocrinol. Metab. 57: 1226-1232.

Ghatei, M. A., Stratton, M. R., Allen, J. M., Joplin, G. F., Polak, J. M. and Bloom, S. R. (1987). Co-secretion of calcitonin gene-related peptide, gastrin-releasing peptide and ACTH by a carcinoid tumour metastasizing to the cerebellum. Postgrad Med J. 63: 123-30.

Gherardi, E. and Stoker, M. (1991). Hepatocyte growth factor - Scatter factor: mitogen, motogen and m e t Cancer Cells. 3: 227-232.

Giaccone, G., Battey, J., Gazdar, A. F., Oie, H., Draoui, M. and Moody, T. W. (1992). Neuromedin B is present in lung cancer cell lines. Cancer Res. 52s: 2732s-2736s.

Gil, J., Higgins, T. and Rozengurt, E. (1991). Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation. Journal of Cell Biology. 113: 943-950.

Goedert, M., Reeve, J. G., Emson, P. C. and Bleehen, N. M. (1984). Neurotensin in human small cell lung carcinoma. Br. J. Cancer. 50: 179-183. 183

Gorden, P., Comi, R. J., Maton, P. N. and Go, V. L (1989). Somatostatin and somatostatin analogue (SMS 201-995) in treatment of hormone-secreting tumors of the pituitary and gastrointestinal tract and non-neoplastic diseases of the gut. Ann Intern Med. 110: 35-50.

Green, J. A., Trillet, V. N. and Manegold, C. (1991). R-methuG-CSF (G-CSF) with CDE chemotherapy (CT) In small-cell lung cancer (SCLC): interim results from a randomized, placebo controlled trial (meeting abstract). Proc Annu Meet Am Soc Clin Oncol. 10: A832

Greenberg, M. E. and Ziff, E. B. (1984). Stimulation of 3T3 cells induces transcription of the c-/bs proto-oncogene. Nature. 311: 433-8.

Gross, A. J., Steinberg, S. M., Reilly, J. G., Bliss, D. P., Brennan, J., Tram Le, P., Simmons, A., Phelps, R., Mulshine, J. L., Ihde, D. C. and Johnson, B. E. (1993). Atrial natiuretic factor and arginine vasopressin production in tumor cell lines from patients with lung cancer and their relationship to serum sodium. Cancer Res. 53: 67-74.

Haeder, M., Rotsch, M., Bepler, G., Hennig, C., Havemann, K., Heimann, B. and Moelling, K. (1988). Epidermal growth factor receptor expression in human lung cancer cell lines. Cancer Res. 48: 1132-6.

Hall, A. (1990). RAS AND GAP-WHO’S CONTROLLING WHOM? (17 Refs). Cell. 61: 921-3.

Hansen, H. H. and Kristjansen, P. E. (1991). CHEMOTHERAPY OF SMALL CELL LUNG CANCER (54 Refs). Eur J Cancer. 27: 342-9.

Harbour, J. W., Lai, S. L, Whang-Peng, J., Gazdar, A. F., Minna, J. D. and Kaye, F. J. (1988). Abnormalities in structure and expression of the human retinoblastoma gene in sclc. Science. 241: 353-7.

Havemann, K., Rotsch, M., Schoneberger, H. J., Erbil, C., Hennig, C. and Jaques, G. (1990). Growth regulation by insulin-like growth factors in lung cancer. J Steroid Biochem Mol Biol. 37: 877-82. 184

Heald, E. B., Kramer, S. T. and Smith, J. P. (1992). Trophic effects of unsulfated cholecystokinin on mouse pancreas and human pancreatic cancer. Pancreas. 7: 530- 5.

Heighway, J., Thatcher, N., Cerny, T. and Hasleton, P. S. (1986). Genetic predisposition to human lung cancer. Br J Cancer. 53: 453-7.

Heikkila, R., Trepel, J. B., Cuttitta, F., Neckers, L. M. and Sausville, E. A. (1987). Bombesin-related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines. J. Biol. Chem. 262: 16456-16460.

Heimbrook, D. C., Boyer, M. E., Garsky, V. M., Balishin, N. L, Kiefer, D. M., 01 iff, A. and Riemen, M. W. (1988). Minimal ligand analysis of gastrin releasing peptide, receptorbinding and mitogenesis. J Biol Chem. 263: 7016-9.

Hendler, F. J., Shum-Siu, A., Oechsli, M., Nanu, L, Richards, C. S. and Ozanne, B. W. (1989). Increased egf-rl binding predicts a poor survival in squamous tumors. Cancer Cells. 7: 347-51.

Herget, T., Brooks, S. F., Broad, S. and Rozengurt, E. (1992). Relationship between the major protein kinase C substrates acidic 80-kDa protein-kinase-C substrate (80K) and myristoylated alanine-rich C-kinase substrate (MARCKS). Eur. J. Biochem. 209: 7-14.

Hibi, K., Takahashi, T., Sekido, Y., Ueda, R., Hida, T., Ariyoshi, Y., Takagi, H. and Takahashi, T. (1991). Coexpression of the stem cell factor and the c-kit genes in small-cell lung cancer. Oncogene. 6 : 2291-6.

Higginbottom, M., Horwell, D. C. and Roberts, E. (1993). Selective ligands for cholecystokinin receptor subtypes CCK-A and CCK-B within a single strucural class. Bio. Med. Chem. Lett, (in press).

Honegger, A. M., Schmidt, A., Ullrich, A. and Schlessinger, J. (1990). Evidence for epidermal growth factor (egf)-induced intermolecular autophosphorylation of the egf receptors in living cells. Mol Cell Biol. 10: 4035-44.

Hoosein, N. M., Kiener, P. A., Curry, R. C. and Brattain, M. G. (1990). Evidence for autocrine growth stimulation of cultured colon tumor cells by a gastrin/cholecystokinin-like peptide. Experimental Cell Research. 186: 15-21. 185

Hoosein, N. M., Kiener, P. A., Curry, R. C., Rovati, L C., McGilbra, D. K. and Brattain, M. 6 . (1988). Antiproliferative effects of gastrin receptor antagonists and antibodies to gastrin on human colon carcinoma cell lines. Cancer Res. 48: 7179-7183.

Howlett, T. A., Price, J., Hale, A. C., Doniach, I., Rees, L H., Wass, J. A. and Besser, G. M. (1985). Pituitary ACTH dependent Cushing's syndrome due to ectopic production of a bombesin-like peptide by a medullary carcinoma of the thyroid. Clin Endocrinol Oxf. 22: 91-101.

Huang, H. J., Yee, J. K., Shew, J. Y., Chen, P. L, Bookstein, R., Friedmann, T., Lee, E. Y. and Lee, W. H. (1988). Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 242: 1563-6.

Huang, S. C., Zhang, L., Chiang, H. C. V., Wank, S. A., Maton, P. N., Gardner, J. D. and Jensen, R. T. (1989). Benzodiazepine analogues L365,260 and L364,718 as gastrin and pancreatic CCK receptor antagonists. American Journal of Physiology. 257: G169-G174.

Huffman, W. P., Kinter, L. B., Moore, M. L. and Stassen, F. L. (1988). Vasopressin antagonists. Ann. Rep. Medicinal Chem. 23: 91-99.

Hunt, N. H., Perris, A. D. and Sandford, P. A. (1977). Role of vasopressin In the mitogenic response of rat bone marrow cells to haemorrhage. J. Endocrinol. 72: 5- 16.

Isobe, M., Emanuel, B. S., Givol, D., Oren, M. and Croce, C. M. (1986). Localization of gene for human p53 tumour antigen to band 17pl 3. Nature. 320: 84-5.

Issandou, M. and Rozengurt, E. (1990). Bradykinin transiently activates protein kinase C in Swiss 3T3 cells: distinction from activation by bombesin and vasopressin. J. Biol. Chem. 265: 11890-11896.

Jaques, G., Kiefer, P., Rotsch, M., Hennig, C., Goke, R., Richter, G. and Havemann, K. (1989). Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines. Exp Cell Res. 184: 396-406. 186

Jaques, G., Rotsch, M., Wegmann, C., Worsch, U., Maasberg, M. and Havemann, K. (1988). Production of immunoreactive insulin-like growth factor I and response to exogenous IGF-I in small cell lung cancer cell lines. Exp Cell Res. 176: 336-43.

Jard, S., Gillard, R. C., Guillon, G., Marie, J., Schoenenberg, P., Muller, A. F., Manning, M. and Sawyer, W. H. (1986). Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol. 30: 171-177.

Jensen, R. T., Jones, S. W., Folkers, K. and Gardner, J. D. (1984). A synthetic peptide that is a bombesin receptor antagonist. Nature. 309: 61-63.

Jensen, R. T., Wank, S. A., Rowley, W. H., Sato, S. and Gardner, J. D. (1 9 8 9 ). Interaction of CCK with pancreatic acinar cells. Trends. Pharmacol. Sci. 10: 418- 423.

Johnson, B. E., Ihde, D. C., Makuch, R. W., Gazdar, A. F., Carney, D. N., Oie, H., Russell, E., Nau, M. M. and Minna, J. D. (1987). myc family oncogene amplification in tumor cell lines established from small cell lung cancer patients and its relationship to clinical status and course. J Clin Invest. 79: 1629-34.

Johnson, B. E., Makuch, R. W., Simmons, A. D., Gazdar, A. F., Burch, D. and Cashell, A. W. (1988). Myc family DNA amplification in small cell lung cancer patients’ tumors and corresponding cell lines. Cancer Res. 48: 5163-6.

Johnson, B. E., Sakaguchi, A. Y., Gazdar, A. F., Minna, J. D., Burch, D., Marshall, A. and Naylor, S. L. (1988). Restriction fragment length polymorphism studies show consistent loss of chromosome 3p alleles in small cell lung cancer patients’ tumors. J Clin Invest. 82: 502-7.

Johnson, L. R. (1984). Trophic effects of gastrin on the colon. New York, Raven Press.

Johnson, L. R. and Guthrie, P. D. (1984). Proglumide inhibition of trophic action of pentagastrin. Am. J. Physiol. 246: G62-G66.

Julius, D., Livelli, T. J., Jessell, T. M. and Axel, R. (1989). Ectopic expression of the serotonin Ic receptor and the triggering of malignant transformation. Science. 244: 1057-62. 187

Julius, D., Macdermott, A. B., Axel, R. and Jessell, T. M. (1988). Molecular characterization of a functional cDNA ancoding the serotonin 1 c receptor. Science. 241: 558-564.

Kado-Fong, H. and Malfroy, B. (1989). Effects of bombesin on human small cell lung cancer cells: Evidence for a subset of bombesin non-responsive cell lines. J Cell Biochem. 40: 431-7.

Kamata, N., Chida, K., Rikimaru, K., Horikoshi, M., Enomoto, S. and Kuroki, T. (1986). Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res. 46: 1648- 53.

Kaye, P., Battey, J., Nau, M., Brooks, B., Seifter, E., De-Greve, J., Birrer, M., Sausville, E. and Minna, J. (1988). Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing. Mol Cell Biol. 8: 1 BB­ SS.

Kiefer, P., Jaques, G., Schoneberger, J., Heinrich, G. and Havemann, K. (1991). Insulin-like growth factor binding protein expression in human small cell lung cancer cell lines. Exp Cell Res. 192: 414-7.

Kiefer, P. E., Bepler, G., Kubasch, M. and Havemann, K. (1987). Amplification and expression of protooncogenes in human small cell lung cancer cell lines. Cancer Res. 47: 6236-42.

Kiefer, P. E., Wegmann, B., Bacher, M., Erbil, C., Heidtmann, H. and Havemann, K. (1990). Different pattern of expression of cellular oncogenes in human non-small- cell lung cancer cell lines. J Cancer Res Clin Oncol. 116: 29-37.

Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G., Schlessinger, J. and Rhee, S. G. (1991). PDGF stimulation of inositol phospholipid hydrolysis requires PLC-yl phosphorylation on tyrosine residues 783 and 1254. Cell. 65: 435-441.

Klastersky, J. A. and Sculier, J. P. (1989). Intensive chemotherapy of small cell lung cancer. Lung Cancer The Netherlands. 5: 4-6. 188

Knigge, U., Holst, J. J., Knutsen, S., Bach, F. W. and Bang, P. (1987). Corticotrophin-releasing activity of gastrin-releasing peptide in normal men. J. Clin. Endocrinol. Metab. 65: 1291-1295.

Knigge, U., Holst, J. J., Knutsen, S., Petersen, B., Krarup, T., Holst-Pedersen, J. and Christiansen, P. M. (1984). Gastrin-releasing peptide: Pharmacokinetics and effects on gastro-entero-pancreatic hormones and gastric acid secretion in normal men. J. Clin. Endocrinol. Metab. 59: 310-315.

Knutsen, S., Holst, J. J., Baldissera, F. G. A., Skak-Nielsen, T., Poulsen, S. S., Jensen, S. L. and Nielsen, 0. V. (1987). Gastrin-releasing peptide in the porcine pancreas. Gastroenterology. 92: 1153-1158.

Kopin, A. S., Lee, Y.-M., McBride, E. W., Miller, L. J., Lu, M., Lin, H., Kalowski, L. F. and Beinborn, M. (1992). Expression cloning and characterization of the canine parietal cell gastrin receptor. Proc. Natl. Acad. Sci. USA. 89: 3605-3609.

Korman, L. Y., Carney, D. N., Citron, M. L. and Moody, T. W. (1986). Secretin/vasoactive intestinal peptide-stimulated secretion of bombesin/gastrin releasing peptide from human small cell carcinoma of the lung. Cancer Res. 46: 1214-8.

Krane, I. M., Naylor, S. L., Helin-Davis, D., Chin, W. W. and Spindel, E. R. (1988). Molecular cloning of cDNAs encoding the human bombesin-like peptide neuromedin B. Chromosomal localization and comparison to cDNAs encoding its amphibian homolog ranatensin. J. Biol. Chem. 263: 13317-13323.

Krystal, G., Birrer, M., Way, J., Nau, M., Sausville, E., Thompson, C., Minna, J. and Battey, J. (1988). Multiple mechanisms for transcriptional regulation of the m yc gene family in small-cell lung cancer. Mol Cell Biol. 8: 3373-81.

Kumar, R., Shepard, H. M. and Mendelsohn, J. (1991). Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a mammary carcinoma cells. Mol Cell Biol. 11: 979-986.

LaForgia, S., Morse, B., Levy, J., Barnea, G., Cannizzaro, L A., Li, F., Nowell, P. C., Boghosian, S. L., Click, J., Weston, A. and et, a. I. (1991). Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21. Proc Natl Acad Sci USA. 88: 5036-40. 189

Lai, S. L., Goldstein, L J., Gottesman, M. M., Pastan. I., Tsai, C. M., Johnson, B. E., Mulshine, J. L., Ihde, D. C., Kayser, K. and Gazdar, A. F. (1989). MDR1 gene expression in lung cancer. J Natl Cancer Inst. 81:1144-50.

Lamers, C. B. H. W. and Jansen, J. B. M. J. (1988). Role of gastrin and cholecystokinin in tumours of the gastrointestinal tract. Eur. J. Cancer Clin. Oncol. 24: 267-273.

Land, H., Parada, L. F. and Weinberg, R. A. (1983). Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 304: 596-602.

Landis, C. A., Masters, S. B., Spada, A., Pace, A. M., Bourne, H. R. and Vallar, L. (1989). GTPase inhibiting mutations activate the alpha chain of G(s) and stimulate adenylyl cyclase in human pituitary tumours. Nature. 340: 692-6.

Lane, D. (1992). p53, guardian of the genome. Nature. 358: 15-16.

Lane, D. (1993). A death in the life of p53. Nature. 362: 786-787.

Langdon, S., Sethi, T., Ritchie, A., Muir, M., Smyth, J. and Rozengurt, E. (1992). Broad spectrum neuropeptide antagonists inhibit the growth of small cell lung cancer in vivo. Cancer Res. 52: 4554-7.

Langdon, S. P., Rabiasz, G. J., Anderson, L., Ritchie, A. A., Fergusson, R. J., Hay, F. G., Miller, E. P., Mullen, P., Plumb, J., Miller, W. R. and et, a. I. (1991). Characterisation and properties of a small cell lung cancer cell line and xenograft WX322 with marked sensitivity to alpha-interferon. Br J Cancer. 63: 909-15.

Lavigueur, A., Maltby, V., Mock, D., Rossant, J., Pawson, T. and Bernstein, A. (1989). High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene. Mol Cell Biol. 9: 3982-91.

Lazarus, L. H. and Hernandez, 0. (1985). Physalaemin-like immunoreactivity from human lung small cell carcinoma: isocratic reversed-phase HPLC analysis of the chemically modified peptide. Recent Results Cancer Res. 99: 56-66. 190

Lee, M., Draoui, M., Zia, F., Gazdar, A., Oie, H., Bepler, G., Bellot, P., Tarr, C., Kris, R. and Moody, T. W. (1992). Epidermal growth factor receptor monoclonal antibodies inhibitthe growth of lung cancer cell lines. Monogr Natl Cancer Inst.

Lehy, T., Accary, J. P., Labeille, D. and Dubrasquet, M. (1983). Chronic administration of bombesin stimulates antral gastrin cell proliferation in rat. Gastroenterology. 84: 914-919.

Leonard, R. C., Duncan, L. W. and Hay, F. G. (1990). Immunocytological detection of residual marrow disease at clinical remission predicts metastatic relapse in small cell lung cancer. Cancer Res. 50: 6545-8.

Leonard, R. C., MacKay, T., Brown, A., Gregor, A., Crompton, G. K. and Smyth, J. F. (1988). Small-cell lung cancer after retinoblastoma [letter]. Lancet. 2:

Levitzki, A. (1992). Tyrphostins: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction. FASEB. 6 : 3275-3282.

Lewin, B. (1991). Oncogenic conversion by regulatory changes in transcription factors. Cell. 64: 303-312.

Lezoche, E., Basso, N. and Speranza, V. (1981). Actions of bombesin in man. London, Churchill Livingstone.

Lhoste, E. F. and Longnecker, D. S. (1987). Effect of bombesin and caerulein on early stages of carcinogenesis induced by azaserine in the rat pancreas. Cancer Res. 47: 3273-7.

Lin, S. W., Holladay, M. W., Barrett, R. W. and al., e. (1989). Distinct requirements for activation at CCK-A and CCK-B/gastrin receptors: Studies with a C-terminal hydrazide analogue of cholecystokinin tetrapeptide (30-33). Mol. Pharmacol. 36: 881-886.

Little, C. D., Nau, M. M., Carney, D. N., Gazdar, A. F. and Minna, J. D. (1983). Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature. 306: 194-6.

Lopez, R. A., Mendoza, S. A., Nanberg, E., Sinnett-Smith, J. and Rozengurt, E. (1987). Ca^+-mobilizing actions of platelet-derived growth factor differ from those of 191 bombesin and vasopressin in Swiss 3T3 mouse cells. Proc Natl Acad Sci USA. 64: 5768-72.

Lotti, V. J. and Chang, R. S. L (1989). A new potent and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-B) ligand: L365,260. European Journal of Pharmacology. 162: 273-280.

Lotz, M., Carson, D. A. and Vaughan, J. H. (1987). Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis. Science. 235: 893-895.

Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D. and Schlessinger, J. (1992). The SHz and SH 3 domain-containing protein Crb 2 links receptor tyrosine kinases to ras signaling. Cell. 70: 431-42.

Luster, W., Gropp, C., Kern, H. F. and Havemann, K. (1985). Lung tumour cell lines synthesizing peptide hormones established from tumours of four histological types: Characterization of the cell lines and analysis of their production. Br J Cancer. 51: 865-75.

Mabry, M., Nakagawa, T., Baylin, S., Pettengill, 0., Sorenson, G. and Nelkin, B. (1989). Insertion of the v-Ha-ras oncogene Induces differentiation of calcitonin- producing human small cell lung cancer. J Clin Invest. 84: 194-9.

Mabry, M., Nakagawa, T., Nelkin, B. D., McDowell, E., Gesell, M., Eggleston, J. C., Casero, R., Jr. and Baylin, S. B. (1988). V-ha-ras oncogene insertion: a model for tumor progression of human small cell lung cancer. Proc Natl Acad Sci USA. 85: 6523-7.

Mabry, M., Nelkin, B. D., Falco, J. P., Barr, L. F. and Baylin, S. B. (1991). Transitions between lung cancer phenotypes - Implications for tumor progression. Cancer Cells. 3: 53-8.

Macaulay, V. M., Everard, M. J., Teale, J. D., Trott, P. A., Van-Wyk, J. J., Smith, I. E. and Millar, J. L. (1990). Autocrine function for insulin-like growth factor I in human small cell lung cancer cell lines and fresh tumor cells. Cancer Res. 50: 2511-7. 192

Macaulay, V. M., Smith, I. E., Everard, M. J., Teale, J. D., Reubi, J. C. and Millar, J. L (1991). Experimental and clinical studies with somatostatin analogue octreotide in small cell lung cancer. Br J Cancer. 64: 451-6.

Macauly, V. M., Teale, J. D., Everard, M. J., Joshi, G. P., Smith, I. E. and Millar, J. L (1988). -C/insulin-like growth factor-1 is a mitogen for human small cell lung cancer. Br J Cancer. 57: 91-3.

Madsen, 0. D., Larsson, L I., Rehfeld, J. F., Schw artz, T. W., Lernmark, A., Labrecque, A. D. and Steiner, D. F. (1986). Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones. J Cell Biol. 103: 2025-34.

Mahmoud, S., Staley, J., Taylor, J., Bogden, A., Moreau, J. P., Coy, D., Avis, I., Cuttitta, F., Mulshine, J. L. and Moody, T. W. (1991). [psi 13T4] bombesin analogues inhibit growth of small cell lung cancerin vitro and in vivo. Cancer Res. 51: 1798- 802.

Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J., Jr., Nelson, C. E., Kim, D. H., Kassel, J., Gryka, M. A., Bischoff, F. Z., Tainsky, M. A. and Friend, S. H. (1990). Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 250: 1233-8.

Maneckjee, R. and Minna, J. D. (1990). Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines. Proc Natl Acad Sci USA. 87: 3294-8.

Maneckjee, R. and Minna, J. D. (1992). Nonconventional opioid binding sites mediate growth inhibitory effects of methadone on human lung cancer cells. Proc Natl Acad Sci U S A. 89: 1169-73.

Mantyth, P. W., Johnson, D. J., Boehmer, C. G., Catton, M. D., Vinters, H. V., Maggio, J. E., Too, H.-P. and Vigna, S. R. (1989). Substance P receptor binding sites are expressed by glia in vivo after neuronal injury. Proc. Natl. Acad. Sci. USA. 8 6 : 5193-5197.

Maruno, K., Yamaguchi, K., Abe, K., Suzuki, M., Saijo, N., Mishima, Y., Yanaihara, N. and Shimosato, Y. (1989). Immunoreactive gastrin-releasing peptide as a specific tumor marker in patients with small cell lung carcinoma. Cancer Res. 49: 629-32. 193

Matsumura, Y., Kimura, M., Kato, H., Yamamoto, T. and Maeda, H. (1989). Quantification, isolation and structural determination of bradykinin and hydroxyprolyl-bradykinin in tumor ascites. Adv Exp Med Biol. 247a. 587-592.

Matthews, M. J., Kanhouwa, S., Pickren, J. and Robinette, D. (1973). Frequency of residual and metastatic tumor in patients undergoing curative surgical resection for lung cancer. Cancer Chemother. Rep. (part 3). 4: 63.

Mattson, K., Niiranen, A., Pyrhonen, S., Holsti, L. R., Holsti, P., Kumpulalnen, E. and Cantell, K. (1992). Natural interferon alfa as maintenance therapy for small lung cancer. Eur J Cancer. 28a.

Maurer, L. H. (1985). Ectopic hormone syndrome in small cell carcinoma of the lung. Clinics. Oncol. 4: 1289-1296.

Mehmet, H. and Rozengurt, E. (1991). Regulation of c-fos expression in Swiss 3T3 cells: an interplay of multiple signal transduction pathways. Br Med Bull. 47: 76- 86 .

Mendoza, S. A., Schneider, J. A., Lopez, R. A., Sinnett-Smith, J. W. and Rozengurt, E. (1986). Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells, ii. changes in Na+ and Ca^+ fluxes, NaVK+ pump activity, and intracellular ph. J Cell Biol. 102: 2223-33.

Millar, J. B. and Rozengurt, E. (1989). Heterologous desensitization of bombesin- induced mitogenesis by prolonged exposure to vasopressin: a post-receptor signal transduction block. Proc Natl Acad Sci USA. 86 : 3204-8.

Millar, J. B. and Rozengurt, E. (1990a). Arachidonic acid release by bombesin, a novel postreceptor target for heterologous mitogenic desensitization. J Biol Chem. 265: 19973-9.

Millar, J. B. and Rozengurt, E. (1990b). Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells, distinction from acute desensitization. J Biol Chem. 265: 12052-8.

Millar, J. B. A. and Rozengurt, E. (1 9 8 8 ). Bombesin enhancem ent of cAMP accumulation in Swiss 3T3 cells: evidence of a dual mechanism of action. Journal of Cell Physiology. 137: 214-222. 194

Miller, R. P., Husain, F., Svensson, M. and Lohin, S. (1977). Enhancement of [^H- methyl]thymidine incorporation and replication of rat chondrocytes grown in tissue culture by plasma, tissue extracts and vasopressin. Endocrinology. 100: 1365- 1375.

Miller, Y. E., Minna, J. D. and Gazdar, A. F. (1 9 8 9 ). Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p. J Clin Invest. 83: 2120-4.

Minna, J. D., Higgins, G. A. and Galstein, G. A., Ed. (1985). Cancer of the lung. Principles and practice of oncology. Philadelphia, Lippinscott.

Minuto, F., Del, M. P., Barreca, A., Alama, A., Cariola, G. and Giordano, G. (1988). Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor I on an established human lung cancer cell line. Cancer Res. 48: 3716-9.

Mitsuhashi, M. and Payan, D. G. (1987). The mitogenic effects of vasoactive neuropeptides on cultured smooth muscle cell lines. Life Sci. 40: 853-861.

Moody, T. W. (1984). Bombesin-like peptides in the normal and malignant lung. Philadelphia, W.B. Saunders.

Moody, T. W. (1988). Neuropeptide receptors on small cell lung cancer cells. Lung cancer. 4: 186.

Moody, I. W., Carney, D. N., Cuttitta, F., Quattrocchi, K. and Minna, J. D. (1985a). High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Sci. 37: 105-13.

Moody, T. W., Carney, D. N., Korman, L. Y., Gazdar, A. F. and Minna, J. D. (1985b). Neurotensin is produced by and secreted from classic small cell lung cancer cells. Life Sci. 36: 1727-32.

Moody, I . W., Lee, M., Kris, R. M., Bellot, F., Bepler, G., Ole, H. and Gazdar, A. F. (1990). Lung carcinoid cell lines have bombesin-like peptides and EGF receptors. J Cell Biochem. 43: 139-47. 195

Moody, T. W., Murphy, A., Mahmoud, S. and Fiskum, G. (1987). Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells. Biochem Biophys Res Commun. 147: 189-95.

Moody, T. W., Pert, C. B., Gazdar, A. F., Carney, D. N. and Minna, J. D. (1981). High levels of intracellular bombesin characterize human small-cell lung carcinoma. Science. 214: 1246-1248.

Moody, T. W., Staley, J., Zia, F., Coy, D. H. and Jensen, R. T. (1992). Neuromedin B binds with high affinity, elevates cytosolic calcium and stimulates the growth of small-cell lung cancer cell lines. J Pharmacol Exp Ther. 263: 311-7.

Moody, T. W., Zia, F., Goldstein, A. L , Naylor, P. H., Sarin, E., Brenneman, D., Koros, A. M. C., Reubi, J. C., Korman, L Y. and Fridkin, M. (1992). VIP analogues inhibit small cell lung cancer growth. Biomed. Res. 13: 131-135.

Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T. M. and Williams, L. T. (1989). Direct activation of the serine/threonine kinase activity of raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 58: 649- 57.

Morrison, D. K., Kaplan, D. R., Rapp, U. and Roberts, T. M. (1988). Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase raf -1 phosphorylation and associated protein kinase activity. Proc Natl Acad Sci USA. 85: 8855-9.

Moyer, J. D., Trepel, J. B. and Sausville, E. A. (1988). Bombesin induces phosphatidylinositol 4,5-bisphosphate hydrolysis in H345 human small cell lung cancer (SCLC) cells (meeting abstract). Proc Annu Meet Am Assoc Cancer Res.

Mukai, H., Munekata, E. and Higashijima, T. (1 9 9 2 ). G protein antagonists (A novel hydrophobic peptide competes with receptor for G protein binding). J. Biol. Chem. 267: 16237-16243.

Mulshine, J., Avis, I., Carrasquillo, J., Merchant, B., Boland, C., Perentesis, P., Reynolds, J., Larson, S., Treston, A., Scott, F. and et, a. I. (1990). Phase I study of an anti-gastrin releasing peptide (GRP) monoclonal antibody in patients with lung cancer (meeting abstract). Proc Annu Meet Am Soc Clin Oncol. 9: A 888 196

Murphy, W. A., Lance, V. A., Moreau, S., Moreau, J. P. and Coy, D. H. (1987). Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin. Life Sci. 40: 2515-22.

