CCKA and CCKB Receptors Are Expressed in Small Cell Lung Cancer Lines and Mediate Ca2+ Mobilization and Clonal Growth

Home , Cholecystokinin receptor, Neuromedin B, Neuropeptide receptor

[CANCER RESEARCH 53,5208-5213, November 1. 1993] CCKA and CCKB Receptors Are Expressed in Small Cell Lung Cancer Lines and Mediate Ca2+ Mobilization and Clonal Growth

Tariq Sethi,1 Thomas Herget, S. Vincent Wu, John H. Walsh, and Enrique Rozengurt2

Imperial Cancer Research Punti, London. England [T. S.. T. H., E. R.Â¡,and Department of Medicine, UCl^A School of Medicine, University' of California. Los Angeles, California W073 /S. V. W. J. H. W.I

ABSTRACT growth factors for the SCLC cell line H510. Gastrin and CCK also induce a rapid mobilization of Ca2+ in other SCLC cell lines (e.g., Castrili, cholecystokinin (CCK), and CCK-related peptides comprise a H69 and H345). but the magnitude of the Ca2+ response was mark Imimonili family characterized by an identical carboxy-terminal amino edly lower than in the H510 SCLC cell lines (8). In a previous study, acid sequence, a domain critical for receptor binding. The addition of radiolabeled CCK has been shown to bind to SCLC cell lines (9). gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium i|(';r'|,i. Further Gastrin and CCK share a common COOH-terminal pentapeptide and more, gastrin acts as a direct growth factor through CCKn/gastrin recep bind to at least two different receptor subtypes (10, 11). The CCK[}/ tors. We report here that the expression of the mRNA coding for CCKu/ gastrin receptors, which are found mainly in the central nervous gastrin receptors correlates with the responsiveness of SCLC cells to system and in the gastrointestinal tract, bind either CCK or gastrin gastrin in terms of ( ';r ' mobilization and stimulation of clonal growth in with approximately equal affinities, whereas the CCKA receptors ex semisolid medium. The GLC19 SCLC cell line had no detectable expres hibit a 500-fold higher affinity for CCK than for gastrin (11). These sion of CCKn/gastrin receptor mRNA. Accordingly, gastrin (1-100 IIMIdid not cause any measurable increase in |( 'Â¡i'' |Â¡-Incontrast, the addition of receptor subtypes can also be distinguished by their sensitivity to specific antagonists (12, 13). The receptors that mediate the effects of cholecystokinin residues 26â€”33(CCK-8) caused a rapid and transient increase in |<'Â¡r'1],in this cell line. CCK-8 mobilized Â«;i'' in a dose- gastrin and CCK in the SCLC cell lines studied so far exhibit phar dependent manner in the nanomolar range (half-maximal stimulatory macological properties of the CCKi(/gastnn receptor (8, 14). Although concentration = 12 nvi). Furthermore, the selective CCKA antagonist cDNAs encoding the CCKÃŸ/gastrin receptors from various sources CAM-1481 inhibited the increase in |(':i-'-|, induced by CCK-8 (half- have been cloned and sequenced (15-18), direct evidence demonstrat maximal inhibitory concentration = 3 n>i) in GLC19 but not in H510 cells. ing the expression of CCKn/gastrin receptor in SCLC is not yet The selective CCKn/gastrin antagonist blocked the increase in |( ;r ' |, available. Furthermore, none of the previous studies provided evi induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in dence indicating that SCLC cell lines also express CCKA receptors. H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated The results presented here provide direct evidence demonstrating through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors the expression of CCKÃŸ/gastrin receptors in SCLC cell lines, as in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 shown by Northern blot analysis and by the molecular cloning and cells in a dose-dependent manner in the nanomolar range, whereas over sequencing of this receptor from SCLC cDNA using PCR methodol the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but ogy. In addition, our findings also show, for the first time, that certain not in H510 cells. Our results show, for the first time, that CCKA receptors SCLC cell lines express CCKA rather than CCKe/gastrin receptors. can mediate ( a'' mobilization and growth in SCLC cells and that SCLC CCK-8 acting through either CCKH or CCKA receptors promotes rapid Ca2 + mobilization and stimulates the clonal growth of several cells express two distinct functional CCK receptor subtypes. SCLC cell lines. INTRODUCTION MATERIALS AND METHODS Lung cancer remains the commonest fatal malignancy in the de veloped world. SCLC3 constitutes 25% of the total and follows an Amplification, Cloning and Sequencing of the Human CCKn/Gastrin Receptor from Brain and SCLC aggressive clinical course, despite initial sensitivity to chemotherapy and radiotherapy (1). Novel therapeutic approaches are needed, and Cloning of the Human CCKn/Gastrin Receptor. In order to generate a they will arise, most likely, from a better understanding of the factors DNA probe by PCR for isolating cDNAs encoding the human CCKVgastrin receptor two oligonucleotides were synthesized. Primer no. 2 (5'-GAG C/AGA and intracellular events that are responsible for stimulating the rapid TAOT A/GGC GCC ATC TGC-3') was deduced from a consensus sequence growth of SCLC cells. In this context, recent studies have shown that SCLC cell lines express receptors for multiple Ca2^-mobilizing neu- of the second cytoplasmic domain of the dog CCKu/gastrin (16) and the rat CCKA receptor (17). Primer no. 4 (5'-CGC TTC TTC GCCAT AA/TC AGG/C ropeptides (2-5), which, in turn, are known to act as potent growth TTG G-3') was reverse complement to a consensus sequence of the cDNAs factors (6, 7) for a variety of cell types, including SCLC (4, 5). coding for the third cytoplasmic domain. Recently, we reported that gastrin and cholecystokinin (CCK) cause PCR experiments were performed under the following conditions. Plaque- a rapid mobilization of Ca2 + from internal stores and act as direct forming units (3 X IO7) of a human fetal brain cDNA library in AZAP II were used as templates and incubated with 50 pmol of each of the appropriate primers (nos. 2 and 4 for the cDNA library) at 10()Â°Cfor 8 min and then Received 5/10/93; accepted 8/26/93. quickly chilled on ice. PCR was carried out with 2.5 units Jaq DNA polym The costs of publication of this article were defrayed in part by the payment of page erase in a final volume of 50 \u as recommended by the manufacturer (Boeh- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ringer Mannheim). The reaction was allowed to proceed on a thermal cycler 1 Present address: Department of Medicine (RIE), Respiratory Medicine Unit. Edin (Perkin-Elmer) for 30 cycles, each cycle consisting of a 1-min denaturing step burgh University, Edinburgh, Scotland. at 94Â°C,a 1-min annealing step at 60Â°C,and a 3-min polymerization step at - To whom requests for reprints should be addressed, at the Imperial Cancer Research Fund. P. O. Box 123, Lincoln's Inn Fields, London WC2A 3PX England. 72Â°C(19). In this way a product of about 550 nucleotides was amplified. This ' The abbreviations used are: SCLC. small cell lung cancer; cDNA. complemetary PCR product was trimmed by treatment with the Klenow-fragment (Amer- DNA; HITESA. RPMI medium supplemented with 10 nM hydrocortisone. 5 n-g/ml insu sham), isolated from an agarose gel by the GeneClean (Bio 101) method, and lin, 10 jig/ml transferrin, 10 nw estradiol, 30 nM selenium, and 0.25% bovine serum cloned directly by blunt-end ligation into the Hindll site of the pBluescript- albumin; AME, tetraacetoxymethylester; [Ca- +]Â¡,intracellular concentration of Ca2+; CCK, cholecystokinin; CCK-8, cholecystokinin residues 26-33; PCR, polymerase chain (SK-) vector (Stratagene). Plasmids containing the PCR fragment were se reaction. quenced by the dideoxy chain termination method (20) using a modified T7 520S

