REGULATION OF ADRENOCORTICOTROPHIN AND ITS PRECURSORS IN SMALL CELL LUNG CANCER Mary Felicity Stewart, BSc, MB BS A Thesis submitted to the University of London for the degree of DOCTOR OF MEDICINE ProQuest Number: U553788 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U553788 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT The ACTH precursor gene, pro-opiomelanocortin (POMC) is expressed in many non-pituitary tumours. In this thesis, small cell lung cancer (SCLC) cell lines have been studied as an in vitro model of the regulation of human, non-pituitary POMC. In contrast to the aggressive growth of this tumour in patients, SCLC cell lines are slow growing and difficult to establish and maintain in culture. ACTH and its precursors were quantitated by two- site immunoradiometric assays based on monoclonal antibodies. Ten cell lines (56%) secreted predominantly ACTH precursors (POMC and pro-ACTH) in the range 37-1405 pmol/1, with very little, if any, processing to ACTH. POMC RNA was demonstrated in 5 cell lines and a major transcript of approximately approximately 1350 bases was identified by Northern blot analysis in the cell line, COR L103, slightly larger than normal human pituitary (approximately 1100 bases). Incubation of SCLC cells for 10 days with hydrocortisone (500 and 1000 nM) did not inhibit intracellular POMC mRNA or ACTH precursor secretion, while this protocol produced significant inhibition in pituitary adenoma cells (AtT20). However, the dopamine agonist, bromocriptine (lOpg/ml) markedly suppressed ACTH precursor secretion following 24h and 7 days incubation. Corticotrophin releasing factor (CRF, 1000 ng/ml) had little effect on SCLC cells but significantly stimulated AtT20 and primary cultures of human pituitary cells. ACTH precursor secretion by SCLC cells was increased by dibutyryl cyclic AMP indicating that this intracellular signalling pathway is functional. These results are consistent with the clinical features of the ectopic ACTH syndrome, where ACTH circulates in precursor forms and is relatively resistant to glucocorticoid inhibition or stimulation with CRF. Thus the POMC gene is regulated in SCLC but it appears that the mechanisms differ from those operating in the anterior pituitary. 2 To: Stephen, Eleanor and Peter 3 The results presented in this thesis have not been submitted by the author for any other degree and to the best of her knowledge are her own work, except where due acknowledgement is made. 4 i ACKNOWLEDGEMENTS I am indebted to Dr. Anne White for her careful supervision and direction during the preparation of this Thesis. I wish to acknowledge the generous donations of small cell lung cancer cell lines by the following: Dr. Peter Twentyman (MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, UK). Dr. Frank Cuttitta (National Cancer Institute, Bethesda, Maryland, USA). Dr. M. Brouwer (Netherlands Cancer Institute, Amsterdam, The Netherlands). Dr. Lou de Leij (University of Groningen, The Netherlands) Dr. Gill Duchesne (Ludwig Institute for Cancer Research, Sutton, UK). I am grateful to the many clinicians at Hope Hospital, Salford and Wythenshawe Hospital, Manchester, for providing tumour samples from their patients. During the course of this project a number of collaborative studies were carried out and the contribution of others to the work described in this Thesis is detailed below. 1. The immunoradiometric assay for ACTH precursors was developed by Dr. Steven Crosby. My own contribution was to culture the pituitary and small cell lung cancer cells from which we were able to purify a POMC standard for the assay. 2. The chromatography of ACTH-related peptides was carried out jointly by Dr. Steven Crosby and me. 3. Studies of the molecular biology of POMC in small cell lung cancer cell lines were undertaken with Dr. Adrian Clark and Professor L.H. Rees (Department of Chemical Endocrinology, St. Bartholomew’s Hospital, London). 5 !f Northern and slot blot analysis of POMC, glucocorticoid receptor and ii, tyrosine amino transferase RNA were carried out by Dr. Adrian Clark and Dr. Paul Lavender (St. Bartholomew’s Hospital) and Dr. Bill Farrell (Hope Hospital). I thank Dr. Adrian Clark and Paul Lavender for carrying out one set of cell culture experiments on AtT20 cells. These experiments are identified in the text. 4. The dexamethasone binding assay was carried out by Dr. Bill Farrell and Dr. Adrian Clark with help and advice from Professor Gavin Vinson (St. Bartholomew’s Hospital). 5. Immunoradiometric assays for ACTH precursors and ACTH and cortisol radioimmunoassays were performed by Mrs Sarah Gibson, Ms Naomi Simpson and Ms Eileen Glover for which I am very grateful. 