Myers, M. J., Backer, J. M., Sun, X. J., Shoelson, S., Hu, P., Schlessinger, J., Yoakim, M., Schaffhausen, B. and White, M. F. (1992). IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci U S A. 89: 10350-4.

Nachbaur, D., Denz, H., Zwierzina, H., Schmalzl, F. and Huber, H. (1 9 9 0 ). Stimulation of colony formation of various human carcinoma cell lines by rhGM-CSF and rhlL-3. Cancer Lett. SO: 197-201.

Nakanishi, Y., Cuttitta, F., Kasprzyk, P. G., Avis, I., Steinberg, S. M., Gazdar, A. F. and Mulshine, J. L. (1988). Growth factor effects on small cell lung cancer cells using a colorimetric assay: can a transferrin-like factor mediate autocrine growth? Exp Cell Biol. 56: 74-85.

Nakanishi, Y., Mulshine, J. L., Kasprzyk, P. G., Natale, R. B., Maneckjee, R., Avis, I., Treston, A. M., Gazdar, A. F., Minna, J. D. and Cuttitta, F. (1988). Insulin-like growth factor -1 can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro. J Clin Invest. 82: 354-9.

Nakata, H., Matsui, T., Ito, M., Taniguchi, T., Naribayashi, Y., Nakamura, A., Kinoshikazu, K., Chihara, K., Hosoda, S. and Chiba, T. (1992). Cloning and characterization of Gastrin receptor from ECL carcinoid Tumor of Mastomys Natalensis. Biochem. Biophys. Res. Com. 187: 1151-1157.

Naldini, L., Weidner, K. M., Vigna, E., Gaudino, G., Bardelli, A., Poncetto, C., Narsimhan, R. P., Hartmann, G., Zarnegar, R., Michalopoulos, G. K., Birchmeier, W. and Comoglio, P. M. (1991). Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 10: 2867-2878.

Nanberg, E. and Rozengurt, E. (1988). Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca^"*" in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin. EMBO. J. 7: 2741-2748. 197

Nau, M. M., Brooks, B. J., Battey, J., Sausville, E., Gazdar, A. F., Kirsch, I. R., McBride, 0. W., Bertness, V., Hollis, G. F. and Minna, J. D. (1985). L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature. 318: 69-73.

Naylor, S. L , Johnson, B. E., Minna, J. D. and Sakaguchi, A. Y. (1 9 8 7 ). Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature. 329: 451-4.

Naylor, S. L., Sakaguchi, A. Y., Spindel, E. and Chin, W. W. (1987). Human gastrin- releasing peptide gene is located on chromosome 18. Somat Cell Mol Genet. 1 3: 87- 91.

Nemunaitis, J. and Singer, J. W. (1989). The effect of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and recombinant human interleukin-1 (rhlL-1) on proliferation of human tumor cell lines. Cancer J. 2: 369-372.

Nilsson, J., von, E. A. and Dalsgaard, C. J. (1985). Stimulation of connective tissue cell growth by substance p and substance k. Nature. 315: 61-3.

Nilsson, T., Arkhammar, P., Rorsman, P. and Berggren, P. 0. (1989). Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca^"*" concentration. J. Biol. Chem. 264: 973-980.

Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 334: 661-65.

North, W. G., Maurer, L. H., Valtin, H. and O’Donnell, J. F. (1980). Human neurophysins as potential tumor markers for small cell carcinoma of the lung: application of specific radioimmunoassays. J. Clin. Endocrinol. Metab. 51: 892- 896.

Osann, K. E. (1991). Lung Cancer in women: The importance of smoking , family history of cancer and medical history of respiratory disease. Cancer Res. 51: 4893- 4897. 198

Ozols, R. F. and Cowan, K. (1986). New aspects of clinical drug resistance: the role of gene amplification and the reversal of resistance in drug refractory cancer. Import Adv Oncol, 129-157.

Pagani, A., Papotti, M., Sanfilippo, B. and Bussolati, G. (1991). Expression of the gastrin-releasing peptide gene in carcinomas of the breast. Int J Cancer. 47: 371-5.

Palazzi, E., Fisone, G., Hokfelt, T., Bartfai, T. and Consolo, S. (1988). Galanin inhibits the muscarinic stimulation of phosphoinositide turnover In rat ventral hippocampus. Eur. J. Pharmacol. 148: 479-480.

Panula, P. (1986). Histochemistry and function of bombesin-like peptides. Med Biol. 64: 177-92.

Pawson, T. (1 9 9 2 ). SH 2 and SH 3 domains. Current Opinion Struct. Biol. 2: 432- 437.

Payan, D. G. (1989). Neuropeptide and inflammation: the role of substance P. Annu. Rev. Med. 40: 341-352.

Payan, D. G., Brewster, D. R., Missirian, B. A. and Goetzl, E. J. (1984). Substance P recognition by a subset of human T lymphocytes. J Clin Invest. 74: 1532-9.

Petruzzelli, S., Camus, A. M., Carrozzi, L, Ghelarducci, L, Rindi, M., Menconi, G., Angeletti, C. A., Ahotupa, M., Hietanen, E., Aitio, A. and et, a. I. (1988). Long-lasting effects of tobacco smoking on pulmonary drug-metabolizing enzymes: a case-control study on lung cancer patients. Cancer Res, 48: 4695-700.

Pfeifer, A. M., Mark, G. E., Malan-Shibley, L., Graziano, S., Amstad, P. and Harris, C. C. (1989). Cooperation of c-raf -1 and c-m yc protooncogenes in the neoplastic transformation of simian virus 40 large tumor antigen-immortalized human bronchial epithelial cells. Proc Natl Acad Sci USA. 86 : 10075-9.

Pisegna, J. R., deWeerth, A., Huppi, K. and Wank, S. A. (1992). Molecular cloning of the human brain and gastric cholecystokinin receptor Structure, function, expression and chromosomal localization. Biochem. Biophys. Res. Com. 189: 269-303.

Price, J., Kruseman, A. C., Doniach, I., Howlett, T. A., Besser, G. M. and Rees, L H. (1985). Bombesin-like peptides in human endocrine tumors: Quantitation, 199 biochemical characterization, and secretion. J Clin Endocrinol Metab. 60: 1097- 103.

Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J. and Sealfon, S. C. (1992). Sequence alignment of the G-protein coupled receptor superfamily. DNA and Cell Biol. 11: 1- 20.

Putnam, E. A., Yen, N., Gallick, G. E., Steck, P. A., Fang, K., Akpakip, B., Gazdar, A. F. and Roth, J. A. (1992). Autocrine growth stimulation by transforming growth factor- alpha in human non-small cell lung cancer. Surg Oncol. 1: 49-60.

Quinn, K. A., Treston, A. M., Scott, F. M., Kasprzyk, P. G., Avis, I., Siegfried, J. M., Mulshine, J. L and Cuttitta, F. (1991). alpha-Amidation of peptide hormones in lung cancer. Cancer Cells. 3: 504-510.

Rabiasz, G. L, Langdon, S. P., Bartlett, J. M., Crew, A. J., Miller, E. P., Scott, W. N., Smyth, J. F. and Miller, W. R. (1992). Growth control by epidermal growth factor and transfoming growth factor-alpha in human lung squamous carcinoma cells. Br. J. Cancer. 66 : 254-259.

Reeve, J. G. (1991). Expression of insulin-like growth factor (IGF) and IGF binding protein genes in human lung tumor cell lines (meeting abstract). Br J Cancer.

Regoli, D., Drapeau, G., Dion, S., D’ Orleans-Juste, P. and Barabe, J. (1986). The actions of antagonists on B^ and B£ receptor system s. Eur. J. Pharmacol. 1 2 3 :

61-65.

Regoli, D., Drapeau, G., Dion, S. and D’O rleans-Juste, P. (1 9 8 9 ). Receptors for substance P and related tachykinins. Pharmacology. 38: 1-15.

Rehfeld, J. F., Bardram, L. and Hilsted, L. (1989). Gastrin in human bronchogenic carcinomas: Constant expression but variable processing of . Cancer Res. 49: 2840-3.

Reubi, J. C., Laissue, J., Krenning, E. and Lamberts, S. W. (1992). Somatostatin receptors in human cancer: Incidence, characteristics, functional correlates and clinical implications. J Steroid Biochem Mol Biol. 43: 27-35. 200

Reubi, J. C., Waser, B., Sheppard, M. and Macaulay, V. (1990). Somatostatin receptors are present in small-cell but not in non-small-cell primary lung carcinomas: Relationship to EGF-receptors. Int J Cancer. 45: 269-74.

Rokaeus, A. (1987). Galanin - a newly isolated biologically-active neuropeptide. Trends Neurosci. 10: 158-164.

Ronsin, C., Muscatelli, F., Mattei, M.-G. and Breathnach, R. (1993). A novel putative receptor protein tyrosine kinase of the met family. Oncogene. 8: 1195-1202.

Rosen, N., Yee, D., Lippman, M. E., Paik, S. and Cullen, K. J. (1 9 9 1 ). Insulin-like growth factors in human breast cancer. Breast Cancer Res Treat.

Ross, R., Raines, E. W. and Bowen-Pope, D. F. (1986). The biology of platelet-derived growth factor. Cell. 46: 155-169.

Roth, K. A. and Barchas, J. D. (1986). Small cell carcinoma cell lines contain opioid peptides and receptors. Cancer. 57: 769-773.

Rozengurt, E. (1985). The mitogenic response of cultured 3T3 cells: integration of early signals and synergistic effects in a unified framework. Amsterdam, Elsevier Science Publishers B.V.

Rozengurt, E. (1986). Early signals in the mitogenic response. Science. 234: 161- 66 .

Rozengurt, E. (1991a). NEUROPEPTIDES AS CELLULAR GROWTH FACTORS: ROLE OF MULTIPLE SIGNALLING PATHWAYS (105 Refs). Eur J Clin Invest. 2 1 :1 2 3 -3 4 .

Rozengurt, E. (1991b). A role for arachidonic acid and its metabolites in the regulation of p2K*® activity. Cancer Cells. 3: 397-8.

Rozengurt, E., Fabregat, I., Coffer, A., Gil, J. and Sinnett-Smith, J. (1990). Mitogenic signalling through the bombesin receptor: role of a guanine nucleotide regulatory protein. J Cell Sci Suppl. 13: 43-56.

Rozengurt, E., Legg, A. and Pettican, P. (1979). Vasopressin stimulation of 3T3 cell growth. Proc. Natl. Acad. Sci. USA. 76: 1284-87. 201

Rozengurt, E., Rodriguez-Pena, A. and Smith, K. A. (1983). Phorbol esters, phopholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells. Proc. Natl. Acad. Sci. USA. 80: 7244- 48.

Rozengurt, E. and Sinnett- Smith, J. W. (1987). Bombesin induction of c-fbs and c- myc proto-oncogenes in Swiss 3T3 cells: Significance for the mitogenic response. J Cell Physiol. 131: 218-25.

Rozengurt, E. and Sinnett-Smith, J. (1983). Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA. 80: 2936-40.

Rozengurt, E. and Sinnett-Smith, J. (1988). Early signals underlying the induction of th e c-fos and c-myc genes in quiescent fibroblasts: studies with bombesin and other growth factors. Prog. Nucl. Acid. Res. Mol. Biol. 35: 261-295.

Rozengurt, E. and Sinnett-Smith, J. (1990). Bombesin stimulation of fibroblast mitogenesis: specific receptors, signal transduction and early events. Philos Trans R Soc Lond Biol. 327: 209-21.

Rudiger, H. W., Nowak, D., Hartmann, K. and Cerutti, P. (1985). Enhanced formation of benzo(a)pyrene:DNA adducts in monocytes of patients with a presumed predisposition to lung cancer. Cancer Res. 45: 5890-4.

Ruff, M. R., Farrar, W. L. and Pert, C. B. (1986). Interferon gamma and granulocyte/macrophage colony-stimulating factor inhibit growth and induce antigens characteristic of myeloid differentiation in small-cell lung cancer cell lines. Proc Natl Acad Sci USA. 83: 6613-7.

Russell, W. E. and Bucher, N. L. R. (1983). Vasopressin modulates liver regeneration in th e Brattleboro rat. Am. J. Physiol. 245: G321-324.

Ryberg, B., Axelson, J., Hakanson, R., Sundler, F. and Mattson, H. (1990). Trophic effects of continuous infusion of [Lysl 5]-gastrin-17 in the rat. Gastroenterology. 98: 33-40. 202

Rygaard, K., Nakamura, T. and Spang, T. M. (1993). Expression of the proto­ oncogenes c-m et and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts. Br J Cancer. 67: 37-46.

Safi, A., Sadmi, M., Martinet, N., Menard, 0., Vaillant, P., Gallati, H., Hosang, M. and Martinet, Y. (1992). Presence of elevated levels of platelet-derived growth factor (PDGF) in lung adenocarcinoma pleural effusions. Chest. 102: 204-7.

Sager, R. (1989). Tumor suppressor genes: the puzzle and the promise. Science. 246: 406-412.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press.

Sameshima, Y., Matsuno, Y., Hirohashi, S., Shimosato, Y., Mizoguchi, H., Sugimura, T., Terada, M. and Yokota, J. (1992). Alterations of the p53 gene are common and critical events for the maintenance of malignant phenotypes in small-cell lung carcinoma. Oncogene. 7: 451-457.

Sanders, B. M., Jay, M., Draper, G. J. and Roberts, E. M. (1989). Non-ocular cancer in relatives of retinoblastoma patients. Br J Cancer. 60: 358-365.

Sanger, F., Nicklen, S. and R., C. A. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467.

Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della-Porta, G. and Barbacid, M. (1984). Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science. 223: 661-4.

Satoh, T., Endo, M., Nakafuku, M., Nakamura, S. and Kaziro, Y. (1990). Platelet- derived growth factor stimulates formation of active p21 GTP complex in Swiss mouse 3T3 cells. Proc. Natl. Acad. Sci. USA. 87: 5993-5997.

Satoh, T., Nakafuku, M. and Kaziro, Y. (1992). Function of Ras as a molecular switch in signal transduction. J. Biol. Chem. 267: 24149-24149.

Sausville, E., Carney, D. and Battey, J. (1985). The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J Biol Chem. 260: 10236-41. 203

Sausville, E. A., Lebacq-Verheyden, A. M., Spindel, E. R., Cuttitta, F., Gazdar, A. F. and Battey, J. F. (1986). Expression of the gastrin-releasing peptide gene in human small cell lung cancer. Evidence for alternative processing resulting in three distinct mRNAs. J Biol Chem. 261: 2451-7.

Saxon, P. J., Srivatsan, E. S. and Stanbridge, E. J. (1986). Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells. Embo J. 5: 3461-6.

Scambia, G., Panici, P. B., Baiocchi, G., Perrone, L, lacobelli, S. and Mancuso, S. (1988). Antiproliferative effects of somatostatin and the somatostatin analog SMS 201-995 on three human breast cancer cell lines. J Cancer Res Clin Oncol. 114: 306-8.

Schally, A. V. (1988). Oncological applications of somatostatin analogues. Cancer Res. 48: 6977-6985.

Schardt, C., Rotsch, M., Erbil, C., Goke, R., Richter, G. and Havemann, K. (1993). Characterization of insulin-like growth factor II receptors inhuman small cell lung cancer cell lines. Exp Cell Res. 204: 22-9.

Scholar, E. M. and Paul, S. (1991). Stimulation of tumor cell growth by vasoactive intestinal peptide. Cancer. 67: 1561-4.

Schuller, H. (1991). Receptor-mediated mitogenic signals and lung cancer. Cancer Cells. 3: 496-503.

Schultz, A. M., Copeland, T., Oroszlan, S. and Rapp, U. R. (1988). Identification and characterization of c-ra7 phosphoproteins in transformed murine cells. Oncogene. 2: 187-93.

Schwab, M., Varmus, H. E. and Bishop, J. M. (1985). Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature. 316: 160-2.

SeckI, M. and Rozengurt, E. (1993). Tyrphostin inhibits bombesin stimulation of tyrosine phosphorylation, c-fos expression and DNA synthesis in Swiss 3T3 cells. J. Biol. Chem. 268: 9548-9554. 204

Seglen, P. 0., Skomedal, H., Saeter, G., Schwarze, P. E. and Nesland, J. M. (1989). Neuroendocrine dysdifferentiation and bombesin production in carcinogen-induced hepatocellular rat tumours. Carcinogenesis. 10: 21-5.

Sehested, M., Hirsch, F. R., Osterlind, K. and Olsen, J. E. (1986). Morphological variations on small cell lung cancer. A histopathologic study of pretreatment and posttreatment specimens in 104 patients. Cancer. 57: 804-807.

Seifter, E. J., Sausville, E. A. and Battey, J. (1986). Comparison of amplified and unamplified c-myc gene structure and expression in human small cell lung carcinoma cell lines. Cancer Res. 46: 2050-5.

Sekido, Y., Takahashi, T., Ueda, R., Takahashi, M., Suzuki, H., Nishida, K., Tsukamoto, T., Hida, T., Shimokata, K., Zsebo, K. M. and Takahashi, T. (1993). Recombinant human stem cell factor mediates chemotaxis of small cell lung cancer cell lines aberrantly expressing the c-/f/t protooncogene. Cancer Research. 53: 1709-1714.

Sellers, T. A., Bailey-Wilson, J. E., Elston, R. C., Wilson, A. P., Elston, G. Z., Ooi, W. L. and Rothschild, H. (1990). Evidence of mendelian inheritance in the pathogenesis of lung cancer. J Natl Cancer Inst. 82: 1272-9.

Sellers, T. A., Elston, R. C., Atwood, L. D. and Rothschild, H. (1992). Lung cancer histologic type and family history of cancer. Cancer. 69: 86-91.

Sharoni, Y., Viallet, J., Trepel, J. B. and Sausville, E. A. (1990). Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells. Cancer Res. 50: 5257-62.

Sharp, G. W., Le-Marchand-Brustel, Y., Yada, T., Russo, L. L., Bliss, C. R., Cormont, M., Monge, L. and Van-Obberghen, E. (1989). Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase. J. Biol. Chem. 264: 7302-7309.

Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B. and Costa, J. (1992). Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA. 89: 4495-9. 205

Shiota, G., Rhoads, D. B., Wang, T. C., Nakamura, T. and Schmidt, E. V. (1992). Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc Natl Acad Sci USA. 89: 373-7.

Simms, E., Gazdar, A. F., Abrams, P. G. and Minna, J. D. (1980.). Growth of human small cell (oat cell) carcinoma of the lung in serum-free growth factor-supplemented medium. Cancer Res. 40: 4356-4363.

Singh, P., Walker, J. P., Townsend, C. M. and Thompson, J. C. (1986). Role of gastrin and gastrin receptors on the growth of a transplantable mouse colon carcinoma (MC- 26) in Balb/c mice. Cancer Res. 46: 1612-1616.

Sithanandam, G., Dean, M., Brennscheidt, U., Beck, T., Gazdar, A., Minna, J. D., Brauch, H., Zbar, B. and Rapp, U. R. (1989). Loss of heterozygosity at the c-raf locus in small cell lung carcinoma. Oncogene. 4: 451-5.

Smith, J. P., Kramer, S. T. and Cheung, J. Y. (1991). Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells. Regul Pept. 36: 299-310.

Smyth, J. P., Fowlie, S. M., Gregor, A., Crompton, G. K., Busutill, A., Leonard, R. C. F. and Grant, I. W. B. (1986). The impact of chemotherapy on small cell carcinoma of the bronchus. Quart. J. Med. 61: 969-976.

Snider, R. M., Constantine, J. W., Lowe, J. A. I., Longo, K. P., Lebel, W. S., Woody, H. A., Drozda, S. E., Desai, M. C., Vinick, F. J., Spender, R. W. and Hess, H.-J. (1991). A potent nonpeptide antagonist of the substance P (NK1) receptor. Science. 251: 435- 437.

Snyder, S. H. (1980). Brain peptides as neurotransmitters. Science. 209: 97 6 - 983.

Soderdahl, G., Betsholtz, C., Johansson, A., Nilsson, K. and Bergh, J. (1988). Differential expression of platelet-derived growth factor and transforming growth factor genes in small- and non-small-cell lung carcinoma cell lines. International Journal of Cancer. 41: 636-641.

Sorenson, G. D., Pettengill, 0. S., Brinck-Johnsen, T., Cate, C. C. and Maurer, L. H. (1981). Hormone production by cultures of small-cell carcinoma of the lung. Cancer. 47: 1289-1296. 206

Spindel, E. R., Giladi, E., Brehm, P., Goodman, R. H. and Segerson, T. P. (1990). Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Mol. Endocrinol. 4: 1956- 1963.

Spindel, E. R., Sunday, M. E., Hofler, H., Wolfe, H. J., Habener, J. F. and Chin, W. W. (1987). Transient elevation of messenger RNA encoding gastrin-releasing peptide, a putative pulmonary growth factor in human fetal lung. J Clin Invest. 80: 1172-9.

Spiro, S. G., Souhami, R. L, Geddes, D. M., Ash, C. M., Quinn, H., Harper, P. G., Tobias, J. S., Partridge, M. and Eraut, D. (1989). Duration of chemotherapy in small cell lung cancer A cancer research campaign trial. Br J Cancer. 59: 578-83.

Staddon, J. M., Barker, C. J., Murphy, A. C., Chanter, N., Lax, A. J., Michell, R. H. and Rozengurt, E. (1991). Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca^+ in Swiss 3T3 cells. J Biol Chem. 266: 4840-7.

Staley, J., Fiskum, G., Davis, T. P. and Moody, T. W. (1989). Neurotensin elevates cytosolic calcium in small cell lung cancer cells. Peptides. 10: 1217-1221.

Staley, J., Jensen, R. T. and Moody, T. W. (1990). CCK antagonists interact with CCK- B receptors on human small cell lung cancer cells. Peptides. 11: 1033-1036.

Stam, N. J., Kast, W. M., Voordouw, A. C., Pastoors, L B., van, d. H. F., Melief, C. J. and Ploegh, H. L. (1989). Lack of correlation between levels of MHC class I antigen and susceptibility to lysis of small cellular lung carcinoma (SCLC) by natural killer cells. J Immunol. 142: 4113-7.

Stephens, L. R., Hughes, K. T. and Irvine, R. F. (1991). Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature. 351: 33-9.

Steranka, L R., Farmer, S. G. and Burch, R. M. (1989). Antagonists of bradykinin receptors. FASEB J. 3: 2019-2025.

Sternweis, P. C. and Smrcka, A. V. (1992). Regulation of phospholipase C by G proteins. TIBS. 17: 502-506. 207

Suda, Y., Aizawa, S., Hirai, S., Inoue, T., Furuta, Y., Suzuki, M., Hirohashi, S. and Ikawa, Y. (1987). Driven by the same Ig enhancer and SV40 T promoter nas induced lung adenomatous tumors, myc induced pre-p cell lymphomas and SV40 large T gene a variety of tumors in transgenic mice. Embo J. 6 : 4055-65.

Sunday, M. E., Choi, N., Spindel, E. R., Chin, W. W. and Mark, E. J. (1991). Gastrin- releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas. Hum Pathol. 22: 1030-9.

Sunday, M. E., Wolfe, H. J., Roos, B. A., Chin, W. W. and Spindel, E. R. (1988). Gastrin-releasing peptide gene expression in developing, hyperplastic, and neoplastic human thyroid C-ceils. Endocrinology. 122: 1551-1558.

Suzuki, Y., Orita, M., Shiraishi, M., Hayashi, K. and Sekiya, T. (1990). Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 5: 1037- 43.

Takahashi, T., Nau, M. M., Chiba, I., Birrer, M. J., Rosenberg, R. K., Vinocour, M., Levitt, M., Pass, H., Gazdar, A. F. and Minna, J. D. (1989). p53: A frequent target for genetic abnormalities in lung cancer. Science. 246: 491-4.

Takahashi, T., Takahashi, T., Suzuki, H., Hida, T., Sekido, Y., Ariyoshi, Y. and Ueda, R. (1991). The p53 gene is very frequently mutated in small-cell lung cancer with a distinct nucleotide substitution pattern. Oncogene. 6 : 1775-8.

Takuwa, N., Takuwa, Y., Ohue, Y., Mukai, H., Endoh, K., Yamashita, K., Kumada, M. and Munekata, E. (1990). Stimulation of calcium mobilization but not proliferation by bombesin and tachykinin neuropeptides in human small cell lung cancer cells. Cancer Res. 50: 240-4.

Takuwa, N., Takuwa, Y., Yanagisawa, M., Yamashita, K. and Masaki, T. (1989). A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J. Biol. Chem. 264: 7856-61.

Tamura, K., Palmer, J. M., Winkelmann, C. K. and Wood, J. D. (1988). Mechanism of action of galanin on myenteric neurons. J. Neurophysiol. 60: 966-979. 208

Tatemoto, K., Rokaeus, A., Jornvall, H., McDonald, T. J. and Mutt, V. (1983). Galanin - a novel biologically active peptide from porcine intestine. FEES Lett. 124-128.

Taylor, J. E., Bogden, A. E., Moreau, J. P. and Coy, D. H. (1988). In vitro and in vivo inhibition of human small cell lung carcinoma (NCI-H69) growth by a somatostatin analogue. Biochem Biophys Res Commun. 153: 81-6.

Taylor, J. E., Moreau, J. P., Baptiste, L. and Moody, T. W. (1991). Octapeptide analogues of somatostatin inhibit the clonal growth and vasoactive intestinal peptide- stimulated cyclic amp formation in human small cell lung cancer cells. Peptides. 12: 839-43.

Thomas, P., Arvelo, P., Antoine, E., Jacrot, M. and Poupon, M. P. (1992). Antitumoral activity of bombesin analogues on small cell lung cancer xenografts: Relationship with bombesin receptor expression. Cancer Res. 52: 4872-7.

Thumwood, C. M., Hong, J. and Baldwin, G. S. (1991). Inhibition of cell proliferation by the cholecystokinin antagonist L-364,718. Exp. Cell Res. 192: 189-192.

Treisman, R. (1992). The serum response element. TIBS. 17: 423-426.

Trepel, J. B., Moyer, J. D., C uttitta, P., Prucht, H., Coy, D. H., Natale, R. B., Mulshine, J. L., Jensen, R. T. and Sausville, E. A. (1988a). A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line. Biochem Biophys Res Commun. 156: 1383-9.

Trepel, J. B., Moyer, J. D., Heikkila, R. and Sausville, E. A. (1988b). Modulation of bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung-cancer cell line. Biochem J. 255: 403-10.

Tsarfaty, I., Resau, J. H., Rulong, S., Key da r. I., Paletto, D. L. and Vande, W. G. (1992). The m et proto-oncogene receptor and lumen formation. Science. 257: 1258-61.

Tsien, R. Y., Pozzan, T. and Rink, T. J. (1982). T-cell mitogens cause early changes in cytoplasmic free Ca^'*' and membrane potential in lymphocytes. Nature. 295: 68 - 71. 209

Uddman, R. and Sundler, F. (1987). Neuropeptides in the airways: a review. Am. Rev. Respir. Dis. 136: 3-8.

Ullrich, A. and Schlessinger, J. (1990). SIGNAL TRANSDUCTION BY RECEPTORS WITH TYROSINE KINASEACTIVITY(91 Refs). Cell. 61: 203-12.

Valius, M. and Kazlauskas, A. (1993). Phospholipase C-yl and phosphatidylinositol 3 kinase are the down stream mediators of the PDGF receptor’s mitogenic signal. Cell. 73: 321-334.

Vangsted, A. J., Andersen, E. V., Nedergaard, L. and Zeuthen, J. (1991). Gastrin releasing peptide GRP( 14-27) in human breast cancer cells and in small cell lung cancer. Breast Cancer Res Treat. 19: 119-28.

Veale, D., Kerr, N., Gibson, G. J. and Harris, A. L. (1989). Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer. Cancer Research. 49: 1313-1317.

Vellenga, E., Biesma, B., Meyer, C., Wagteveld, L., Esselink, M. and de-Vries. E. (1991). The effects of five hematopoietic growth factors on human small cell lung carcinoma cell lines: Interleukin 3 enhances the proliferation in one of the eleven cell lines. Cancer Res. 51: 73-6.

Verbeeck, M. A., Elands, J. P., de-Liej, L., Buys, C. H., Carney, D. N., Bepler, G., Roebroeck, A. J., Van-de Yen, W.J. and Burbach, J. P. (1992). Expression of the vasopressin and gastrin-releasing peptide genes in small cell lung carcinoma cell lines. Pathobiology. 60: 136-42.

Vostrejs, M., Moran, P. L. and Seligman, P. A. (1988). Transferrin synthesis by small cell lung cancer cells acts as an autocrine regulator of cellular proliferation. J Clin Invest. 82: 331-9.

W.H.O. (1982a). Reappraisal of the present situation in prevention and control of lung cancer. Bull World Health Organ. 60: 809-19.

W.H.O. (1982b). The World Health Organization. Histological typing of lung tumours. Neoplasma. 29: 111-23.

W.H.O. (1988. ). World Health Statistics Annual. Geneva, World Health Organisation. 210

Wallace, M. R., Marchuk, D. A., Andersen, L B., Letcher, R., Odeh, H. M., Sauiino, A. M., Fountain, J. W., Brereton, A., Nicholson, J., Mitchell, A. L, Brownstein, B. H. and Collins, F. S. (1990). Type 1 neurofibromatosis gene: Identification of a large transcript disrupted in three NF1 patients. Science. 249: 181-6.