DNA polymerase (Sequenase Version 2.0; USB). Double-stranded plasmid Determination of Intracellular < :r ' Concentration DNA was sequenced from both strands using reverse and forward vector Aliquots of 4-5 X IO6 SCLC cells were cultured in HITESA for 3-5 days, primers and PCR primers that corresponded to the CCKÃŸ/gastrin receptor. washed, and incubated for 2 h at 37Â°Cin 10 ml fresh HITESA medium. Then Using this PCR fragment, two cDNA libraries were screened under standard conditions (19) with the cloned 550-base pair PCR fragment as the probe: first, 1 /XMfura-2/AME from a stock of 1 mw in dimethyl sulfoxide was added, and a cDNA library synthesized from the mRNAof a 17-18-week-old human fetal the cells were incubated for a further 5 min. The cell suspension was centri- brain, cloned in the Ã€ZAPII vector (Stratagene); second, a 5'-stretch cDNA fuged at 2000 rpm for 15 s, and the cells were resuspended in 2 ml of library from a 26-week-old human fetal brain cloned in the Ã€gtll vector electrolyte solution (140 mM NaCl. 5 mM KC1, 0.9 mM MgCl2, 1.8 mM CaCl, 25 mM glucose, 16 mM 4(-2-hydroxyethyl)-piperazineethane-sulfonic acid, 16 (Clontech). Both libraries were oligodeoxythymidine and random primed. Inserts of positive bacteriophages were cloned by in vivo excision (Ã€ZAP1I)or mMTris, and a mixture of amino acids at pH 7.2), transferred to a quartz cuvet, and stirred continuously at 37Â°C.Fluorescence was recorded continuously in a after gel purification of the insert (Agt11) into the EcoRI site of the pBluescript- (SK-) plasmid. A full-length CCKÃŸ/gastrin receptor clone was isolated; the Perkin-Elmer LS5 luminescence spectrometer with an excitation wavelength of 336 nm and an emission wavelength of 510 nm. [Ca2+]Â¡was calculated as complete sequence of this clone was obtained (as described for the PCR fragment) by custom primer-directed sequencing using specific primers previously described (28). complementary to internal cDNA sequences. Clonogenic Assay Cloning of the CCKVGastrin Receptor from SCLC Cell Lines H510 and H345. The following primers were used for PCR analysis of the coding SCLC cells, 3-5 days postpassage, were washed and resuspended in region of SCLC CCKu/gastrin receptor cDNA: 24185 (5'-CGG GCG CTG HITESA. Cells were then disaggregated into an essentially single cell suspen CAG TOC GTG CAT CGTTGG CCC-3'); 3F (5'-GTC GCC TAC GGG TTG sion by two passes through a 19-gauge needle and then through 20-fAm nylon ATC TC-3'); 6F (5'-AAC GGG CGT TGC CGG CCT GAG-3'); 6R (5'-CTC gauze. Cell number was determined with a Coulter counter, and IO4viable cells AGG CCG OCA ACG CCC GTT-3'); 7R (5'-GGC ACT ATA AAC TGG CAA were mixed with HITESA containing 0.3% agarose and agonist/antagonist at CC-3'); and 10R (5'-CTA CTC CTC AGC CAG GGC CCA GTG TGC-3'). the concentrations indicated and layered over a solid base of 0.5% agarose in These primers were deduced from the sequence of the cloned human CCK.^/ HITESA with agonist/antagonist at the same concentration, in 33-mm plastic gastrin receptor cDNA obtained in this study (see above). Primers were ethanol dishes. The cultures were incubated in humidified 10% CC>2/90% air at 37Â°C precipitated, adjusted to a concentration of 50 pmol/fj.1 in H2O, and were used for 21 days and then stained with the vital stain nitroblue tetrazolium. Colonies directly for PCR. of >120 fj.m diameter (16 cells) were counted with a microscope. First-strand cDNA was synthesized from total RNA from each SCLC cell line (H510 and H345). The reverse transcription mixture consisted of 10 fig of Materials cellular RNA. 100 ng of oligonucleotide primer 10R, 1 IBMeach deoxynucleo- The CCKe/gastrin and CCKA antagonists (CAM 2200 and CAM 1481, tide triphosphate, 4 triMdithiothreitol, 50 ITIMTris (pH 8.3), 75 mM KC1. 3 mM respectively) were the generous gift of Dr. J. Hughes (Parke Davis, Cambridge, MgCl2, and 40 units of RNase inhibitor. The reaction was initiated by adding England). Gastrin sulfated on tyrosine residue 12 and cholecystokinin residues 200 units of Moloney murine leukemia virus reverse transcriptase (Superscript kit; BRL) and carried out at 42Â°Cfor 1 h. 26-33 sulfated on tyrosine residue 27 (CCK-8) were purchased from Sigma (Poole, Dorset, England). 32P-labeled nucleotides a-dATP and a-dCTP and Using PCR with appropriate primers and 1 ng SCLC first-strand cDNA as Hybond N+ filters were purchased from Amersham (Aylesbury, Buckingham a template, we amplified DNA fragments, which were cloned and sequenced as shire, England). pBluescript vector and a cDNA library synthesized from described above. mRNA of a 17-18-week-old human fetal brain, cloned in the Ã€ZAPI1vector, were purchased from Stratagene (Cambridge. England); a 5'-stretch cDNA Northern Blot Analysis library from a 26-week-old human fetal brain cloned in the Ã€gili vector was For performing Northern blot analyses we used PCR to obtain a probe from purchased from Clontech (Los Angeles, CA). EcoRI and Taq polymerase were the start codon to the third cytoplasmic domain. Using the upstream primer no. purchased from Boehringer Mannheim (Lewes, East Sussex, England). 38 (5'-GCC ATG GAG CTG CTA AAG CTG AAC-3') in combination with Sequenase 2.0 was purchased from USB (Cambridge, England). All other primer no. 4 and 1 ng of the full-length CCKu/gastrin receptor cDNA (digested reagents were of the highest grade commercially available. by EcoRI) as a template resulted in the amplification of a region of about 1 kilobase, as expected. RESULTS RNA was isolated from different SCLC lines according to the method described by Chirgwin (21). Thirty /xg of total RNA were separated on a 1% Cloning and Expression Analysis of the SCLC CCKn/Gastrin agarose/2.2 Mformaldehyde gel, transferred onto Hybond N+ membrane (Am- Receptor. Initially, we cloned part of the coding region of the human ersham), hybridized with this 1-kilobase human CCKÃŸ/gastrinreceptor probe, brain CCKE5/gastrin receptor to analyze its expression at the mRNA which was radiolabeled by random priming (22) using both [a-12P|dATP and level in SCLC cell lines. Primers were designed to amplify the region [a-32P]dCTP. Membranes were washed at high stringency (final washes: 3 X between the second and third intracellular loops. The primer se 0.5 h with 0.1X standard saline citrate/0.2% sodium dodecyl sulfate at 60Â°C) quences were deduced according to the nucleotide sequences of the and exposed to preflashed Kodak XAR-5 films for 3 days at -70Â°C with recently cloned cDNAs encoding the canine CCKn/gastrin (16) and intensifier screens. [1 X SSC = 0.15 MNaCl, 0.015 Msodium citrate] The blots the rat CCKA receptor (17). PCR using primers nos. 2 and 4 (see were dehybridized and probed with a human 28S rRNA probe (23). "Materials and Methods") and a human fetal brain cDNA library