6. I carried out statistical analysis using the ’Minitab’ package with helpful advice from Sally Hollis (University of Manchester, Department of Statistical and Computational Studies). I thank Peter Crowe and Robert Weinkove for teaching me to use computer graphics. I particularly wish to thank Sarah Gibson, Steven Crosby and Bill Farrell for many useful discussions, and other staff in the Department of Medicine (Clinical Biochemistry) at Hope Hospital. Mrs. Christine Atkinson’s expert secretarial skill was invaluable in producing this manuscript and I also thank the Department of Medical Illustration for their help with photographs and figures. Finally, I wish to acknowledge Professor John G. Ratcliffe for supporting me at the outset of my project and Professor Pankaj Vadgama for continuing that support. 6 CONTENTS INTRODUCTION PAGE NOS I. ACTH IN NORMAL TISSUES 18 1. THE POMC GENE 18 2. BIOSYNTHESIS AND RELEASE OF ACTH AND RELATED PEPTIDES 20 i) Structure of the ACTH precursor, Pro-opiomelanocortin 20 (POMC) ii) Processing of POMC in the Anterior Pituitary 23 iii) The Neurointermediate Lobe 27 iv) POMC peptides in non-pituitary tissues 28 3. BIOLOGICAL ACTIONS OF POMC DERIVED PEPTIDES 29 i) ACTH 29 ii) N-POC 31 iii) The Lipotrophins and Beta-endorphin 32 4. REGULATION OF POMC EXPRESSION IN THE PITUITARY 32 i) Glucocorticoids 34 ii) Corticotrophin Releasing Factor 36 iii) Other Regulators of POMC expression 38 iv) Regulation of POMC in the Neurointermediate Lobe 40 II. MEASUREMENT OF POMC PEPTIDES 42 1. ACTH 42 2. Precursors of ACTH: POMC and Pro-ACTH 45 3. Other POMC derived peptides 46 III. HORMONES IN TUMOURS 47 1. Origin of Hormones in Lung Cancer 47 2. Nature of Hormones in Lung Cancer 50 3. Tumorigenesis: Links between Oncogenes, Autocrine 52 Growth Factors and Tumour Hormones 7 PAGE NOS IV. ACTH IN SMALL CELL LUNG CANCER (SCLC) 55 1. Clinical Aspects 55 2. Nature of ACTH related peptides produced by SCLC 56 3. The POMC gene in SCLC 58 4. Regulation of ACTH in SCLC 59 V. SCLC CELL LINES 61 1. Establishment and Characterisation of SCLC Cell Lines 61 2. Cytogenetic abnormalities and oncogene expression 65 3. Hormone Production 66 4. Autocrine growth factors 68 5. Advantages and limitations 69 VI. SUMMARY AND AIMS OF THIS THESIS 71 MATERIALS AND METHODS I Primary culture of human lung tumour cells 73 II Culture of established SCLC cell lines 77 III Culture of pituitary cells 80 IV Freezing down and retrieving cells from liquid nitrogen 82 V Methods for quantitation of tumour cells 83 VI Extraction of cell pellets for ACTH and precursors 85 VII Immunoradiometric assays for ACTH and ACTH precursor 86 peptides VIII Cortisol radioimmunoassay 91 IX Analysis of RNA 92 X Glucocorticoid binding assay 94 XI Statistical analysis 95 8 RESULTS Primary culture of lung and pituitary tumours 99 Secretion of ACTH and its precursors by SCLC cell lines 109 POMC gene expression in SCLC cell lines 121 Regulation of POMC gene expression and peptide secretion 127 in SCLC cell lines Glucocorticoids 127 Bromocriptine 167 Corticotrophin releasing factor (CRF) 177 Effect of glucocorticoid and CRF on human corticotroph 184 cells Second messenger mechanisms in SCLC cells 188 DISCUSSION Primary culture of human lung and pituitary tumours 204 ACTH precursors in SCLC cell lines 205 ACTH and ACTH precursor secretion by pituitary cells 206 Expression of POMC RNA in SCLC cells 208 Regulation of POMC gene expression and peptide secretion 209 in SCLC cells Summary and conclusions 218 9 PAGE NOS REFERENCES 220 APPENDIX I Materials 257 APPENDIX II Tables of data 272 Section 1. Peptides 273 Section 2. RNA 292 APPENDIX III Normal Probability Plots 295 APPENDIX IV Summary of results of statistical 317 analysis APPENDIX V Publications arising from this Thesis 325 10 LIST OF TABLES Page Table 1 Regulators of POMC in the anterior pituitary. 33 Table 2 Peptide hormones associated with SCLC. 51 Table 3 Composition of HITES medium for SCLC. 62 Table 4 Classification of SCLC cell lines based on 64 in vitro biological properties. Table 5 Hormone secretion by SCLC cell lines. 67 Table 6 SCLC cell lines. 78 Table 7 Primary culture of lung tumour samples. 100 Table 8 Levels of ACTH precursors and ACTH secreted 107 by human pituitary cells. Table 9 Levels of ACTH and precursor peptides in 112 supernatant medium from SCLC cell lines. Table 10 Levels of ACTH and precursor peptides in cell 113 pellet extracts from SCLC cell lines. Table 11 Levels of precursors in medium following 24h 117 incubation of SCLC cells. Table 12 ACTH precursors secreted by SCLC cells at different 119 stages of growth.