Walsh, J. H. (1987). Gastrointestinal hormones. New York, Raven Press.

Wank, S. A., Pisegna, J. R. and deWeerth, A. (1992). Brain and gastrointestinal cholecystokinin receptor family: Structure and functional expression. Proc. Natl. Acad. Sci. USA. 89: 8691-8695.

Watson, S., Durrant, L. and Morris, D. (1989). Gastrin: growth enhancing effects on human gastric and colonic tumour cells. Br. J. Cancer. 59: 554-558.

Weber, S., Zuckerman, J. E., Bostwick, D. G., Bensch, K. G., Sikic, B. I. and Raffin, T. A. (1985). Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro. J Clin Invest. 75: 306-9.

Weiner, D. B., Liu, J., Cohen, J. A., Williams, W. V. and Greene, M. I. (1989). A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature. 339: 230-1.

Weiner, D. B., Nordberg, J., Robinson, R., Nowell, P. C., Gazdar, A., Greene, M. I., Williams, W. V., Cohen, J. A. and Kern, J. A. (1990). Expression of th e neu gene- encoded protein (P185(neu)) in human non-small cell carcinomas of the lung. Cancer Res. 50: 421-5.

Westermark, B. and Heldin, C.-H. (1991). Platelet-derived growth factor in autocrine transformation. Cancer Res. 51: 5087-5092.

W eston, A., Willey, J. C., Modali, R., Sugimura, H., McDowell, E. M., Resau, J., Light, B., Haugen, A., Mann, D. L, Trump, B. F. and Harris, C. C. (1989). Differential DNA sequence deletions from chromosomes 3, 11, 13, and 17 in squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma of the human lung. Proc Natl Acad Sci U S A. 86 : 5099-103.

Weynants, P., Wauters, P., Coulie, P. G., Van-den Eynde B., Symann, M. and Boon, T. (1988). Cytolytic response of human T cells against allogeneic small cell lung 211 carcinoma treated with interferon gamma. Cancer Immunol Immunother. 27: 228- 32.

Whang-Peng, J., Bunn, P., Jr., Kao-Shan, C. S., Lee, E. C., Carney, D. N., Gazdar, A. and Minna, J. D. (1982). A nonrandom chromosomal abnormality, del 3p(14-23), in human small cell lung cancer (SCLC). Cancer Genet Cytogenet. 6 : 119-34.

Whang-Peng, J., Knutsen, T., Gazdar, A., Steinberg, S. M., Oie, H., Linnoila, I., Mulshine, J., Nau, M. and Minna, J. D. (1991). Nonrandom structural and numerical chromosome changes in non-small-cell lung cancer. Genes Chromosom Cancer. 3:

168 - 88 .

Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A. and Harlow, E. (1988). Association between an oncogene and an anti-oncogene: the adenovirus El a proteins bind to the retinoblastoma gene product. Nature. 334: 124-9.

Willey, J. C., Lechner, J. F. and Harris, C. C. (1984). Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells. Exp. Cell Res. 153: 245-248.

Williams, D. W., Williams, E. D. and Wynford, T. D. (1989). Evidence for autocrine production of IGF-1 in human thyroid adenomas. Mol Cell Endocrinol. 61: 139-43.

Woll, P. J. and Rozengurt, E. (1988). Two classes of antagonist Interact with receptors for the mitogenic neuropeptides bombesin, bradykinin and vasopressin. Growth Factors. 1: 75-83.

Woll, P. J. and Rozengurt, E. (1988a). Bombesin and bombesin antagonists: studies in Swiss 3T3 cells and human small cell lung cancer. Br J Cancer. 57: 579-86.

Woll, P. J. and Rozengurt, E. (1988b). [D-Arg^,D-Phe5,D-Trp^*®,Leu^^]substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, Inhibits the growth of human small cell lung cancer cells in vitro. Proc Natl Acad Sci USA. 85: 1859-63.

Woll, P. J. and Rozengurt, E. (19 8 8 c). [Leu^ 3_psi(CH 2NH)LeuT 4] bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells. Biochemical and Biophysical Research Communications. 155: 359-365. 212

Woll, P. J. and Rozengurt, E. (1989a). Multiple neuropeptides mobilise calcium in small cell lung cancer: Effects of vasopressin, bradykinin, cholecystokinin, galanin and neurotensin. Biochem Biophys Res Commun. 164: 66-73.

Woll, P. J. and Rozengurt, E. (1989b). Neuropeptides as growth regulators. Br Med Bull. 45: 492-505.

Woll, P. J. and Rozengurt, E. (1990a). Multiple neuropeptides mobilize calcium in small-cell lung cancer (meeting abstract). Br J Cancer. 62:

Woll, P. J. and Rozengurt, E. (1990b). A neuropeptide antagonist that inhibits the growth of small cell lung cancer in vitro. Cancer Res. 50: 3968-73.

Wood, S. M., Wood, J. R., Ghatei, M. A., Lee, Y. C., O’Shaughnessy, D. and Bloom, S. R. (1981). Bombesin, somatostatin and neurotensin-like immunoreactivity in bronchial carcinoma. J. Clin. Endocrinol. Metab. 53: 1310-1312.

Yano, T., Pinski, J., G root, K. and Schally, A. V. (1992). Stimulation by bombesin and inhibition by bombesin/gastrin-releasing peptide antagonist RC-3095 of growth of human breast cancer cell lines. Cancer Res. 52: 4545-4547.

Yapp, R., Modlin, I. M., Kumar, R. R., Binder, H. J. and Dubrow, R. (1992). Gastrin and colorectal cancer: Evidence against an association. Dig Dis Sci. 37: 481-4.

Yoakum, G. H., Lechner, J. F., Gabrielson, E. W., Korba, B. E., Malan-Shibley, L., Willey, J. C., Valerio, M. G., Shamsuddin, A. M., Trump, B. F. and Harris, C. C. (1985). Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene. Science. 227: 1174-9.

Yoder, D. G. and Moody, T. W. (1987). High affinity binding of cholecystokinin to small cell lung cancer cells. Peptides. 8: 103-7.

Yokota, J., Wada, M., Shimosato, Y., Terada, M. and Sugimura, T. (1987). Loss of heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc Natl Acad Sci USA. 84: 9252-6.

Yoshimoto, K., Yamasaki, R., Sakai, H., Tezuka, U., Takahashi, M., lizuka, M., Sekiya, T. and Saito, S. (1989). Ectopic production of parathyroid hormone by small cell lung cancer in a patient with hypercalcemia. J Clin Endocrinol Metab. 68: 976-81. 213

Yoshinaga, K., Evers, B. M., Izukura, M., Parekh, D., Uchida, T., Townsend, C. J. and Thompson, J. C. (1992). Neurotensin stimulates growth of colon cancer. Surg Oncol. 1: 127-134.

Young, D., Waitches, G., Birchmeier, C, Fasano, 0. and Wigler, M. (1986). Isolation and characterization of a new cellular oncogene encoding a protein with multiple potential transmembrane domains. Cell. 45: 711-9.

Zachary, I., Gil, J., Lehmann, W., Sinnett-Smith, J. and Rozengurt, E. (1991). Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact swiss 3t3 cells. Proc Natl Acad Sci USA. 88: 4577-81.

Zachary, I. and Rozengurt, E. (1985). High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells. Proc Natl Acad Sci USA. 82: 7616-20.

Zachary, I. and Rozengurt, E. (1986). A substance P antagonist also inhibits the specific binding and mitogenic effects of vasopressin and bombesin-related peptides in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 137: 135-141.

Zachary, I. and Rozengurt, E. (1987). Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking. J. Biol. Chem. 262: 3947-3950.

Zachary, I. and Rozengurt, E. (1992). Focal Adhesion Kinase (pi 25^^): A point of convergence in the action of neuropeptides, integrins and oncogenes. Cell. 71: 891- 894.

Zachary, I., Sinnett-Smith, J. and Rozengurt, E. (1991). Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells. JBiol Chem. 266: 24126-33.

Zachary, I., Sinnett-Smith, J. and Rozengurt, E. (1992). Bombesin, vasopressin and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells: Identification of a novel tyrosine kinase as a major substrate. J. Biol. Chem. 267: 19031-19034.

Zachary, I., Woll, P. and Rozengurt, E. (1987). A role for neuropeptides in the control of cell proliferation. Dev. Biol. 124: 295-308. 214

Zagon, I. S. and McLaughlin, P. J. (1987). Modulation of murine neuroblastoma in nude mice by opioid antagonists. J Natl Cancer Inst. 78: 141-7.

Zhu, Y. X., Hsi, K. L., Chen, Z. G., Zhang, H. L., Wu, S. X., Zhang, S. Y., Fang, P. P., Guo, S. Y., Kao, Y. S. and Tsou, K. (1986). Neo-kyotorphin, an analgesic peptide isolated from human lung carcinoma. Febs Lett. 208: 253-7.

Zijistra, J. G., de, V. E. and Mulder, N. H. (1987). Multifactorial drug resistance in an Adriamycin-resistant human small cell lung carcinoma cell line. Cancer Res. 47: 1780-4.

Zimmerman, K. A., Yancopoulos, G. D., Collum, R. G., Smith, R. K., Kohl, N. E., Denis, K. A., Nau, M. M., Witte, 0. N., Toran-Allerand, D., Gee, C. E. and et, a. (1986). Differential expression of m y c family genes during murine development. Nature. 3 1 9 :7 8 0 -3 .

Zurier, R. B., Kozma, M., Sinnett-Smith, J. and Rozengurt, E. (1988). Vasoactive Intestinal Peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, CaZ+ and protein kinase C. Exp. Cell. Res. 176:155-61. 1< \N( ^K KIM \KCII SI. I 674-If,-'V M .in h !<•. M V I| Galanin Stimulates Ca^"^ Mobilization, Inositol Phosphate Accumulation, and Clonal Growth in Small Cell Lung Cancer Cells lariq Sctlii and Enrique Ro/enyurt'

Imperial Cancer Raearch Fund. P O Ho\ 12.1. I.incoln'i Inn f ields. Ijindini II ( 2-1 .IP \. I niied Kingdom

ABSTRACT blocks Ca-* influx via voltage-gated Ca-" channels (2, 10). Furthermore, galanin inhibits muscarinic agonist-stimulated Addition of I he neuropeptide xalanin to small cell lung cancer (SCI.C) breakdown of inositol phospholipids in tissue slices of ventral cells loaded with the fluorescent Ca’* indicator fura-2-letraaceto\yme- hippocampus (II). To date, galanin has been found to neither th)lester causes a rapid and transient increase in the intracellular con­ centration of Ca'^ (|Ca'*|,) followed by homologous desensiti7.ation. Gal­ stimulate inositol phosphate production or Ca^* mobilization anin increased |Ca’*|, in a concentration-dependent fashion with half from internal stores in target cells nor act as a direct regulator maximum effect (EC*) at 20-22 n\i in 1169 and U5I0 SCLC cells. of proliferation in any cell type. Galanin mobilized Ca'" from intracellular stores since its effects on |Ca'^|, Evidence is rapidly accumulating that neuropeptides acting were not blocked by chelation of extracellular Ca’". Pretreatment with through distinct receptors and signal transduction pathways pertussis toxin (200 ng/ml for 4 h) did not prevent galanin-induced Ca'* can control the proliferation of a variety of cell types (12-14). mobilization. In contrast, direct activation of protein kinase C with SCLC' cells are known to produce and respond to a variety of phorbol esters attenuated the Ca’" response induced by galanin. The neuropeptides (15-19). Bombesin-like peptides including GRP effects of galanin could be dissociated from changes in membrane poten­ induce a rapid increase in |Ca^"], and act as autocrine growth tial: galanin did not increase membrane potential in SCLC cells loaded factors for certain SCLC cell lines (15, 16). Recently, it has with bis(l,3-diethyltiobarbiturate)-trimethineoxonol and induced Ca'^ been shown that multiple neuropeptides stimulate Ca’" mobi­ mobilization in depolarized SCLC cells, i.e., in cells suspended in a solution containing 145 m\t K* instead of Na'*. Galanin also caused an lization in a variety of SCLC cell lines (17, 18, 20). Although increase in the formation of inositol phosphates in a time- and dose- the precise role of (Ca’*], in the control of cell proliferation dependent manner (EC» 10 nxi). A rapid increase in the inositol Iris- remains undefined, this ionic response is part of a mitogenic phosphate fraction was followed by a slower increase in the inositol signaling cascade identified in Swiss 3T3 cells (12-14), a cell monophosphate fraction. Galanin stimulated clonal growth of both H69 line that has provided a model system for the response of SCLC and H 5I0 cells in semisolid (agarose-containing) medium. This growth- to neuropeptides. In view of the fact that galanin opposes Ca’’ promoting effect was sharply dependent on galanin concentration (EC» signals and modulates the action of other neuropeptides in 20 nxt) and markedly inhibited by |Arg‘,t>Trp'” ,M ePhe'|substance P, a various cellular systems (see above), it was important to deter­ recently identified broad spectrum neuropeptide antagonist. The results mine whether galanin could reduce |Ca^*), and antagonize the show for the first time that galanin receptors a coupled to inositol Ca^’-mobilizing effects of other neuropeptides in SCLC cell phosphate and |Ca'*|, responses in SCLC cells a in particular, that this neuropeptide can act as a direct growth factor k , these human cancer lines. Surprisingly, a preliminary result indicated that galanin ce lls . increased rather than decreased (Ca^’j, in certain SCLC cell lines (17). Hence, the elucidation of the signal transduction pathways activated by galanin in SCLC cell lines warranted LNTRODUCTION further experimental work. It is increasingly recogni d that neuropeptides act as molec­ In the present study we demonstrate that galanin stimulates ular messengers in a comp! network of information exchange a rapid mobilization of Ca'’ from intracellular stores and by cells throughout the bod,.. Galanin, a 29-amino acid peptide induces an increase in the production of inositol phosphates in (1), has widespread distribution and occurs in central and SCLC cell lines. Furthermore, we also show that galanin is a peripheral neurones (2). It elicits a variety of rapid biological grow th factor for responsive SCLC cell lines, stim ulating clonal responses including modulation of the release of several hor­ growth in semisolid medium. mones (3), stimulation of smooth muscle contractility, and inhibition of neuronal excitability (4). Since galanin may play MATERIALS AND METHODS an important role in the regulation of endocrine, neuronal, and smooth muscle function, its mechanism of action is attracting SCLC Cell Culture. SCLC cell lines H345 and H 5I0 were the kind gift of Dr. Adi Gazdar (National Cancer Institute, Bethesda. M D). H69 considerable attention. was purchased from the American Type Culture Collection. Stocks In the endocrine pancreas and in pancreatic (3-cell models in were maintained in RPMI 1640 medium supplemented with 10% (v/v) vitro, galanin inhibits the release of insulin (for review see Ref. fetal bovine serum (heat inactivated at 57‘C for I h) in a humidified 5). Galanin activates an ATP-sensitive K" channel, hyperpolar- atmosphere of 10% COj:90% air at 37’C. They were passaged every 7 izes the plasma membrane (6, 7), and thereby inhibits the days. For experimental purposes, the cells were grown in RPMI 1640 activity of voltage-dependent Ca’" channels (8, 9). In this m an­ medium with HITESA (21). ner, galanin reduces Ca’" influx and blocks the activity of Determination of |Ca” k Concentration. Aliquots of 4-5 x 10* SCLC various agents that increase the intracellular concentration of cells, cultured in HITESA for 3-5 days, were washed and incubated for Ca’’ (|Ca^“],) in the pancreatic /3-cell. These effects are induced 2 h at 37’C in 10 ml fresh HITESA medium. Then, I »i M fura-2 AME via a pertussis toxin-sensitive G protein (7). In myenteric neu­ ' The abbreviations used are: SCLC. small cell lung cancer. GRP. gastrin rons, galanin also hyperpolarizes the plasma membrane and releasing peptide; HITESA. 10 nw hydrocortisone. 5 *ig/ml insulin. 10 *ig/ml transferrin. 10 nsi estradiol. .10 nxt selenium, and 0.25% bovine serum albumin. Received 8./24/90; accepted 1/9/91. AME. tetraacetoxymethylester. Hepes. 4-(2-hydroxyethyl) l-piperazineethane The costs of publication of this article were defrayed in part by the payment sulfonic acid. ECTA. ethyleneglycol bis(d-aminoclhyl ether)-A'.A.,\'VV-leiraa of page charges This article must therefore txr hereby marked advcriiscmeni in cctic arid, his oxonol. bis(l.1-dieihyltiobarbiturate)-trimethineoxonol; TC A. tri­ accordance with 18 tl.S .C . Section 17.14 solely to indicate this fact chloroacetic acid; FPLC. fast protein liquid chromatography; InsP. inositol ' To whom requests for reprints should he addressed phosphate; ECv,. half maximum stimulation; PBt,. 12,11-dibutyrate

1674 C.AI.AMN STIMULATES EAR1.\ SKiVAI.S AND <.ROW III from a stock of I nisi in dimcihyl sulfoxide was added, and the cells gradient used was 25-min buffer A (1005(). a 17 min gradient to 15% were incubated for a further 5 min. The cell suspension was centrifuged buffer B, and then an isocratic elution at 15% for 40 min followed by at 2000 rpm for 15 s. and the cells were rcsuspcnded in 2 ml of 25-min elution at 100% buffer B. Radioactivity peaks were identified clcctrolyie solution containing 140 niM NaCl, 5 him KCI. 0.9 mM by use of |'H|inositol standards added to controls not pretreated w ith MgCl:, 1.8 niM CaCh, 25 niM glucose. 16 mM Hepes. 6 mM Tris, and myo-|'H]inositol which were then lysed and treated as previously de­ a mixture of amino acids at pH 7.2, transferred to a quartz cuvette, and scribed or by coelution of P” -labcled inositol standards w ith samples. stirred continuously. Fluorescence was recorded continuously in a Clonogenir Assay. SCLC cells, 3-5 days post passage, were washed Perkin-Elmer I.S5 luminescence spectrometer with an excitation wave­ and resuspended in HITESA. Cells were then disaggregated by two length of 5)6 nm and an emission wavelength of 510 nm. |Ca-'|. was passes through a 19 gauge needle into an essentially single cell suspen­ calculated using the formula (22. 25): sion as judged by microscopy. Cell number was determined using a Coulter Counter and 10' cells were mixed with HITESA containing A'(F - fmm) 0.3% agarose and galanin. at the concentrations indicated, and layered |Ca^*l, nxi = (Fn.,, - F) over a solid base of 0.5% agarose in HITESA with galanin at the same concentration, in 33-mm plastic dishes. The cultures were incubated in w here F is the fluorescence at the unknown [Ca'*|„ F„„, is the fluores­ humidified 10% CO>:90% air at 37’C for 2 1 days and then stained with cence after the trapped fluorescence is released by the addition of 0.02% the vital stain nitro-blue tétrazolium. Colonies of >120 /im in diameter Triton-X-100. and F„,„ is the fluorescence remaining after the Ca'" in (16 cells) were counted using a microscope. the solution is chelated with 10 m.M ECTA. The value of A' was 220 Materials. Radiochcmicals were obtained from Amersham Interna­ for fura-2 (22). tional (Amersham, United Kingdom). Galanin was purchased from Measurement of Membrane Potential. Membrane potential was mon­ Sigma Chemical Co. (St. Louis, MO), antagonist (Arg*. i) Trp ". itored with the lipophilic fluorescent dye bis-oxonol (22). Cells cultured MePhe")substance P from Peninsula Laboratories (Belmont. CA). fura- in HITESA were washed and incubated for 2 h in fresh HITESA. The 2-AME from Calbiochem Corp. (La Jolla. C.A), agarose from Seaketn cells were then resuspended in 2 ml electrolyte solution (see above), (Rockland, ME). Dowex (mesh size. 200-400) from Bio-Rad Labora­ placed in a quartz cuvette, and stirred continuously. Bis-oxonol was tories (Richmond. C.A). and pertussis toxin from List Biological Lab­ added at a (Inal concentration of 100 nxt from a stock solution of 1 oratories (Campbell. CA). Bis-oxonol was obtained from Molecular mxt in dimethyl sulfoxide to the cell suspension in electrolyte solution Probes (Eugene, OR). Fetal bovine serum was from Gibco Europe at 37‘C for 5 min before starting the experiment. Fluorescence was (Paisley, United Kingdom). All other reagents were of the highest grade monitored in a Perkin-Elmcr Ls5 luminescence spectrometer at 37*C. commercially available. Excitation and emission wavelengths were 540 and 580 nM, respectively (22). Accumulation of Inositol Phosphates. The SCLC cell line H69 was RESULTS maintained in culture as previously described. Cells (2 x 10’) were Galanin Increases |Ca'*|,inSCLC. .Addition of 100 nM galanin labeled in 20 ml HITESA with 10 pCi/ml myo-['H|inositol for 24 h. to either H69 or H 5I0 cells loaded with the fluorescent Ca'" For determination of the production of total inositol phosphates, cells indicator fura-2 AM E increased [Ca'"], without any measurable were washed twice in HITESA 0.02 M-Hepes-Na, pH 7.2, at 37’C. delay (Fig. 1). At this concentration, galanin increased [Ca'"], Approximately 1.5 x 10"" cells were resuspended in 1 ml HITESA plus Hepes and incubated with 20 mxt LiCI for 20 min prior to the addition from 81 ± 4.5 (SEM) (n = 35) to 115 ± 6.3 (rt = 10) nxi in of galanin at the concentrations and times as indicated. Following the H69 cells and from 107 ± 5.9 (n = 27) to 152 ± 9 (« = 10) nxi incubation at 37’C the cells were lysed using 200 /il 18% perchloric in H 5I0 cells. [Ca'")i reached peak values at 20-30 s and acid and left at 4’C for 30 min. The supernatant was collected after subsequently declined toward the basal level. The magnitude centrifugation and neturalized with 0.5 m KOH-25 mM Hepes-10 mxt and kinetics of the [Ca'"]* response induced by galanin were EOTA using 0.01% phenol red as an indicator. The precipitated salts were removed by centrifugation. The supernatant was diluted in water r H 6 9 H 5 1 0 and loaded on Dowex columns which were subsequently washed four times with water, and the total inositol phosphates were eluted using 5 mIO.I M formic acid and 1 xi ammonium formate (24). Aliquots (1 ml) of eluates were transferred to scintillation vials containing 10 ml Picofluor and radioactivity was determined in a.Beckman (3-Counter. V P t Separation of Inositol Phosphate by FPLC. Cells were maintained in culture as previously described, washed at 3-5 days postpassage, and labeled in HITESA with 50 ;/Ci/ml myo|'H|inositol for 24 h. Cells were then washed twice in HITESA at 37’C, pH 7.2, and 3-5 x 10* VP cells were resuspended in I ml electrolyte solution and 20 mM LiCI for 'g .I 'BK 'Gal 20 min before addition of galanin (100 nxi). The cells were then 'Gal incubated at 37’C for various times as indicated. The cells were then lysed with 250 /il 25% TCA, cooled rapidly on ice, and left at 4’C for 30 min. The extract was centrifuged and the TCA in the supernatant was removed by six extractions w ith water-saturated ether. Excess ether BK lyp w as blown off under nitrogen and the sample was diluted to 10 ml with 'Gal buffer A (10 mxt Hepes 100 /ixt EDTA, pH 7.4). The inositol phos­ 1 Min phates were separated by anion-exchange chromatography using a Fig. I Effect of galanin on |Ca'*|i in SCLC celh . SCLC cell lines H69 ilefi) Mono Q column fitted in a Pharmacia FPLC system (24). The inositol and H 5 I 0 {.right) were cultured in HITESA for .^-5 days. Aliquots of 4-5 x 10* phosphates were eluted w ith a gradient of sodium sulfate at a flow rate cells were wushed and incubated in 10 ml fresh HITESA medium for 2 h at tt'C Then, I pxi fura 2 AXIE was added for 5 min. The cells were washed and of I ml/min at pH 7.4. The gradient used was from 0 (buffer A) to 0.5 resuspended in 2 ml of electrolyte solution. This cell suspension was placed in a M sodium sulfate (buffer B) as follows: 25 min buffer A 100%, a 20- quartz cu vette. Fluorescence w as m onitored and jCa-’" |, w as calculated as deserihed min gradient to 20% buffer B; a 25-min gradient to 32.5% buffer B; a in “Xiatcrials and Xlelhods." .Agonists and antagonists were added either inde­ 15-min gradient to 50% buffer B: and a 25-min elution at 100% buffer pendently {lo p )nr sequentially {m iJ illr and hotioni) at the following litial concen­ trations: BA. 10 nxt bradskinin: BA-*-. 100 nst hradykinin: I'P. 10 nsi lasopressin; B. Fractions (I ml) were collected and counted in 4.5 ml Picofluor in a f ’P-*. 100 nxt vasopressin: BA.4, 10 >iM |t>-.-\rg".H>p'.Thi**.t)-l’lic|hradykinin: Beckman /3-Counter. Separation of |'H|lns(1.4.5)P, from |'H) PI P. 100 nst jPmp'.f/Xtc. I yr’.Arg'|sasoprcssin: galanin (f»'o/) was added at Ins(1.3,4)P, was carried out by the same method except that the either 100 nst {lo p )or 2,s nst {m id d le and lower).

1675 CM.AMN STIMULATES EARL\ SIC;S\LS AND GROWTH

H69 Control EGTA 120 H 6 9 H 5 1 0

1 00 1 Min

60 1 00 Fig .3. Effect of EGTA. pertussis toxin, and PBt, on galanin-induced Ca’* mobilization in SCLC cell lines H69 (top) and H 5I0 (bottom ) Cells were Galanin (nM) preloaded «ith fura-2 AME and fluorescence «as monitored continuously as previously described EGTA: The Ca" chelator EGTA was added to a final Fig 2. Dose-dfpondeni effcci of galanin on |Ca’*|, in SCLC cells Left. H 69. concentration of 1.8 mxt 1-2 min prior to the addition of galanin. Pertussis toxin: r;*/». H5I0. Cells (4-5 x 10‘) «ere loaded «ilh fura 2 AME (1 mm) and resus- Cells cultured in HITESA for 3-5 days were «ashed and incubated in 10 ml fresh pended in 2 ml elecirolyie solution. Galanin was added ai I he concentrations HITESA. Pertussis toxin (P .T x.) was added to a final concentration of 200 ng/ indicated Fluorescence «as monitored continuously as described in “Materials ml and incubated for 4 h at 37‘C Fura 2 AME (I um) «as then added for 5 min and Methods ■ Basal |C a''|, and peak (Ca'*|. «ere calculated at the concentrations and the cells «ere then washed and the fluorescence was monitored as previously- indicated The results represent peak |Ca^*|, values obtained at the given conccn described PBt, Cells «ere pretreated with PBt, at a final concentration of 500 iraiions of 5-5 independent experiments Point, mean; bar, ±SE M . nxt for 3 min prior to the addition of galanin. In all cases, the final concentration of galanin (C a l) «as 25 nxi The results obtained in 3-b independent experiments are shown in Table I . comparable to those induced by other Ca^’-mobiiizing neuro­ peptides such as bradykinin (H69) or vasopressin (H5I0). Similarly, direct activation of protein kinase C with phorbol Repeated additions of galanin (at 25 nM ) caused homologous desensitization of Ca’" mobilization but did not prevent the PBt] inhibited the Ca’" response to galanin in both H69 and H510 cells (Fig. 3). increase in (Ca’"), induced through other neuropeptide recep­ tors such as bradykinin and vasopressin (Fig. 1). Accordingly, Dissociation of Ca’" mobilizing Effects of Galanin from Changes in Membrane Potential. It has been suggested that the addition of specific bradykinin and vasopressin antagonists inhibitory effects of galanin on pancreatic secretion and neu­ blocked the effect of the corresponding neuropeptides but did ronal excitability are mediated by increases in membrane po­ not interfere with the rapid increase in |Ca^"j, induced by tential (2, 10). Therefore, we examined whether galanin exerts galanin (Fig. I). G anin increased the peak level of |Ca^"), in a concentration- any effect on membrane potential of SCLC cell lines using cells loaded with the membrane potential sensitive dye bis-oxonol. dep dent manner in both H69 and H5I0 cells (Fig. 2). The Fig. 4 shows that 25 nM galanin did not cause any detectable concentrations of galanin required to induce ECso of [Ca^"j, change in membrane potential, as Judged by bis-oxonol fluores­ increase were 22 and 20 nM in H69 and H 510 cells, respectively. cence. As expected, addition of 80 m M K" caused a striking Maximum stimulation was achieved at 100 n.M galanin in both depolarization of the cells. We next determined whether galanin SCLC cell lines. can induce Ca’" mobilization in depolarized cells. Galanin Effect of EGTA, Pertussis Toxin, and Phorbol 12,13-Dibutyr- increased [Ca^*|, in either H69 or H510 cells suspended in ate. Since the effect of galanin on (Ca’"], in the S C " cell lines medium in which the extracellular Na" was substituted by K" was entirely different from that observed in other llular sys­ (Fig. 4; Table I ). Thus, the effects of galanin on |Ca^"j, in SCLC tems (see “Introduction"), we characterized the Ca " response cells can be dissociated from changes in membrane potential. to galanin in more detail (Fig. 3; Table 1). The increase of [Ca’"|, results from Ca’" mobilization from internal stores since it still occurred after the addition of 1.8 m M EGTA to chelate H 6 9 K *1 4 5 m M Na+ 140m M extracellular Ca’" just prior to the addition of galanin (Fig. 3). UJ- In pancreatic d-cells, galanin receptors are coupled to the ATP- sensitive K" channel via a pertussis toxin-sensitive G protein (7). Treatment with pertussis toxin (200 ng/m l for 4 h) did not prevent galanin-induced Ca^" mobilization in SCLC cells (Fig. 'G a l 3). In other cellular systems activation of protein kinase C H 5 1 0 attenuates Ca^" mobilization from intracellular stores (25). o Q. 0> Tablf I Effect of ECTA. pertussis toxin, and membrane depolarization on the o increase in {C a'' j, induced by galanin Experimental conditions are identical to those described in the legend to Figs 'G a l 1 Min 'G a l 'G a l 3 and 4. The increase in jCa'*), caused by 25 nxt galanin in the absence or presence of various additions «as calculated by subtracting the basal (Ca’*), from Fig 4. Effect of galanin on membrane potential and |Ca"), in SCLC cells. the |C a’% peak Top. H 69. bottom . H 510. Left: Cells cultured in HITESA were washed and Increase in |Ca"), (nxi) incubated for 2 h in fresh HITESA. The cells were then resuspended in 2 ml electrolyte solution and placed in a quartz cuvette. Bis-oxonol at a final concen­ Addition H69 cells H5I0 cells tration of ICO nxt was then added to the cell suspension which was continuously stirred for 5 min prior to the sequential addition of galanin 25 nxt (G al) and 80 31 ± 3.5* 37 ± 3.8 mxt KCI IK*) Fluorescence was monitored as described in “Materials and EGTA 24 ± 1.8 26 ± 2 M ethods." Right: Cells preloaded with fura-2-AME were resuspended in electro­ Penussis toxin 30 ± 2.8 37 ± 3.6 lyte solution (140 mxt, Na‘) or in a modified electrolyte solution in which Na* K*. 145 mxt 31 ± 3.9 41 ± 3.8 was replaced by K". giving a concentration of 145 mxt K*. |Ca"), was calculated ' Mrant ± SEM of 3-6 indcprndcnl dcirrminations as described in “Materials and Methods."

