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Influences of endocrine and autocrine factors in normal and neoplastic ovarian surface epithelium Choi, Kyung-Chul 2001

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INFLUENCES OF ENDOCRINE AND AUTOCRINE FACTORS IN NORMAL AND NEOPLASTIC OVARIAN SURFACE EPITHELIUM B Y KYUNG-CHUL CHOI D . V . M . , SEOUL N A T I O N A L UNIVERSITY, 1990 M . S c , SEOUL N A T I O N A L UNIVERSITY, 1992  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  IN THE FACULTY OF GRADUATE STUDIES REPRODUCTIVE & D E V E L O P M E N T A L SCIENCES P R O G R A M D E P A R T M E N T OF OBSTETRICS & G Y N E C O L O G Y THE UNIVERSITY OF BRITISH C O L U M B I A  W E A C C E P T THIS THESIS A S C O N F O R M I N G TO THE REQUIRED S T A N D A R D  THE UNIVERSITY OF BRITISH C O L U M B I A J U L Y 2001 © K Y U N G - C H U L CHOI, 2001  UBC  Special  Collections  - Thesis Authorisation  Form  Page 1 o f 1  In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make it f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n .  KYUKKT-CHOL Department of  C H O I  O B S & (xYrsI  The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  Date  Tuly \J*,  2-OQl  http://www.library.ubc.ca/spcoll/thesauth.html  17/07/01  ABSTRACT The common epithelial ovarian tumors appear to arise from the ovarian surface epithelium (OSE), which is a simple squamous-to-cuboidal meso^helium covering the ovary. The exact mechanism of ovarian tumorigenesis is not well known even though this disease is the most frequent cause of cancer death in gynecological malignancies. Repeated ovulation contributes to neoplastic transformation of OSE, indicating that the process of healing ruptured OSE may contribute to the disease. Therefore, it has been hypothesized that endocrine and autocrine factors may influence the occurrence of ovarian tumors in women. Recently, non-tumorigenic and tumorigenic immortalized OSE (IOSE) cells were generated by sequentially introducing simian virus 40 (SV40)-large T antigen (IOSE-29) and E-cadherin (IOSE-29EC) into normal OSE. These IOSE-29EC cells were found to be anchorage  independent  and formed  transplantable, invasive subcutaneous and intraperitoneal adenocarcinomas in SCID mice. Thus, two additional cell lines, designated IOSE-29EC/T4 and IOSE-29EC/T5 were established from tumors that arose in IOSE-29EC-inoculated SCID mice. This experimental culture model provides a unique system to examine the influences of endocrine and autocrine factors in OSE at progressive stages of neoplastic transformation. In the present study, the effects of activin, transforming growth factor (TGF)-(3, estradiol (E2), follicle-stimulating hormone (FSFf) and gonadotropin-releasing hormone-II (GnRH-II) were investigated in the growth-stimulation or -inhibition and regulation of apoptosis in normal and neoplastic OSE cells. Different levels of activin/inhibin and activin receptor isoforms were expressed in normal and neoplastic OSE cells. In addition, the altered expression of the activin/inhibin subunits, as well as the cell proliferative effect of activin observed in OVCAR-3 but not in normal OSE cells, indicate that activin may act as an autocrine regulator of neoplastic OSE progression. Interestingly, activin and TGF-(3 inhibited growth and induced apoptosis in early neoplastic (IOSE-29) and tumorigenic OSE (IOSE-29EC) cells. Furthermore, the antiapoptotic bcl-2 protein was down-regulated by TGF-p\ whereas no difference was observed in bax protein by activin or TGF-(3 treatment and in bcl-2 protein by activin. These results suggest that activin and TGF-P may play a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic stages of ovarian cancer. In terms of regulation of apoptosis by E2, it has been demonstrated that IOSE cell lines expressed both ERoc and ERp at the mRNA and  11  protein levels. In addition, treatment with E2 prevented tamoxifen induced-apoptosis through ERs. The mechanism of E2 action may be associated with up-regulation of bcl-2 gene at the mRNA and protein levels in IOSE-29EC cells. These results suggest that estrogen may play a role in ovarian tumorigenesis by preventing apoptosis in tumorigenic OSE cells. In addition, FSH receptor (FSH-R) was expressed and F S H induced a growth-stimulation in normal and neoplastic OSE cells. Interestingly, F S H stimulated the activation of the M A P K cascade and activated M A P K phosphorylated Elk-1 in neoplastic OSE cells. These results suggest that the M A P K cascade may be involved in cellular function such as growth stimulation in response to FSH in neoplastic OSE cells. GnRH-II mRNA is expressed in normal OSE, immortalized OSE (IOSE), ovarian tumors from the patients and ovarian cancer cell lines, suggesting that GnRH-II Q  7  exerts an autocrine/paracrine effect in these cells. Treatments with increasing doses (10" - 10" M) of GnRH-I and -II resulted in growth-inhibition and induction of apoptosis in IOSE-29 and IOSE-29EC cells. These results suggest that GnRH-II may be an integral regulator similar to GnRH-I in normal OSE physiology and may play a role in the induction of a growth-inhibitory response in neoplastic OSE cells. Taken together, these results suggest that these endocrine and autocrine factors may play a role in ovarian tumorigenesis in the regulation of growthstimulation or -inhibition and/or apoptosis of normal and neoplastic OSE cells via their specific receptors.  iii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  ix  LIST OF FIGURES  x  LIST OF A B B R E V I A T I O N S  xiii  LIST OF PUBLICATIONS  xvi  ACKNOWLEDGMENTS  xix  I. BACKGROUND  1  1. Ovarian surface epithelium  1  1.1. Prologue  1  1.2. Structure  2  1.3. Functions  5  1.4. Differentiation  7  1.5. OSE in culture  :  1.5.1. Culture methods  9 9  1.5.2. Extension of the life-span of OSE cells 1.6. Regulation by hormones, growth factors and cytokines  11 14  1.6.1. Gonadotropin-releasing hormone and gonadotropins  14  1.6.2. Steroids  15  1.6.3. Growth factors  15  1.6.3.1. Transforming growth factor-^ family  15  1.6.3.2. Epidermal growth factor (EGF) family  17  1.6.3. Cytokines  18  2. Epithelial ovarian carcinomas  18  2.1. Prologue  18  2.2. Genetic changes  19  2.3. Regulation by hormones, growth factors and cytokines  20  iv  2.3.1. Gonadotropin-releasing hormone (GnPvH)  '.  2.3.2. Gonadotropins 2.3.3. Sex steroids  20 22  1  24  2.3.4. Activin/inhibin".  ...27  2.3.5. Growth factors  29  2.3.5.1. Transforming growth factor-^  29  2.3.5.2. Epidermal growth factor and transforming growth factor-a  32  2.3.5.3. Hepatocyte growth factor  34  2.3.5.4. Insulin-like growth factors  35  2.3.5.5. Vascular endothelial growth factor  37  2.3.5.6. Other growth factors  38  2.3.6. Cytokines  39  2.3.7. Lysophosphatidic acid  41  2.4. Apoptosis and bcl-2 gene family  42  2.4.1. Apoptosis and cancer  42  2.4.2. Bcl-2 family  43  2.4.3. Regulation of apoptosis and bcl-2 gene family  47  2.5. Mitogen-Activated Protein Kinases 3. Rationale and Objectives  48 51  Specific aim 1 (EXPERIMENT A)  52  Specific aim 2 (EXPERIMENT B)  52  Specific aim 3 (EXPERIMENT Q  53  Specific aim 4 (EXPERIMENT D)  54  Specific aim 5 (EXPERIMENTE)  55  II. MATERIALS AND METHODS  57  1. Materials 2. Cell cultures  57 :  57  2.1. Normal OSE cells  57  2.2 Primary cultred ovarian tumors (PC-OVC) from the patients  58  2.3. Immortalized ovarian surface epithelium cell lines (IOSE cell lines)  59  v  2.4. Established epithelial ovarian cancer cell lines  59  3. Treatments  61  4. R N A extraction and RT-PCR procedures  62  5. Southern blot analysis  62  6. Cloning and sequencing  64  7. Northern blot