DEVELOPMENT OF AN IN-VITRO MODEL OF HUMAN PROSTATE By Bernadette Daly-Burns B.Sc. A Thesis Submitted For The Degree Of Masters Of Philosophy To The Faculty Of Medicine University College London Institute of Urology and Nephrology Research Laboratories University College London I UMI Number: U602662 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 complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602662 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The mechanisms controlling the growth and development of Benign Prostate Hyperplasia (BPH), the most common non-malignant disorder found in men over the age of 70, are poorly understood. There is a shortage of relevant “in vitro” models suitable for studying this disease. The aim of this project was to develop a representative “in vitro” model system for the study of BPH. Epithelial and fibroblast cell lines (Pre2.8 and S2.13 respectively) were derived from the same biopsy of BPH and immortalised using the temperature sensitive SV40 large-T antigen construct. At 33°C the cells grow progressively under the influence of the SV40 large T-antigen, but at 39°C the conformation of the protein changes and the protein is no longer functional, so the cells stop dividing and are able to differentiate. When monolayer cultures were switched from 33°C to 37°C or 39°C, changes in morphology, cell size distribution and a reduction in cell proliferation were observed. Both cell lines expressed the 5oc-reductase type I enzyme, but did not express androgen receptor (AR) and did not secrete PSA and PAP, hence they are likely to be undifferentiated cells. Pre2.8 epithelial cells have a basal cell phenotype at 33°C and undergo limited differentiation at 37°C and 39°C. DNA profiling was used to confirm the origin of the cells. The cell lines were shown to be Mycoplasma-free. Both cell lines showed amplification of DNA on chromosome 20q, a region known to be associated with cell immortalisation. In order to develop a representative model of BPH, Pre2.8 and S2.13 cells were mixed in 3-dimensional matrigel cultures and optimised in order to obtain the most suitable culture conditions to produce prostate-specific characteristics. The three-dimensional culture system consisted of epithelial cells surrounded by a layer of stromal cells and in some cases with the formation of acinus like structures. The 3-D model showed some prostate-specific characteristics, for instance the epithelial cells expressed the androgen receptor. The epithelial cells differentiated towards a luminal phenotype, expressing K8 and 18. In conclusion, a matched pair of epithelial and stromal BPH cell lines has been established which show some typical characteristics of prostate cells and have been grown in a three-dimensional culture. The model may be useful for studying the cell interactions that control the growth of benign prostate Hyperplasia. m This thesis is dedicated to the wonderful men in my life, my husband Michael and son Ciaran. Thank you for all your support and patience during this long period of time. IV ACKNOWLEDGEMENTS I am indebted to my supervisor Prof. John Masters for the training, encouragement and support he has provided during my MPhil studies at the Institute of Urology. Special thanks to Dr David Hudson, my second supervisor for his invaluable training and support throughout the duration of the project. To other colleagues involved in this area of research, Dr. Pat Fry, Ms Tahirah Alam, Dr Rodger Tatoud, Dr Istvan Laczko and Dr Qin Wang thank you for all your technical input, advice, expertise, help and support. To all other work colleagues who have come and gone. Thank you for your support and patience during those stressful moments. I would like to thank Dr. Mike O’Hare for transducing the primary cultures with the temperature sensitive SV40 construct. Finally, I would like to thank my family and friends for their encouragement and support. Michael thank you for acting as father and house husband at weekends, while I was working on my MPhil. Completion of this thesis would not have been possible without your understanding, encouragement and unquestioning support. V TABLE OF CONTENTS Page CHAPTER Is INTRODUCTION 1 1.1 The Prostate 2 1.1.1 Development And Hormone Control 2 1.1.2 Anatomy 3 1.1.3 Morphology And Histology 5 1.1.4 Cell Types 6 1.1.5 Biochemistry 9 1.2 Benign Prostatic Hyperplasia (BPH) 10 1.2.1 Etiology Of BPH 11 1.2.2 Clinical Presentation 12 1.2.3 Treatment 14 1.3 In Vitro Models 17 1.3.1 Primary Culture Systems 17 