1676 <;M.ANIN STIMULATES L \K L \ SKJNAl.S AND GROWTH

Galanin Stimulates Accumulation of Inositol Phosphates. The or tum or cells, including SCLC. arc able to form colonics in binding of a variety of ligands to their specific receptors causes scmisolid media (27, 28). Consequently, the ability of H69 and breakdown of phosphatidylinositol 4.5-bisphosphatc by a phos- H 5I0 cells to form colonies in this assay was tested in the pholipase C. yielding lns(l,4,5)P» which is released into cytosol presence of increasing concentrations of galanin. Galanin and mobilizes Ca’* from intracellular stores (for review see Ref. caused a marked stimulation of colony formation in a concen­ 26). Consequently, we determined whether galanin stimulates tration-dependent fashion (Fig. 7). The concentrations required the formation of inositol phosphates in SCLC cell lines. The to promote half-maximum stimulation were approximately 20 inositol phosphate response was amplified by adding LiCI for PM for H69 and H 510 cells. The maximum effect was achieved 20 min prior to the termination of the incubation (26). As within a narrow range of galanin concentration (about 50 p m ). shown in Fig. 5. addition of galanin to H69 cells labeled with At higher concentrations, the growth-promoting effect of gal­ myo-['H]inositol stimulated the accumulation of total inositol anin was sharply reduced, presumably due to homologous de­ phosphates in a time- and dose-dependent manner. The re­ sensitization. The half-maximum concentrations required to sponse can be detected at a concentration of 5 n M and the ECw induce clonal grow th were sim ilar to those required to stim ulate value was 10 nM . Similar results were obtained when H510 Ca’* mobilization (Fig. 2) or inositol phosphate accumulation cells were used instead of H69 cells. (Fig. 5). As reported previously (17), galanin did not increase In order to characterize the inositol response in more detail, (Ca’*), in H345, a SCLC cell line responsive to GRP. In this the major inositol phosphate fractions were separated by FPLC cell line, galanin failed to prom ote clonal grow th, whereas G R P, after various times of galanin treatm ent. As shown in Fig. 6, added to parallel assays, caused a marked stimulation (results an increase in InsP, and InsP? was detectable within seconds of not shown). Thus, the growth-promoting effects of galanin are galanin addition. This was followed by a marked and slower clearly associated with the ability of this neuropeptide to induce increase in the InsP, fraction. A marked increase in ('HJ early signaling events. lns(l,4,5)P, was observed after 30 s of galanin treatment, Recently, |A rg\D-Trp^\M ePhe')substance P (6-11) has been whereas [’H]Ins(l,3.4)P., was predominant after 20 min of identified as a broad spectrum neuropeptide antagonist (29). incubation (Fig. 6. bottom ). In both H69 and H510 there was a rapid increase in InsP.,, which was detectable at 30 s (increase of 40-60% above control) and maintained for up to 20 min H 5 1 0 1 (increase 5 0 -190% above control) (data not shown). The results 100 shown in Figs. 5 and 6 demonstrate that galanin stim ulates an I n sP inositol phosphate response in SCLC cell lines. .A y Galanin Stimulates Clonal Growth in SCLC. The rapid stim ­ ulation of Ca’* mobilization and inositol phosphate production SO induced by galanin in SCLC cells prom pted us to test the effect I n sP of this neuropeptide on the growth of these cells. Transformed N

60 I n sP

8 0 f x 1 10 20 1 10 20 Tim e (Min)

H 6 9 t»-tn*(1,4,S)P,

4 0 tns(L3.4)Ç

25 30 Fraction Number 0 100 Fig 6. Top, changes in the level of InsP,. InsP,. and InsP, ingalanin-stimulatcd H69 and H5I0 SCLC cells as a function of time H69 and H5I0 cells were Galanin (nM) prelabcled with myo-j’Hjinosiiol and incubated in HITESA containing 20 mxt LiCI for 20 min. Then. 100 nxi galanin was added for various times. Parallel Fig. S. EfTcci of galanin on I he accumulation of inositol phosphates. H69 cultures were incubated in the presence of LiCI but without galanin (controls). SCl.C cells incubated in HITESA for 3-5 days were washed and labeled in Incubations were stopped by addition of 250 it\ ice cold TCA. The samples were HITES A with 10 ,,Ci/ml myo-j’Hjinositol for 24 h. Cells were then washed twice analyzed for their composition of inositol phosphates by anion exchange chro­ in HITESA at 37'C. .Approximately 1.5 x 10* cells were resuspended in I ml matography on a Mono Q column. All other experimental conditions were as HITESA containing 0.02 M Hepes and incubated at 37’C with 20 mw LiCI for described in "Materials and Methods." Each point has an appropriate control. 20 min before the addition of galanin at the concentrations indicated. Cells were Point, mean percentage change from the control of 3-5 experiments; bar, ± S E M . incubated with galanin for 20 min. The accumulation of total inositol phosphates B ottom, elution profile of lns(l.4,5)Pj and lns(l.3.4)P, in H69 cells stimulated was determined as described in "Materials and Methods." The increase in inositol by galanin. H69 cells were prelabcled with mvo-j'Hjinositol as described above phosphate at a particular galanin concentration is expressed as a percentage and incubated in HITESA containing 20 mxt LiCI for 40 min. Galanin (100 nxi) increase above the control (/'.e.. cultures incubated in the presence of LiCI for 40 was added either for 30 s (#) or for 20 min (A) before the termination of the min) and represents the mean of 4 independent experiments. P oint, m ean; bar, ± experiment. Parallel cultures were incubated for 40 min in the presence of LiCI SEM. Average control value. 1405 cpm {n = 10); average 100 nM galanin value, without galanin (□). |'H|lns(l.4.5)Pj was separated from |^H|lns(l.3.4)P, as 2141 cpm (n - 10). Inset, lime course of the accumulation of inositol phosphates. described in "Materials and Methods." The peak of radioactivity corresponding Galanin (100 nst. #) was added for the times indicated in the presence of LiCI. to lns(l,4,5)P] in the sample was assigned on the basis of coelution with standards Percentage of increase in total inositol phosphates from control is shown. The j’H]lns(l.4.5)P, and |^’P|lns(1.4.5)Pj. In this system, the peak eluting immedi­ control (O) (LiCI onl> for 40 min) value. 2194 cpm: 100 nxt galanin for 20 min ately prior to lns( l.4.5)P, is ascribed to lns(l..3.4)P, (25). A similar profile was value. 33.30 cpm. All other details were as described in "Materials and Methods." obtained in 3 independent experiments. 1677 (, \I.\M \ S1IMU1AII..S t \K1 \ SK.NMS W Dt.KOUIII

H 69 H 5 1 0 A 1 4 7 - 3 0 0 3 0 0

cn 2 0 0 200 Ü 101 - G a l 'G a U

1 M m 100 Galanin (nM)

Fig 7 EITfCi of galanin on coioni formation in H69 (/<>//) and HSIO ^ri^;hl) SCLC cells Cells days postpassage were washed, resuspended in HITESA. and then disaggregated into an essentially single eell suspension Cell number was determined using a Coulter Counter and 10* cells in 0 agarose were layered on top of 0.5% agarose, both layers containing galanin at the same eoncentration in .1.1mm plastic dishes Colonies represent aggregates of cells >16 counted under a microscope after 21 days. H69; poini. mean of 7 experiments (each with 5 replicates); t>ar, ±SEM H5I0: point, mean of 5 experiments (each with 5 replicates), har, *S E M 0 10 25 50 100 200 Galanin (nM) Fig. 8 shows that addition of this antagonist, at 20 ^m, pre­ Fig 8. Top. effci-i (if uniagonisi |.\rg“.D Trp'*.MePhf'|subsiance P on galanin sented the increase in (Ca-*), caused by a subsequent addition slimulaicd Ca’’ mobilization in H69 SCLC cells Addilions: galanin. 25 nxi of galanin in H69 cells. This prompted us to determine whether (Go/); galanin I *im (Go/+ ); |Arg‘.t>Trp” .McPhe*)subsiance P. 20 »jM (.-tni) this antagonist could also prevent galanin from stimulating Bottom. f(Teci of |Arg‘.D Trp” .MePhc*|substance P on galanin induced coloni formation Cells (10') in 0 1% agarose containing galanin at concentrations arc clonal growth in these cells. As shown in Fig. 8 {bottom ), indicated either in the absence (■) or in the presence (D) of 20 o't |Arg‘.r> |Arg'’,D-Trp’‘’,MePhe*)substance P, added at 20 ^m. caused a Trp’*.MePhe*|substance P Colonies of >16 cells were counted after 21 dais under a microscope Colum n, mean of 2 experiments (each with 5 replicates); bar. profound inhibition of colony formation. This inhibitory effect ± S D was reversed by high concentrations of galanin.

nal stores. In this context, H69 and H5I0 SCLC cells max DISCUSSION provide a useful model to study these novel effects of galanin. The results presented here demonstrate that the neuropeptide Lung cancer remains the commonest fatal malignancy in the galanin induces a rapid and transient increase in |Co |, and an developed world. SCLC constitutes nearly 25% of all pulmo­ accumulation of inositol phosphates and stimuh s clonal nary cancers and follows a rapid and aggressive clinical course growth of SCLC cell lines. The findings demonstrate that despite initial chemosensitivity (31). Increased understanding galanin can act as a direct growth factor for cultured human of the signal-transduction pathways that regulate SCLC grow th cells. may identify novel targets for therapeutic intervention. Recent Galanin is widely distributed and elicits a multiplicity of work from this (17) and other ( 18) laboratories has shown that physiological responses (2). However, the only model system a variety of neuropeptides, acting through distinct receptors, in which the signal transduction pathways activated by galanin induces C. mobilization in SCLC cell lines. However, the have been studied in detail is the pancreatic /S-cell (5). In these precise rel. onship between a rapid and transient increase in cells, galanin stimulates an ATP-sensitive K* channel which [Ca'*j, and long-term in SCLC growth remains undefined. In increases the plasma membrane potential, blocks the influx of view of the Ca'*-mobilizing actions of galanin shown in this Ca^" through voltage-gated Ca’* channels, and thereby de­ study, it was im portant to determine whether this neuropeptide creases |C a’*|, (8). These effects are mediated by a pertussis influences the growth of responsive SCLC. Tumor and trans­ toxin-sensitive G protein (7). The results presented here dem­ formed cells including SCLC are able to form colonies in onstrate that galanin initiates an entirely different set of early agarose medium. Indeed, there is a positive correlation between events in SCLC cell lines. Galanin stimulates a rapid increase cloning efficiency of the cells and the histological involvement in [Ca’*], from internal stores through a pertussis toxin-insen­ and invasiveness of the tumor in specimens taken from SCLC sitive pathway. This Ca’*-mobilizing action of galanin can be (27, 28). Consequently, we determined the effect of galanin on completely dissociated from changes in membrane potential. the ability of H69 and H 510 cells to form colonies in semisolid Furthermore, galanin stimulates the production of inositol m e d iu m . phosphates, consistent w ith the hypothesis that galanin-induced In the present study we demonstrate that galanin markedlv Ca’* mobilization is mediated by lns(l,4,5)P». This is the first stimulates the clonal growth of either H69 or H5I0 cells in time that galanin has been shown to evoke inositol phosphate semisolid medium. The EC m, values for promoting colony for­ and Ca'* mobilization responses in any cell type. m ation are in excellent agreement with the EC,n values for Ca'* Pharmacological and molecular cloning studies provided evi­ mobilization and inositol phosphate accumulation. Further­ dence that neuropeptide receptors are frequently expressed in more, (A rg\D -Trp’‘',M ePhe"|substance P (6-11), recently iden­ multiple molecular forms (30). We propose that a galanin tified as a broad spectrum neuropeptide antagonist (29), pre­ receptor exists in at least two different molecular subtypes. One vented Ca'* mobilization induced by galanin and strikingly couples to K* channels via a pertussis toxin sensitive G protein, inhibited basal and galanin-stimulated colony formation. This e.g., in the pancreatic (f-cell. A second subtype, present in certain is the first lime that galanin has been shown to act as a growth SCLC cells, is coupled to phospholipasc C which generates factor for any cell type. Galanin is widely distributed (2) and in lns(l,4.5)Pi and thereby leads to Ca’* mobilization from inter human lung is associated with other peptides in neuroendocrine 678 GAI.ANIN STIM l'l a TI S KARIA SKiNALS AND (iROWTH cells (32) from which it is presumed that SCLCs derive (33). In cell line, B iochcm . J., 25.5. 4 0 1 - 4 10. 1988. 16. C uttitta. r.. Carney. D. N.. Mulshinc. J.. Moody. T. W . Fcdorko. J.. Fischler. view of its widespread distribution, it is likely that galanin A . and Minna, J. D. Bombesin like peptides can function as autocrine growth regulates the proliferation of other cell types, a possibility that factors in human small cell lung cancer. Nature (Lond ). 3 1 6 : 821-826, 1985. 17. Woll, P. J., and Rozengurt. E Multiple neuropeptides mobilise calcium in warrants further experimental work. The finding that galanin small cell lung cancer: effects of vasopressin, bradykinin. cholecystokin. can act as a direct growth factor for SCLC cells supports the galanin and neurotensin. Biochem. Biophys. Res. Commun . 164: 6 6 -7 1 . proposition that the growth of these tumors may be regulated 1989. 18 Bunn. P. A. Jr., Dicnhart, D. G.. Chan. D . Puck. T. T.. Tagawa. M.. Jewett, in a complex manner by multiple autocrine/paracrine interac­ P. B.. and Braunschweiger. E. Neuropeptide stimulation of calcium flux in tions involving neuropeptides. human lung cancer cells: delineation of alternative palhwavs Proc. Natl. Acad. Sci. USA. «7. 2162-2166. 1990. 19. Abe, K., Kameya, T., 1 amaguchi. k . Kikuchi. k . Adachi. !.. Tanaka, M., kimura. S., Kodama, T., Shimosato. 1'., and Ishikawa, S. Hormone-produc­ REFERENCES ing lung cancers. Endocrinologie and morphologic studies. In: K. L. Becker and A. F. Gazdar (eds.). The Endocrine Lung in Health and Disease, pp. 1 Taicmoio. K.. Rokarus. A,. Jornsall. H., McDonald. T. J.. and Mull. V. 549-595. London: W . B Saunders. 1984. Galanin— a novel biologically aciivc pcpiidc from porcine inicsline. FEBS 20. Heikkila. R.. Tre pel. J. B.. Cuttitta. F.. Neckers. L M . and Sausville. E. A. L cil . 164: 1 2 4 -1 2 8 . 198.1. Bombesin-related peptides induce calcium mobilization in a subset of human 2 Rokacus. A. Galanin—a newly isolaied biologically aclive neuropeplide. small cell lung cancer cell lines. J. Biol. Chem.. 262: 16456-16460. 1987. Trends Ncurosci.. K>: 158-164. 1987. 21. Simms, E„ Gazdar, A. F.. Abram s. P. G , and .Minna. J. D. G row th o f .V Fisonc. G . Wu. C. F.. Consolo, S.. Nordsirom. O.. Brynne. N.. Banfai. T., human small cell (oat cell) carcinoma of the lung in serum free growth factor- Mclander. T.. and Hokfell. T. Galanin inhibils aceiylcholinc release In the supplemented medium. Cancer Res . 40: 4156-4163. 1980. veniral hippocampus of the rai: hisiochemical. autoradiographic, in riro . and 22. Tsien, R. V.. Pozzan, T.. and Rink. T. J T-cell mitogens cause early changes in liiro studies. Proc. Natl. Acad. Sci. US A.. S4: 7.1.19-7.14.1. 1987. in cytoplasmic free Ca’* and membrane potential in lymphocytes. Nature 4. Ekblad. E . Hakanson. R . Sundler. F.. and Wahlestedt. C. Galanin: neuro- (Lond ), 295. 68-71, 1982. modulatory and direct contractile effects on smooth muscle preparations. Br. 23. Mendoza. S. A., Schneider. J. A.. Lopez Rivas. A.. Sinnett-Smith. J. W.. J. Pharmacol . #6. 241-246. 1985. and Rozengurt, E. Early events elicited by bombesin and structurally related 5. Ahren. B . Rorsman. P.. and Berggren. P-O. Galanin and the endocrine peptides in quiescent Swiss 1T1 cells. II. Changes in Na* and Ca’* fluxes. pancreas. FEBS Lett.. 229: 211-237. 1988. Na*/K* pump activity and intracellular pH. J. Cell Biol., 102: 2221-2233. 6 de W eille. J.. Schmid Antomarchi. H . Fosset. M.. and l-azdunski. M. ATP 1986. sensitise K' channels that are blocked by hypoglycemia-inducing sulfonylu- 24. Nânberg. E., and Rozengurt. E. Temporal relationship between inositol reas in insulin secreting cells are activated by galanin. a hyperglycemia- polyphosphate formation and increases in cytosolic Ca’* in quiescent 1T.1 inducing hormone. Proc. Natl. Acad. Sci. USA. 85: 1312-1316. 1988. cells stimulated by platelet-derived growth factor, bombesin and vasopressin. 7. Dunne. M J.. Bullett, M. J.. Li. G. D , Wollheim. C. B . and Petersen, O. EMBO J., 7. 2741-2748, 1988. H. Galanin activates nucleotide-dependent K* channels in Insulin-secreting 25. Lopez Rivas, A., Mendoza, S. A.. Nânberg. E.. Sinnett-Smith, J., and Roz­ cells sia a pertussis to\in-sensilivc G-protein. EM BO J.. 8: 413-420, 1989. engurt, E. The Ca’*-mobilizing actions of platelet-derived growth factor 8. Nilsson. T.. Arkhammar. P.. Rorsman, P., and Berggren, P. O. Suppression differ from those of bombesin and vasopressin in Sw iss 3T3 cells. Proc Natl. of insulin release b> galanin and somatostatin is mediated by a G-protein: an Acad. Sci. USA, 84: 5768-5772. 1987. effect involving re polarization and reduction in cytoplasmic free Ca’* con­ 26. Berridge, M. J., and Irvine, R. F. Inositol phosphates and cell signalling. Nature (Lond.), 341: 197-205. 1990. centration. J. Biol. Chem., 264: 973-980. 1989. 27. Carney, D. N„Gazdar, A. F„ and Minna. J. D. Positive correlation between 9. Sharp. G. W ., Le Marchand Brusiel, Y., Vada, T.. Russo. L. L., Bliss, C. R , histological tumor involvement and generation of tumor cell colonies in Cormont. M., Monge. L.. and Van Obberghen, E Galanin can inhibit insulin agarose in specimens taken directly from patients with small cell carcinoma release by a mechanism other than membrane hyperpolarization or inhibition of the lung. Cancer Res., 40: 1820-1823. 1980. of adenylate cyclase. J. Biol. Chem., 264: 7102-7109, 1989. 28. Carney, D. N., Cuttitta, F., Moody. T. W .. and Minna. J. D. Selective 10. Tamura. K.. Palmer, J. M., Winkelmann, C. K , and Wood, J. D. Mechanism stimulation of small cell lung cancer clonal growth by bombesin and gastrin- of action of galanin on mventeric neurons. J. Neurophysiol., 60: 9 6 6 -9 7 9 , releasing peptide. Cancer Res., 47: 821-825, 1987. 1988. 29. Woll, P. J., and Rozengun, E. A neuropeptide antagonist that inhibits the 11. Palazzi. E , Fisone, G . Hokfelt. T., Bartfai. T., and Consolo, S. Galanin grow th of small cell lung cancer. Cancer Res.. 50: 3968-3973. 1990. inhibits the muscarinic stimulation of phosphoinosilide turnover in rat ven­ 30. Woll, P. J., and Rozengurt, E. Neuropeptides and growth regulators. Br. tral hippocampus. Eur. J. Pharmacol.. 148: 419-480. 1988. Med. Bull., 45. 492-505, 1989. 12. Rozengurt, E. Early signals in the mitogenic response. Science (Washington 31. Smyth, J. F., Fowlie, S. M.. Gregor, A., Crompton, G. K , Busuttil, A., D C ). 234: 161-166. 1986. Leonard, R. C., and Grant, I. W The impact of chemotherapy on small cell II. Zachary. I.. Woll. P.. and Rozengurt, E. A role for neuropeptides in the carcinoma of the bronchus. Quant. J. Med., 61: 969-971, 1986. control of cell proliferation. Dev. Biol., 124: 295-308. 1987. 32. Uddman, R., and Sundler, F. Neuropeptide in the airways: a review. Am 14 Rozengurt. E . and Sinnett-Smith. J. Bombesin stimulation of Fibroblast Rev. Respir. Dis., /3d. S3-8, 1987. mitogenesis: specific receptors, signal transduction and early events. Philos. 33. Gazdar, A. F., and McDowell. M. Pathobiology of lung cancer. In: S. T. Trans. R. Soc. Lond Biol. Sci.. 327: 209-221. 1990. Rosen, J. L. Mulshine, F. Cuttitta. and P. G. Abrams (eds ). Biology of Lung 15 Trepel. J. B., Moyer. J. D . Heikkila, R , and Sausville. E. A. Modulation of Cancer Diagnosis and Treatment, pp. 1-42. New York: Marcel Dekker Inc.. bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung cancer 1988.

1679 |( \NC ER RESEARCH 51. .'(>2 I-U:.V Jul> I. I99l|

Advances in Brief

Multiple Neuropeptides Stimulate Clonal Growth of Small Cell Lung Cancer: Effects of Bradykinin, Vasopressin, Cholecystokinin, Galanin, and Neurotensin lariq Sethi and Enrique Rozengurt'

liiiperial Cancer Research Fund. P.O. Box I2.i. JJncoln's Inn Fields. London H 'O .4 i P \ . I n iie d k in n d o m

A b s tra c t SCLC growth is regulated by multiple autocrine and paracrine c irc u its. We tested whether Ca’*-mobilizing neuropeptides can function as growth factors for small cell lung carcinoma cells. The neuropeptides bradykinin, neurotensin, cholecystokinin, and vasopressin at nanomolar Materials and Methods concentrations stimulated a rapid and transient increase in the intracel­ Cell Culture. SCLC cell lines H345 and H5I0 were the kind gift of lular concentration of Ca’*. Crucially, these peptides in the same concen­ Dr Adi Gazdar (National Cancer Institute, Bethesda, MD). H69 was tration range also caused a marked increase in colony formation in purchased from the American Type Culture Collection. Stocks were semisolid medium in responsive small cell lung carcinoma cell lines. At maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine optimal concentrations bradykinin, neurotensin, cholecystokinin, vaso­ serum (heat inactivated at 57*C for 1 h) in a humidified atmosphere of pressin, galanin, and gastrin-releasing peptide were equally effective in 10% COj:90% air at 37'C. They were passaged every 7 days. For promoting clonal growth. These findings support the hypothesis that experimental purposes, the cells were grown in HITESA (17). small cell lung carcinoma grow th is sustained by an extensive network of D eterm ination of |C a’*|, Concentration. A liquots o f 4 -5 x 10* SC L C autocrine and paracrine interactions involving multiple neuropeptides. cells cultured in HITESA for 3-5 days were washed and incubated for 2 h at 37'C in 10 ml of fresh HITESA medium. Fura-2-tetra aceioxy Introduction methyl ester (1 fiSi) in dimethyl sulfoxide was then added and the cells were incubated for a further 5 min. The cell suspension was centrifuged Lung cancer is the commonest fatal malignancy in the devel­ at 2000 rpm for 15 s, and the cells were resuspended in 2 ml of electrolyte solution (140 mxt NaCl, 5 mM KCI, 0.9 mM MgClj, 1.8 mM oped world. SCLC’ constitutes 25% of the total and follows a CaC I, 25 mM glucose, 16 mM 4-(2-hydroxycthyl)-l-piperazineethane- rapid and aggressive clinical course, despite initial chemosen­ sulfonic acid, 16 mM Tris, and a mixture of amino acids at pH 7.2), sitivity (I). Identification of the factors that stim ulate the pro­ transferred to a quartz cuvete, and stirred continuously at 37'C. Fluo­ liferation of SCLC cells will be important in the design of rescence was recorded continuously in a Perkin-Elmer LS5 ' ,mines- alternative and more effective therapeutic strategies. SCLC is cence spectrometer with an excitation wavelength of 336 n^ tnd an characterized by the presence of intracytoplasmic neurosecre­ emission wavelength of 510 nm. (Ca’*]i was calculated as t viously tory granules and by its ability to secrete many hormones and described ( 12). neuropeptides (2, 3), including bombesin, neurotensin, chole­ Clonogenic Assay. SCLC cells, 3-5 days |X>stpassage, were washed cystokinin, and vasopressin (2, 4-9). Among these, only bom­ and resuspended in HITESA. Cells were then disaggregated into a besin-like peptides, which include GRP, have been shown lo single cell suspension by two passes through a 19-gauge needle and act as autocrine growth factors for certain SCLC cell lines (10). then through a 15-fim pore size nylon gauze. Viability was judged by trypan blue exclusion on a standard hemocytometer. Approximately The role of other neuropeptides in the proliferation of SCLC lO'* viable cells/ml, as determined using a Coulter Counter, were cells remains poorly understood. suspended in culture medium and 0.3% agarose. One ml of the mixture M ultiple neuropeptides, including bradykinin, vasopressin, was plated in 5 replicate 35 mM plastic dishes containing a 2-ml base cholecystokinin, galanin, neurotensin, and G RP, stimulate a layer of 0.5% agarose in culture medium that had hardened. Both layers rapid, transient increase in the intracellular concentration of contained neuropeptide at the same concentration. Cultures were in­ Ca’* (jCa’*]i) in SCLC cell lines (II-I4). Since a rise in cubated at 37'C in a humidified atmosphere at 10% CO::90% air for [Ca’*)i is one of the early signals in a com plex signaling cascade 21 days and then stained with the vital stain Nitro blue tétrazolium. leading to mitogenesis in fibroblast model systems (15, 16), it Colonies with diameters of >120 um (16 cells) were counted under a has been hypothesized that SCLC growth is regulated by mul­ microscope. Materials. Bradykinin, vasopressin, neurotensin, cholecystokinin, tiple autocrine and/or paracrine circuits involving Ca’*-mobi- GRP, and galanin were purchased from Sigma Chemical Co., St. Louis, lizing neuropeptides (II, 12). A crucial test of this hypothesis MO; fura-2-tetraaceioxy methyl ester from Calbiochem Corp., La Jolla, is to determine whether Ca’*-mobilizing neuropeptides increase CA; and agarose from SeaKem, Rockland, ME. All the other reagents the ability of SCLC cell lines to form colonies in semisolid were of the highest grade commercially available. medium. We now report that bradykinin, vasopressin, chole­ cystokinin, neurotensin, galanin, and G RP, at optimal concen­ Results and Discussion trations, are equally effective in stim ulating clonal growth of responsive SCLC cell lines. This supports the hypothesis that Addition of bradykinin to H69, H510, or H345 cells loaded