analysis  66  8. Immunoblot analysis  66  9. [ H]thymidine incorporation assay  67  10. D N A fluorometric assay  70  3  11. Quantification of apoptotic cells  :  12. In vitro M A P K assay  71 r  71  13. RIA for intracellular cAMP  72  14. Statistical analysis  72  III. RESULTS 1. EXPERIMENT  73 A  73  1.1. Expression of activin/inhibin subunits  73  1.2. Expression of activin receptors  73  1.3. Effects of activin on cell proliferation  77  1.4. Effects of activin on activin/inhibin subunits and activin receptors  77  2. EXPERIMENTB  85  2.1. Expression of activin/inhibin subunit mRNAs  85  2.2. Expression of activin receptor mRNAs  85  2.3. Expression of activin receptor proteins  88  2.4. Effects of activin on cell number  88  2.5. Effects of TGF-(3 on cell proliferation  91  2.6. Effects of activin and TGF-P on apoptosis  91  vi  2.7. Expression of pro- and anti-apoptotic gene mRNAs  96  2.8. Effects of activin and TGF-p on apoptotic proteins  96  3. EXPERIMENT  C  100  3.1. Expression of ERa and ER(3 mRNAs  100  3.2. Expression of E R a and ERp proteins  100  3.3; Effects of E2 on cell proliferation  103  3.4. Effects of E2 on apoptosis  108  3.5. Expression of pro- and anti-apoptotic gene mRNAs and proteins  108  3.6. Effect of E2 on pro- and anti-apoptotic mRNAs  Ill  3.7. Effect of E2 on pro- and anti-apoptotic proteins  Ill  4. EXPERIMENTD  116  4.1. Expression of FSH-R mRNA  116  4.2. Effects of FSH on proliferative index  116  4.3. Expression of M A P K s in normal and neoplastic OSE cells  121  4.4. Effects of FSH and/or PD98059 on M A P K activation  122  4.5. Effects of FSH and/or P K C inhibitor staurosporin on M A P K activation  126  4.6. Effects of FSH and/or PD98059, staurosporin on M A P K activation  131  4.7. Effect of FSH and/or. PD98059 on Elk-1 phosphorylation  131  4.8. Effects of M A P K and P K C inhibitors on FSH-stimulated cell growth  134  4.9. Effects of FSH on intracellular cAMP accumulation  134  5. EXPERIMENTS  137  5.1. Expression of GnRH-II mRNA  137  5.2. Expression of GnRH-R mRNA  137  5.3. Effects of GnRH-I and -II on proliferative index  137  5.4. Effects of GnRH-I and -II on apoptosis  140  5.5. Effects of GnRH-I and -II on the regulation of bax and bcl-2 proteins  144  vii  IV. DISCUSSION  146  1. Experiment A  146  2. Experiment B  150  3. Experiment C  155  4. Experiment D  160  5. Experiment E  165  V. S U M M A R Y A N D F U T U R E STUDIES  ...168  1. Summary  168  2. Future Studies  174  VI. B I B L I O G R A P H Y  177  viii  LIST OF TABLES TABLE 1. Development of early neoplastic, tumorigenic and late neoplastic OSE cells from human normal OSE  60  TABLE 2. Oligonucleotide sequences of PCR primers for human  63  ix  LIST OF FIGURES Figure 1. Morphology of normal OSE and inclusion cyst (IQ  3  Figure 2. Epithelio-mesenchymal conversion of OSE cells  10  Figure 3. Morphology of OSE in culture  12  Figure 4. Summary of anti-apoptotic and pro-apoptotic bcl-2 family members  44  Figure 5. Activation of apoptotic pathway through homodimerization (bax/bax) of pro-apoptotic bcl-2 gene family, release of cytochrome-c, and activation of caspases  46  Figure 6. Signal-transduction pathways of receptors or stress-activated M A P K s . .  50  Figure 7. A standard curve for the protein assay  68  Figure 8. Differential expression level of activin/inhibin subunits in normal OSE and OVCAR-3 cells.  74-75  Figure 9. Differential expression level of type II activin receptors in normal OSE and OVCAR-3 cells.  76  Figure 10. Effects of rh-activin A on OVCAR-3 cell proliferation  78  Figure 11. Effect of rh-activin A on a subunit expression in OVCAR-3 cells  79  Figure 12. Effect of rh-activin A on p\A subunit expression in OVCAR-3 cells  80  Figure 13. Effect of rh-activin A on  81  subunit expression in OVCAR-3 cells  Figure 14. Effects of rh-activin A on type II activin receptors expression in OVCAR-3 cells  83  Figure 15. A time-dependent increase of a and (3B subunit by rh-activin A in OVCAR-3  84  Figure 16. The mRNA expressions of activin/inhibin subunits in IOSE cell lines  86  Figure 17. The mRNA expressions of activin receptors in IOSE cell lines  87  Figure 18. The expression of activin receptor proteins in IOSE cell lines  89  Figure 19. Effect of activin on cell proliferation in IOSE cell lines  90  x  ' Figure 20. Effects of TGF-p on cell proliferation in IOSE cell lines  92-93  Figure 21. Effect of activin in the induction of apoptosis  94  Figure 22. Effect of TGF-p in the induction of apoptosis  95  Figure 23. The expression of bax and bcl-2 mRNAs in IOSE cell lines  97  Figure 24. Effect of activin on bax and bcl-2 proteins  98  Figure 25. Effect of TGF-p on bax and bcl-2 proteins  99  Figure 26. The mRNA levels of E R a and ERp in IOSE cell lines  101  Figure 27. The protein levels of E R a and ERp in IOSE cell lines  102  Figure 28. Effect of E2 on cell proliferation/apoptosis in normal OSE and OVCAR-3 cells  104  Figure 29. Effect of E2 on cell proliferation/apoptosis in IOSE-29 cells  105  Figure 30. Effect of E2 on cell proliferation/apoptosis in IOSE-29EC cells  106  Figure 31. Effect of E2 on cell proliferation/apoptosis in IOSE-29EC/T4 and /T5  107  Figure 32. Effect of E2 on apoptosis in IOSE-29EC cells  109  Figure 33. Expression of bcl-2 protein in IOSE-29 and IOSE-29EC cells  110  Figure 34. Effect of E2 on bax and bcl-2 mRNA levels in IOSE-29EC cells  112  Figure 35. Effect of E2 on bcl-2 mRNA level in IOSE-29EC cells  113  Figure 36. Effect of E2 on bax and bcl-2 proteins in IOSE-29EC cells  114  Figure 37. Effect of E2 on bcl-2 protein in IOSE-29EC cells  115  Figure 38. Expression of FSH-R mRNA in normal OSE and IOSE cell lines  117  Figure 39. Expression of FSH-R mRNA in IOSE and ovarian cancer cell lines  118  Figure 40. Effects of FSH on cell proliferation in normal OSE and O V C A R - 3 cells  119  Figure 41. Effects of FSH on cell proliferation in IOSE-29 and IOSE-29EC cells  120  Figure 42. Basal expression level of P - M A P K and T - M A P K in normal and neoplastic OSE cells  123  Figure 43. Effects of FSH in the presence or absence of PD98059 on M A P K activation IOSE-29 cells (dose-dependent)  124  xi  Figure 44. Effects of FSH in the presence or absence of PD98059 on M A P K activation IOSE-29EC cells (dose-dependent)  125  Figure 45. Effects of FSH in the presence or absence of PD98059 on M A P K activation in IOSE-29 cells (time-dependent)  127  Figure 46. Effects of FSH in the presence or absence of PD98059 on M A P K activation in IOSE-29EC cells (time-dependent)  128  Figure 47. Effects of FSH in the presence or absence of staurosporin on M A P K activation in IOSE-29 cells  129  Figure 48. Effects of FSH in the presence or absence of staurosporin on M A P K activation in IOSE-29EC cells  130  Figure 49. Effects of FSH in the presence or absence of PD98059 or staurosporin on M A P K activation in ovarian cancer cell lines  132  Figure 50. Effect of FSH in the presence or absence of PD98059 on Elk-1 phosphorylation  133  Figure 51. Effects of M A P K and P K C inhibitors on FSH-stimulated cell growth  135  Figure 52. Effect of FSH on intracellular c A M P accumulation  136  Figure 53. Expression of GnRH-II mRNA in normal and neoplastic OSE cells  138  Figure 54. Expression of GnRH-R mRNA in IOSE cell lines and O V C A R - 3 cells  139  Figure 55. Effects of GnRH-I on proliferative index in IOSE cell lines  141  Figure 56. Effects of GnRH-II on proliferative index in IOSE cell lines  142  Figure 57. Effects of GnRH-I and -II on apoptosis  143  Figure 58. Effects of GnRH-I and -II on the regulation of bax and bcl-2 proteins  145  Figure 59. Proposed