Cell Culture cloned in AZAPII as a template resulted in the amplification of a single DNA fragment of the expected size of about 550 base pairs. The SCLC cell lines H510, H69, and H345 were generously donated by Dr. A. PCR product was cloned and the nucleotide sequence was determined. Gazdar (Bethesda, MD) and purchased from the American Type Culture Col The corresponding deduced peptide sequence showed homology with lection. H5IO is derived from an extrapulmonary SCLC in the brain (24). the rat and canine receptor sequence but also revealed a surprising GLC19 and GLC28 were the kind gift of Dr. de Leij (Groningen, the Nether diversity. Specifically, the pentapeptide Ala/Thr-Ala/Gly-Pro-Gly-Pro lands) (25, 26). All of these lines have been well characterized and were (residues 272-276) of the canine/rat CCKÃŸ/gastrin receptors corre established from patients who had undergone chemotherapy. Stocks were sponding to the third cytosolic loop (Fig. 1), a region thought to play maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (heat-inactivated at 57Â°Cfor 1 h) in a humidified atmosphere of 10% CC>2/90% a critical role in signal transduction, was absent in the amplified air at 37Â°C.They were passaged every 7 days. For experimental purposes, the human sequence. Using this PCR product we screened two human cells were grown in HITESA consisting of RPMI 1640 medium supplemented fetal brain cDNA libraries and isolated a full-length CCK|,/gastrin receptor clone (see "Materials and Methods"). The nucleotide and with 10 nM hydrocortisone, 5 fig/ml insulin, 10 ng/ml transferrin, 10 nM estradiol, 30 nM selenium, and 0.25% bovine serum albumin (27). deduced amino acid sequence of the full-length human CCKu/gastrin 5209