Received 3/28/91: accepted 5/16/91. with the Ca’*-sensitive indicator fura-2 increased (Ca’*)i w ith ­ The costs of publication of this article were defrayed in part by the payment out any measurable delay (Fig. I). Peak [Ca’*]; was reached 20- of page charges. This article must therefore be hereby marked advenisement In 30 s after addition of the peptide. Bradykinin, at 100 nM, accordance with 18 L'.S.C. Section 17.34 solely lo indicate this fact. ' To w hom requests for reprints should be addressed. in c re a s e d |Ca’*)j from 100 ± 8 (/i = 6) to 192 ± 9 (n = 6) nM ’ The abbreviations used are: SCLC. small cell lung carcinoma: GRP. gastrin in H69 cells, from 134 ± 17 (n = 5) to 206 ± 17 (n = 5) nM in releasing peptide: |Ca’*|.. intracellular concentration of Ca’*; HITESA, RPMI 1640 with 10 nst hydrocortisone. 5 ug/ml insulin. 10 ug/ml transferrin. 10 nst H510 cells, and from 89 ± 7 (n = 4) to 126 ± 9 (« -- 4) nM in estradiol. .30 nst selenium, and 0.25'r bovine scrum albumin. H345 cells. In each cell line bradykinin increased [Ca’*), in a 3621 Ml I I ICI I M I KOI'I ri 11)1 s \s I.KI )\v ill I \( lOKs

H69 the cIToci of brtids kinin on ilic abiiils of H69. H5 10. and H.345 200 200 Il20 edits lo form eolonies in senitsolid mcdiimi. f i^ I (hoiioiii) 5 shosss liiai brads kinin niarkcdis increased colons grossih of these SCLC cell lines in a steeply dose-dependent manner. 150' 100 Optimal colons stimulation ssas achiescd at 10 nst bradykinin o in H69 and H.345 cell lines and at 5-10 n\i bradykinin in H510 cells. .At higher concentrations the stimulators effect decreased, 100 presumably due to homologous desensitization in this long­ I term assay (Fig. I). Time-dependent mitogenic desensitization 100 has been reported in other cellular systems (19. 20). I 200 100- The role of brads kinin reeeptors in mediating Ca’* mobili­ 0 zation and cell grossth ssas tested using (n-A rg".H sp'.Thy' " .d - Phe jbradskinin. a specific competitise antagonist of the /T 1 100. so u rceeptor (2 I ). The antagonist, at 10 pM. completels bloeked the i ! increase in both |Ca'*|, and colons formation induced bs 10 n\t bradykinin in either H69 cells or H.345 cells (Fig. 2). |d - Bradykinin nM Arg".FIyp’.T hy'’‘.i>-Phe'lbradykinin at 10 p\t had no effeet on

Fig 1 Dose dependent cffccis of bradskinin on |Ca’'|, (lop) and on colons the basal |Ca-*), or on spontaneous colony formation in the form aiion (hoiioni) in SCLC cells SCLC cell lines H69 i/c/i), 115Id (nni/i/U ). absence of bradykinin (Fig. 2). and H 145 (nfiht) were cultured in HITFSa for 5-5 dnss |( a''|, and colons The results obtained ssith bradykinin prompted us to test formaiion sscre deiermined ai the conceniraiions of peptide indicated as dcscrilx. d in “Materials and Methods “ /nscl. the fluorescncc tracing obtained s s hen lOd other neuropeptides for their effects on Ca’’ mobilization and nst brads kinin s»as added to cells loaded ss iih fura-2. Tspical |( a’*), dose resisonse colons gross th. The peptides neurotensin, cholecystokinin. and curses arc shossn for each cell line The basal |Ca''), is the mean salue for that esperiment r SEM The increases in |Ca'"|, induced bs ICO nst brads kinin sscre sasopressin are secreted by SCLC (5-9. 22). and increase |C a’*|, repeated in seseral independent esperimenis and the data arc giscn in the test in responsise SCLC cell lines through distinct receptors (II, Each point in the colons formation assas represents the mean ± SEM (Aurs) of 14). Fig 5 shosss that these peptides increased |C a’*], in H69. 3-4 independent esperimenis (each with 5 replicates) H 510, and H345 cells, respectisels. in a dose-dependent fash­ ion. Crucially, neurotensin, cholecystokinin. and sasopressin at nanomolar concentrations stimulated clonal gross th in semi­ H 3 4 5 175 solid medium (Fig. 3). Cholecystokinin. sasopressin. and GRP in H69 cells or galanin in H345 cells caused little or no rise in |Ca’*], and did not stimulate colons formation in these cell lines (Table I) The abiiit' >f multiple Ca’*-mobilizing neuropeptides to promote cion gross th in semisolid medium in different SCLC cell lines is shossn in Table 1. The neuropeptide galanin. re­ — BKA — BKA — BKA — BKA cently shossn to stimulate inositol phosphate accumulation. BK BK Ca'* mobilization, and colons formation in H69 and H5I0 cells (12), and G RP ssere also included in parallel experiments

OÏ 200 100 g H 6 9 H510 H 345 100- 220- y * * .1 7 0 n 5 200 " ' n i p 180 120 — BKA — BKA — BKA — BKA “ bk“ BK Fig 2. Effect of the brad>kinin antagonist (D-Arg“.Ftyp',Th\’ *.D-Phe’|brad\ Itinin on bradskinin induced |Ca'*|, mobilization and colony formation in SC'LC 0 10 100 1000 10 100 1000 1 0 " 100 1 0 0 0 cells Ft69 iie/l) and Ft345 (rtg/il) Bradykinin (BA) and |D-Arg“.FIyp'. Thy” ) bradykinin (BA14) were added at tO nst and tO »iM. respectisels. Top I TOO |Ca’*|, ssas determined as described in “Materials and M ettiods.'Cl Basal |Ca’*|, I lO o' Each column represents the mean ± SEM of 3-6 experiments B o tw m . 10* cells « I {' in 0.39c agarose were layered onto 0 5% agarose containing bradykinin either in Z 200- the absence (— ) or presence (BK.-*) of the brads kinin antagonist. After 21 dass colonies >16 cells were counted under a microscope. CL spontaneous colons formation Each Aor represents the mean ± SD (b a n ) o f 5 replicates 1 - 1 100 dose-dependent fashion in the nanomolar range; typical dose Neurotensin nM Cholecystokinin nM Vasopressin nM response relationships are depicted in Fig. I. Fig 3 Effect of neurotensin. cholee>stukinin. and \asupressin on jf'a’T, and colon) formation in 1169. H5IÜ. and H345 SC LC i-ell lines. |Ca’'|. and colon) Tumor and transformed cells, including SCLC, arc able to formation were determined as described in “Materials and Ntethods" /n\er. form colonies in agarose medium. Indeed, there is a positive fluorescence tracing obtained when ICO nst concentrations of the peptide indi correlation between cloning efficiency of the cells and the cated were added to eclls loaded with fura-2. T)piial jCa’T. dose response relationships are shown I he basal |('a’’|. is the mean \alue lor that esperiment histological involv ement and invasiveness of the tumor in spec­ ± SEM (hors). Each pou u in the colon) assa) represents the mean ± SEM of 2- imens taken from SCLC (18). Consequently, we determined 3 independent espertnients (each with 5 replicates) .3622 MULTM’I.K Ni;iiRÜI>i:i'IIOKS AS C R O U TH HACTORS

T ;ihli‘ I Multiple Ca^'-mohiUzinn ncuropeptidea stimulate clonal urowth of SCLC cell lines |C'j’*|, w;is measured «iili the fliioresivnl indiealor fura 2 as described in “Maicrials and Moihods." The posiii\ii\ of ihc lC'a’*|, response reflects a productiee lifiaiid-reeepior comptes. Colony formaiion was determined using the clonogenic assay as described in "Materials and Methods." Spontaneous colony formation, i.e.. in the absence of an> esogenonsis added peptide (— ). 98 ± 4. 57± 4. and 56 ± 6 in 1469. H510. and H.445. respect is el), is normalized lo 100%. The perecntage of colons formation is e\ pressed as the mean z SL.M The number of .15 nim dishes counted are indicated in parentheses at the concentration or range of concentrations indicated. In H69 cells. GRI’. sasopressin. and cholecystokinin had little or no effect on both |Ca’*|, and colons formation. In H.145 cells, galanin neither increased |Ca’'|, nor stimulated clonal growth.

Cell line Peptide |n M ) |C a'*l. % o f colony form ation

H 69 100 (80) Bradykinin 10 ■¥ .360 ± 19(24) G alanin 50 -t- 252 ± 8(34) Neurotensin 5 0 455 i 37 (9)

11510 __ 100 (70) Bradykinin 5 - 1 0 359 ±23 (19) G alanin 50 368 ± 21 (23) Vasopressin 100 344 ± 12(18) Cholecystokinin 25 291 ± 8 ( 9 )

H.Ï45 100 (50) Bradykinin 10 + 321 ± 37 (20) Vasopressin 150 -t- 257 ± 19(9) GRP 5 -1 0 + 232 ± 7(10)

for comparison. The results demonstrated that, at optimal 8. Sausville, E., Carney, D., and Battes. J. The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung concentrations, bradykinin. vasopressin, cholecystokinin, gal­ cancer cell line. J. Biol. Chem.. 260: 10 2 3 6 - 10 2 4 1. 1985. anin. neurotensin, and GRP induce comparable increases of 9. Bepler, G , Rotsch, M., Jaques, G., Haeder. M.. Hermanns, J.. Hartogh. G , SCLC clonal growth in responsive cell lines (Table 1). Thus, Kiefer, P., and Havemann, K. J. Peptides and growth factors in small cell lung cancer: production, binding sites, and growth effects. Cancer Res. Clin. multiple Ca’^-mobilizing neuropeptides, via distinct receptors, O ncol.. 114: 235-244, 1988. can act directly as growth factors for SCLC. 10. Cuttitta, F.. Carney, D. N., Mulshine, J., M oody,T. W., Fedorko. J., Fischler, It is known that GRP, vasopressin, cholecystokinin, and A., and Minna, J. D. Bombesin like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature (Lond.), 3 1 6 : 823-826.1985. neurotensin are secreted by some SCLC tum ors (5-9,22). O ther 11. Woll, P. J„ and Rozengurt, E. Multiple neuropeptides mobilise calcium in peptides may be released by a variety of normal cells in the lung small cell lung cancer: effects of vasopressin, bradykinin. cholecystokinin. galanin and neurotensin. Biochem. Biophys. Res. Commun.. 164: 6 6 -7 3 . or. like bradykinin, produced extracellulary as a result of the 1989. proteolytic cleavage of plasma precursors in the damaged tissue 12. Sethi, T„ and Rozengurt, E. Galanin stimulates Ca'" mobilization, inositol surrounding tumors (21). Collectively, these findings support phosphate accumulation and clonal growth in small cell lung cancer cells. Cancer Res., 5/; 1674-1679, 1991. the hypothesis that SCLC growth is sustained by an extensive 13. Heikkila, R , Trepel, J. B , Cuttitta. F„ Neckers, L. M., and Sausville, E. A. network of autocrine and paracrine interactions involving mul­ Bombesin-related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines. J. Biol. Chem., 262: 16456-16460,1987. tiple neuropeptides. Broad spectrum neuropeptide antagonists 14. Bunn, P. A., Dienhart, D. G„ Chan, D„ Puck, T. T„ Tagawa, M., Jewett, P. (23) provide a strategy to block SCLC growth which takes into B., and Braunschweiger, E. Neuropeplide stimulation of calcium flux in account this mitogenic complexity. human lung cancer cells: delineation of alternative pathways. Proc. Natl. Acad. Sci. USA, f 7. 2162-2166, 1990. 15. Rozengurt, E. Early signals in the mitogenic response. Science (Washington D C ), 23 4 : 161-166, 1986. P T eren ces 16. Rozengun, E. Neuropeptides as cellular growth factors: role of multiple signalling pathways. Eur. J. Clin. Invest., 21: 123-134, 1991. Smyih. J. F.. Fowlie. S. M.. Gregor. A.. Crompion. G. K.. Busulill. A.. 17. Simms, E., Gazdar, A. F„ Abrams, P. G , and Minna, J. D. Growth of Leonard. R. C. F.. and Grant. I. \V. B. The Impact of chemotherapy on small human small cell (oat cell) carcinoma of the lung in serum-free growth factor- cell carcinoma of the bronchus. Quart. J. Med.. 61: 969-976. 1986. supplemented medium. Cancer Res , 40: 4356-4363, 1980. 2. Sorenson. G. D.. Pettengill. O. S.. Brinck-Johnsen. T., Cate. C. C . and 18. Carney, D. N., Gazdar, A. F„ and Minna, J. D. Positive correlation between .Maurer. L. H Hormone production bv cultures of small-cell carcinoma of histological tumor involvement and generation of tumor cell colonies in the lung. Cancer (Phila.). 47.- 1 2 8 9 -1 2 9 6 . 1981. agarose in specimens taken directly from patients with small cell lung .1. Maurer. L. H Ectopic hormone syndrome in small cell carcinoma of the carcinoma of the lung. Cancer Res., 40: 1820-1823,1980. lung. Clin. Oncol.. 4. 67-83. 1985. 19. Millar, J. B. A„ and Rozengurt, E. Chronic desensitization to bombesin by 4. \\ ood. S. M.. Wood. J. R . Ghatei. M. A.. Le. Y. C . G'Shaughnessy. D. and progressive down-regulation of bombesin receptors in Swiss 3T3 cells: dis­ Bloom. S. R. Bombesin, somatostatin and neurotensin like immunoreactivity tinction from acute desensitization. J. Biol. Chem., 26S: 12052-12058,1990. in bronchial carcinoma. J. Clin. Endocrinol. Mctab., S 3 : 13I0-I3I2, 1981. 20. Millar, J. B. A., and Rozengurt, E. Heterologous desensitization of bombesin- 5. North. W G . Maurer. L. H.. Valtin. H . and O'Donnell, J. F. H um an induced mitogenesis by prolonged exposure to vasopressin: a post-receptor neurophysins as potential tumor markers for small cell carcinoma of the signal transduction block. Proc. Natl. Acad. Sci. USA, 8 6 :3204-3208,1989. lung: application of specific radioimmunoassays. J. Clin. Endocrinol. Metab.. 21. Steranka, L. R„ Farmer, S. G.. and Burch, R. M. Antagonists of 0 j bradykinin SI: 892-896. 1980. receptors. FASEB J„ J. 2019-2025, 1989. 6. Gazdar, A. F., and Carnes. D. N. In: K. Becker and A. F. Gazdar (eds.). The 22. Davis. T. P.. Burgess, H. S., Crowell, S., Moody, T. W„ Culling-Berglund. Endocrine Lung in Health and Disease, pp. 501-508. New York: W B A., and Liu, R. H. d-Endorphin and neurotensin stimulate in vitro clon al Saunders Co., 1984. growth of human SCLC cells. Eur. J. Pharmacol., 161: 283-285, 1989. 7. Goedert. M., Reeve, J. G.. Emson. P. C . and Bleehen, N. M. Neurotensin 23. Woll, P. J„ and Rozengurt, E. A neuropeptide antagonist that inhibits the in human small cell lung carcinoma. Br. J. Cancer. SO: 179-183, 1984. growth of small cell lung cancer in vitro. Cancer Res , SO: 3968-3973, 1990.

.3623 ICA N CER r e s e a r c h (SUPPL.) 52. 2732s 274:s. Ma> I. I992| Growth of Small Cell Lung Cancer Cells; Stimulation by Multiple Neuropeptides and Inhibition by Broad Spectrum Antagonists in Vitro and in Vivo^

Tariq Sethi, Simon Langdon, John Sm yth, and Enrique Rozengurt^

Imprrial Cancer Research Fund, P. O. Box I2Î, Lincoln's Inn Fields, London H'C2A iPX, England (T. S., E. R.f. and ICRF Medical Oncolog)- Unit, Hestern General Hospital. Cren-e Road, Edinburgh EH4 2XU, Scotland /S. L , J. S.]

A b stra c t be stim ulated to reinitiate DNA synthesis and cell division by the addition of various neuropeptide growth factors in serum- Neuropeptides are increasingly implicated in the control of cell prolif­ free medium (12). In particular, bombesin (14), vasopressin eration and their mechanisms of action are attracting intense interest. (15), bradykinin (16), vasoactive intestinal peptide (17), endo­ The early complex cascade of events initiated by peptides of the bombesin family including gastrin-releasing peptide is increasingly understood. The thelin (18), and vasoactive intestinal contractor (19) can act as cause-effect relationships and temporal organization of these early signals growth factors for cultured 3T3 cells. In what follows some and moleclular events provide a paradigm for the study of other growlh fundamental features of the mechanism of action of neuropep­ factors and mitogenic neuropeptides and illustrate the activation and tides as growth factors in 3T3 cells will be discussed and interaction of a variety of signaling pathways. These peptides may also subsequently the evidence for multiple neuropeptide growth act as autocrine growth factors for certain small cell lung cancer cells. factor action In SCLC will be considered. The results discussed here strongly suggest that the autocrine growlh The present article is not intended as an extensive review of loop of bombesin-like peptides may be only a part of an extensive netw ork the rapidly expanding literature, but rather as a presentation of of autocrine and paracrine interactions involving a variety of C a '- specific topics and ideas under investigation in our laboratories. mobilizing neuropeptides in small cell lung cancer including bradykinin, cholecystokinin, galanin, neurotensin, and vasopressin. In this context, broad spectrum antagonists that prevent the function of multiple Ca'*- Elarly Signaling Events mobilizing receptors are of special interest. These antagonists block neuropeptide mediated signals and inhibit small cell lung cancer growth The early cellular and molecular responses elicited by bom­ in vitro and in vivo. Thus, broad spectrum neuropeptide antagonists besin and structurally related peptides in 3T3 cells (Fig. I) have constitute potential anticancer agents. been elucidated in detail (20). The cause-effect relationships and tem poral organization of these early signals and molecular events provide a paradigm for the study of other growth factors Introduction and mitogenic neuropeptides and illustrate the activation and interaction of a variety of signaling pathways (21). Lung cancer is the commonest fatal malignancy in the devel­ Bom besin/G RP binds to a single class of high affinity recep­ oped world. SCLC^ con itutes 25% of the total and follows an tors in Swiss 3T3 cells (22, 23). The receptors are M, 7 5 ,0 0 0 - aggressive clinical cour ' despite initial chemosensitivity (I). 85,000 glycoproteins with a M, 43,000 core (24-26). The Identification of the factors that stimulate the proliferation of receptor is coupled to one or more G proteins as judged by the SCLC cells will be im portant in the design of alternative and modulation of ligand binding in either membrane preparations more effective therapeutic strategies. SCLC is characterized by or receptor-solubilized preparations and of signal transduction the presence of intracytoplasmic neurosecretory granules and in permeabilized cells (23,27-29). The bombesin/GRP receptor by its ability to secrete many hormones and neuropeptides (2, has recently been cloned and sequencr (30, 31) and shown to 3) including bombesin, neurotensin, cholecystokinin, and va­ be a member of the G protein-couple receptor family. These sopressin (2-9). Among these, only bombesin-like peptides, receptors have seven predicted transmembrane domains which which include G RP, have been shown to act as autocrine growth cluster to form a ligand-binding pocket (32, 33). O ther neuro­ factors for certain SCLC cell lines (9, 10). In contrast, the role peptide mitogens with receptors of this type include angioten­ of other neuropeptides in the proliferation of SCLC cells re­ sin, endothelin, serotonin, substance K, and substance P (34, mains poorly understood. Consequently, it is important to 3 5 ). understand in detail the receptor and signal transduction path­ Binding of bom besin/GRP to its receptor initiates a cascade ways that mediate the mitogenic action of bombesin and GRP of intracellular signals (summarized in Fig. I) culminating in as well as to elucidate the role played by other neuropeptides DNA synthesis 10-15 h later (13,21). One of the earliest events in SCLC growth. to occur after the binding of bombesin to its specific receptor Neuropeptides are increasingly recognized to act as cellular is a rapid m obilization of Ca^* from internal stores, which leads growth factors (11) and their mechanisms of action arc attract­ to a transient increase in the intracellular concentration of Ca'* ing considerable attention. M any studies to identify the molec­ ([Ca'*)i) and subsequently to Ca'* efflux and decreased Ca'* ular pathways by which neuropeptide mitogens elicit cellular content of the cells (36, 37). The mobilization of Ca'* by growth have exploited cultured murine 3T3 cells as a model bombesin is mediated by (Ins(I,4,5)Py], which, as a second system (12, 13). These cells cease to proliferate when they messenger, binds to an intracellular receptor and induces the deplete the medium of its growth-promoting activity and can release of Ca'* from internal stores. Bombesin causes a rapid

' Presented it the NCI Workshop “Investigational Strategies for Detection increase in Ins(l,4,S)Py, which coincides with the increase in and Intervention in Early Lung Cancer," April 21-24, 1991, Annapolis, MD. cytosolic Ca'* (38). Ins(I,4,5)Py is formed as a result of PLC- ’ To whom requests for reprints should be addressed. catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate ' The abbreviations used are: SCLC, small cell lung cancer, GRP, gastrin- releasing peptide; |lns( 1,4,5)P,|, inositol 1,4,5-trisphosphaie; PLC, phospholipase in the plasma membrane, a process tha( also generates 1,2- C: |Ca’*j„ intracellular |Ca'“|; G. guanine nucleotide binding; PKC, protein kinase diacylglycerol. Diacylglycerol can also be generated from other C; fura-2/A.ME. fura-2-tetraacetoxy methyl ester. HITESA. RPMI 1640 with 10 nst hydroconisone. 5 ng/ml insulin, 10 ng/ml transferrin. 10 nst estradiol, JO nst sources, such as phosphatidylcholine hydrolysis (39), and acts selenium, and 0.25% bovine serum albumin. as a second messenger in the activation of PKC by bombesin. 2737s ICANCER RESEARCH (Sl'PPL.) 52. 2237j-2742!., Ma> I. 1992) Growth of Small Cell Lung Cancer Cells: Stimulation by Multiple Neuropeptides and Inhibition by Broad Spectrum Antagonists in Vitro and in Vivo^

Tariq Sethi, Simon Langdon, John Sm yth, and Enrique Rozengurt^

Imperial Cancer Research Fund. F. O. Box 123, Lincoln's Inn Fields. London H’C2A JFX. England[T. S.. E. R.j. and ICRF Medical Oncolog)- Vnii. H'esiern General Hospital. Crewe Road. Edinburgh EH4 2XU. Scotland {S. L.. J. S.J

A b s tra c t be stimulated to reinitiate DNA synthesis and cell division by the addition of various neuropeptide growth factors in serum- Neuropeptides are increasingly implicated in the control of cell prolif­ free medium (12). In particular, bombesin (14), vasopressin eration and their mechanisms of action are attracting intense interest. (15), bradykinin (16), vasoactive intestinal peptide (17), endo­ The early complex cascade of events initiated by peptides of the bombesin thelin (18), and vasoactive intestinal contractor (19) can act as family including gastrin-releasing peptide is increasingly understood. The cause-effect relationships and temporal organization of these early signals growth factors for cultured 3T3 cells. In what follows some and moleclular events provide a paradigm for the study of other growlh fundamental features of the mechanism of action of neuropep­ factors and mitogenic neuropeptides and illustrate the activation and tides as growth factors in 3T3 cells will be discussed and interaction of a variety of signaling pathways. These peptides may also subsequently the evidence for multiple neuropeptide growth act as autocrine growth factors for certain small cell lung cancer cells. factor action in SCLC will be considered. The results discussed here strongly suggest that the autocrine growlh The present article is not intended as an extensive review of loop of bombesin-like peptides may be only a part of an extensive netw ork the rapidly expanding literature, but rather as a presentation of of autocrine and paracrine interactions involving a variety of Ca *- specific topics and ideas under investigation in our laboratories. mobilizing neuropeptides in small cell lung cancer including bradykinin, cholecystokinin, galanin, neurotensin, and vasopressin. In this context, broad spectrum antagonists that prevent the function of multiple Ca'*- Early Signaling Events mobilizing receptors are of special interest. These antagonists block neuropeptide mediated signals and inhibit small cell lung cancer growth The early cellular and molecular responses elicited by bom­ in vitro and in vivo. Thus, broad spectrum neuropeptide antagonists besin and structurally related peptides in 3T3 cells (Fig. 1 ) have constitute potential anticancer agents. been elucidated in detail (20). The cause-effect relationships and temporal organization of these early signals and molecular events provide a paradigm for the study of other growth factors Introduction and mitogenic neuropeptides and illustrate the activation and interaction of a variety of signaling pathways (21). Lung cancer is the commonest fatal malignancy in the devel­ Bom besin/GRP binds to a single class of high affinity recep­ oped world. SCLC^ con itutes 25% of the total and follows an tors in Swiss 3T3 cells (22, 23). The receptors are M, 7 5 ,0 0 0 - aggressive clinical cour despite initial chemosensitivity (I). 85,000 glycoproteins with a M, 43,000 core (24-26). The Identification of the factors that stim ulate the proliferation of receptor is coupled to one or more G proteins as judged by the SCLC cells will be im portant in the design of alternative and modulation of ligand binding in either membrane preparations more effective therapeutic strategies. SCLC is characterized by or receptor-solubilized preparations and of signal transduction the presence of intracytoplasmic neurosecretory granules and in permeabilized cells (23,27-29). The bom besin/GRP receptor by its ability to secrete many hormones and neuropeptides (2, has recently been cloned and sequence (30, 31) and shown to 3) including bombesin, neurotensin, cholecystokinin, and va­ be a member of the G protein-couple receptor family. These sopressin (2-9). Among these, only bombesin-like peptides, receptors have seven predicted transm^mbranc domains which which include G RP, have been shown to act as autocrine growth cluster to form a ligand-binding pocket (32, 33). O ther neuro­ factors for certain SCLC cell lines (9, 10). In contrast, the role peptide mitogens with receptors of this type include angioten­ of other neuropeptides in the proliferation of SCLC cells re­ sin, endothelin, serotonin, substance K, and substance P (34, mains poorly understood. Consequently, it is important to 3 5 ). understand in detail the receptor and signal transduction path­ Binding of bom besin/GRP to its receptor initiates a cascade ways that mediate the mitogenic action of bombesin and GRP of intracellular signals (summarized in Fig. I) culminating in as well as to elucidate the role played by other neuropeptides DNA synthesis 10-15 h later (13,21). One of the earliest events in SCLC growth. to occur after the binding of bombesin to its specific receptor Neuropeptides are increasingly recognized to act as cellular is a rapid mobilization of Ca^* from internal stores, which leads growth factors (II) and their mechanisms of action are attract­ to a transient increase in the intracellular concentration of Ca.^* ing considerable attention. M any studies to identify the molec­ ([Ca**]i) and subsequently to Ca** efflux and decreased Ca** ular pathways by which neuropeptide mitogens elicit cellular content of the cells (36, 37). The mobilization of Ca** by growth have exploited cultured murine 3T3 cells as a model bombesin is mediated by (Ins(l,4,5)Py], which, as a second system (12, 13). These cells cease to proliferate when they messenger, binds to an intracellular receptor and induces the deplete the medium of its growth-promoting activity and can release of Ca** from internal stores. Bombesin causes a rapid

' Presented at the NCI Workshop “Investigational Strategies for Detection increase in Ins(l,4,5)P3, which coincides with the increase in and Intervention in Early Lung Cancer,” April 21-24, 1991, Annapolis, MD. cytosolic Ca** (38). Ins(l,4,5)Pj is formed as a result of PLC ' To whom requests for reprints should be addressed. catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate ' The abbreviations used are; SCLC, small cell lung cancer, GRP, gastrin- releasing peptide; |lns(l ,4,5)Pi|, inositol 1,4,5-trisphosphaie; PLC, phospholipase in the plasma membrane, a process that also generates 1,2- C; (Ca’*),. intracellular |Ca’*|; G, guanine nucleotide binding; PKC, protein kinase diacylglycerol. Diacylglycerol can also be generated from other C; fura-2/A.ME, fura-2-tetraacetoxy methyl ester, HITESA. RPMI 1640 with 10 sources, such as phosphatidylcholine hydrolysis (39), and acts n.M hydrocortisone, 5 «ig/ml insulin, 10 «ig/ml transferrin. 10 nw estradiol. 30 nM selenium, and 0.25% bovine serum albumin. as a second messenger in the activation of PKC by bombesin. 2737s STIMlil.ATlON OF SCLC CELl GROWTH NtUROPEPTlDES

Bombesin. GRP Tablf I Effect of multiple peptide hormones and neuropeptides on ICa^'], Broad Spectrum mobilization in SCLC cell lines A ntagonists Antagonists Inlraccllular Ca’* »as measurfd in SCLC cell lines NCI H69, H510, H345. H209, and H 128 with the indicator fura-2/AME as described previously (53. 54). EITcciise peptides resulted in consistent large responses at nanomolar concentra­ tions; the response in the sarious cell lines were heterogeneous (53. 54).’ Gp PLC PIP, Effeciivc Noneffective Tyf Kinase Ararhidonir Acifvaiion Acid Rrkasr Bradykinin ACTTH Cholecystokinin Angiotensin 1. II. Ill C* ’• mowiuaiion Galanin Atrial natriuretic peptide Bombesin/GRP Calcitonin Neurotensin Chorionic gonadotrophin M8*rrl * tc '-l, / \ Vasopressin -Endorphin lH*lj (r-1 . Endothelin Epinephrine Follicle stimulating hormone , Transcriptional Growth hormone-releasing hormone A ctivation G lucose 1-phosphate G lucagon 5-FIydroxytrypumine Fig I Bombesin mediated signal transduction. Initiation of cell proliferation L.euenkephalin in Swiss 3T3 cells is stimulated by multiple signal transduction pathways that act Neuropeptide Y in a synergistic and combinatorial fashion. The interactions have been well defined Parathyroid hormone in these cells and provide experimental evidence for a model that involves multiple Substance K pathways. The mechanism of action of neuropeptide grow th factors are explained Substance P within the framework, of this model. FLA,, phospholipase A,. PIP,, phosphali Thyrotropin-releasing hormone dylinositol 4.5 bisphosphate. DAC. diacylglycerol. PC. phosphatidylcholine. PCE,. prostaglandin E,; ECFr, epidermal growth factor receptor.