intracellular signaling cascades of activin, TGF-p\ E2, FSH....  173  and GnRH-II in neoplastic OSE cells  xii  LIST OF ABBREVIATIONS ANOVA AR ATP BFGF  BH1-4 bp C Ca cAMP cDNA 2 +  c-fms cGMP Ci Cpm DDT DEPC DHT DNTP DNA DNase EDTA  E2 EGF • EGF-R ELISA ER E R o/p ERE  ERK1/2 Fas L FBS FSH FSH-R G GDP GnRH GnRHa GnRH-H GnRH-R G-protein GPCR G-CSF GM-CSF  Analysis o f variance Androgen receptor Adenosine 5'-triphosphate Basic fibroblast growth factor Bcl-2 homology regions 1-4 Base pairs Celcius Calcium Cyclic adenosine monophosphate Complementary deoxyribonucleic acid Receptor for colony stimulating factor-1 Cyclic guanosine monophosphate Curie Counts per minute Dithiothreitol Diethylpyrocarbonate 5a-dihydrotestosterone Deoxynucleoside triphosphate Deoxyribonucleic acid Deoxyribonuclease Ethylene diaminetetraacetic acid 17p-estradiol Epidermal growth factor E G F receptor Enzyme-linked immunosorbant assay Endoplasmic reticulum Estrogen receptor a/p Estrogen response element Extracellular signal-regulated kinase 1/2 Fas ligand Fetal bovine serum Follicle-stimulating hormone F S H receptor Acceleration o f gravity Guanosine diphosphate Gonadotropin-releasing hormone Gonadotropin-releasing hormone agonist Gonadotropin-releasing hormone-II Gonadotropin-releasing hormone receptor GTP-binding protein G-protein coupled receptors Granulocyte colony-stimulating factor Granulocyte-macrophage colony stimulating factor  xiii  GTP H HBSS HCG HGF HGF-R HGLCs HMG IC ICE IGF IGFBP IGF-R IL IOSE IP IP3  IU JNK/SAPK Kb KDa LH LH-R LMP LP A mAb M-CSF MAPK M A P K K s (=MKK) MAPKKKs MAPKKKKs MEK1/2  Ml Min MMP MRNA Mw n (as in nM) OCAF OSE p (as in pM) P4  PAGE PBS  Guanosine triphosphate Hour Hank's balanced salt solution Human chorionic gonadotropin Hepatocyte growth factor HGF receptor Human granulosa-luteal cells Human menopausal gonadotropin Inclusion cyst Interleukin-lp (IL-1P) converting enzyme Insulin-like growth factor Insulin-like growth factor binding protein IGF receptor Interleukin Immortalized ovarian surface epithelium Inositol phosphate Inositol 1, 4 , 5-triphosphate International unit c-jun terminal kinase/stress-activated protein kinases Kilobase Kilodaltons Luteinizing hormone L H receptor Low malignant potential Lysophosphatidic acid Micro Monoclonal antibody Macrophage colony-stimulating factor Mitogen-activated protein kinase M A P K kinases M A P K K kinases M A P K K K kinases M A P K / E R K kinase 1/2 Mililiters Minutes Matrix metalloproteinases Messenger ribonucleic acid Molecular weight Nano Ovarian cancer activating factor Ovarian surface epithelium Pico Progesterone Polyacrylamide gel electrophoresis Phosphatase buffered saline  xiv  PCD PCR PD-ECGF PDGF PDGF-R PGF2oc PI PIP PIP 2  PIP3  PI3K PKA PKC PLA PLC PLD PMA PMSF PR RIA rpm RT RT-PCR SCID sec SD SDS Taq TCF TE TEMED TGF-a TGF-p TpRII TIMP TM TNF-a Tris Txf UV VEGF VPF v/v w/v  Programmed cell death Polymerase chain reaction Platelet-derived endothelial cell growth factor Platelet-derived growth factor PDGF receptor Prostaglandin F2oc Phosphatidylinositol Phosphatidylinositol 4-phosphate Phosphatidylinositol 4, 5-phosphate Phosphatidylinositol 3,4,5-triphosphate Phosphatidyl inositol 3 kinase Protein kinase A Protein kinase C Phospholipase A Phospholipase C Phospholipase D Phorbol 12-myristate 13-acetate Phenylmethylsulfonyl fluoride Progesterone receptor Radioimmunoassay Revoultions per min Room temperature Reverse transcription polymerase chain reaction Severe combined immunodeficient Seconds Standard deviation Sodium dodecyl sulphate Thermus aquaticus, source of a D N A polymerase Ternary complex factor Tris-EDTA N , N , N ' , N'-tetramethylethlenediamine Transforming growth factor-a Transforming growth factor-p TGF-P receptor Tissue inhibitor of metalloproteinase Transmembrane Tumor necrosis factor-a Trisfhydroxy methyl) aminomethane Tamoxifen Ultraviolet Vascular endothelial growth factor Vascular permeability factor/ Volume per volume Weight per volume  XV  LIST OF PUBLICATIONS PEER-REVIEWED PAPERS 1. Choi K-C, Kang SK, Nathwani PS, Cheng K W , Auersperg N , Leung P C K 2001 Differential expression of activin/inhibin subunit and activin receptor mRNAs in normal and neoplastic ovarian surface epithelium (OSE). Mol Cell Endocrinol 174:99-110 2. Choi K-C, Kang SK, Tai C-J, Auersperg N , Leung P C K 2001 The regulation of apoptosis by activin and TGF-P in early neoplastic and tumorigenic ovarian surface epithelium (OSE). J Clin Endcrinol Metab 86:2125-2135 3. Choi K-C, Kang SK, Tai C-J, Auersperg N , Leung P C K 2001 Estradiol up-regulates antiapoptotic bcl-2 mRNA and protein in tumorigenic ovarian surface epithelium (OSE). Endocrinology 142:2351-2360 4. Choi K-C, Kang SK, Tai C-J, Auersperg N , Leung P C K 2001 Follicle stimulating hormone (FSH) activates mitogen-activated protein kinases (MAPKs) in neoplastic ovarian surface epithelium (OSE) cells. J Clin Endcrinol Metab Submitted 5. Choi K-C, Auersperg N , Leung P C K 2001 Expression and role of second form of gonadotropin-releasing hormone (GnRH) in normal and neoplastic ovarian surface epithelium (OSE) cells. J Clin Endcrinol Metab In press 6. Choi K-C, Tai C-J, Auersperg N , Leung P C K 2001 Adenosine triphosphate (ATP) activates mitogen-activated protein kinases (MAPKs) in neoplastic ovarian surface epithelium (OSE) cells. Endocrinology Submitted 7. Kang SK, Choi K-C, Cheng K W , Nathwani PS, Auersperg N , Leung P C K 2000 Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium. Endocrinology 141:72-80 8. Kang SK, Choi K-C, Tai C-J, Auersperg N , Leung P C K 2001 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and modulates the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology 142:580-588 9. Tai C-J, Choi K-C, Fluker M , Leung P C K 2001 Extracellular adenosine triphosphate induces apoptosis in human granulosa-luteal cells. J Clin Endcrinol Metab Submitted 10. Auersperg N , Wong AST, Choi K-C, Kang SK, Leung P C K 2001 Ovarian surface epithelium: biology, endocrinology and pathology. Endocr Rev 22:255-288 11. Tai C-J, Kang SK, Choi K-C, Leung P C K 2001 Prostaglandin F 2 a activates mitogenactivated protein kinase in human granulosa-luteal cells. J Clin Endcrinol Metab 86:375-380  xvi  12. Tai C-J, Kang SK, Choi K-C, Tzeng C-R, Leung P C K 2001 Antigonadotropic action of ATP in human granulosa-luteal cells: Involvement of PKCoc. J Clin Endcrinol Metab In press 13. Kang SK, Cheng K W , Nathwani PS, Choi K-C, Leung P C K 2000 Autocrine role of gonadotropin-releasing hormone (GnRH) and its receptor in ovarian cancer cell growth. Endocrine 13: 297-304 14. Nathwani PS, Kang SK, Cheng K W , Choi K-C, Leung P C K 2000 Regulation of gonadotropin-releasing hormone (GnRH) and its receptor gene expression by 17(3-estradiol in cultured human granulosa-luteal cells. Endocrinology 141:1754-1763 15. Tai C-J, Kang SK, Cheng K W , Choi K-C, Nathwani PS, Leung P C K 2000 Expression and regulation of purinergic receptor (P2UR) in human granulosa-luteal cells. J Clin Endo Metab 85:1591-1597 16. Kang SK, Tai C-J, Nathwani PS, Choi K-C, Leung P C K 2001 Stimulation of mitogenactivated protein kinase by gonadotropin-releasing hormone in human granulosa-luteal cells. Endocrinology 142:671-679 17. Kang SK, Cheng K W , Ngan ESW, Chow B K C , Choi K-C, Leung P C K 2000 Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. Mol Cell Endocrinol 162:157-166  A B S T R A C T S A N D O R A L PRESENTATIONS 1. Choi K-C, Kang SK, Nathwani PS, Cheng K W , Tai C-J, Auersperg N , Leung P C K (Poster Presentation) The expression levels of activin/inhibin subunits and activin receptors: Autocrine function of activin in ovarian surface epithelium and ovarian cancer. 