244 Fig. 1. Deduced amino acid sequence of the third cytoplasmic domain of SCLC CCKn/gastrin SCLC CCKB: ELYLGLRFDG DSDSDSQSRV RNQGGLPGA- VHQNGR CRPETGAVGE receptor cDNA. mRNAfrom SCLC cell lines H5ÃŒO HumCCKB: ELYLGLRFDG DSDSDSQSRV RNQGGLPGA- VHQNGR CRPETGAVGE and H345 was prepared, and cDNAs were synthe RatCCKB: ELYLGLHFDG ENDSE TQSRA RNQGGLPGGA APGPVHQNGG CRPVTSVAGE sized by reverse Iranscriptase with primer IOR. Amplification by PCR with appropriate internal MasCCKB: ELYLGLRFDG DNDSDTQSRV RNQGGLPGGT APGPVHQNGG CRHVT-VAGE primers produced DNA fragments, which were cloned and sequenced (see "Materials and Meth DogCCKB: ELYLGLRFDE DSDSE--SRV RSQGGLRGGA GPGPAPPNGS CRPEGGLAGE ods"). The amino acid sequence was deduced. 323 Alignment of the third cytoplasmic domain of [he SCLC CCKB: DSDGCYVQLP RSRPA LELTA LTAPGPG--S GSRPTQAKLL AKKRWR SCLC, human brain (this study and Ref. 15), canine (16), ral (17). and Masiomys (18) CCKn/gaslrin HumCCKB: DSDGCYVQLP RSRPA LELTA LTAPGPG--S GSRPTQAKLL AKKRWR receptor is shown. The indicated amino acid posi RatCCKB: DSDGCCVQLP RSR--LEMTT LTTPTPGPVP GPRPNQAKLL AKKRWR tion is nos. 244-323 according to the human se MasCCKB: DNDGCYVQLP RSR--LEMTT LTTPTPGPGL ASA-NQAKLL AKKRWR quence. DogCCKB: DGDGCYVQLP RSRQT LELSA LTAPTPGPGG GPRPYQAKLL AKKRWR receptor cDNA clone confirmed the sequence of the PCR product were analyzed by scanning densitometry (Fig. 2, bottom). The strong (Fig. 1) and was identical to the sequence of the human CCKÃŸ/gastrin est signal was detected in H510. Intermediate signals were observed receptor from brain and stomach recently published (15). The se in H345 and H69, whereas only a faint band was detected in GLC28. quence comparison shown in Fig. 1 also emphasises the important GLC19 had no detectable expression of CCKÃŸ/gastrin receptor difference in the region corresponding to the less conserved third mRNA. cytosolic domain. Cultures parallel to those used to prepare RNA were used to deter We then proceeded to analyze the expression of CCKn/gastrin mine the ability of gastrin to induce Ca2+ mobilization. The addition receptor mRNA in SCLC by Northern blot hybridization using spe of gastrin (100 HM)to either H510, H69, or H345 and GLC28 SCLC cific probes. Total RNA was isolated from different SCLC lines grown lines, loaded with the fluorescent Ca2+ indicator fura-2/AME, caused in serum-free HITESA medium (27), gel-fractionated, and hybridized a rapid and transient increase in [Ca2+]Â¡.Gastrin induced a prominent with the radiolabeled 1-kilobase human CCKB/gastrin receptor probe Ca2+ mobilization in the SCLC cell line H510. The magnitude of the (see "Materials and Methods"). The autoradiogram (Fig. 2, top) shows [Ca2+]Â¡response induced by gastrin in this cell line was greater than a single hybridizing transcript of about 2.4 kilobases. These bands the responses induced in the cell lines H345, H69, and GLC28 (A- [Ca2+]j was 155, 30, 25, and 10 DM,respectively, in response to 100 nM gastrin). In the GLC19 SCLC line gastrin did not cause any increase in [Ca2+]Â¡(Fig. 2, bottom). Tlz-xa^.... To provide direct evidence that SCLC cell lines express CCKÃŸ/ gastrin receptors, mRNAs isolated from H510 and H345 cells were -28S reverse transcribed, and the resulting cDNAs were used for PCR analysis. Amplification of these cDNAs using primers 24185 and 6R, - 18S 3F and 7R, 6F and 7R, and 6F and IOR (see "Materials and Methods") resulted in PCR DNA fragments of 243, 140, 231, and 528 base pairs, respectively, exactly as predicted from the human brain CCKÃŸ/gastrin â€”¿28S receptor sequence (this study and Ref. 15). These overlapping PCR fragments from each cell line were subcloned and sequenced. The deduced amino acid sequence of the CCKÃŸ/gastrin receptor from -100 SCLC cells was identical to that of the CCKÃŸ/gastrin receptor from human brain. In particular, the nucleotide sequence of the third cyto O) plasmic loop (the most variable region between species) of the CCK.R/ m gastrin receptors in both SCLC cell lines lack residues 272-276 pre O 50 sent in the canine/rat CCKVgastrin receptors (Fig. 1). Hence, we provide direct evidence that CCKÃŸ/gastrinreceptors are expressed in SCLC cells and that these receptors are identical to those in human brain. S S S o o V o o Effect of CCK and Gastrin on [Ca2+]Â¡Â¡nH510 and GLC19 i i i o o x i I O C5 SCLC Cell Lines. The expression of the CCKÃŸ/gastrin receptor in Fig. 2. Top: Northern blot analyses of different human SCLC lines. Thirty fxg of total RNA from the SCLC lines HS10, H345. H69, GLC28, and GLC19 were separated on a SCLC cell lines H510, H345, and H69 can account for the respon \% agarose/2.2 M formaldehyde gel. blotted, then hybridized with a radiolabeled 1-kilo- siveness of these cell lines to both gastrin and CCK-8. Thus, the basc human CCKB receptor cDNA probe and washed under high stringent conditions addition of gastrin to H510 cells attenuated the increase in [Ca2+]Â¡ (top). Exposure time was 3 days. The migration positions of the 28S and 18S rRNA are labeled on the right. The blot was reprobed with a 28S rRNA probe to demonstrate equal induced by CCK-8, and reciprocally, brief exposure to CCK-8 pre loading of all lanes of the gel. Bottom left: effect of 100 nvi gastrin on [Ca2^ ], in SCLC vented the [Ca2+]Â¡response induced by gastrin (Fig. 3A). Neither cells. SCLC cell lines H510, H345. H69, GLC28, and GLC19 were cultured in HITESA gastrin nor CCK-8 prevented the increase in [Ca2+]Â¡induced through for 3-5 days. Aliquots of 4-5 X 10(t cells were washed and incubated in 10 ml fresh HITESA medium for 2 h at 37Â°C.Then, l mw fura-2/AME was added for 5 min. The cells a distinct neuropeptide receptor such as bradykinin. Similar results were washed and resuspended in 2 ml of electrolyte solution. This cell suspension was were obtained in the SCLC cell lines H345 and H69 (results not placed in a quartz cuvet. 100 nM gastrin was added, fluorescence was monitored, and [Ca2 *]Â¡wascalculated as described in "Materials and Methods." Bars, mean Â±SD of 4-8 shown). independent experiments. Bottom right: scanning densitometry of Northern blots. The In contrast to the results obtained with H510, H345, and H69, the density of each band shown at flit' top was determined by scanning densitometry. The level of H510 expression of CCKe/gastrin receptor mRNA was taken as 100%. Each bar addition of gastrin (100 nM) to the SCLC cell line GLC19 did not represents the mean Â±SD of 3 independent experiments. stimulate Ca2+ mobilization, whereas the addition of 100 nM CCK-8 5210