have been characterized as mitogens for Swiss 3T3 cells includ­ In accordance with this, bombesin strikingly increases the phos­ ing vasopressin, bradykinin, and endothelin-related peptides, phorylation of the acidic M, 80,000 protein (28, 40, 41), a and their signaling pathways have also been defined in detail. major substrate of PKC which has been recently purified from These neuropeptide receptors are also linked to phosphoinosi- Swiss 3T3 cells (42) and molecularly cloned (43). Bombesin/ tide breakdown and Ca'* mobilization but the intensity, dura­ GRP also stimulates a rapid exchange of Na'’, H*, and K* ions tio n (e.g., PKC activation), and even the occurrence of early across the cell membrane, leading to cytoplasmic alkalinization sig n a ls (e.g., arachidonic acid release) differ substantially (21, and increased intracellular |K*), (36) and induces a striking 4 5 , 50). PKC-dependent transmodulation of the epidermal growth fac­ tor receptor (40). Ca'* Mobilization in SCLC Cell Lines Recently, bombesin, vasopressin, and endothelin have been shown to induce a rapid and potent stimulation of tyrosine Studies with SCLC have demonstrated a similar set of early phosphorylation of several substrates in quiescent 3T3 cells events to those previously elucidated in murine 3T3 cells. (44). This response is not mediated by either PKC activation Specifically, GRP stimulates mobilization of intracellular Ca'* or Ca^* mobilization (44). The mechanism by which neuropep­ and inositol phosphate turnover in SCLC cells (10, 51, 52). In tide receptors elicit this novel pathway as well as the precise a subsequent study, multiple neuropeptides were screened for role of tyrosine phosph -ylation in neuropeptide-mediated sig­ their ability to induce a rapid increase in [Ca'*Ji in different nal transduction are in guing issues that warrant further ex­ SCLC cell lines (53). This assay should be regarded as an perimental work. indicator of a productive ligand-receptor interaction. Ca'* mo­ In addition, bombesin, but not vasopressin, induces a marked bilization is, as discussed in the preceding section and shown and sustained release of arachidonic acid and its cyclooxygenase in Fig. 1, one of the com ponents of a complex array of signaling metabolite prostaglandin Ey into the medium (45). Thus, bom­ events rather than the signal that promotes cell growth. Woll besin receptors may be coupled both to PLC activation through and Rozengurt (53) demonstrated that bradykinin, cholecysto­ a putative G protein (Gp) and to arachidonic acid release kinin, galanin, neurotensin, and vasopressin induce a rapid and possibly via phospholipase Aj, although other possibilities re­ transient increase in (Ca'*]j in SCLC cell lines (Table 1). The main open. Considerable evidence indicates that the liberation expression of these receptors is heterogeneous among these of arachidonic acid is an early signal that contributes to bom­ lines. These neuropeptides increased [Ca'*J, in a dose-dependent besin-mediated mitogenesis (45, 46). fashion in the nanomolar range; typical dose-response relation­ In common with many other growth factors, bombesin/GRP ships are depicted in Fig. 2. The Ca'*-mobilizing effects are stimulates transient expression of the nuclear oncogenes c-fos mediated by distinct receptors as shown by the use of specific an d c-m yc (47). It is likely that the induction of c-fos by antagonists and by the induction of homologous desensitization bombesin is mediated by the coordinated effects of PKC acti­ (53, 54).* Studies carried out in other laboratories are in agree­ vation, Ca'* mobilization, and an additional pathway dependent ment with these findings (55-57). on arachidonic acid release (47-49). Furthermore, additional The observation that galanin, a 29-amino acid neuropeptide, pathways of control of c-fos expression that are completely causes Ca'* mobilization in SCLC is of special interest. In independent of activation of PKC have also been shown (47). pancreatic cells galanin activates an ATP-sensitive K* channel, Indeed, bombesin can initiate DNA synthesis via PKC-depend­ hyperpolarizes the plasma membrane, and inhibits the activity ent and -independent pathways (47). This complex network of of voltage-dependent Ca'* channels (58). In this manner it signals (Fig.l; Ref. 21) involves a degree of redundancy and reduces Ca'* influx and blocks the activity of various agents ensures the amplification of the stimulus. In addition to bombesin, several other regulatory peptides * Unpublished results 2738s STIMULATION o r SCLC CELL GKOVKTH H\ NEUROPEPTIDES

[BfaOyhininJ (WeufOlenstn) (Cholecyslokinin) galanin to SCLC cell lines did not alter membrane potential. I"" • J Thus, these studies suggest that SCLC express a novel type of / galanin receptors that are coupled to Ca’* mobilization. Collectively, the studies discussed in this section indicate that ISO SCLC exhibit receptors for multiple neuropeptides and that the ______' itoLc_____ expression of these receptors is heterogeneous among SCLC 10 100 0 10 100 1000 6 ' 10 100 cell lines. H 5 10 -« . . . ‘H345 ■|^rH345 ~T\

Multiple Neuropeptides Stimulate Clonal Growth in SCLC C ells

In view of the findings discussed in the preceding section, it 0 10 100 10 100 |G jla n in | IVasopitssin] has been hypothesized that SCLC growth is regulated by mul­ tiple autocrine and/or paracrine circuits involving Ca^*-mobi-

Fig. 2. Effeci of brad> kinin, neuroiensin. rholecyslokinin, galanin vasopressin lizing neuropeptides (53, 54, 59, 60). A crucial test of this and GRP on (Ca’*), in SCLC cells. SCLC cell lines H69, H5I0. and H345 were hypothesis is to determine whether Ca**-mobilizing neuropep­ cultured in HITESA for 3-5 days. Aliquots of 4-5 x 10* cells were washed and tides can act as growth factors for SCLC cell lines. Conse­ incubated in ID ml fresh HITESA medium for 2 h at 3 7 ‘C. Then. I pM fura-2/ AME was added for 5 min. The cells were washed and resuspended in 2 ml of quently, we determined the effect of multiple Ca’*-mobilizing electrolyte solution (53. 54) This cell suspension was placed in a quartz cuvet neuropeptides to promote clonal growth in semisolid medium and stirred continuously. Fluorescence was monitored in a Perkin Elmer Ls5 luminescence spectrophotometer and basal and peak |Ca’*), values were deter­ in different SCLC cell lines (Fig. 3) (60).The results shown in mined as described (54). Each curve represents a typical dose-response relation­ Fig. 3 dem onstrate that, at optimal concentrations, bradykinin, ship for the neuropeptide in the SCLC cell line indicated. The basal |C a ’*|, neurotensin, vasopressin, cholecystokinin, galanin, and GRP represents the mean value for that experiment ± SEM (bars). induce comparable increases of SCLC clonal growth in respon­ sive cell lines. Thus, multiple Ca^’-mobilizing neuropeptides, via distinct receptors, can act directly as growth factors for H69 H69 H510 H510 H345 H345 ' 200[ I SCLC. 5200 It is known that GRP, vasopressin, cholecystokinin, and neurotensin are secreted by some SCLC tumours (2-10), Other peptides may be released by a variety of normal cells.in the lung E 100 or, like bradykinin, produced extracellularly as a result of the proteolytic cleavage of plasma precursors in the damaged tissue — 10 — 50 — 25 — 50 surrounding tumors (61). Collectively, these findings support BK NT CCK Gal the hypothesis that SCLC growth is sustained by an extensive Fig 3, Effect of bradykinin (BK). neurotensin (AT), cholecystokinin (CCK), network of autocrine and paracrine interactions involving mul­ galanin (C o l), vasopressin (I'P), and GRP on colony formation in SCLC cells. SCLC cell line H69, H5I0, and H345 were used as indicated. Cells 3-5 days tiple neuropeptides. Approaches designed to block SCLC postpassage were washed and resuspended in serum-free medium. Cells were then growth must take into account this mitogenic complexity. disaggregated into an essentially single cell suspension, judged by microscopy, by passing the cells through a 19-gauge needle and then through a 20-pm nylon gauze Viability was judged by trypan blue exclusion on a standard haemocyto- Blocking the Action of Multiple Neuropeptides: Broad meter. Cell number was determined using a Coulter Counter and approximately 10* viable cells/ml were suspended in culture medium and 0.3% agarose. One ml Spectrum Antagonists of the mixture was plated in 5 replicates in 35-mm plastic dishes containing a base layer of 0.5% agarose in culture medium that had hardened. Both layers As understanding of the effects of growth factors in cancer contained neuropeptide at the following concentrations: bradykinin. 10 nxt; neurotensin, 50 nxt; cholecystokinin, 25 nxi, galanin, 50 nxt; vasopressin, 100 increases, it has become possible to plan rational therapeutic nxi, GRP, 10 nxi These concentrations were found to be optimal in stimulating interventions. If an autocrine growth loop is considered, in colony formation in many full dose-response experiments.’ Cultures were incu­ which cells synthesize, secrete, bind, and respond to the same bated at 37 ‘C in a humidified atmosphere at 10% C O j/90% air C olon ies represent aggregates of cells >120 um counted under a microscope after 21 days. growth factor, it is evident that interruption of this cycle at any Colum ns, mean of 2-5 experiments. Bars, SEM . point will block mitogenesis. Paracrine growth could be blocked in the same way. As discussed in the preceding sections, SCLC

Table 2 Bombesin/CRP and broad spectrum antagonists constitutes a special case in which unrestrained proliferation Bombesin: appears driven, at least in part, by multiple autocrine and pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHj paracrine circuits involving Ca’*-mobilizing neuropeptides. Secreted factors can be cleared by antibodies, such as the Broad spectrum antagonists (substance P analogues): Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NHj bombesin monoclonal antibody 2AII used to retard the growth of SCLC xenografts in nude mice (9). We have directed our Antagonist A: DArg-DPro-Lys-Pro-Gln-Gln-DTrp-Phe-oTrp-Leu-Leu-NHj Antagonist D: DArg-Pro-Lys-Pro-DPhe-Gln-oTrp-Phe-DTrp-Leu-Leu-NH, effort to develop peptide antagonists which are not antigenic Antagonist G; Arg-oTrp-MePhe-DTrp-Leu-Met-NHj and should have higher tissue penetration than antibody pro­ teins. We have characterized neuropeptide antagonists in the model Swiss 3T3 fibroblast system and then tested their effects that increase the intracellular concentration of Ca^*. Surpris­ o n S C L C in vitro a n d in vivo. ingly, in SCLC cell lines galanin caused a rapid and transient The first antagonist to be studied was an analogue of sub­ increase in [Ca^*)i (53). Recent studies showed that galanin stance P, [DArg',DPro\DTrp^ \Leu"]substance P (antagonist induced rapid mobilization of Ca^* from internal stores and A (Table 2)]. Substance P is structurally unrelated to the bom- stimulated early production of inositol phosphates, particularly besin-like peptides, but antagonist A, which is a substance P lns(l,4,5)Pj (54). In contrast to the pancreatic cells addition of antagonist, was found to block the secretory effects of bombesin 2739s ST IM ll ATION ()l s r i c (til (.k O U IH in ML^ROIM I’TIDES

4 0 0 H 6 9 H 3 4 5 be necessary to test the effect of the broad spectrum antagonists on ligand binding to purified receptors. The availability of

Î 3 0 0 purified preparations of the bombesin receptor from Swiss 3T3 cells (66, 67) should make it possible to distinguish between ^ 200 these different molecular models.

Broad Spectrum Antagonists Block SCLC Growth

— G ,— G Galanin Vasopressin The compounds characterized as broad spectrum antagonists

Fig 4. EfTeci of anijgonisi C on basal and neuropeptide stimulated colonx in Swiss 3T3 cells were tested as inhibitors of neuropeptide- formation. SCLC cell lines H69 (/e/r) and H345(ng/ir) were cultured in HITESA mediated signals and grow th in SCLC cell lines. Because SCLC for 3-5 da>s, washed, resuspended in HITESA, and then disaggregated into an is a heterogeneous group of tumors, each compound was tested essentially single cell suspension. Cells (10') in 0 3% agarose were layered onto 0.5% agarose both layers containtng (H69) galanin at 50 nst or (H 345) vasopressin in several cell lines. The broad spectrum antagonists inhibited 100 n.si in the presence or absence of 20 ust antagonist C Colonies >120 um Ca’* mobilization stimulated by GRP, vasopressin, bradykinin, were counted after 21 dass under a microscope. Colonies represent the mean ± S E M (H 6 9 , n = 10, H 34% n = 5). cholecystokinin, and galanin in diverse cell lines (54, 59) and inhibited the grow th of SCLC cell lines in liquid and semisolid media (54, 59, 64). Antagonists D and G were equipotent, with on a pancreatic preparation (62), It was subsequently found to half-maximal effect at about 20 m m , w hereas antagonist A was block '-M-GRP binding and bombesin-stimulated early signal­ 5-fold less potent. ing events and mitogenesis in Swiss 3T3 cells (22, 23, 28. 36, The broad spectrum antagonists (D and G) caused a dramatic 47), It did not affect mitogenesis stimulated by polypeptide decrease of the cloning efficiency of these cells in the absence growth factors, such as epidermal and platelet-derived growth of any exogenously added peptide (i.e., basal colony formation). factor, but was found to block vasopressin-stimulated mitoge­ Broad spectrum antagonists also decrease clonal growth in the nesis (63), Further substance P analogues were therefore studied in order to identify more potent antagonists that could be tested presence of neuropeptide stimulation (54), For example, antag­ in SCLC (59, 64)’ onist G profoundly inhibited the clonal growth of SCLC H69 Two interesting compounds were |DArg',DPhe\DTrp^\Leu"| or H345 cells in the absence as well as in the presence of either substance P (antagonist D (Table2)| and |Arg‘,DTrp'’ galanin or vasopressin (Fig, 4), The striking finding that antag­ MePhe*jsubstance P(6-l 1 ) (antagonist G (Table 2)], Both an­ onists D and G inhibit the basal and stimulated clonal growth tagonists reversibly inhibited GRP-stimulated mitogenesis in of so many cell lines (54, 59), regardless of positivity for Swiss 3T3 cells, and antagonist D was 5-fold more potent than bombesin receptors, suggests that broad spectrum antagonists antagonist A, although antagonist G was less potent than A CO 'd be more useful anticancer drugs than ligand-specific (59), In contrast, when tested as competitive inhibitors of gi 'th factor antagonists. vasopressin-stimulated mitogenesis, antagonists D and G were ,vS a first step to test this possibility, we examined the effect equipotent, with half-maximal effect at 1 (59), In addition, of antagonist G on the growth of a H69 SCLC xenograft. the antagonists were found to block mitogenesis stimulated by Fragments of the H69 xenograft were implanted s.c. in the the neuropeptides bradykinin and endothelin (16, 59, 65). It is flanks of nude mice and allowed to grow to a measurable size important to note that the antagonists neuher block DNA (30 mm ’). Then, a group of animals were treated with antago­ synthesis by platelet-derived growth facto: vhich stimulates nist G given peritumorally once a day for I week. Fig. 5 shows Ca^" mobilization through a difTerent mech .ism from neuro­ that the antagonist profoundly inhibited the growth of the peptides (i.e., mediated by tyrosine phosphorylation rather than tumor, as compared with the control group. The inhibitory by a G protein) nor inhibit mitogenesis stim ulated by vasoactive effect was clearly maintained beyond the duration of adminis­ intestinal peptide which induces cyclic AMP accumulation tration. These results demonstrate that antagonist G can inhibit without Ca’* mobilization (59, 64). Thus, the substance P SCLC growth in vivo as well as in vitro. analogue antagonists showed broad spectrum specificity against the neuropeptide mitogens bombesin/GRP, vasopressin, bra­ dykinin, and endothelin, which act through distinct receptors H69 Xenograft in Swiss 3T3 cells but activate common signal transduction I 1000 / p a th w a y s (e.g.. Fig, 1). The molecular mechanism by which broad spectrum antago­ nists interfere with the action of Ca^*-mobilizing neuropeptides remains to be defined. Antagonists D and G competed with the radiolabeled ligands '-'I GRP, (^HJvasopressin and '^'I-endo- thelin for binding in a dose-dependent fashion and inhibited

Ca’* mobilization stimulated by each of these peptides, in Oay addition to other early intracellular signals triggered by them Fig 5. EfTcci of antagonist G on H69 SCLC xenograft growth in nude mice. (23, 59, 64, 65), It is plausible that the antagonists recognize a Fragments of the NCI-H69 xenograft (previously established from the cell line) common domain on these Ca’*-mobilizing neuropeptide recep­ were implanted s.c. into female nude mice. After 6 weeks, when tumors had tors, each of w hich is probably coupled to a common G protein reached a mean volume of 30 mm*, groups of 7 mice were given injections of either PBS containing 0.9 mg of antagonist G (•) or phosphate-buffered saline (Gp) responsible for the regulation of phosphatidylinositol 4,5- alone (O) s.c. adjacent to the tumor once a day for 7 days. Tumor volume was bisphosphate-specific PLC (see Fig, I), Alternatively, the an­ determined by means of vernier calipers and estimated according to the formula 0.5 X length x width'. For each individual tumor; the change in volume was tagonists might bind to a separate protein (Gp?) that regulates compared lo the value at the start of the treatment. Points, mean of 7 values is receptor activity. To distinguish between these models, it will indicated 'P < 0.05 (; test) 2740s STIMULATION CI S( L( (TXI CKOWTII NEUROPEPTIDI'.S

Conclusions 16. Woll. P. J.. and Rozengurt E. Two classes of antagonist interact with receptors for the mitogenic neuropeptides bombesin, bradykinin and vaso­ pressin. Growth Factors. I: 75-83. 1988. Neuropeptides are increasingly implicated in the control of 17. Zurier. R. B . Kozma. M.. Sinnett-Smith. J.. and Rozengurt, E. Vasoactive cell proliferation and their mechanisms of action are attracting intestinal peptide synergistically stimulates D \.-\ synthesis in mouse 3T3 cells: role of cAMP. Ca’* and protein kinase C. E\p. Cell Res., 176: 1 5 5 - intense interest. The peptides of the bombesin family including 161. 1988. GRP bind to specific surface receptors and initiate a complex 18. Takuwa. N.. Takuwa. V.. Vanagisawa. M.. Vamashita. K.. and Masaki. T. A cascade of signaling events (Fig. I) that culminates in the novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J. Biol. Chem.. 264: 7856-7861. stimulation of DNA synthesis and cell division in Swiss 3T3 1989. cells in the absence of other growth-promoting factors. These 19 Fabregat. I . and Rozengurt, E. Vasoactive intestinal contractor, a novel peptides may also act as autocrine growth factors for certain peptide, shares a common receptor with endothelin-1 and stimulates Ca’* mobilization and DNA synthesis in Swiss 3T3 cells. Biochem. Biophys Res SCLC cells. The results discussed here strongly suggest that C om m u n .. 167: 161-167. 1990. the autocrine growth loop of bombesin-like peptides may be 20. Rozengurt, E.. and Sinnett-Smith, J. Bombesin stimulation of fibroblast mitogenesis: specific receptors, signal transduction and early events. Philos only a part of an extensive network of autocrine and paracrine Trans. R. Soc. Lond. B Biol. Sci., 327: 209-221. 1990. interactions involving a variety of Ca^*-mobilizing neuropep­ 21. Rozengurt, E. Neuropeptides as cellular growth factors. Eur. J. Clin. Invest.. tides in SCLC including bradykinin, cholecystokinin, galanin, 21: 123-134. 1991. 22. Zachary, I., and Rozengurt, E. High-affinity receptors for peptides of the neurotensin, and vasopressin. In the context of the multistage bombesin family in Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA, 82: 7 6 1 6 - evolution of cancer, neuropeptide mitogenesis may play a role 7620, 1985. at an early stage in SCLC as tum or prom oters in initiated cells 23. Sinnett-Smith. J., Lehmann, W.. and Rozengurt. E. Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling or later as growth factors in the unrestrained growth of the and modulation by guanine nucleotides. Biochem J.. 265: 485-493, 1990. fully developed SCLC tumor. A detailed understanding of the 24. Kris, R. M.. Hazan. R., Villines. J., Moody. T. W„ and Schlessinger. J. Identification of the bombesin receptor on murine and human cells by cross- receptors and signal transduction pathways that mediate the linking experiments. J. Biol. Chem., 262: 11215-11220, 1987. mitogenic action of neuropeptides may identify novel targets 25. Zachary, 1., and Rozengurt, E Identification of a receptor for peptides of the for therapeutic intervention. In this context, broad spectrum bombesin family in Swiss 3T3 cells by affinity cross-linking. J. Biol. Chem . 262: 3947-3950, 1987. antagonists that prevent the function of multiple Ca'*-mobiliz- 26. Sinnett-Smith. J., Zachary. I., and Rozengurt. E. Characterization of a ing receptors are of special interest. These antagonists block bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking. J. Cell. neuropeptide-mediated signals in the 3T3 and SCLC cells and Biochem., 38. 237-249, 1988. 27. Coffer, A., Fabregat, I., Sinnett-Smith, J., and Rozengurt, E. Solubilization inhibit SCLC growth in vitro a n d in vivo. Thus, broad spectrum of the bombesin receptor from Swiss 3T3 cells membranes: functional asso­ neuropeptide antagonists constitute potential anticancer agents. ciation to a guanine nucleotide regulatory protein. FEBS Lett., 263: 8 0 -8 4 . 1990. 28. Erusalimsky. J. D , Friedberg, I., and Rozengun. E. Bombesin, diacylglyc- R e fe re n c e s erols and phorbol esters rapidly stimulate the phosphorylation of an M, 80,000 protein kinase C substrate in permeabilized 3T3 cells: effect of guanine nucleotides. J. Biol. Chem., 263: 19188-19194. 1988. 1. Smyth, J. F.. Fowlie. S. M., Gregor, A., Crompion, G. K , Busulill, A.. 29. Rozengun, E., Fabregat. 1., Coffer, A., Gil, J , and Sinnett-Smith, J. M ito­ Leonard, R. C. F., and Grant, I W. B. The impact of chemotherapy on small genic signalling through the bombesin receptor role of a guanine nucleotide cell carcinoma of the bronchus. Q. J. Med., 61: 969-976. 1986. regulatory protein. J. Cell. Sci., Suppl. 13, 43-56, 1990. 2. Sorenson, G. D . Pettengill. O. S.. Brinck-Johnsen, T., Cate. C. C., and 30. Battey, J. F., Way, J. M., Corjay, M. H., Shapira. H., Kusano. K , H arkins. Maurer, L. H. Hormone production by cultures of small-cell carcinoma of R , W'u, J. M., Slattery, T., Mann, E., and Feldman, R. I. Molecular cloning the lung. Cancer (Phila.), 47: 1289-1296. 1981. of the bombesin/GRP receptor from Swiss 3T3 cells. Proc. Natl. Acad. Sci 3. Maurer, L. H. Ectopic hormone syndrome in small cell carcinoma of the USA, 88. 395-399,1990. lung. Clin. Oncol.. 4: 1289-1296, 1985. 31. Spindel, E. R., Ciladi, E.. Brehm, P., Goodman, R. H., and Segerson, T. P. 4. Wood, S. M.. Wood, J. R., Ghatei, M. A., t-ee, Y. C , G’Shaughnessy. D., Cloning and functional characterization of a complementary DNA encoding and Bloom, S. R. Bombesin, somatostatin and neurotensin-like immuno­ the murine fibroblast bombesin/gastrin-releasing iteptide receptor. M ol. En­ reactivity in bronchial carcinoma. J. Clin. Endocrinol. Metab., 53: 1 3 1 0 - docrinol.. 4: 1956-1963, 1990. 1312, 1981. 32. Dohlman, H. G . Caron, M. G , and L-efkowitz, R. J. A family of receptors 5. North, W. G.. Maurer, L. H.. Valtin. H , and O'Donnell. J. F. Human coupled to guanine nucleotide regulatory proteins. Biochem. J., 26: 2 6 5 7 - neurophysins as potential tumor markers for small cell carcinoma of the 2664, 1987. lung: application of specific radioimmunoassays. J. Clin. Endocrinol. Metab., 33. Lefkowitz, R. J., and Caron. M. G. Adrenergic receptors: models for the j;.-892-896, 1980. study of receptors coupled to guanine nucleotide regulatory proteins. J. Biol. 6 Gazdar. A. F.. and Carney. D. N. In: K. Becker and A. F Gazdar (eds.). The C h em ., 263: 4993-4996, 1988. Endocrine Lung in Health and Disease, pp. 501-508. Eastbourne, England: 34. Arai, H., Hori, S., Aramori, I., Ohkubo, H., and Nakanishi, S. Cloning and W. B. Saunders Co., Ltd., 1984. expression of a cDN'A encoding an . Nature (Lond ). 348: 7. Goeden, M., Reeve, J. G.. Emson, P. C., and Bleehen, N. M. Neurotensin 730-732, 1990. in human small cell lung carcinoma. Br. J. Cancer, 50: 179-183, 1984. 35. Sakurai, T., Vanagisawa, M., Takuwa, V., Miyazaki, H., Kimura. S., Goto, 8. Sausville, E., Carney. D., and Battey, J. The human vasopressin gene is K., and Masaki, T. Cloning of a cDNA encoding a non-isopeptide-selective linked lo the oxytocin gene and is selectively expressed in a cultured lung subtype of the endothelin receptor. Nature (Lond ), 348: 732-735, 1990. cancer cell line. J. Biol. Chem.. 2 6 0 : 10236-10241, 1985. 36. Mendoza, S. A., Schnedier, J. A., Lopez Rivas. A., Sinnett-Smith. J. W.. 9. Cuttitta. F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko. J., Fischler. and Rozengurt, E. Early events elicited by bombesin and structurally related A . and Minna, J. D. Bombesin-like peptides can function as autocrine growth peptides in quiescent Swiss 3T3 cells. II. Changes in Na* and Ca’* fluxes. factors in human small-cell lung cancer. Nature (Lond.), 3 1 6 : 8 2 3 - 8 2 6 ,19 8 5 . Na*/K* pump activity and intracellular pH. J. Cell Biol., 102: 2223-2233. 10. Mahmoud, S.. Staley, J.. Taylor, J., Bogden, A.. Moreau, J. P., Coy, D., 1986. Avis, 1.. Cuttitta. F., Mulshine. J. L . and Moody, T. W. |Psi" "jbombesin 37. Lopez-Rivas, A., Mendoza, S. A., Nanberg. E., Sinnett-Smith. J., and Roz­ analogues inhibit growth of small cell lung cancer in vitro and in vivo. Cancer engurt, E. The Ca’*-mobilizing actions of platelet-derived growth factor Res., 5/; 1798-1802, 1991. differ from those of bombesin and vasopressin in Swiss 3T3 cells. Proc. Natl. 11. Zachary, I , Woll, P., and Rozengurt, E. A role for neuropeptides in the Acad. Sci. USA, 84. 5768-5772, 1987. control of cell proliferation. Dev Biol.. 124: 295-308, 1987. 38. Nanberg, E., and Rozengurt, E Temporal relationship between inositol 12. Rozengurt, E The mitogenic response of cultured 3T3 cells: integration of polyphosphate formation and increases in cytosolic Ca’* in quiescent 3T3 early signals and synergistic effects in a unified framework. In: P. Cohen and cells stimulated by platelet-derived growth factor, bombesin and vasopressin. M. Houslay (eds.). Molecular Mechanisms of Transmembrane Signalling, E M B O J .. 7. 2 7 4 1 -2 7 4 8 , 1988. pp. 429-452. Amsterdam: Elsevier Science Publishers BV, 1985. 39. Cook, S. J . and Wakelam, J. O. Analysis of the water-soluble products of 13. Rozengurt, E. Earlv signals in the mitogenic response. Science (Washington phosphatidylcholine breakdown by ion-exchange chromatography. Biochem. D C ). 234: 161-166. 1986. J., 263. 581-587, 1989. 14. Rozengurt, E.,and Sinnett-Smith. J. Bombesin stimulation of DNA synthesis 40. Zachary. I.. Sinnett-Smith, J. W., and Rozengurt, E. Early events elicited bv and cell division in cultures of Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA. bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. 80 2936-2940. 1983. Activation of protein kinase C and inhibition of epidermal growth factor 15. Rozengurt. E.. Legg. A., and Peitican P. Vasopressin stimulation of 3T3 cell binding. J. Cell Biol., 102: 2211-2222. 1986. growth. Proc. Natl. Acad. Sci. US.A. 76.- 1284-1287. 1979. 41. Rozengurt. E.. Rodriguez Pena. A., and Smith. K. A. Phorbol esters, phos- 2741s SlIMlil ATIOS ()l SCLC Ctl.L GROWTH NFI ROPEI’HOES