1999 Health Sciences Student Research Forum Poster Presentation, Woodward Instructional Resources Center, University of British Columbia, Vancouver, B C , Canada (Oct 13, 1999) 2. Choi K-C, Kang SK, Tai C-J, Auersperg N , Leung P C K (Oral Presentation) Endocrine influences on normal and neoplastic ovarian surface epithelium (OSE) cell growth. Fertil Steril 74 (3S): S5 3. Choi K-C, Tai C-J, Auersperg N , Leung P C K (Poster Presentation) Estradiol up-regulates anti-apoptotic bcl-2 mRNA and protein in tumorigenic ovarian surface (OSE) cells. UBC/C&W Student Research Forum Poster Presentation, Children's & Women's Health Center of British Columbia, Poster #13 (March 5, 2001) 4. Tai C-J, Kang SK, Choi K-C, Leung P C K (Oral Presentation) Prostaglandin F2oc activates mitogen-activated protein kinase in human granulosa-luteal cells. Fertil Steril 74 (3S): S2S3  xvn  5. Choi K-C, Auersperg N , Leung P C K (Oral Presentation) Estradiol up-regulates antiapoptotic bcl-2 mRNA and protein in tumorigenic ovarian surface (OSE) cells. UBC/Dept of OB & GYNResearch Day Presentation, Chan Auditorium, B C Women's Hospital (April 19, 2001) 6. Tai C-J, Kang SK, Choi K-C, Leung P C K (Poster Presentation) Role of mitogen-activated protein kinase in prostaglandin F 2 a action in human granulosa-luteal cells. UBC/C&W Student Research Forum Poster Presentation, Children's & Women's Health Center of British Columbia, Poster #47 (March 5, 2001) 7. Kang SK, Cheng K W , Choi K-C, Leung P C K (Poster Presentation) Combinations of cellspecific factors may explain the differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. UBC/Dept of OB & GYN Research Day Presentation, Chan Auditorium, B C Women's Hospital (April 19, 2001) 8. Tai C-J, Cheng K W , Nathwani PS, Choi K-C, Leung P C K (Poster Presentation) The role of protein kinase C in regulating ATP-evoked intracellular calcium oscillations and enhancing progesterone production in cultured human granulosa-luteal cells. Biology of Reproduction. 60(Supp 1): 131, 1999 9. Tai C-J, Kang SK, Cheng K W , Choi K-C, Nathwani PS, Leung P C K (Oral Presentation) Expression of purinergic receptor (P2UR) in human granulosa-luteal cells. ASRM/CFAS '99 Annual Meeting Program Supplement S-47, 1999  AWARDS 1. Excellent Student Scholarship of Seoul National University (Sep. 1986 -Feb. 1990) 2. Seoul National University Alumni Association Scholarship (Mar. 1990-Feb. 1992) 3. Graduate Student Travel Award from U B C for A S R M Annual Meeting (Oct. 21-26 2000) 4. Gynecological Oncology Research Award of U B C Department of Obstetrics and Gynaecology Residents' Research Day (Apr. 19 2001) th  th  xviii  ACKNOWLEDGMENTS M y deepest gratitude and appreciation goes to my supervisor, Dr. Peter C.K. Leung for his supervision and resources throughout my studies. His advice and guidance have proven valuable to my research and future career pursuits. In addition, I really appreciate to Dr. N. Auersperg for giving a lot of advice and guidance through all studies. I am always encouraged by her enthusiasm, wisdom and perseverance in the research. I would like to extend my sincere gratitude to the supervisory committee members, Drs. A. Perks, C. Roskelley, A. Cheung for their directions and criticisms for this thesis. Furthermore, my great thanks are extended to the final examiners, Drs. Y. S. Moon, B. Verchere, M . Bally and M . H . Melner (School of Medicine, Vanderbilt University) for their criticisms and revisions for the thesis.  I am thankful to Ms. S. Maines-Bandiera and Ms. C. Salamanca for providing human normal and immortalized OSE cells. In addition, I wish to thank members of the Department of Obstetrics and Gynecology, especially, Drs. E . Mitchell, T. Ehlen, J. Pikes, M . Bertrand, D.M. Miller for their cooperation in providing surgical specimens of the ovarian surface epithelium and primary tumors of ovarian cancer from patients. I offer my appreciation to my colleagues, S.K. Kang, C-J. Tai, M . Woo, K.W. Cheng, P.S. Nathwani, and other members of Dr. Leung's and Auersperg's labs for encouragements and instructive suggestions. I will never forget what we shared and what we learned from each other during Ph.D course. I must also acknowledge Ms. R. Nair and Ms. M-A Rampf for their administrative and secretarial help.  A grateful acknowledgement must be extended in particular to my lovely family, Sung-Sook (Farrah), Joon-Bin (Kelvin) and Yoon-Bin (Robin) for their encouragement, support, and love throughout my studies. Without their sacrifice and love, this work would not be finished and completed. As well as my family, this thesis is dedicated to my parents and parent-in-laws in Korea who unlimitedly supported me to follow my dreams and who were my source of strength and inspiration.  I also wish to express my gratitude to the Canadian Institutes of Health Research and National Cancer Institute of Canada, which provided financial support during these studies.  xix  I. BACKGROUND  1. Ovarian surface epithelium 1.1. Prologue The ovarian surface epithelium (OSE), also referred to in the literature as ovarian mesothelium (OM) (Nicosia et al, 1991; 1997), is the modified pelvic mesothelium that covers the ovary. It is composed of a single layer of flat-to-cuboidal epithelial cells with few distinguishing features (Nicosia et al, 1991). The OSE used tp be referred to as the 'germinal epithelium' as it was once mistakenly believed that it could give rise to new germ cells. Since this hypothesis was disclaimed, ovarian research has centered on the components of the ovary that carry out its important and highly complex endocrine and reproductive functions. Interest in the OSE revived when it became apparent that approximately 90% of human ovarian cancers, epithelial ovarian carcinomas, might arise in the OSE (Auersperg et al., 1998; Herbst, 1994 Nicosia et al, 1991). The implication of OSE as the source of epithelial ovarian cancers was questioned  (Dubeau,  1999)  because  it  was  based  mainly  on  histopathologic  and  immunocytochemical observations. Animal models were not available because, except in aging hens (Fredrickson, 1987), ovarian tumors in species other than human do not arise in OSE but in follicular, stromal or germ cells, and the biology of these tumors is fundamentally different from that of epithelial ovarian cancer. Because of the resulting lack of experimental models, the etiology and early events in ovarian carcinogenesis are still among the least understood of all major human malignancies. The first tissue culture systems for OSE from human (Auersperg et al., 1984; Siemens and Auersperg, 1988) were developed. Subsequently, information about the normal functions of OSE and its relationship to ovarian cancer expanded rapidly and, recently, 1  the capacity of cultured OSE to give rise to ovarian adenocarcinomas was demonstrated experimentally (Auersperg et al, 1999; Ong et al, 2000). The results of these studies indicate that OSE is physiologically much more complex than would be predicted from its inconspicuous appearance, and they support the hypothesis that the ovarian epithelial cancers arise in this simple epithelium.  