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. CCKA AND CCKu RECEPTORS IN SCLC caused a rapid and transient increase in [Ca2+]Â¡.Furthermore, prior addition of gastrin did not attenuate the increase in [Ca2+]Â¡induced by CCK-8 in this cell line (Fig. 3A). These results suggest that the H510 GLC19 GLC19 SCLC cell line expresses CCKA rather than CCKâ€ž/gastrin 210 - 200 receptors. This conclusion was further substantiated by the dose responses of CCK-8 and gastrin on [Ca2+]Â¡in H510 and GLC19 cells (Fig. 3ÃŸ). CCK-8 and gastrin increased [Ca2+]Â¡in H510 cells at identical con 170- centrations (half-maximal stimulatory concentration = 5 nM). In con trast, CCK-8 caused a dose-dependent increase in [Ca2+]Â¡in the Gastrin CCK BK Gastrin CCK BK GLC19 cell line (half-maximal stimulatory concentration = 15 nM), 195 - while gastrin, up to 100 nM, had no effect. Effect of Specific CCKA and CCKn/Gastrin Antagonists. Next, we attempted to distinguish the CCK receptors expressed by the 85 ~J 150 - SCLC cell lines H510 and GLC19 by using selective antagonists. Fig. 3C shows that the specific CCKÃŸ/gastrinantagonist CAM-2200 (29) CCK Gastrin 'BK CCK Gastrin BK profoundly inhibited the increase in [Ca2+]Â¡stimulated by 10 nM CCK-8 in H510 cells (half-maximal inhibitory concentration = 80 1 Min PM). In contrast, this antagonist had little effect on the Ca2+ mobili zation mediated by 10 nM CCK-8 in GLC19. Conversely, the CCKA antagonist CAM-1481 (30) inhibited the increase in [Ca2+]Â¡induced by 10 nMCCK-8 in GLC19 (half-maximal inhibitory concentration = 3 nM) but had little effect on the Ca2+ mobilization stimulated by 10 HMCCK-8 in H510. As shown in Fig. 2, the GLC28 SCLC cell line shows detectable expression of CCKn/gastrin receptor mRNA. In agreement with this, the addition of gastrin caused a rapid and transient increase in [Ca2+]Â¡ (Fig. 4). The addition of CCK-8 also stimulated Ca2+ mobilization in this cell line, but the magnitude of the response (A[Ca2+]Â¡= 30 nM) was consistently higher than that induced by gastrin (A[Ca2+]Â¡= 10 1 10 100 1000 1 10 100 1000 nM). Repeated additions of gastrin caused homologous desensitization [Agonist] nM of Ca2+ mobilization (results not shown). However, the addition of 100 nM gastrin did not prevent CCK-8 from increasing [Ca2+]Â¡, whereas prior exposure to 100 nM CCK-8 prevented the [Ca2+]Â¡ response induced by gastrin (Fig. 4, top). Fig. 4 also shows that prior treatment with CAM 1481 did not block the gastrin-induced [Ca2+]Â¡ response but markedly depressed the Ca2 * mobilization in response to CCK-8. Conversely, the addition of CAM 2200 prevented the [Ca2+]Â¡ response to gastrin but affected only slightly the increase in [Ca2+]Â¡ induced by CCK-8. These results strongly suggest that the SCLC cell line GLC28 expresses both CCKA and CCK,,/gastrin receptors. CCKA and CCIÃ•B/Gastrin Receptor Subtypes Stimulate Clonal Growth in SCLC. Tumor and transformed cells are able to form colonies in agarose medium. There is a positive correlation between 0.01 0.1 1 1 10 CAM-2200 CAM-1481 cloning efficiency of the cells and the histolÃ³gica! involvement and invasiveness of the tumor in specimens taken from SCLC (31). Fig. 5 [Antagonist]nM (left) shows that in the GLC19 SCLC cell line, CCK-8 markedly Fig. 3. A: effect of sequential additions of gastrin and CCK-8 on [Ca2 *]Â¡inSCLC cell stimulated clonal growth, over the concentration range 10-100 HM, lines H5IO and GLC19. Cells loaded with fura-2/AME were resuspended in electrolyte while at the same concentrations gastrin had no effect. solution and placed in a quartz cuvet. Fluorescence was monitored, and basal and peak In the SCLC cell line H510, the CCKâ€ž/gastrin antagonist (CAM- [Ca24]i was calculated as described previously. Gastrin. CCK-8 (CCK), and bradykinin 2200) at 1 nMinhibited the colony-stimulating effect of 50 nMCCK-8, (BK) were added sequentially at the following final concentrations (nM): KM), 1(K),and 5, respectively. B: dose-dependent increase in [Ca2 ' ]Â¡induced by gastrin and CCK-8 in whereas the CCKA antagonist (CAM-1481) at 10 nM had no effect H510 and GLC19 SCLC cell lines. Peak [Ca2+]Â¡was measured and calculated as de scribed in "Materials and Methods." A representative dose-response curve to gastrin (results not shown). In the GLC19 SCLC cell line, the CCKA antago P, D) and CCK-8 (â€¢)is shown for each cell line. C: effect of CCKn/gastrin and CCKA nist CAM-1481 at 10 nMabolished the colony-stimulating effect of 50 receptor antagonists on the increase in [Ca2 *]Â¡inducedby CCK-8 in the H510 and GLC19 nMCCK-8. The CCKe/gastrin antagonist (CAM-2200) at 1 nMhad no SCLC cell lines. H510 (â€¢)and GLC19 (O) cells loaded with fura-2/AME were resus effect on CCK-8-mediated growth (Fig. 5, righi). These results show pended in electrolyte solution and placed in a quart/ cuvet. Fluorescence was monitored, and basal and peak [Ca2 f ]Â¡was calculated as described previously. Dose-dependent that the occupancy of the CCKA receptor can also stimulate clonal inhibition of Ca2+ mobilization induced by 10 nMCCK-8 in H510 and GLC19 SCLC cell lines, by the CCKB/gastrin receptor antagonist CAM-2200 (left) and by the CCKA growth in SCLC. receptor antagonist CAM-1481 (right) was determined. A[Ca2*]Â¡(i.e., peak [Ca2+]Â¡- basal [Ca2*]/) was calculated at each antagonist concentration. A[Ca2 +]i induced by 10 nM CCK-8 is taken as 100%. DISCUSSION