pholipasc C. and growth factors rapidl\ stimulate the phosphorylation of a 54. Set hi. T.. and Rozengurt. E. Galanin stimulates Ca'" mobilization, inositol M, 80.000 protein in intact quiescent .1T.1 cells. Proc. Natl. Acad. Sci. USA. phosphate accumulation and clonal growth in small cell lung cancer cells. « 0 .-7 2 4 4 -7 2 4 8 . 198.1 Cancer Res . 5/ 1674-1679. 1991. 42. Brooks. S. F.. Erusalimsky. J. D..Totly. N. F .and Rozengurt. E. Purification 55. Staley. J.. Fiskum. G., and Moody. T. W . Cholecystokinin elevates cytosolic and internal amino acid sequence of the 80kDa protein kinase C substrate calcium in small cell lung cancer cells. Biochcm. Biophvs. Res. Commun., from Swiss IT 3 fibroblasts. FEBS Lett.. 26H: 291-295. 1990. 763. 605-610. 1989. 43 Erusalimsky. J. D . Brooks. S. F.. Hcrget. T., Morris. C.. and Rozengurt. E. 56. Staley. J., Fiskum. G., Davis. T. P., and Moody. T. W. Neurotensin elevates Molecular cloning and characterization of the acidic 80 kDa protein kinase cvlosolic calcium in small cell lung cancer cells. Peptides. 20: 1217-1221, substrate from rat brain. J. Biol. Chem.. 266: 7073-7080. 1991. 1989. 44. Zachary. !.. Gil. J.. Lehmann. W . Sinnett-Smith. J.. and Rozengurt. E. 57. Bunn. P. A.. Dienhart. D, G., Chan, D.. Puck. T. T.. Tagawa, M.. 3ewett. P. Bombesin, vasopressin and endothelin rapidly stimulate tyrosine phosphor­ B., and Braunschweiger. E. Neuropeptide stimulation of calcium flux in ylation in intact Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA. 8 8 :4 5 7 7 -4 5 8 1 . human lung cancer cells: delineation of alternative pathways. Proc. Natl. 1991. Acad. Sci. USA, 87. 2162-2166, 1990. 45. Millar. J. B. A., and Rozengurt. E. Arachidonic acid release by bombesin: a 58. Dunne, M. 3.. Bullett. M. J.. Li, G. D . Wollheim, C B.. and Petersen, O. novel post receptor target for heterologous mitogenic desensitization. J. Biol. H. Galanin activates nucleotide-dependent K' channels in insulin-secreting C hem ., 265: 19973-19979. 1990. cells via a penussis toxin-sensitive G protein. EMBO J.. 8: 413-420, 1989. 46. Gil, J.. Higgins. T.. and Rozengurt. E. Mastoparan. a novel mitogen for 59. Woll. P. J.. and Rozengun. E A neuropeptide antagonist that inhibits the Swiss 3T3 cells, stimulates penussis toxin-sensitive arachidonic acid release growth of small cell lung cancer in vitro. Cancer Res , SO: 3968-3973, 1990. 60. Sethi. T.. and Rozengurt, E. Multiple neuropeptides stimulate clonal grow th without inositol phosphate accumulation. J. Cell B iol. 113: 943-950. 1992. of small cell lung cancer: effects of bradykinin. vasopression. cholecystokinin. 47. Rozengurt. E.. and Sinnett-Smith. J. Early signals underlying the induction galinin. and neurotensin. Cancer Res , 52: 3621-3623, 1991. o f the c-fos and c-m yc genes in quiescent fibroblasts: studies with bombesin 61. Steranka. L. R . Farmer, S. G .and Burch. R. M. Antagonists of di bradykinin and other growth factors. Prog Nucl. Acid. Res. Mol. Biol.. 35. 261-295. receptors. FASEB J., 3. 2019-2025. 1989. 1988. 62. Jensen. R. T., Jones, S. W.. Folkers, K.. and Gardner, J. D. A synthetic 48. Mehmet, H . Morris. C.. and Rozengurt. E. Multiple synergistic that is a bombesin receptor antagonist. Nature (Lond.), 309: 6 1 -6 3 . transduction pathways regulate c-fos expression in Swiss 3T3 cells: the role 1984. of cyclic AMP. Cell Growth Differ.. I: 292-298. 1990. 63. Zachary. I., and Rozengurt, E. A substance P antagonist also inhibits the 49. Mehmet. H.. Millar. J. B. A.. Lehmann. \ \ .. Higgins. T.. and Rozengurt, E. specific binding and mitogenic effects of vasopressin and bombesin-related Bombesin stimluation of c-fos expression and mitogenesis in Sw iss 3T3 cells: peptides in Swiss 3T3 cells. Biochem. Biophys. Res. Commun., 237: 1 3 5 - the role of prostaglandin E;-mediated cyclic AMP accumulation. Exp. Cell 141, 1986. R es., 190: 265-270. 1990. 64. Woll. P. J., and Rozengurt. E. |DArg'.DPhe’,DTrp’*,Leu"|Substance P. a 50. Issandou, M., and Rozengurt. E. Bradykinin transiently activates protein potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth kinase C in Swiss 3T3 cells: distinction from activation by bombesin and of human small cell lung cancer cells in vitro. Proc Natl. Acad. Sci. USA. vasopressin. J. Biol. Chem., 265. 11890-11896. 1990. 85: 1859-1863. 1988. 51. Heikkila. R., Trepel. J. B.. Cuttitta, F.. Neckers. L. M., and Sausville, E. A. 65. Fabregat. I., and Rozengurt, E |DArg',DPhe’.DTrp’-*.Leu"|Substance P, a Bombesin-related peptides induce calcium mobilization in a subset of human neuropeptide antagonist, blocks binding. Ca'*-mobilizing, and mitogenic small cell lung cancer cell lines. J. Biol. Chem.. 262: 16456-16460, 1987. effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells. 52. Trepel, J. B., Moyer. J. D.. Heikkila. R . and Sausville, E. A. Modulation of J. Cell. Physiol., 745. 88-94, 1990. bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung-cancer 66. Feldman, R !.. W'u, J. M.. Jenson, J. C., and Mann. E. Purification and cell line. Biochem. J.. 255. 403-410, 1988. characterization of the bombesin/gastrin-releasing peptide receptor from 53. Woll, P. J., and Rozengurt, E. Multiple neuropeptides mobilise calcium in Swiss 3T3 cells. J. Biol. Chem., 265: 17364-17372, 1990. small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin, 67. Coffer. A.. Sinnett-Smith, J.. and Rozengun, E. Bombesin receptor from galanin and neurotensin. Biochem. Biophvs. Res. Commun., 264: 6 6 -7 3 , Swiss 3T3 cells: affinity chromatography and reconstitution into phospho­ 1989. lipid vesicles. FEBS Lett., 275. 159-164. 1990.

2742s ICANCER RESEARCH 60H-A0U. NmcnitKri I. I9<)2| Gastrin Stimulates Ca^^ Mobilization and Clonal Growth in Small Cell Lung Cancer Cells

Tariq Sethi and Enrique Rozengurt'

Imperial Cancer Research t'unj. P. O. Box I2J, 44 IJncoln's Inn Fields, London lI'CiA M’X. F.nitlund

ABSTRACT tion. Nevertheless, compelling evidence that gastrin acts as a cellular growth factor or as an autocrine factor in tumors has Gastrin has t>een postulated to be a physiological growth factor, but been difficult to document in clonal cell populations. Indeed, compelling in ritro evidence of this has been difficult to obtain. In the studies using receptor antagonists and colon carcinoma cell present study we investigated whether small cell lung carcinoma cell lines resulted in controversial results (6-8). The lack of a con­ lines could provide a useful model system lo study the effects of gastrin on signal transduction and cell proliferation in rilro. We found that the venient model system has impeded the elucidation of the pos­ addition of gastrin to small cell lung cancer cells loaded with the fluo­ sible action of gastrin as a direct growth factor in vitro. rescent C a" indicator fura 2-tetraacetoxymethylester causes a rapid Cell lines established from SCLC’ provide a useful model and transient increase in the intracellular concentration of Ca' system to study the effects of hormonal peptide agonists and (|C a'*|j) followed by homologous desensitization. The (Ca'*|j response antagonists on early signaling events and on cell proliferation was especially prominent in the small cell lung carcinoma cell line (9-13). The SCLC cell line H.345 has been shown to express H5I0. In this cell line, gastrin I, gastrin II, cholecystokinin residues receptors for gastrin (14. 15), but the effect of this peptide on 26-33 (CCK-8). and unsulfated CCK-8 increased ICa'^ji in a concen­ H345 growth was not determined. In the present study we tration-dependent fashion with half-maximum effects at 7, 2.5, 3, and 5 report that gastrin, at nanom olar concentrations, stimulates the n\i. respectively. clonal growth of the SCLC cell line H5I0. identified as an The Ca'*-mobilizing effects of gastrin and CCK-8 were prevented by proglumide, benzotript, and the specific gastrin/CCKn receptor excellent model system for studying the effects of gastrin in antagonist L365260. Gastrin stimulated the clonal growth of H510 cells vitro. The results demonstrate that gastrin acts as a direct in semisolid (agarose-containing) medium, increasing both the number growth factor and show, for the first time, that this hormone and the size of the colonies. Gastrin and CCK agonists were equally can stimulate the proliferation of cells outside the gastrointes­ effective in promoting clonal growth. The broad-spectrum neuropeptide tinal tract. antagonists |D-Arg',o-Phe\D-Trp'\Leu"| substance P and |Arg*,r>- Trp'-’,MePhe*| substance P (6-11) markedly inhibitetl gastrin-stimu­ lated C a" mobilization and clonal growth. These results show that MATERIALS AND METHODS gastrin acts as a direct growth factor through gastrin/CCK, receptors Cell Culture. SCLC cell line H 510 w: purchased from the An and demonstrate, for the first time, that these peptides can stimulate the can Type Culture Collection. Slocks wei maintained in RPMI It.-i0 proliferation of cells outside the gastrointestinal tract. supplemented with 10% (v/v) fetal bovine serum (heat inactivated at 57*C for I h) in a humidified atmosphere of 10% COj/90% air at 37'C. INTRODUCTION They were passaged every 7 days. For experimental purposes, the cells were grown in HITESA which consists of RPMI 1640 supplemented The possibility that the gastrointestinal peptide gastrin could with 10 nM hydrocortisone, 5 ng/ml insulin, lO^g/ml transferrin. 10 nst act as a hormonal growth factor has attracted considerable in­ estradiol, 30 nxi selenium, and 0.25% bovine serum albumin (16). terest. A considerable body of evidence has demonstrated that Determination of Intracellular C a'' Concentration. Aliquots of 4-5 the adm inistration of gastrin induces growth-promoting effects X 10* SCLC cells cultured in HITESA for 3-5 days were washed and in the digestive tract and exocrine pancreas (I). In particular, an incubated for 2 h at 37'C in 10 ml fresh HITESA medium. Then I p\i increase in the circulating levels of gastrin has been related to fura-2-tetraacetoxymethylester AME from a stock of I mm in dimethyl hyperplasia of the gastric enierochromaffin-like cells (2). Fur­ sulfoxide was added, and the cells were incubated for a further 5 min. The cell suspension was centrifuged at 2000 rpm for 15 s, and the cells thermore, a decrease in circulating gastrin induced by antrec­ were resuspended in 2 ml of electrolyte solution (140 m,\i NaCl. 5 m\i tomy resulted in atrophy of the colonic mucosa in the rat, an KCI, 0.9 mM M g C lj, 1.8 mM C aC I. 25 mxt glucose, 16 mxi 4( 2 hydros effect reversed by the administration of pentagastrin (3). G as­ yethyl)-piperazineethanesulfonic acid. 16 mw Tris, and a mixture of trin also appears to be a growth-promoting hormone for ma­ amino acids at pH 7.2), transferred to a quartz cuvette, and stirred lignant cells grown as xenografts in nude mice (4, 5). While continuously at 37'C. Fluorescence was recorded continuously in a these observations strongly suggest that gastrin acts as a growth Perkin-Elmer LS5 luminescence spectrometer with an excitation wave­ factor, it is dilTicult to obtain unam biguous evidence for a direct length of 336 nm and an emission wavelength of 510 nm. (Ca'*|, was growth-promoting effect of gastrin in vivo because the adm in­ calculated as previously described (13). istration of this peptide could stimulate the release of other Clonogenic Assay. Cultures of the SCLC cell line H5I0. 3-5 davs biologically active peptides or growth factors which could act as post passage in HITESA, were washed and resuspended in the same medium. Cells were then disaggregated into a single cell suspension bv the proximal effectors of the action of gastrin. two passes through a 19-gauge needle and then through 20 «m nylon Cultured cells have provided useful experimental systems for gauze. Viability was determined by Trypan blue exclusion on a standard elucidating the extracellular factors that promote cell growth hemocytometer. Approximately 10' viable cells/ml. measured using a without the many complexities of whole animal experimenta­ Coulter counter, were suspended in HITESA containing 0.3% agarose

Received 4/30/92; accepted 8/24/9.2. ' The abbreviations used are: SCl.C. small cell lung cancer; (iRIV gastrin rc Tiie costs of publication of this article were defrayed in part by the payment of leasing peptide; HITESA. 10 nsi hydrocortisone, s «ig/ml insulin. 10 «g/nil tranv page charges. This article must therefore be hereby marked advertisement in accord­ fcrrin. 10 nw estradiol. 30 nw selenium, and 0.2S% bovine serum albumin. A M I., ance with 18 U.S.C. Section I 7.14 solely to indicate this fact. tetraacetoxymethylester; |C a" |,. intracellular concentration nfC a"; CCK. eholc ' To whom requests for reprints should be addressed. cystokinin; CCK-8. cholecystokinin residues 26-.13 6031 (.AMHIN SIIMUl MION <)l

Tlic'n. I ml of (Ins mi\(iirc «as plnicd in Tnc rcplicalc 15 mni plaslic Gastrin-I GastFin-ll i dishes coniainmp a 2-ml base layer of 0.5% agarose in HITESA lhal had liardened 1 he i«o layers contained neuropeplide at the same con t s o centration Cultures «ere incubated at 17’C tn a humidiricd atm osphere at 10% CO:/90'hi air for 21 days and then stained with the vital stain to o nitrobliie tetrarolitim Colonies of >120 *im diameter (16 cells) were counted under a microscope, and, using a x <1 lens, the image is relayed via a TV camera and anakzed on a Macintosh Ilex computer running the digital image processing and analysis program Image. M aterials. Gastrin 171 (unsulfated), gastrin 17-11 (sulfated on po ^ 0 siiion 6 from the COOH terminus), CCK residues 26-,11 sulfated on / the 7th position from the COOH terminus (CCK-8), desulfaied CCK-8 OJ des-CCK-8 CCK-8 |des(SOOCCK-8|. and CCK 10-27 were purchased from Sigm a Chem Ü “ 150 ical Co (St. Louis. MO). |u Arg'.o Ehe'.o Trp^ ".Lett"] substance L and |Arg",n Trp' ".MePhe") substanee P (6-11) were purchased from ^ * Peninsula Laboratories (Pelmont, CA); L164.7I8 and L165.260 were a to o kind gift from John W alsh (University of California, Los Angeles, CA). fura-2-AME «as from Calbiochem Corporation (La Jolla, CA); and agarose «as from SeaKem (Rockland, ME) All of the other reagents «ere of the highest grade commercially available.

10 to o 10 100 RESULTS [Peptitde] nM Gastrin Stimulates Ca’’ Mobilization. The addition of gas­ trin I or CCK-8 to either H69 or H345 SCLC lines loaded with Fig 2 Dose doprndoni cffrol of gasirin I. gasirin II, C( k X. and dcs(SOi) CCK-8 on |C a-• I. M510 rolls wrrr prrloadrd «iih fura 2/AM F and fluorrsccnoo the fluorescent Ca-" indicator fura-2 AME caused a rapid and was nionitorrd as previously drscrihod J c s C C K /I. d o s ( S O ,) C f K 8 A |C a - • |, (/ <•, transient increase in |Ca^"),, These findings (Fig. 1) are in peak |Ca-*|, - basal |C a'"|,) «as ralrulaird ai e.irh ronrrniranon P o in ts, m e a n of 1-5 indrprndrni rxprrim rnis bars. S I M agreement with other reports that demonstrated that gastrin induces Ca’" mobilization in the SCLC cell line H 345 ( 14, 15). However, the increase of |C a’"), by gastrin or CCK-8 (A|Ca^"]„ including bradykinin, vasopressin, or galanin (A|Ca^"|, '00, 20-30 nst) was considerably smaller than that induced by other 70, and 50 nst, respectively), identical results were obi ncd peptides including bradykinin (H69) or GRP (H345) (A|Ca^"|„ when CCK-8 was added instead of gastrin I (Fig 1). The in­ 80-100 and 85-120 nst, respectively). crease in ICa^*), induced by gastrin 1 in H 510 cells results from The salient feature shown in Fig. 1 is that gastrin I and Ca^* mobilization from internal stores, since it still occurred CCK-8 induced a prominent Ca’ mobilization in the SCLC after the addition of ethyleneglycol-bis-(g-aminoethyl ether) cell line H510. The magnitude of :e |Ca^"), r e s p o n s e in d u c e d /V./V,A^,/V-telraacetic acid to chelate extracellular Ca^*, just by gastrin I in this cell line (A|Ca’"|„ 150 nst) was gr ter than prior to the addition of gastrin 1 (results not shown). In view of the responses induced by other Ca’"-mobilizing neui peptides the findings depicted in Fig I, the H510 SCLC line has been used in additional experiments to characterize the Ca’"-mobi- H 6 9 H 3 4 5 lizing effects of gastrin I and CCK-8. Repeated additions of gastrin I caused the homologous de sensitization of Ca^* mobilization. Furthermore, the addition of gastrin I attenuated the increase in (Ca’"|, induced by CCK-8, and reciprocally, brief exposure to CCK-8 prevented the |C a’"|j response induced by gastrin I. Neither gastrin I nor CCK-8 prevented the increase in |Ca’"|, induced through a CCKE C C K 8 BK distinct neuropeptide receptor such as bradykinin (data not - H 5 1 0 shown). These results suggest that gastrin 1 and CCK-8 induced Ca’" mobilization in H5I0 through a common receptor. Gastrin I, gastrin II, dcs(S0,)CCK-8. and CCK-8 increased the peak level of |C a’"|, in a concentration-dependent fashion (Fig 2). The concentrations required to induce half-maximum stim ulation by these agonists were 7, 2.5, 5, and 2.5 nxi, respec­ tively. Thus, the receptors expressed by H5I0 cells recognize C C K 8 BK gastrin and CCK agonists wiih approximately equal apparent

I ig I Lfh-ci ot g.isirin I ;ind C'Ck 8 on |C:r ' |, in ioinp;irison lo other ncii affinities. ropcpiidcs in vjrious S( l.( veil linos .S( I.C or 11 linos 1169 (to ph jl) . I I 1 4 S (top Effect of Various CCK/Gaslrin Antagonists. In order to gain riphi). ;ind IISIO (liinci) woro ruliurrd in tllTESA for 1-S d;iys. A hqools of 4-5 V 10* rolls woro « iislird ,ind moiih.iiod in 10 nil frosh HI fF..SA iiiodium for 2 li insight into the receptor that mediates the |C a’*|, response lo III .'7'C . Tlion. I (JSI fiir.i 2/.\.M f w;is ;iddod for 5 nun 1 ho orlls woro wnslird :in

2 1 6 - 0

- 1 8 0 0 \\' io [L365,260] nM

131 - - rig 4 Effect of an- , ,nisi L365.260 on gasirin induced C a-‘ moliili/aiion in I tie SCLC cell tine U.- T o p . U 5I0 cells preloaded «iiti fura T/A.ME: fluorés 2 Pioglu cence «as monitored as previously described The following final conceniraiions Gasilin w e re u se d C i a s i r w l l. gasirin It (5 n»i). BK. bradylinin (10 nn). L365.260 (15 n si): f u s i r i n l* l , gasirin It (100 nsi). B o iio n i.dose-dependent inhibition of Ca^' ' 191 niobilizalion induced by 5 nsi gasirin It and 5 nsi CCK 8 bs L365.260 1 8 0 -

- 1 5 1 that gastrin I and CCK-8 caused a striking increase in the ability of these cells lo form colonies in semisolid medium. Gastrin increased both the number and the size of colonies, in a dose- 'Gal Gasilin II CCK-l dependent manner (Fig. 6). This increase in clonal growth was comparable to that induced by other growth factors. Gastrin I. I Mm gasirin II, des(S0.,)CCK-8, and CCK-8 stimulated colony for mation at comparable concentrations (Fig. 7). In contrast, 1 ilOO CCK-10“^", which lacks the COOH-terminal sequence critical 8 0 for receptor binding, neither increased (Ca’”], nor stimulated colony formation. The ability of gastrin to induce colony f 6 0 I growth was attenuated by the addition of the specific gastrin/ . 4 0 CCK,, receptor antagonist L-365260 (results not shown). i 20 The peptides ID-Arg|,D-Phe‘',tvTrp'’ ’.Leu"! substance P and r 201 |Arg'’,D-Trp'’'’.M ePhe''] substance P (6-11) have been shown 'W to inhibit signal transduction and cell proliferation by multi­ 100 1000 ple neuropeptides that induce Ca* ' mobilization in SCLC cells [Benzotript] itM [Proglumide] mM (11. 13, 20). These broad-spectrum neuropeptide antagonists inhibited both the |Ca^”J, response and the stimulation of col I IK 3 f.tTcci of fi.isirm II and CCK-8 ;md ilicir unuipnniAls proglumidr and iK-u/oiripi on |( a ''|, in itic H.SIO SCl.C cctl line T o p . IISIO celts, cullurcd. on y formation induced by gastrin (data not shown). «iisticd iind loiided wuti furj 2/AM t'. «ere rcsuspcnded in cicclrolylc soluiion ;md pliiecd in x guaro cu\cuc riuorcsecnce «:is monitored, and tias.il and peal |( a-'), «as caleulaicd as deseritied previousli I Itc following afihrcvialions and DISCUSSION final conceniraiions «ere used naiirin II. gasirin II (S no). C O -^1. CCK 8 (S nsi). 1‘io y h i. proglumide tIO ms,I g m lr in 11^ , g ,islrin II ( I 0 0 n o ) . WA. I ir a d y lin in ( 10 IIS,). I lc m . txnzoiripi (I m\i). G ul. galanin (25 n\,). ( ( K ctiolecysiokinin X In the present study we demonstrate that the addition of ( 1 0 0 n s i) llo lio n i.dose dependeni intiiUiiion of ( a' ' moliiliraiion induced h\ 5 gastrin at nanomolar concentrations to the SCLC line H510 ns, gasirin II b> tien/olripi and proglumide -S|( a'" |, (ir. peal |( a-'|, - basal |f a - • j.) «as caleulaicd ai each aniagoiiisi conccnlralioii -l|Ca^* |, induced hi S os, causes a rapid mobilization of Ca-* from intracellular stores gasiiin II IS la l e n a s lOO'S. The magnitude of the |C a’* j, signal evoked by gastrin was more

60.3 (.AMHIN S IIM tllA llO N OI ( I ON.M (,K()\V1 I|

Gastrin-I

l ig S Lficii of g.isinii I and ( C'K K on colon) lonnaiion in IISlO SC L( cells ( ul C'O lines 1-S dass posipassajjr in HI! I S \. were «ashed ind 10"' siable cells'nil «ere plalcd in m l I SA medium euniainin*! 0 I'S .i(:arosc on lop of .1 base of 0 “i'Vi. agarose in euliure me diuni as described in "Maierials and Meih ods “ Uoih lasers eoniained eiiher g.isirin I or ( ( K 8 at sarious eonceniraiions as indicaied ( iiliures ssere ineubaied ai 17'C in a hum idi fled aimosphere ai 10% C O j /90 "i. air for 21 CCK-8 d a ss an d I hen siained ss nh nilroieira/olium blue Colonies on a eonirol dish and from dishes ss Ilh 10. >0 and 200 nsi g.isirin I and ( ( K X are shossn V V ---- 10 50 200 [P eptide] nM