1.2. Structure In the mature woman, OSE is an inconspicuous monolayered squamous-to-cuboidal epithelium (Fig. 1). It is characterized by the keratin types 7, 8, 18 and 19 which represent the keratin complement typical for simple epithelia. It expresses mucin antigen M U C 1 , 17(3hydroxysteroid dehydrogenase and cilia, which distinguish it from extraovarian mesothelium, apical microvilli and basal lamina (Auersperg et al, 1994; Blaustein and Lee 1979; Siemens and Auersperg 1988). Intercellular contact and epithelial integrity of OSE are maintained by simple desmosomes, incomplete tight junctions (Siemens and Auersperg 1988), several integrins (Cruet et al, 1999; Kruk et al, 1994) and cadherins (Davies et al, 1998; Sundfeldt et al, 1997). The OSE is separated from the ovarian stroma by a basement membrane and, underneath, by a dense collagenous connective tissue layer, the tunica albuginea, which is responsible for the whitish color of the ovary. It is thinner and less resilient than the tunica albuginea in the testis, but likely provides a partial barrier to the diffusion of bioactive agents between the ovarian stroma and the OSE. The OSE differs from all other epithelia by its tenuous attachment to its basement membrane, from which it is easily detached by mechanical means. Until recently, the resulting loss of OSE in surgical specimens was responsible for the widely held opinion that OSE is frequently absent in ovaries of older women.  2  Figure 1. Morphology o f normal O S E and inclusion cyst (ic). Section through a normal adult ovarian cortex, showing O S E on top as a cuboidal monolayer and an epithelial inclusion cyst lined with O S E . The inset illustrates an inclusion cyst that has undergone tubal metaplastic changes as indicated by the densely arranged, columnar epithelial cells. Hematoxylin & eosin, X 80 (Auersperg et al, 2001).  3  Whether this loose attachment has any physiological consequences is not known. With age, the human ovary assumes increasingly irregular contours and forms OSE-lined surface invaginations (clefts) and epithelial inclusion cysts in the ovarian cortex. It has been suggested that the squamous and cuboidal forms of OSE cells on the ovarian surface represent cell groups that, respectively, have or have not undergone postovulatory proliferation (Gillett et al., 1991). In addition, OSE cells tend to assume columnar shapes, especially within clefts and inclusion cysts. Whether these shape changes are the result or crowding of whether they reflect genetically determined metaplastic changes is not always clear, but they may be derived by either process. The importance of surface invaginations and inclusion cysts lies in the propensity of the OSE in these regions to undergo metaplastic changes, i.e. to take on phenotypic characteristics of Mullerian (usually tubal) epithelium, which include columnar cell shapes and several markers found in ovarian neoplasms, including CA125 and E-cadherin (Maines-Bandiera and Auersperg 1997; Mittal et al, 1995; van Niekerk et al, 1991; Sundfeldt et al, 1997) Furthermore, OSElined clefts and inclusion cysts, rather than surface OSE, are not only common sites of benign metaplasia but also of early neoplastic progression (Deligdisch et al, 1995; Scully, 1995a; 1995b). It has been suggested that the inclusion cysts form from OSE fragments that are trapped in or near ruptured follicles at the time of ovulation (Murdoch, 1994). However, inclusion cysts have been reported to be more numerous in ovaries of multiparous women than in nulliparous women who ovulate more frequently, and the cysts are particularly numerous in women with polycystic ovarian disease, a condition which is characterized by anovulation or infrequent ovulation (Scully, 1995b), proposing as an alternative that inclusion cysts arise through inflammatory adhesions of surface OSE which becomes apposed at sites of surface invaginations, combined with localized stromal proliferation.  4  There is currently no definitive explanation for the predilection of inclusion cysts as preferred sites of neoplastic progression of OSE but these preferential locations strongly suggest the presence of specific tumor-promoting microenvironmental factors in these sites. Two different scenarios can be envisaged: (1) OSE within inclusion cysts is not separated from underlying stroma by the tunica albuginea. Therefore, this OSE likely has more access to stromally derived growth factors and cytokines as well as to blood-born bioactive agents which may promote neoplastic progression. This hypothesis is supported by the observation that, in inclusion cysts located near the ovarian surface, metaplastic and dysplastic changes tend to be more pronounced on the side near the stroma than on the side adjacent to the tunica albuginea (Scully, 1995a; 1995b). (2) Neoplastic progression in OSE-lined cysts and clefts may be promoted by autocrine mechanisms through OSE-derived cytokines and hormones, since these agents may accumulate to bioactive levels in such confined sites but not on the ovarian surface where they diffuse into the pelvic cavity. The hypothesis that these factors participate in autocrine loops is supported by the capacity of normal OSE to secrete bioactive cytokines including interleukin (IL)-l and IL-6 (Ziltener et al., 1993) and by reports that IL-1 and IL-6 enhance the proliferation of ovarian carcinomas (Berchuck et al., 1993), and that IL-1 causes changes in gene expression including the induction of tumor necrosis factor (TNF)-oc which is a mitogen for OSE (Wu et al., 1992; 1993). Within inclusion cysts, such cytokines and hormones might act as immediate autocrine growth regulators, or they might cause secondary changes in gene expression which promote neoplasia.  1.3. Functions The OSE transports materials to and from the peritoneal cavity and takes part in the cyclical ovulatory ruptures and repair. Most of these functions vary with the reproductive cycle and thus  5  are likely hormone dependent (Nicosia et al, 1991; Osterholzer et al, 1985). There have been several reports based on electron microscopy and histochemistry, suggesting that the OSE contains lysosome-like inclusions and produces proteolytic enzymes which may contribute to follicular rupture (Bjersing and Cajander, 1975). These reports were supported by direct observations of protease secretion by cultured OSE (Kruk et al, 1994). However, this concept has been questioned because of inconsistencies in the timing of the appearance of the dense lysosome-like granules in the OSE, their biochemical nature, and the observation that follicles denuded of overlying OSE can also rupture (Espey and Lipner, 1994). Furthermore, electron microscopy in various species has revealed that OSE cells degenerate and slough off the follicular surface shortly prior to ovulatory rupture. There is evidence that this cyclic, localized loss of OSE near the time of ovulation is due to apoptosis which is induced by prostaglandins (Ackerman and Murdoch, 1993; Murdoch, 1995) and perhaps mediated by the Fas antigen (Baldwin et al, 1999; Quirk et al, 1997). It is possible that, as the tunica albuginea in the area of the stigma thins and ultimately disappears prior to ovulation, the OSE in this region is exposed to stromal influences which induce apoptosis. However, the possibility cannot be ruled out that the OSE alters the tunica albuginea and underlying stroma in the area of incipient ovulation just prior to its disappearance. The proteolytic capacity of OSE might contribute to the remodeling of the ovarian cortex, as well as the breakdown. OSE likely also takes part in the restoration of the ovarian cortex by the synthesis of both epithelial and connective tissue-type components of the extracellular matrix (Auersperg et al, 1994; Kruk et al, 1994; Kruk and Auersperg, 1994) and by its contractile activity which resembles the contractile capacity exhibited by connective tissue fibroblasts during wound healing (Kruk and Auersperg, 1992). Like fibroblasts, which convert to myofibroblasts when engaged in tissue repair, OSE cells in culture contain smooth muscle actin.  