The results presented in this study establish that the expression of the mRNA coding for CCKI}/gastrin receptor correlates extremely 5211

support this conclusion: (a) GLC28 cells show a low level of expres sion of the CCKÃŸ/gastrin receptor mRNA; (b) CCK-8 induced an GLC 28 increase in [Ca2+]Â¡ofhigher magnitude than that stimulated by gastrin 150- in these cells; (c) prior exposure to gastrin did not prevent CCK-8 from increasing [Ca2+]Â¡,whereas CCK-8 completely prevented the response to a subsequent addition of gastrin; and (d) CCKA and CCKI}/gastrin selective antagonists were effective in preventing the 120-â€”; increase in [Ca2+]Â¡induced by CCK-8 and gastrin, respectively. The results presented here show that SCLC cell lines can express the two C Gastrin CCK BK distinct CCK receptor subtypes, CCKA and CCKB/gastrin, either in-' dependently or coexisting in the same cell. CCK has been reported to exert trophic effects on normal pancreas J60- and to stimulate the growth of rat stomach in vivo and has also been implicated in the growth of gut tumors (34, 35). While these obser 140 - vations suggest that CCK can act as a growth factor, it is difficult to 13Â°-M l T 140- obtain unambiguous evidence that CCK acts through CCKA receptors tilt to stimulate growth. Consequently, we determined the effect of CCKA Ant A Gastrin CCK BK AntB Gastrin CCK BK receptor occupancy on the ability of GLC19 cells to form colonies in agarose-containing medium. We found that CCK-8 markedly stimu 1 Min lates colony formation in GLC19 cells in a dose-dependent manner in Fig. 4. Top: effect of sequential additions of gaslrin and CCK-8 on [Ca2 *]Â¡inthe SCLC cell line GLC28. Cells loaded with fura-2/AME were resuspended in electrolyte solution the nanomolar range. The selective CCKA antagonist CAM-1481 and placed in a quartz cuvet. Fluorescence was monitored, and basal and peak [Ca2*]i was inhibited the CCK-stimulated colony formation in GLC 19 but not in calculated as described previously. Bottom: effect of CCKa/gaslrin and CCKA receptor antagonists on CCK-8 and gastrin-mcdiated Ca2 + mobilization in the GLC28 SCLC cell H510 cells, whereas the selective CCKB/gastrin antagonist CAM- line. GLC28 cells loaded with fura-2/AME were resuspended in electrolyte solution and 2200 inhibited the CCK-stimulated colony formation in H510 but not placed in a quartz cuvet. Fluorescence was monitored and [Ca2*]Â¡was calculated as in GLC 19 cells. These results demonstrate, for the first time, that described previously. Agonists and antagonists were added sequentially at the following CCK-8 acting through CCKA receptors can stimulate Ca2+ mobiliza final concentrations (nM): gastrin, 1(X);CCK, UX);bradykinin, 20 (BK); CCKA antagonist CAM-1481. 25 (Ant A); CCKa/gastrin antagonist CAM-2200. 3.5 (Ant B). tion and clonal growth in a SCLC cell line. SCLC is characterized by its ability to secrete many hormones and neuropeptides including gastrin-releasing peptide, neurotensin, and well with the responsiveness of SCLC cells to gastrin and provide vasopressin (36-38). Results from our laboratory show that, at optimal direct evidence for the expression of CCKÃŸ/gastrinreceptors in SCLC cells. Nucleotide sequencing of PCR-derived fragments from SCLC concentrations, neurotensin, vasopressin, gastrin-releasing peptide, lines H510 and H345 cDNAs were identical to the brain CCKu/gastrin galanin, and bradykinin induce comparable increases of SCLC clonal receptor (Ref. 15 and this study). In particular, the predicted amino growth in responsive cell lines (4, 5). Consequently, it has been hypothesized that SCLC growth is regulated by multiple autocrine acid sequence of the third cytoplasmic loop of the CCKn/gastrin and/or paracrine circuits involving Ca2+-mobilizing neuropeptides receptors in both SCLC cell lines lack residues 272-276 present in the (2-5). Interestingly, certain SCLC cell lines have also been shown to canine/rat CCK1}/gastrin receptors. express gastrin and CCK peptides (39, 40). Thus, the findings pre- Our 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 CCKn/gastrin receptor. Similarly, vari ability in the response to gastrin-releasing peptide (32) and neuromedin B (33) among SCLC cell lines can also be explained in terms of .52 300 differential expression of the corresponding receptor mRNAs. We conclude that a major mechanism leading to the heterogeneity of o> neuropeptide responsiveness among SCLC cell lines is the differential expression of the genes encoding for the neuropeptide receptors. U The GLC19 SCLC cell line had no detectable expression of CCKÃŸ/ 100 gastrin receptor mRNA. Accordingly, gastrin, at nanomolar concen trations, did not cause any increase in [Ca2+]Â¡in this cell line. In contrast, CCK-8 caused a rapid and transient increase in [Ca2+]Â¡,and â€”¿ 10 100 10 25 50 100 â€”¿ pretreatment with gastrin did not attenuate the increase in [Ca2 +]Â¡ [Gastrin] nM [CCK] nM induced by CCK-8. In GLC19 cells, CCK-8 mobilized [Ca2^]Â¡in a CCK Fig. 5. Left: effect of gastrin and CCK-8 on colony formation in GLC19 SCLC cells. dose-dependent manner in the nanomolar range, whereas over the GLC19 cell cultures, 3-5 days postpassage in HITESA, were washed, and IO4 viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base of 0.5% same concentration range gastrin had no measurable effect. Further agarose in culture medium as described in "Materials and Methods." Both layers con more, the selective CCKA antagonist CAM-1481 blocked the increase tained no additions (control; D). gastrin (W), or CCK-8 â€¢¿atthe concentrations indicated. in [Ca2+]Â¡induced by CCK-8 in GLC19 but not in H510 cells. Cultures were incubated at 37Â°Cin a humidified atmosphere at 10% CC>2/90% air for 21 Conversely, the selective CCKÃŸ/gastrin antagonist CAM-2200 days and then stained with nitrotetrazolium blue. After 21 days colonies were counted. blocked the increase in [Ca2+]Â¡induced by CCK-8 in H510 but not in Each point represents the mean Â±SD of 2 experiments each of 5 replicates. Righi: effect of CCK-8 in the presence or absence of CCK antagonists on colony formation in GLC19 GLC19 cells. Taken together, these results indicate that the Ca2"1"- SCLC cells. GLC19 cultures, 3-5 days postpassage in HITESA, were washed, and IO4 viable cells/ml were plated in HITESA medium containing 0.3% agarose on top of a base mobilizing effects of CCK-8 are mediated through a CCKA receptor of 0.5% agarose in culture medium as described in "Materials and Methods." Both layers in GLC19 cells and via a CCKB/gastrin receptor in H510 cells. contained either no addition, 10 nM CCKA antagonist CAM-1481 (A), or 1 nM CCKe/ gastrin antagonist CAM-2200 (B), in the presence or absence of 50 nM CCK-8, as In addition to cell lines that express either CCKÃŸ/gastrinor CCKA indicated. Cultures were incubated at 37Â°Cin a humidified atmosphere at 10% CC>2/90% receptors, we also found that the SCLC cell line GLC28 expresses air for 21 days and then stained with nitrotetrazolium blue. Bars, mean Â±SD of 5 both CCKA and CCKg/gastrin receptors. Several lines of evidence replicates. 5212