Gastrin-I iGastrin-

0, 600 desCCK-8 CCK-8

[Gastrin-I] nM Area (mm^2' 0.61

I Ig 6 Effcel of dose dependent siimulalion of gasirin I on colony number and 0 10 50 too 0 10 50 100 sire in H 5 I0 SCLC cells Cultures. .1-5 days posipassage. svere washed and resuspended in HITESA. Single cells (IO') in 0 1% agarose were layered on top [Peptide] nM ol 0 .s'hi agarose, both layers containing gasirin I at the eoncentration indicated Fig, 7. Gasirin I. gasirin It, CCK-8. and dfS-(S0,)CCK-8 slimutaic colony C olonics ssere incubated and stained as described Colonies represcni aggregates formaiion in H 510 SCLC cells. Culiurcs. 3-5 dayi posipassage, were washed and of cells >0.01 mm- sisualired bs a T\ camera and analyred using ihe .Macintosh rcsuspcnded in HITESA. Single cells (10") in 0.3% agarose were layered on lop Image program of 0.5% agarose; traih layers eoniained pepiide ai ihc same concentraiion as indicalcd. Colonies were ineubaied and siained as described in ‘ Maierials and Mcihods ■ Colonies represent aggregaies of cells >16 cells (120 um diameier) counicd under a microscope. C olum m . mean of 3-4 indcpendeni experimenis pronounced than that elicited by other Ca^‘'-mobiiizing pep (each wiih 5 replicaies i SEM). tides in this cell line. Thus, the SCLC cell line H 5I0 has been idcntiHed as a useful model system for studying the effects of Gastrin has been postulated as a cellular growth factor, but g a strin in vitro. compelling in vitro evidence of this has been difficult to obtain. Gastrin and CCK share a common COOH-terminal pen SCLC cell lines, including H510, are able to form colonies in tapeptide and consequently bind to common cell surface recep­ scmisolid medium, and their clonogenic ability is markedly in­ to rs. T h e CCKa receptors have a 500-fold higher affinity for creased by multiple neuropeptides (21). The SCLC cell line CCK-8 than for gastrin. In contrast, CCK,, receptors bind ei­ H510 provides an excellent model system in which to deter­ ther CCK-8 or gastrin with approximately equal apparent af­ mine whether gastrin can act as a direct growth factor. Conse­ finities (17). Here we demonstrate that gastrin I, gastrin 11, quently, we determined the effect of gastrin on the ability of CCK-8, and des (S0y)CCK-8 induce rapid Ca*" mobilization these cells to form colonies in agarose-containing medium. in SCLC H5I0 at comparable concentrations. Furthermore, Here we demonstrate that gastrin markedly stimulates the the selective gastrin/CCK„ antagonist L360265 (18, 19) clonal growth of H510 cells. Gastrin 1, gastrin 11, des-(SOy)- blocked the increase in |Ca*"|, induced by either gastrin or CCK-8, and CCK-8 induce colony formaiion at comparable CCK-8. Thus, the effects of gastrin and CCK in SCLC H 5I0 concentrations, in agreement with the effects obtained on Ca^" are mediated by gastrin/CCK„ receptors. mobilization. |n-Arg,,i>-Phe^,D-Trp’ '',L eu"| Substance P and 6034 CASIKIN M IM Iil M IO N Ol Ci.ONAI (;KOW III lArg‘’,D-Trp^ ''.McPlic'*l substance P (6-11), wliicii have inrcii 9 Carney. I). N . (ia/dai. A I . and Minna. J. D. Positive correlation Ik'Iwcch identified as broad-spectrum neuropeptide antagonists (20), histological tumor insoltcnicnl and generation of tumor cell colonies in agarose in specimens taken directly from patients with small cell carcinoma prevented the Ca^* mobilization induced by gastrin and strik of the lung. Cancer Res . 40: 1820-1823. 1980. ingly inhibited basal and gastrin-stimulated colony rormation. 10 Heikkila. R . Trepel. J II . Cuttitta. P.. Neckers. I. M.. and Sausville I' A. These results demonstrate that gastrin acts as a direct growth Bombesin related ptides induce calcium niuhiliration in a siilisel of liiinian small cell lung cancer cell lines. J. Biol. Client.. 262: 16456-1646(1. 1987. factor in vitro and show, for the first time, that this hormonal 11 W o ll. P. J . and Ro/engurt. E A neuropeptide antagonist that inliihits the peptide can stimulate the proliferation of cells outside the gas­ growth of small cell lung cancer in vitro. C an cer R es . .50. 3 9 6 8 -3 9 7 3 . 199(1. trointestinal tract. 12. Woll. P. J . and Ro/engurt. F Multiple neuropeptides mobilise caleiuni in Lung cancer remains the commonest fatal malignancy in small cell lung cancer effects of vasopressin, hradykinin. cholecystokinin. galanin and neurotensin Bios hem Biophys Res. Commun.. 164 6 6 -7 3 . the developed world. SCLC constitutes 25% of the total and 1989. follows an aggressive clinical course, despite initial chem o­ 13. Sethi. I.. and Ro/engurt. E. (iaianin stimulates Ca’" mohili/ation. mositul sensitivity (22). Identification of the factors that stimulate phosphate accumulation and clonal growth in small eell lung cancer cells C an ce r R es . S t: 1674-1679. 1991. the proliferation of SCLC cells will be im portant in the design 14. Staley. J . Jensen. R. I . and Mimdy. I . \\ CCK antagonists inter.let with of alternative and more effective therapeutic strategics. SCLC CCK-B receptors on human small cell lung cancer cells. Peptides (N3 |. // is characterised by its ability to secrete many hormones and 1033-1036. 1990 I 5. l.in. S. W . Molladay. M. W . Barrett. R. W . ri a l Distinct reguirements for neuropeptides including GRP, neurotensin, and vasopressin activation al CCK-A and ( CK B/gastrin recepiurs. studies with a f term in al (23-25). Results from our laboratory show that, at optimal hydra/.idc analogue of cholecystokinin tetrapi-pnde (30-3.3). Mol. Plt:irma concentrations, neurotensin, vasopressin, GRP, galanin, and col.. J6. 881-886. 1989 16 S im m s. E . Cia/dar. A. I . .Abrams. P. (i . and Minna. J I), (irowth ol human bradykinin induce comparable increases of SCLC clonal growth small cell (oat cell) carcinoma of the lung in serum free growth factor sup in responsive cell lines (21). Consequently, it has been hypoth­ plemenied medium. Cancer Res.. 40 4356-4363. 1980. esized that SCLC growth is regulated by multiple autocrine 17. Jensen. R T.. Wank. S. A.. Row les. W. IE. Sato. S.. Gardner. J. D. Inter and/or paracrine circuits involving Ca^*-mobilizing neuropep­ action of CCK with pancreatic acinar cells I rends Pharmacol Set.. Ill 418-42.3. 1989 tides (12, 21, 26). Interestingly, SCLC cells have been shown to 18. Lotti. V. J .. and Chang. R. I. A ne» potent and selective nonpcptide gastrm express gastrin and CCK peptides (27, 28). Thus, the findings antagonist and brain cholecystokinin receptor hgand .365.260. Eur. J. Pliar presented here demonstrating that gastrin and CCK-8 can act m acoE. 162: 27.3-280. 1984. 19. Bock. M. G . D iPardn. R. M .. Evans. B. F ...r ia t Hcn/.odia/.epine gastrin and as direct growth factors for the SCLC cell line H 5I0 further brain cholecystokinin receptor ligands: L .365.260. J Med. Client.. .12: 13- extend the hypothesis that SCLC growth may be regulated by 16. 1989. multiple autocrine and paracrine loops involving neuropep­ 20. Woll. P. J . and Ro/engurt. E. |n.Arg'.iiPhe'.iiTrp’ *.Lcu"|substance P. a potent bombesin antagonist in Swiss 3T3 cells, inhibits the growth of human tid e s . small cell lung cancer cells in vitro. Proc. Natl. .Acad. Sci. I A. fl.V 1859- 1863. 1988. REFERENCES 21. Sethi. T.. and Rozengurt. E. Multiple neuropeptides stimulât clonal growth of small cell lung cancer: effects of bradykinin. vasopressin, cholecystokinin. 1. Johnson. L. R. Trophic efTecis of gasirin on the colon. In: S. R. Woinian and galanin and neurotensin. Cancer Res . f t : 3621-3623. 1991. A. J. Masiromarino (eds.). Progress in Cancer Research and Therapy. Vol. 22. Smyth. J. F.. Fowlie, S. M.. Gregor, A .. Crompton. C. K.. Busuttil. A.. 29. pp. 327-.136. Ne» York: Raven Press, 1984. Leonard, R. C., and Grant. I W. The impact of chemotherapy on small cell 2. Ryberg. B.. Axelson. J .. Hakanson. R.. Sundler. F . and M alison. H. Trophic carcinoma of the bronchus. Quant J. Med., 61: 969-973. 1986. etTecis of continuous infusion of |Lys'*j-gastrin-l7 in Ihc rat. Caslrncnicr- 2.3. C u ttitta. F.. Carney, D. N .. M uh te. J . ef ol. Bombesin like peptides can olo g y . 98: 33-40. 1990. function as autocrine growth fact in human small-cell lung cancer. Nature 3. Johnson. L. R., and Guihrie. P. D. Proglumide inhibition of trophic action of (Lond.). J/6; 82.3-826. 1985. pentagastrin. Am. J. Physiol.. 246.-C62-C66. 1984. 24. Goedert. M.. Reeve. J. G . Emson. P. C.. and Bleehen. N. M. Neurotensin in 4. Singh, P.. Walker. J. P.. To»nsend, C. M., and Thompson. J . C. H ole o f gastrin and gastrin receptors on the growth of a transplantable mouse colon human small cell lung carcinoma. Br. J. Cancer. 50: 179-183, 1984. carcinoma (MC-26) in Balb/c mice. Cancer R es. 46; I6I2-I6I6. 1986. 25. Sausville. E.. Carney, D.. and Battey. J. The human vasopressin gene is linked 5. Watson, S., Durrani. L., and Morris, D. Gastrin: growth enhancing effects on to the oxytocin gene and is selectively expressed in a cultured lung cancer cell human gastric and colonic tumour cells. Br. J. Cancer., JV; 554-558. 1989. line. J. Biol. Chem.. 260: 10236-10241. 1985. 6. Hoosein. N. M.. Kiener. P. A . Curry. R. C . Rovati. L. C . McGilhra. I). K . 26. Sethi. T., Langdon. S., Smyth. J . and Rozengurt. E. Growth of small cell and Brattain. M. G. Antiproliferative effects of gastrin receptor antiigonisis lung cancer cells: stimulation by multiple neuropeptides and inhibition by and antibodies to gastrin on human colon carcinoma cell lines. Cancer Res . broad spectrum antagonists in vitro and in vivo. Cancer Res.. 52: 2737s 48: 7179-7183. 1988. 2742s. 1992. 7. Hoosein. N. M.. Kiener. P. A.. Curry. R. C.. and Brattain. M G. Evidence for 27. Rehfeld. J F.. Bardrum. L . and Hilsted. L Gastrin in human bronchogenic autocrine growth stimulation of cultured colon tumor cells hy a gastrin/ carcinomas: constant expression but variable processing of progastrin cholecystokinin like peptide. E.\p. Cell Res., 186: 15-21, 1990. Cancer Res.. 49: 2840-284.3. 1989. 8. Thumwood. C. M.. Hong. J.. and Baldwin. G. S. Inhibition of cell prolifer­ 28. Geijer. T.. Folkesson. R . Rehfeld. J F.. and Monstein. H-J. Expression of ation by the cholecystokinin antagonist L-364.718. Exp. Cell Res . /V2 the cholecystokinin gene in a human (small-celll lung eareiiioma eell line 189-192.1991 FEBS 1-ctt.. 270: 30-32. 1990.

6 0 3 5 ICANCER RESEARCH 52. 4554-4557, Aufiusi 15. I992|

Advances in Brief

Broad Spectrum Neuropeptide Antagonists Inhibit the Growth of Small Cell Lung Cancer in Vivo

Simon Langdon, Tariq Sethi, Alison Ritchie, Morwenna Muir, John Smyth, and Enrique Rozengurt'

Imperial Cancer Research Fund Medical Oncology Unit, H'esiern General Hospital, Edinburgh EH4 2XL' /S. I... A. R.. M. M.. J. S.J. and Imperial Cancer Research Fund Laboratories. 44 Lincoln's Inn Fields. London H'C2A JPX JT. S.. E. R.J. United Kingdom

A b s tra c t venting agonist receptor binding in a reversible fashion (13,14). In SCLC cell lines, these neuropeptide antagonists also blocked The proliferation of small cell lung cancer (SCLC) cells appears Ca^* mobilization by multiple neuropeptides, inhibited cell sustained by multiple autocrine and paracrine circuits involving Ca’" proliferation in liquid culture, and markedly reduced colony mobilizing neuropeptides. Consequently, broad spectrum neuropeptide antagonists w hich inhibit SCLC grow th in vitro have been suggested as formation in semisolid medium either in the absence or in the potential anticancer agents. Here we evaluated this hypothesis using presence of exogenously added stimulating neuropeptides xenografts of \\ X322 cells, a SCLC cell line that responds to multiple (13, 15-17). Thus, broad spectrum neuropeptide antagonists Ca’ mobilizing neuropeptides. The broad spectrum neuropeptide an­ can block multiple autocrine and paracrine growth loops in tagonists |Arg‘,i>-Trp'” ,MePhe*|substance P(6-ll) and |n-Arg',o- SCLC. In order to test whether these neuropeptide antagonists Phe',Trp’ *Leu"lsubstance P were shown to inhibit the growth of could be useful anticancer agents in SCLC, we have evaluated \\ \322 xenografts in nude mice. Similar results were obtained with the effects of (Arg‘’,D-Trp‘^-’,M ePhe*jsubstance P (6 -ll) and xenografts of the SCLC cell line H69. The results indicate that broad [D-Arg',D-Phe*,D-Trp’ ’,Leu")substance P on the growth of spectrum neuropeptide antagonists can inhibit the growth of SCLC in xenografts of the SCLC cell lines WX322 and H69 in nude vivo and suggest that these antagonists could be useful in the treatment of SC LC . m ic e .

Introduction M aterials and Methods

Lung cancer is the most common malignancy in the devel­ Cell Culture. SCLC cell line WX322 was grown in HITESA. The oped world. SCLC^ constitutes 25% of all pulmonary cancers cells were passaged every 7 days. and follows an aggressive clinical course. In spite of initial Determination of Intracellular Ca’" Concentration. Aliquots of 4-5 sensitivity to radio and chemotherapy, the 2-year survival of X JO* SCLC cells cultured in HITESA for 3-5 days were washed, and patients with SCLC remains very low (I). Thus, novel thera­ incubated for 2 h at 37*C in 10 ml fresh HITESA medium. Then, I peutic strategies are needed, and most likely they will arise from fura-2-tetracetoxy methyl ester from a stock of I mw in dimethyl sul­ a better understanding of the factors and signaling pathways foxide was added and the cells were incubated for a further 5 min. The that stimulate the proliferation of SCLC. cell suspension was centrifuged at 2000 rpm for 15 s, and the cells were resuspended in 2 ml of electrolyte solution (140 msi NaCl, 5 mw KCI, SCLC is characterized by the ability to secrete a variety of 0.9 mw MgClj, 1.8 mw CaCI, 25 mw glucose, 16 mw hepes, 16 msiTris, hormonal neuropeptides including G RP, vasopressin, cholecys­ and a mixture of amino acids at pH 7.2), transferred to p quartz cuvet, tokinin, and neurotensin (2-7). Among these, GRP has been and stirred continuously at 37'C. Fluorescence was rec ed continu­ shown to act as an autocrine growth factor for certain SCLC ously in a Perkin-Elmer LS5 luminescence spectromete; vith an exci­ cell lines (8, 9). Furtherm ore, a variety of neuropeptides includ­ tation wavelength of 336 nm and an emission wavelength of 510 nm. ing those secreted by SCLC induce rapid mobilization of Ca^^ |Ca’"]i was calculated as from internal stores of SCLC cell lines (10-12) and promote clonal growth of these cells in semisolid medium (12). Conse­ (C a’*)i nM ■ quently, the emerging view is that SCLC growth appears to be regulated by multiple autocrine and paracrine circuits involving Ca^^ mobilizing neuropeptides. Thus antagonists capable of where Fis the fluorescence at the unknown F„„ is the fluores­ cence after the trapped fluorescence is released by the addition of 0.2% blocking the biological effects of multiple neuropeptides (i.e., Triton X-lOO, and F„i„ is the fluorescence remaining after the Ca’* in broad spectrum neuropeptide antagonists) could provide an ef­ the solution is chelated with 10 mw (ethylenebis(oxyethylenenitrilo)]- fective approach in the treatm ent of SCLC. tetraacetic acid. The value of K was 220 for fura-2 (16). The compounds (i>-Arg',D-Phe®,D-Trp'’ ’,Leu"Jsubstance P Grow th Assay. SCLC cells, 3-5 days postpassage, were washed and and (Arg*,i>-Trp'’’,MePhe*)substance P (6-ll) have been resuspended in HITESA. Cells were resuspended at a density of 5 x 10* shown to inhibit signal transduction and DNA synthesis stim­ cells in I ml HITESA in the presence or absence of antagonists in ulated by bombesin, G RP, bradykinin, and vasopressin by pre­ triplicate. At various times, cell number was determined using a Coulter Counter, after cell clumps were disaggregated by passing the cell sus­ pension through 19- and 21-gauge needles. Received 5/15/92; accepted 7/2/92. Xenografts. The WX322 SCLC xenograft was derived from a s.c. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord­ metastasis of an untreated SCLC tumor (18). The NCI-H69 xenograft ance with 18 U.S.C. Section 1734 solely to indicate this fact. was derived by implantation of 10’ cells of the NCI-H69 SCLC cell line ' To whom requests for reprints should be addressed. into the flanks of female nu/nu (nude) mice. Both xenografts were ' The abbreviations used are: SCLC, small cell lung carcinoma: GRP, gastrin- maintained as s.c. tumors in the flanks of these animals. Histological releasing peptide; |C a ’ " |i. intracellular concentration of Ca’"; HITESA. RPMI 1640 supplemented with 10 nst hydroconisone. 5 >ig/ml insulin, 10 «ig/ml trans­ analysis confirmed the pathology of the xenografts and this was ferrin. 10 nw estradiol. 30 nw selenium, and 0.25% bovine serum albumin. checked every passage. 4554 ANTAGONIST INHIBITION OF CANCER GROWTH /A H iO

Animals. Female n u /n u mice were obtained from OLAC, Ltd. (Ox­ Tablc I Effect of multiple peptide hormones and neuropeptides on ICa^'j, mobilization in the SCLC cell line H'X322 ford, United Kingdom) and maintained in negative pressure isolators |Ca-*|. was measured as described in “Materials and Methods * All peptides (La Calhene, Cambridge, United Kingdom). were tested at a final concentration of 100 nw |Ca’*|, mobilization: -F, increase in Antitumor Testing. The in r iv o propagated cell lines were excised |C a''|, of 20-30 h m ; + + , increase (Ca’*|, of 60-100 nw. from donor animals, cut into small pieces, and implanted as 2-3-mm Effective Increase o f |Ca^“ |, cubed fragments into the flanks of recipient animals. After approxi- Noneffective matel) I month, animals were randomized into control and test groups Angiotensin 1 + -F ACTH* Bradykinin -f-F Atrial natriuretic peptide and given ear tags to allow individual identification. Groups contained Cholecystokinin -F Calcitonin 6-8 mice. Treatment was started when tumors reached a mean diam­ Dynorphin -F Chorionic gonadotropin eter of 4 mm and the first day of treatment was designated Day 0. The Endothelin + a-Endorphin only exception to this was the experiment when (t>-Arg'.D-Phe\D- GHRH ■F Epinephrine Bombesin/GRP ■f Galanin Trp'” ,Leu"Jsubstance P was given as early treatment when treatment Neurotensin GIP was started 3 h after tumor implantation. Tumor growth was assessed Neuromedin B Glucagon by caliper measurement and tumor volumes (F) were estimated as Oxytocin 5-Hydroxytryptamine Substance P L>eu-enkephalin Vasopressin Neuropeptide Y F = - X / X Parathyroid hormone Substance K TRH where / is the longest diameter and w is the perpendicular to this. The * ACTH, adrenoconicotrophic hormone; GHRH, growth hormone releasing relative tumor volume, F/Fo (where F q is the tumor volume at the start hormone; GIP, gastric inhibitory pepiide; TRH. thyrotropin releasing hormone. of the treatment and F, is the tumor volume at any given point) was calculated for each individual tumor at every time point. (ion of substance P, vasopressin, neurotensin, and bradykinin For injection into animals, antagonists were dissolved in sterile wa­ caused rapid and transient increases in (Ca^'^Ji in W X322 cells ter. To imitate continuous infusion, Alzet osmotic minipumps were loaded with the fluorescent Ca^* indicator fura-2. Table I used (model 1007D; pumping rate, 0.5 Fil/h for 7 days; Charles Rivers shows that W X322 cells respond in this assay to a surprisingly UK, Ltd.). The antagonist was diluted in sterile water and 90 u\ s o lu tio n were placed into the pump. The pump was then implanted s.c. in the large number of neuropeptides suggesting that the growth of flank opposite to the tumor of the anesthetized animal. Pumps were these cells could be regulated by multiple autocrine and para­ removed after 7 days, again while animals were anesthetized. crine circuits. M aterials. Antagonists were obtained from Peninsula Laboratories Next, we examined whether broad spectrum antagonists Belmont, CA. Agonists were purchased from Sigma Chemical Co., St. could prevent neuropeptide signal transduction in WX322 Louis, MO; fura 2-tetracetoxy methyl ester from Calbiochem Corpo­ cells. Addition of either (t>-Arg',i>-Phe*,D-Trp’ ’,Leu")sub- ration, La Jolla, CA; and agarose from SeaKem, Rockland, ME. All the stance P or (Arg*,D-Trp‘’-’,MePhe*Jsubstance P (6-ll) pre­ other reagents were of the highest grade commercially available. vented the increase in [Ca^*)i induced either by substance P or Results and Discussion by neurotensin, which acts through a distinct receptor (Fig. IB). The antagonists (20 hm) also blocked the increase in |Ca^*)j Initially we determined whether WX322, a cell line that induced by bradykinin, vasopressin, cholecystokinin, and bom­ readily forms tumors in nude mice (18), expresses receptors for besin in this cell line (results not shown) although the relative multiple neuropeptides. As shown in Fig. lA , sequential addi- affinities of these antagonists for the neuropeptide receptors are different (14). Furthermore, both antagonists added at 20 hm caused a profound inhibition of the proliferation of WX322 cells (Fig. 1C). To test whether broad spectrum neuropeptide antagonists can inhibit SCLC growth in vivo., we examined the effect of 97 - |Arg*,i>-Trp''’,MePhe®)substance P (6-ll) on the growth of WX322 xenograft. Fragments of the xenograft were implanted s.c. in the flanks of nude mice and allowed to grow to a mea­ surable size. Then, a group of animals were treated with the aoi antagonist given peritumorally (45 ng/g) once a day for 1 week.

1 ! In other experiments, we found that this was the maximum tolerated dose that could be administered i.p. to non-tumor bearing nude mice for 14 days without lethality. Fig. 2 shows - 2 5 0 that the antagonist profoundly inhibited the growth of the tu­ 20 mor, as compared with the control group. The inhibitory effect

- 1 3 0 was clearly maintained beyond the duration of administration. NT. 15 The SCLC cell line H69 is also known to express multiple Day 1 lain neuropeptide receptors (10-12). Addition of bradykinin, gala­ Fig. 1. Effect of agonists and broad spectrum neuropeptide antagonists on nin, or neurotensin induced a marked increase in |Ca^+)i in H69 (Cam’ ll and growth in the SCLC cell line WX322. jCa^*]; values were determined as described in "Materials and Methods." A: SP, substance P; FP, vasopressin; cells whereas GRP caused only a slight effect (10-12). Signal AT, neurotensin; BK, bradykinin. All peptides were added at a final concentration transduction and colony formation of this cell line in response o f 100 nw B: SP, substance P (25 niw); SP -r, substance P (100 nw); AT, neuro­ to neuropeptides are markedly inhibited by broad spectrum tensin (5 nw): AT-F, neurotensin (100 nst); D, |i>-Arg',t>-Phe*,D-Trp'’ *,Leu"|- substance P (20 m m ); £7, |Arg‘,o-Trp'’ *,MePhe*)substance P (6-11 ) (20 m m ). C: neuropeptide antagonists (15, 16).’ Fig. 2 (right) shows that effect of broad spectrum antagonists on grow th of WX322 SCLC cells. Cells were (Arg*,o-Trp'' ’,MePhe*)substance P(6-l 1) given peritumorally incubated at a density of 5 x 10* cells in 1 ml HITESA substance P(6-l 1) (O) or (D-Arg',i>Phe’,D-Trp’ *Leu"jsubstance P (O), each at 20 m m . Each point repre­ sents mean ± SD (bars) of 3 determinations. ' Unpublished results. 4 5 5 5 a n t a g o n i s t i n h i b i t i o n Of CANCER GROWTH /A HIP lU

z> 1400 300 Fig 2. Prrilumoral administration of o 800 WX322 500 H69 antagonists to SCLC xenografts. Points, > 1200 mean ± SE {b a n ). ■, untreated control; O, |A rg‘ ,i>-Trp'’ ’ ,M eP he'|sub stan ce P (6 - oc 200 300 11), 45 (ig/g/day for 7 days. Inset, i.p ad­ o 1000 ministration of antagonists to SCLC xe­ s 600 nografts. Points, mean ± SE {ban). 100 3 100 DAY 11 (■), untreated controls; D, (Arg‘,t>- DAY 7 800 Trp’ *,MePhe'|substance P (6-ll), 45 ag i f— ^day. The relative percentage difference ÿ 400 600 in mean body weights between the control and treated groups at any point of the H- treatment was never more than 3% (ac­ < 400 counted for at least in part by smaller tu­ - i 200 mors in the treatment group) for any of LU these experiments. *, P < 0.05 signifi­ CE 200 cantly different from control. Student's t z lest. < LU 20 3 0 2 0 s DAYS DAYS

2000 this antagonist could be administered for 7 days by continuous CO infusion using Alzet osmotic minipumps at doses equivalent to E WX322 E 100 ^g/g/day without any indication of toxicity while a dose of 200 20 ftg/g/day given as i.p. injections produced lethalities in LU 1500 non-tumor bearing nude mice. Fig. 3 shows that administra­ tion of this antagonist at the time of tumor implantation pro­ duced a pronounced inhibition of the growth of the WX322 O xenograft. Furthermore, continuous infusion of [o-Arg',D- > 1000 100 OC DAY 7 Phe*,i>-Trp’ ’,Leu")substance P for 7 days also inhibited o the growth of previously implanted WX322 xenografts (Fig. 3, inset). In other experiments, administration of (D-Arg‘,D- 500 Trp’ ^M ePhe^jsubstance P(6-l 1) by Alzet osmotic minipumps also inhibited the growth of previously implanted H69 xeno­ < grafts (results not shown). In all of the above described antitu­ LU mor experiments, there was no evidence of toxicity as indicated 20 6 0 by lethalities or body weight loss after injection of these antag­ o n ists. DAYS There is currently great interest in developing new treatment strategies for SCLC. It has been proposed that the broad spec­ Fig. 3. Alzet pump administration of [t>-Arg'.D-Phe*,i>-Trp'’ ’,Leu")substance P from the time of tumor implantation. Points, mean ± SE {b a n ) ■ . pumps trum neuropeptide antagonists [D-Arg',D-Phe*,i>-Trp‘' ’,Leu")- containing water alone; O, |t>-Arg'.t>-Phe’,o-Trp’-’,Leu")substance P. A single substance P and [Arg*,D-Trp‘' ’,MePhe®Jsubstance P(6-I1), animal in each of those groups was considered as a no-take if no tumor had appeared by Day 118. These were excluded from the analysis. *, P < 0.05 signif­ provide a novel approach to the treatment of these complex icantly different from control, Student's / test. Inset, effect of continuous infusion tumors, in which multiple neuropeptides interact to stimulate of (i>-Arg',D-Phe*, o-Trp^ *,Leu"|substance P for 7 days on previously implanted growth. Here we evaluated this hypothesis by testing the effect W X322 xenografts. (p-Arg*,D-Phe*,D-Trp'’-*,Leu")substance P was infused at a rale of 100 *ig/g/day for 7 days. There was no difference in mean body weights of these antagonists on the growth of SCLC in vivo. W e u sed between the treated and control group at any point of treatment for either of these the WX322 cell line because we identified that it expresses experiments. receptors for multiple neuropeptides. We found that the broad spectrum neuropeptide antagonists inhibited the growth of the at 45 Mg/g/day for 7 days also produced a marked inhibition of WX322 and H69 SCLC xenografts in nude mice. The results the growth of the H69 xenograft which was maintained beyond support the hypothesis that these compounds could constitute the duration of antagonist treatment. useful antiproliferative agents against SCLC. To determine whether systemic (rather than peritumora!) ad­ ministration of [Arg*,D-Trp’-’,MePhe*)substance P (6 -ll) was References also effective, we tested the effects of i.p. injection of this an­ 1. Smyth, J. F., Fowlie, S. M., Gregor, A., Crompton, G. K., Busutill, A., tagonist on the growth of WX322 and H69 xenografts. Al­ Leonard, R. C. F., and Grant, 1. W. B. The impact of chemotherapy on small though the inhibitory effect of the antagonist administered cell carcinoma of the bronchus. Q. J. Med., 61: 969-976, 1986. 2. Sorenson, G. D., Pettengill, O. S., Brinck Johnson, T., Cate, C. C , and through this route was smaller, the effect was statistically sig­ Maurer, L. H. Hormone production by cultures of small-cell carcinoma of the nificant against W X322 xenografts (Fig. 2, insets). lung. Cancer (Phila ), 47; 1289-1296, 1981. Next we determined whether the broad spectrum antagonist 3. W ood, S M., Wood, J. R , Ghaiei, M. A., l.ee, Y. C , O’Shaughnessy. D., and Bloom, S. R. Bombesin, somatostatin and neurotensin-like immunore- |D-Arg',D-Phe*,Trp’ ’,Leu")substance P(13) could also inhibit aclivity in bronchial carcinoma. J. Clin. Endocrinol. Meiab., 53; 1310-13:2. SCLC growth in vivo. An initial experiment demonstrated that 1981. 455 6 ANTAGONIST INHIBITION OF CANCER GROWTH l.\ FT KO

4. Gazdar, A. F., and Carney, D. N. Endocrine properties of small cell carci­ human lung cancer cells: delineation of alternative pathways. Proc. Nall. n om a o f the lung. In: K. Becker and A. F Gazdar (eds.). The Endocrine Lung Acad. Sci. USA. 87; 2162-2166, 1990. in Health and Disease, pp. 501-508. London: W. B. Saunders Co., 1984. 12. Sethi, T.. and Rozengurt. E. Multiple neuropeptides stimulate clonal growth 5. Goedert, M., Reeve, J. G , Emson, P. C., and Bleehen, N. M. Neurotensin in of small cell lung cancer: effects of bradykinin, vasopressin, cholecystokinin. human small cell lung carcinoma. Br. J. Cancer, 50: 179-183, 1984. galanin and neurotensin. Cancer Res.. 51: 3621-3623, 1991. 6. Sausville, E.. Carney, D., and Battey, J.The human vasopressin gene is linked 13. Woll, P. J., and Rozengurt, E. |i>-Arg',D-Phe’,D-Trp'’-*,Leu"|Subslance P, a to the oxytocin gene and is selectively expressed in a cultured lung cancer cell potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of line. J. Biol. Chem., 2 6 0 : 10236-10241, 1985. human small cell lung cancer cells in riiro. Proc. Natl. Acad. Sci. USA, 85: 7. Bepler, G , Rotsch, M.. Jaques, G., Haeder, M., Heymanns, J., Hanogh, G , 1859-1863, 1988. Kiefer, P., and Havemann, K. J. Peptides and growth factors in small cell 14. W oll, P. J., and Rozengurt, E. Two classes of antagonist interact with recep­ lung cancer: production, binding sites, and growth effects. J Cancer Res. tors for the miiogenic neuropeptides bombesin, bradykinin and vasopressin. Clin. Oncol., 114: 235-244, 1988. Growth Factors, /; 75-83, 1988. 8. Cuttitia, F., Carney, D. N., Mulshine, J., Moody, T. W„ Fedorko, J , 15. Woll, P. J., and Rozengurt, E. A neuropeptide antagonist that inhibits the Fischler, A., and Minna, J. D. Bombesin-like peptides can function as auto­ growth of small cell lung cancer in ritro. Cancer Res., 50: 3968-3973, 1990. crine growth factors in human small-cell lung cancer. Nature (Lond.). 316: 16. Sethi, T., and Rozengurt, E. Galanin stimulates Ca^* mobilization, inositol 823-826, 1985. phosphate accumulation and clonal growth in small cell lung cancer cells. 9. Mahmoud, S., Staley, J., Taylor, J., Bogden, A., Moreau, J-P., Coy, D., Avis, Cancer Res., 51: 1674-1679, 1991. 1., Cuttitia, F., Mulshine, J. L., and Moody, T. W. [f 13,14)-Bombesin ana­ 17. Sethi, T., Langdon, S. P., Smyth, J. F., and Rozengurt, E. Growth of small logues inhibit growth of small cell lung cancer in rilro and in viVo. Cancer cell lung cancer cells: stimulation by multiple neuropeptides and inhibition by R es , SI: 1798-1802, 1991. broad spectrum antagonists in vitro and in vivo. Cancer Res., 5/(Suppl.): 10. Woll, P. J., and Rozengurt, E. Multiple neuropeptides mobilize calcium in 2737-2742, 1991. small cell lung cancer: effects of vasopressin, bradykinin, cholecystokinin. 18. Langdon, S. P., Rabiasz, G. J., Anderson, L., Ritchie, A. A., Fergusson, R J.. galanin and neurotensin. Biochem. Biophys. Res. Commun., 164: 6 6 -7 3 . Hay, F. G , Miller, E. P., Mullen, P., Plumb, J., Miller, W. R., and Smyth. 1989. J. F. Characterisation and properties of a small cell lung cancer cell line 11. Bunn. P. A., Dienhart, D. G., Chan, D., Puck, T. T., Tagawa, M., Jewett. P. W X322 with marked sensitivity to o-interferon. Br. J. Cancer, 63: 9 0 9 -9 1 5 , B., and Braunschweiger, E. Neuropeptide stimulation of calcium flux in 1991.

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