6  This is in keeping with their dual epithelio-mesenchymal phenotype, and with the proposition that OSE cells, like many other cell types, acquire a regenerative rather than stationary phenotype when they are explanted into culture. Contraction by OSE cells may also play a role in the shrinkage of the ovaries that occurs with age and results in their typical convoluted shape and the formation of the OSE-lined clefts and inclusion cysts.  1.4. Differentiation Normal OSE covering a non-ovulating ovary is a stationary mesothelium with both epithelial and mesenchymal characteristics. In contrast to mesothelia, OSE retains the capacity to alter its state of differentiation along pathways leading either to stromal, or to epithelial phenotypes. In response to stimuli that initiate a regenerative (repair) response, such as ovulatory rupture in vivo or explantation into culture, OSE cells assume phenotypic characteristics of stromal cells. Alternatively, OSE acquires complex epithelial characteristics of the Mullerian duct derived epithelia, i.e. of the oviduct, endometrium and endocervix, when it undergoes metaplasia, benign tumor formation, and neoplastic progression. Together, these characteristics show that the differentiation of OSE is not as firmly determined as in other adult epithelia and that OSE is closer to its pleuripotential mesodermal embryonic precursor form than other coelomic epithelial derivatives. Normal stationary OSE has no known tissue-specific differentiation markers. In situ, it can be distinguished from extraovarian mesothelium by the lack of CA125 and by the differential expression of mucin, cilia, 17(3-hydroxysteroid dehydrogenase and several antigenic markers (Auersperg et al, 1998; van Niekerk et al, 1989; 1991; Zeimet et al, 1998). It has classical epithelial features which include desmosomes, tight junctions, basement membrane, keratin and  7  apical microvilli, but other aspects of epithelial differentiation are less defined. For example, Ecadherin and CA125 in human OSE are rare while both markers occur in oviductal and endometrial epithelium, and CA125 is also secreted by extraovarian pelvic mesothelium and by abdominal and pleural peritoneum (Nicosia et al, 1991; van Niekerk et al., 1989; Zeimet et al., 1998). OSE cells also constitutively coexpress keratin with vimentin which is a mesenchymal intermediate filament, expressed by most epithelial cells only in response to wounding, explantation into culture or pathological conditions (Gilles et al., 1999; Hornby et al., 1992). Expression of the connective tissue collagen types I and III has been shown in cultured OSE, but not in situ (Auersperg et al., 1994). During postovulatory repair and in culture, OSE cells have the ability to modulate to a fibroblast-like form which reflects their close developmental relationship to ovarian stromal cells. The exact mechanisms regulating this conversion have not been defined. However, epidermal growth factor (EGF), collagen substrata and ascorbate are all conducive to epitheliomesenchymal conversion of OSE in culture. In addition, transforming growth factor (TGF)-(3, which is an autocrine regulator of OSE growth (Berchuck et al., 1992), causes epitheliomesenchymal conversion in a number of epithelial cell types (Toda et al., 1997). Similar epithelio-mesenchymal conversions occur in vivo in mesodermally derived cell types closely related to OSE, such as pleural mesothelial cells responding to injury (Davila and Crouch, 1993). This capacity of OSE to undergo epithelio-mesenchymal conversion likely confers advantages during the post-ovulatory repair of the ovarian surface: it increases motility, alters proliferative responses and capacities to modify extracellular matrix, and renders the cells contractile. Epithelio-mesenchymal conversion might also function as a homeostatic mechanism to accommodate OSE cells that become trapped within the ovary at ovulation, to allow them to  8  become incorporated into the ovarian stroma as stromal fibroblasts. As a related hypothesis, an inability to undergo epithelio-mesenchymal conversion would preserve the epithelial forms within the ovarian stroma which could lead to OSE cell aggregation and subsequent inclusion cyst formation (Fig. 2). In contrast to epithelio-mesenchymal conversion which is part of normal OSE physiology, the differentiation of metaplastic and neoplastic OSE along the lines of Mullerian duct-derived epithelia is clearly a pathological process, based on complex epigenetic and genetic changes.  1.5. OSE in culture 1.5.1. Culture methods The detailed procedures used for isolating and culturing normal human OSE were summarized previously (Kruk et al., 1990). Briefly, specimens for culture are obtained from overtly normal ovaries at surgery for nonmalignant gynecological diseases. Fragments of OSE are gently scraped from the ovarian surface with a rubber scraper or with the blunt side of a scalpel or other suitable instrument and immediately placed into sterile culture medium, taking care that the tissue remain sterile and does not dry, which happens very rapidly. OSE is also very loosely attached to the underlying ovarian cortex and is easily lost by excessive handling. If the surgery involves the removal of the ovaries, the OSE is obtained either by the surgeon while the ovaries are still in situ, or by a member of the research team after removal from the patient. OSE can also be obtained by the surgeon laparoscopically at the time of minor gynecologic procedures which are carried out by this approach. The OSE fragments are cultured in medium 199:MCDB 105 (1:1) with 15% fetal bovine serum (FBS). In addition, either 50 Lig/ml gentamicin or 100 ixg/ml of penicillin/streptomycin is added for the first few weeks.  9  Ovulation  Incl. cyst with dysplasia Figure 2. Epithelio-mesenchymal conversion o f O S E cells may represent a homeostatic mechanism to incorporate cells that have been displaced from the ovarian surface into the stroma. If such conversion does not take place, the cells are more likely to form epithelial inclusion cysts which are preferred sites o f neoplastic progression. Diagram outlining two paths by which O S E is displaced into the ovarian cortex. O S E fragments are displaced into or near the ruptured follicle at ovulation. O S E also lines surface invaginations, or clefts, which form as the ovary ages. If O S E cells undergo epithelio-mesenchymal conversion, they may migrate into, and become part o f the stroma (str). Alternatively, the cells remain epithelial, aggregate (aggr) and form inclusion cysts (incl cyst). Cysts may also form through the pinching off o f surface clefts. Inclusion cysts are preferred sites o f metaplastic and dysplastic changes which may lead to tumorigenesis. Importantly, the capacity o f O S E to undergo epithelio-mesenchymal conversion is greatly reduced with malignant progression and, to a lesser degree, i n women with a genetic predisposition to develop ovarian cancer (Auersperg et al, 2001).  