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. CCKA AND CCKn RECEPTORS IN SCLC sented here, demonstrating that SCLC expresses two distinct func 20. Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with chain terminating inhibitors. Proc. Nati. Acad. Sci. USA, 74: 5463-5467, 1977. tional CCK receptors, both of which can mediate growth for SCLC 21. Chirgwin, J. N.. Przybyla, A. E., MacDonald. R. J.. and Rutter. W. J. Isolation of cell lines, further extends the hypothesis that SCLC growth may be biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem regulated by multiple autocrine and paracrine loops involving neuro- istry, 18: 5294-5299, 1979. 22. Feinberg. A. P., and Vogelstein, B. A technique for radiolabeling DNA restriction peptides, including CCK and gastrin. Indeed, CCK may constitute a fragments of high specific activity. Anal. Biochem.. 132: 6-13, 1983. unique case in which a single peptide can induce signal transduction 23. Herget. T.. Brooks. S. F.. Broad. S.. and Rozengurt. E. Relationship between the major and clonal growth through two different receptor subtypes (i.e., CCKA protein kinase C substrates acidic 80-kDa protein-kinase-C substrale (80K) and myristoylated alanine-rich C-kinase substrate (MARCKS) Eur. J. Biochem., 209: and CCK,,) w'th equal potency. Broad-spectrum neuropeptide antago 7-14, 1992. nists (41, 42) provide a strategy to block SCLC growth that takes into 24. Johnson, B. E., Whang-Peng, J., Naylor, S. L., Zbar, B., Brauch, H., Lee, E., account the mitogenic complexity of these tumors. Simmons. A.. Russell. E.. Nam. M. H., and Gazdar, A. F. Retention of chromosome 3 in cxtrapulmonary small cell cancer shown by molecular and cytogenetic studies. J. Nail. Cancer Inst.. 81: 1223-1228. 1989. REFERENCES 25. Berendsen, H. H., de Leij. L., de Vries, E. G. E., Mesander, G., Mulder, N. H., de Jong, B.. Buys, C. H. C. M., Postmus, P. E., Poppema, S., Sluiter. H. J., and The, 1. Smylh. J. F., Fowlie, S. M.. Gregor, A., Cromplon, G. K.. Busullil, A., Leonard. R. C, H. T. Characterization of three small cell lines established from one patient during and Grant. I. W. The impact of chemotherapy on small cell carcinoma of the bronchus. longitudinal follow-up. Cancer Res., 48: 6891-6899, 1988. Quant. J. Med., (Â¡1:969-973, 1986. 26. Damstrup. L., Rygaard. K.. Spang-Thomsen, M., and Poulsen, H. S. Expression ot the 2. Woll, P. J., and Rozcngurt, E.. Multiple neuropeptides mobilize Ca- ' in small cell epidermal growth factor receptor in human small cell lung cancer cell lines. Cancer lung cancer: effects of vasopressin. bradykinin. cholecystokinin, galanin and neuro- Res., 52: 3089-3093, 1992. tensin. Biochem. Biophys. Res. Commun.. 164: 66-73. 1989. 27. Simms, E., Gazdar. A. F.. Abrams. P. G.. and Minna, J. D. Growth of human small cell 3. Bunn, P. A., Dienhart. D. G.. Chan, D.. Puck. T. T.. Tagawa, M.. Jewett, P. B.. and (oat cell) carcinoma of the lung in serum-free growth factor-supplemented medium. Braunschweiger. E. Neuropeptide stimulation of calcium flux in human lung cancer Cancer Res., 40: 4356-4363, 1980. cells: delineation of alternative pathways. Proc. Nati. Acad. Sci. USA, 87: 2162-2166, 28. Sethi, T.. and Rozengurt. E. Galanin stimulates Ca- ' mobilization, inositol phosphate 199(1. accumulation and clonal growth in small cell lung cancer cells. Cancer Res., 5/: 4. Sethi. T.. and Rozengurt. E. Multiple neuropeptides stimulate clonal growth of small 1674-1679, 1991. cell lung cancer: effects of bradykinin. vasopressin, cholecystokinin, galanin and 29. Higginbotlom, M.. Horwell, D. C., and Roberts. E. Selective ligands for cholecysto neurotensin. Cancer Res.. 51: 3621-3623, 1991. kinin receptor subtypes CCK-A and CCK-B within a single strucural class. Bio. Med. 5. Selhi. T.. Langdon. S.. Smyth. J.. and Rozcngurl. E. Growth of small cell lung cancer Chem. Lett.. 3: 881-884, 1993. cells: stimulation by multiple neuropeptides and inhibition by broad spectrum an 30. Boden, P., Higginbottom. M.. Hill. D. R., Horwell. D. C., Hughes. J.. Rees, D. C, tagonists i/i vitro and in vivo. Cancer Res.. 52: 2737s-2742s, 1992. Roberts. E.. Singh. L.. Suman-chauhan. N.. and Woodruff. G. N. Cholecystokinin 6. Ro/engurt. E. Early signals in the mitogenic response. Science (Washington DC). dipcploid antagonists: synthesis and anxiolylic profile of some novel CCK-A 234: 161-166. 1986. and CCK-B selective and "mixed" CCK-A/CCK-B antagonists. J. Med Chem., 36: 7. Rozcngurt, E. Neuropeptides as cellular growth factors. Eur. J. Clin. Invest.. 21: 552-565, 1993. 123-134, 1991. 31. Carney, D. N., Gazdar, A. F., and Minna. J. D. Positive correlation between histo 8. Sethi. T.. and Rozengurt, E. Gastrin stimulates Ca2 * mobilization and clonal growth lÃ³gica!tumor involvement and generation of tumor cell colonies in agarose in speci in small cell lung cancer cells. Cancer Res.. .52: 6031-6035, 1992. mens taken directly from patients with small cell carcinoma of the lung. Cancer Res.. 9. Yoder, D. G., and Moody, T. W. High affinity binding of cholecystokinin to small cell 40: 1820-1823, 1980. lung cancer cells. Peptide. (New York), 8: 103-107. 