1.4 Immortalisation And Characterisation Of Prostate Continuous Cell Lines 21 1.4.1 Immortalised Epithelial Cell Lines 22 1.4.2 Immortalised Stromal Cell Lines 26 1.5 3-Dimensional Culture Systems 28 1.6 Immortalisation And Conditional Immortalisation 29 1.7 Rationale For This Study (and key issues to be aware of) 34 CHAPTER 2: MATERIALS AND METHODS 35 2.1 COSHH Regulations 36 2.2 Cell Culture 36 23 Cell Line Authentication and Purity 38 2.3.1 Cross-Contamination 38 2.3.2 Mycoplasma Testing 40 2.3.3 Mycoplasma Testing Of Cell Lines 40 2.4 Cell Line Maintenance 45 2.4.1 Cell Line Passaging 47 VI Page 2.4.2 Haemocytometer Cell Counting 47 2.5 Measurement Of Cell Growth Rates 49 2.5.1 MTT Assay - Growth Curves 49 2.5.2 Cell Counts - Growth Curves 52 2.5.3 Statistics 52 2.6 Cell Size Distribution 54 2.6.1 Graticule And Light Phase Microscope 54 2.6.2 Beckman Z2 Coulter Counter And Size Analyzer 54 2.7 Anchorage Independent Growth In Agar 55 2.8 Colony Forming Assays 56 2.9 Immunohistochemistry 56 2.9.1 Cultures On Coverslips 56 2.9.2 Cutting Paraffin Sections For Immunohistochemistry 57 2.9.3 Antigen Unmasking Solution 57 2.9.4 Monoclonal Antibodies 58 2.9.5 Immunocytochemistry using the Vectastain ELITE ABC kit 59 2.10 Fluorescence Microscopy And Data Capture 61 2.11 Cytogenetics 62 2.12 Flow Cytometry And Fluorescent Activated Cell Sorting (Facs) Analysis 63 2.13 Cell Cycle Distribution 68 2.14 3-Dimensional Cultures 68 2.15 RT-PCR 70 2.15.1 RNA Extraction 71 2.15.2 Measurement Of RNA Concentration 72 2.15.3 Reverse Transcription 72 2.15.4 PCR 73 2.15.4.1 Primer Design 73 2.15.4.2 PCR Analysis 74 2.15.5 Gel Electrophoresis 76 2.16 Appendix: Materials And Sources 77 vn Page CHAPTER 3: RESULTS: DERIVATION AND AUTHENTICATION AND PURITY 81 3.1 Introduction 82 3.2 Derivation OfPre2.8 Epithelial And S2.13 Stromal Cell Lines 83 3.2.1 Prostate Tissue 83 3.2.2 Primary Culture 83 3.2.3 Transduction 84 3.2.4 Development Of Epithelial Cell Line (Pre2.8) 84 3.2.5 Development Of Stromal Cell Line (S2.13) 84 3.3 Morphology Of Living Cells 87 3.3.1 Pre2.8 Cells 87 3.3.2 S2.13 Cells 88 3.4 Presence Of SV40 Large T-Antigen 89 3.5 DNA Profiling 89 3.6 Mycoplasma Screening 91 CHAPTER 4: RESULTS: CHARACTERISATION 94 4.1 Cytogenetics 95 4.1.1 Pre2.8 Cells 95 4.12 S2.13 Cells 95 4.13 Figure Legend For Figure 4.1,4.2,4.3 And 4.5 101 4.2 Cell Proliferation 102 4.2.1 Flow Cytometry 102 4.2.2 Ki-67 Expression 102 4.2.3 Summary 103 4.3 Growth Rates 106 4.3.1 Pre2.8 Growth Assays Using Cell Counts 106 4.3.1.1 Mean Population for Pre2.8 Cells 106 4.3.2 S2.13 Growth Assays Using Cell Counts 110 4.3.2.1 Mean Population for S2.13 Cells 110 vm Page 4.3.3 Comparison Of The Growth Patterns Of Immortalised (1542-NPTX) And Conditionally Immortalised (Pre2.8) Prostate Epithelial Cell Lines 114 4.3.4 Comparison Of The Growth Patterns Of Immortalised (1542-FT) And Conditionally Immortalised (S2.13) 114 4.4 Cell Size Distribution 117 4.4.1 Beckman Coulter Counter 117 4.4.2 Graticule And Inverted Microscope 119 4.4.2.1 Pre2.8 Cells 119 4.4.2.2S2.13 Cells 121 4.5 Colony Forming Effeciency On Plastic And In Soft Agar 123 4.5.1 Colony Forming Efficiency For Pre2.8 Cells On Plastic 123 4.5.2 Anchorage-Independent Growth 123 CHAPTER 5: DIFFERENTIATION OF MONOLAYER CULTURES 124 5.1 Introduction 125 5.2 Stromal Cell Markers 125 5.3 Epithelial Cell Markers 125 5.4 Androgen Expression And Prostate Specific Characteristics Of Pre2.8 And S2.13 Cells 127 CHAPTER 6: RESULTS: 3-DIMENSIONAL CULTURES 135 6.1 Introduction 136 6.2 Comparison Of Growth Of Pre2.8 And S2.13 Cells In Various Tissue Culture Media 137 6.2.1 Growth Of Pre2.8 Cells In Serum-Free PrEGM And RPMI-1640 Medium In The Presence And Absence Of Serum 137 6.2.2 Proliferation Of Pre2.8 Cells In Various Media 139 6.2.3 Proliferation Of S2.13 Cells In Various Media 140 6.3 Effect Of Stromal Cells And Conditioned Medium On The Growth Of Pre2.8 Cells In 3-Dimensional Culture 142 IX Page 6.3.1 Stromal Cell Marker Expression Of 3-Dimensional Cultures 143 6.3.2 Keratin Expression OfPre2.8 Cells In 3-Dimensional Culture 143 6.4 Expression Of Prostate-Specific Characteristics By Pre2.8 Cells In 3-Dimensional Culture 146 6.4.1 Androgen Receptor (AR) 146 6.4.2 PSA 147 6.4.3 PAP 147 6.5 Mibolerone 148 6.6 Cell Proliferation 148 6.7 Summary 167 CHAPTER 7: DISCUSSION 167 7.1 Discussion 170 7.1.1 Derivation and Authentication and Purity 170 7.1.2 Cytogenetic Analysis 172 7.1.3 Differentiation 175 7.1.4 Androgen Receptor 179 7.1.5 3-Dimensional Cultures 181 8.0 REFERENCE LIST 187