10  The cultures are left undisturbed for at least 4 days, grown to confluence and then routinely passaged and split 1:3 when confluent, with 0.06% trypsin (1:250) and 0.01% EDTA. The cultures usually proliferate for 3-4 passages (1:3 splits) and then senesce. They are defined as senescent i f they are composed of large flat cells that do not reach confluence over one month. OSE cells in low passage culture can undergo epithelio-mesenchymal conversion, which tends to extend their life span by a few passages (Fig. 3)(Auersperg et al, 1994). Reduced-serum, and serum-free media were designed for human OSE and used to study mitogenic effects of growth factors and hormones (Elliott and Auersperg, 1993). Markers to distinguish OSE from cell contaminants in culture include keratins 7, 8, 18 and 19 which distinguish OSE from other ovarian cell types (van Niekerk et al., 1991), 17P-OH steroid dehydrogenase and mucin, which distinguish it from extraovarian mesothelial cells, laminin, which together with keratin distinguishes OSE from stromal fibroblasts; and the absence of factor VIII and Ulex lectin receptors which distinguish OSE from the morphologically similar endothelial cells (Auersperg et al., 1994; Nicosia et al, 1991; Siemens and Auersperg, 1988).  1.5.2. Extension of the life-span of OSE cells One of the problems in human OSE research is the small number and short lifespan of cells obtained at surgery. To alleviate this problem, "immortalizing" genes such as SV40 large T antigen (Tag) (Maines-Bandiera et al, 1992) and the H P V genes P6 and P7 (Wan et al, 1997) have been introduced into OSE. Expression of these genes does not truly immortalize human  11  Figure  3. Morphology o f O S E in culture,  a, Primary epithelial culture with a compact,  cobblestone-like growth pattern, b, Passage 2 with flat epithelial O S E cells. Note a small group o f granulosa cells i n the lower right corner, c, Passage 5 with O S E cells that have undergone epithelio-mesenchymal  conversion and have assumed fibroblast-like shapes. Such cells are  initially keratin-positive but tend to lose keratin with time and passages in culture (Siemens and Auersperg, 1988). X 200 (Auersperg et al, 2001).  12  OSE cell lines in that their population doubling capacity is greatly extended but not infinite; however, the lines provide sufficiently large cell numbers for molecular studies. One advantage of these lines is that they tend to retain some, though not all, of the tissue-specific properties of the cells from which they are derived. For example, many of these lines retain keratin, and most, if not all of them, continue to express N-cadherin and lack E-cadherin (in common with normal, and in contrast to neoplastic OSE). Although such lines are nontumorigenic in SCID mice (Ong et al, 2000), their growth controls are profoundly disturbed, which confers on them properties of neoplastic cells such as genetic instability, increased saturation density, reduced serum requirements and variable degrees of anchorage independence. Tag and E6/E7 inactivate the tumor suppressor genes p53 and pl05RB (May and May, 1999; Stiegler et al., 1998). Importantly, 30-80% of epithelial ovarian carcinomas have p53 mutations which disrupt controls of the cell cycle, D N A repair and apoptosis (Wen et al., 1999). Sometimes, a few cells of such "immortalized" OSE cultures survive crisis and become truly immortal, continuous lines. Recently, constitutively expressed E-cadherin was introduced into an SV40 Tag-immortalized line derived from normal OSE. The resulting phenotype closely resembled neoplastic OSE, and the cells formed adenocarcinomas in SCID mice (Auersperg et al., 1999; Ong et al., 2000). These adenocarcinomas resembled Mullerian duct-derived epithelia in that they formed papillae and cysts and expressed CA125 and E-cadherin. The line, IOSE-29EC, became not only tumorigenic but also acquired an indefinite, truly immortal growth potential. While the exact relationships between the introduction of T-antigen and E-cadherin to tumorigenicity need to be examined in additional lines, this is the first experimental transformation of normal human OSE to ovarian adenocarcinoma cells (Table 1) and the first direct confirmation that OSE is capable of such a transformation. The results support the hypothesis that E-cadherin may act as an inducer  13  of the Mullerian epithelial differentiation which accompanies neoplastic conversion of OSE (Wong etal, 1999).  1.6. Regulation by hormones, growth factors and cytokines Normal OSE cells secrete, and have receptors for agents with growth- and differentiation regulatory capabilities.  1.6.1. Gonadotropin-releasing hormone and gonadotropins Recently, gonadotropin-releasing hormone (GnRH) has been shown to be an autocrine growth inhibitor for normal OSE. Using RT-PCR and Southern blot analysis, GnRH and the GnRH receptor in human OSE cells were cloned and found to have sequences identical to those found in the hypothalamus and pituitary, respectively (Kang et al., 2000). It has been shown that gonadotropins stimulate cell proliferation of normal OSE of several species in vivo and in vitro (Davies et al., 1999; Osterholzer et al., 1985). Human OSE cells also have receptors for F S H (Zheng et al., 1996). The presence of these receptors lends support to the hypothesis that the high FSH levels in peri- and postmenopausal women may play a promoting role in ovarian carcinogenesis, since this is the age of the peak incidence of epithelial ovarian carcinomas (te Velde et al., 1998). Human and rabbit OSE express luteinizing hormone (LH) receptors since hCG, which is secreted by human OSE, stimulates their proliferation (Hess et al., 1999) and L H also stimulates rabbit OSE growth in culture (Osterholzer et al., 1985).  14  1.6.2. Steroids Steroidogenesis is a very complex process in the ovary (Song and Melner, 2000). Receptors for estrogen, progesterone and androgen were found at the mRNA and/or protein level in human OSE (Karlan et al, 1995; Lau et al, 1999). SV-40 large T-immortalized OSE cells expressed ERoc but not ER(3 (Brandenberger et al, 1998). No direct effects of these steroids on OSE proliferation have been demonstrated (Karlan et al, 1995), but there is increasing evidence for indirect actions. The expression of GnRH receptor in OSE appears to be reduced by estrogen (Kang et al, 2001) and estrogen also modulates levels of HGF (Liu et al, 1994) and EGF both of which stimulate OSE growth. Furthermore, in ovarian carcinoma cells, estrogen and progesterone markedly influence the steady state levels of mRNA for the H G F receptor Met (Moghul et al, 1994) and 5a-dihydrotestosterone downregulates the expression of mRNA for the TGF-J3 receptors (Evangelou et al, 2000), suggesting that these steroids may also have indirect effects on the growth regulation of normal OSE. Although there is no evidence for a direct mitogenic effect of ovarian steroids on OSE, it has been known for a long time that corticosteroids enhance OSE proliferation in culture, and that combinations of EGF and hydrocortisone are among the most potent mitogens for cultured OSE (Siemens and Auersperg, 1988). The steroidogenic factor 1 (SF-1), a transcription factor which regulates the differentiation of granulosa cells and inhibits their proliferation, is also growth-inhibitory in rat OSE cells (Nash et al, 1998).  