1987. 32. Corjay, M. H., Dorbranski, D. J., Way, J. M.. Viallet. J.. Shapira, H.. Worland. P., 10. Lin, S. W.. Holladay. M. W., Barrett. R. W.. Wolfram, C. A., Miller. T. R.. Witte, Dâ€ž Sausville. E. A., and Battey, J. F. Two distinct bombesin receptor subtypes are Kerwin. J. F., Wagenaar, F., and Nadzan, A. M. Distinct requirements for activation expressed and functional in human lung carcinoma cells. J. Biol. Chem., 266: 18771- at CCK-A and CCK-B/gastrin receptors: studies with a C-terminal hydra/ide ana 18779, 1991. logue of cholecystokinin tctrapeptidc (30-33). Mol. Pharmacol., 3t>: 881-886, 1989. 33. Moody. T. W., Staley, J., Zia, F., Coy, D. H., and Jensen, R. T. Neuromedin B hinds 11. Jensen. R. T., Wank. S. A.. Rowley, W. H., Sato, S., and Gardner, J. D. Interaction of with high affinity, elevates cytosolic calcium and stimulates the growth of small cell CCK with pancreatic acinar cells. Trends Pharmacol. Sci., 10: 418-423, 1989. lung cancer cell lines. J. Pharmacol. Exp. Ther., 263: 311-317, 1992. 12. Lotti. V. J.. and Chang. R. L. A new potent and selective nonpeptide gastrin antagonist 34. Douglas, B. R.. Woutersen. R. A.. Jansen. J. B.. Rovati. L. C.. and Lamcrs, C. B., and brain cholecystokinin receptor ligand 365.260. Eur. J. Pharmacol., 762: 273-280, Study into the role of cholecyslokinin in bomhesin-slimulated pancreatic growth in 1984. rats and hampsters. Eur. J. Pharmacol., 161: 209-214. 1989. 13. Bock, M. G.. DiPardo, R. M., Evans. B. E.. Rillle, K. E., Whitter, W. L., Veber, D. E., 35. Lamers, C. B., and Jansen. J. B.. Role (if gastrin and cholecystokinin in tumours of Anderson, P. S., and Frcidinger. R. M. Bcnzodiazepine gastrin and brain cholecys the gastrointestinal tract. Eur. J. Cancer Clin. Oncol., 24: 267-273, 1988. tokinin receptor ligands: L-365,260. J. Med. Chem., 32: 13-16, 1989. 36. Cuttitta, F., Carney, D. N., Mulshinc, J., Moody, T. W., Fedorko, J., Fischlcr, A., and 14. Slaley, J.. Jensen. R. T., and Moody, T. W., CCK antagonists interact with CCK-B Minna. J. D. Bombesin-like peptides can function as autocrine growth factors in receptors on human small cell lung cancer cells. Pcptides (New York). //: 1033-1036. human small-cell lung cancer. Nature (Lond.), 316: 823-826, 1985. 1990. 37. Goedert, M.. Reeve, J. G., Emson. P. C. and Bleehen. N. M. Neurotensin in human 15. Pisegna. J. R., deWeerth, A., Huppi. K., and Wank. S. A. Molecular cloning of the small cell lung carcinoma. Br. J. Cancer, 50: 179-183, 1984. human brain and gastric cholecystokinin receptor: structure, function, expression and 38. Sausville, E.. Carney. D.. and Battey. J. The human vasopressin gene is linked to the chromosomal localization. Biochem. Biophys. Res. Commun., 189: 296-303, 1992. oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. 16. Kopin. A. S.. Lee. Y-M.. McBride, E. W.. Miller, L. J., Lu, M., Lin. H., Kalowski, L. Chem.. 260: 10236-10241, 1985. F., and Beinhorn, M. Expression cloning and characterization of the canine parietal 39. Rchfeld, J. F., Bardrum, L., and Hilsted, L. Gastrin in human bronchogenic carcino cell gastrin receptor. Proc. Nati. Acad. Sci. USA. 8V: 3605-3609, 1992. mas: constant expression but variable processing of progastrin. Cancer Res.. 49: 17. Wank. S. A.. Pisegna. J. R.. and deWeerth, A. Brain and gastrointestinal cholecysto 2840-2843, 1989. kinin receptor family: structure and functional expression. Proc. Nati. Acad. Sci. 40. Geijer, T., Folkesson, R., Rehfeld. J. F. and Monstein, H-J. Expression of the cho USA, 89: 8691-8695. 1992. lecystokinin gene in a human (small-cell) lung carcinoma cell-line. FEBS Lett.. 270: 18. Nakata. H., Matsui, T.. Ito. M., Taniguchi, T., Naribayashi, Y. Nakamura, A., 30-32, 1990. Kinoshikazu. K., Chihara, K.. Hosoda, S.. and Chiba, T. Cloning and characterization 41. Woll. P. J., and Rozengurt. E. A neuropeplide antagonist that inhibits the growth of of gastrin receptor from ECL carcinoid tumor of Masiomvs naialensis. Biochem. human small cell lung cancer cells in vitro. Cancer Res., 5/: 1674â€”1679.1991. Biophys. Res. Commun., 187: 1151-1157, 1992. 42. Langdon, S., Sethi. T.. Ritchie. A.. Muir. M.. Smyth. J.. and Rozengurt. E. Broad 19. Sambrook, J., Fritsch, E. F., and Maniatis. T. Molecular Cloning: A Laboratory spectrum neuropeptide antagonists inhibit the growth of small cell lung cancer in vivo. Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 1989. Cancer Res., 52: 4554-4557. 1992.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1993 American Association for Cancer Research. CCKA and CCKB Receptors Are Expressed in Small Cell Lung Cancer Lines and Mediate Ca 2+ Mobilization and Clonal Growth

Tariq Sethi, Thomas Herget, S. Vincent Wu, et al.

Cancer Res 1993;53:5208-5213.

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