1.6.3. Growth factors 1.6.3.1. Transforming growth factor-^ family Among agents which inhibit OSE growth are several members of the TGF-[3 family of growth factors (Taipale et al, 1998), which affect and/or are produced by OSE. TGF-(3 itself, a widely  15  distributed growth factor with multiple modes of action, acts as an autocrine growth inhibitor for cultured human OSE (Berchuck et al., 1992) and also counteracts the growth-stimulatory effect of EGF (Vigne et al, 1994). In contrast to some other inhibitory factors, TGF-p does not induce apoptosis in OSE cells (Havrilesky et al, 1995). TGF-p inhibits growth of rabbit OSE (Pierro et al, 1996) and regulates Kit ligand expression in immortalized rat OSE (Ismail et al., 1999). A detailed examination by immunohistochemistry and in situ hybridization of TGF-p subtypes, the related protein endoglin, TGF-P receptors and TGF-p-binding protein demonstrated the presence of all of these in human OSE and, with the exception of the binding protein, levels were lower than in ovarian cancers (Henriksen et al., 1995). Interestingly,  5a-dihydrotestosterone  downregulates the expression of mRNA for the TGF-P receptors I and II in ovarian carcinoma lines (Evangelou et al., 1995), suggesting that it might also counteract growth inhibitory effects of TGF-P in normal OSE. It has been demonstrated that activin, inhibin and follistatin are present in normal and neoplastic ovarian epithelia. OSE, immediately after removal from the ovary, expressed mRNA for follistatin 288 and 315, for the activin receptors IA, IB, II and IIB, as well as for the a subunit and (weakly) the P subunit of the ligands (Welt et al., 1997). At the protein level, OSE produced inhibin only. After 1 month in culture, the a subunit was undetectable while the PA subunit became abundant. Another member of the TGF-P family, anti-Mullerian hormone (AMH), which causes regression of the Mullerian ducts in male fetuses, is produced at low levels by granulosa cells throughout the reproductive life of women (Josso et al., 1998). In view of the close developmental relationship between the Mullerian ducts and OSE, it might be expected that A M H should affect OSE cells.  16  1.6.3.2. Epidermal growth factor (EGF) family Among growth factors, those of the EGF family were among the first reported to stimulate human and rabbit OSE proliferation either with or without co-stimulation by corticosteroids (Berchuck et al., 1993; Pierro et al., 1996; Rodriguez et al., 1991; Siemens and Auersperg, 1988). OSE cells express receptors for EGF and TGF-a, which is a structural homologue of EGF and binds to the EGF receptor (Berchuck et al., 1991). EGF not only stimulates proliferation of human OSE cells but also profoundly affects their differentiation: within a few days of EGF treatment, the cells convert from an epithelial to a spindle-shaped morphology and lose epithelial differentiation markers such as keratin (Siemens and Auersperg, 1988). The resulting localized stimulation of the OSE likely contributes to its rapid postovulatory proliferation and perhaps also to epithelio-mesenchymal conversion of OSE cells trapped within the ruptured follicle. T G F - a has been demonstrated immunohistochemically in human OSE in vivo and in vitro, and found to stimulate thymidine incorporation by cultured human OSE cells. It was also demonstrated immunohistochemically in human theca cells, suggesting that it plays a role in the reproductive functions of the ovary (Jindal et al., 1994). In OSE cells whose lifespan has been extended by transfection with SV40 large T antigen, EGF does not enhance proliferation but promotes survival (McLellan et al., 1999). Amphiregulin, another EGF homologue, is also a potent mitogen for OSE cells and appears to control OSE and ovarian cancer cell proliferation in a complex manner (Gordon et al., 1994; Johnson et al., 1991). Of particular interest for ovarian cancer are the heregulins, including the heregulin/«ew differentiation factor, which are a family of ligands that cause phosphorylation of the HER2/neu receptor, a 185 kD transmembrane protein kinase with extensive homology to the EGF receptor (Aguilar et al., 1999). HER1 (synonymous with E G F receptor), HER2, HER3 and HER4 are  17  members of the type I receptor tyrosine kinase family (RTK I) of epithelial growth factor receptors (Downward et al, 1984). These receptors interact in multiple ways which modify their influence on a variety of cells (Klapper et al, 2000). Though normal OSE cells express EGF receptors, they express little or no HER-2/new (Gordon et al, 1994, Kohler et al, 1992; Owens et al, 1991). However, HER2/neu is amplified and overexpressed in 25-30% of ovarian and breast cancers, and this overexpression is associated with a poor prognosis (Aguilar et al, 1999).  1.6.3. Cytokines Cultured human OSE also secretes bioactive cytokines, including IL-1, IL-6, macrophage colony-stimulating factor  (M-CSF), granulocyte  colony-stimulating factor  (G-CSF) and  granulocyte-macrophage colony stimulating factor (GM-CSF). These agents have regulatory effects on follicular growth and differentiation, ovulation, and the distribution of intraovarian cells of the immune system (Ziltener et al, 1993), and IL-1 enhances OSE proliferation (Marth et al, 1996). Little is known about the regulation of cytokine expression in OSE, but it may be relevant that ovarian steroid hormones regulate GM-CSF production by uterine epithelial cells which are developmentally related to OSE (Robertson et al, 1996).  2. Epithelial ovarian carcinomas 2.1. Prologue The epithelial ovarian carcinomas, i.e. the group derived from the OSE, represent approximately 90% of all human ovarian malignant neoplasms, with the rest originating in granulosa cells or, rarely, in the stroma or germ cells. The poor five year survival (30-40 %) is largely due to the fact that most ovarian carcinomas are inoperable when first discovered and  18  respond poorly to therapy (Herbst, 1994). The common epithelial ovarian tumors appear to arise from the ovarian surface  epithelium (OSE), which is a simple  squamous-to-cuboidal  mesothelium covering the ovary (Auersperg et al, 1998; Herbst, 1994; Nicosia et al, 1991). This group of tumors is the most lethal among ovarian neoplasms and is the prime cause of death from gynecological malignancies. Because of the resulting lack of experimental models, the etiology and early events in ovarian carcinogenesis are still among the least understood of all major human malignancies. The incessant ovulation theory was suggested, which repeated ovulation contributes to (pre)neoplastic change of OSE, suggesting that wound healing process of ruptured OSE may play a role in the disease in women (Fathalla, 1971). Therefore, endocrine and autocrine factors including hormones and multiple growth factors were suggested to influence the occurrence of ovarian tumors during menstrual cycle (Godwin et al, 1993; Hamilton, 1992; Piver etal, 1991; Rao and Slotman, 1991; Risch, 1998; Shoham, 1994; Westerman et al, 1997).  2.2. Genetic changes Amplification, altered expression and mutations in a number of oncogenes and tumor suppressor genes play a role in the development of ovarian epithelial neoplasms.  Oncogenes  which are frequently overexpressed or amplified in ovarian carcinomas include c-myc, in particular in serous adenocarcinomas (Tashiro et al, 1992); K-ras, in particular in mucinous carcinomas which may exhibit enteric mucinous differentiation (Enomoto et al, 1991); and erbB2, EGF-R and c-fms (the receptor for colony stimulating factor-1) all of which are associated with a poor prognosis (Berchuck et al, 1990; Kacinski et al, 1989; Kohler et al, 1989). Recently, phosphatidyl inositol 3 kinase (PI3K) and its downstream effector A K T 2 were also shown to be amplified in a significant proportion of ovarian carcinomas (Bellacosa et al, 1995;  19  Shayesteh et al., 1999). Among tumor suppressor genes, p53 is mutated in about 50% of late stage tumors but rarely in low stage tumors and borderline lesions (Berchuck et al, 1994), and the PI3K inhibitor PTEN is mutated in a significant proportion of endometrioid ovarian carcinomas (Obata et al, 1998). Mutations in the tumor suppressor genes BRCA1 and B R C A 2 appear to form the basis for most cases of familial ovarian cancer. The expression of a recently described tumor suppressor gene, N O E Y 2 (ARHI), is decreased specifically in carcinomas of the ovary and breast (Yu et al, 1999). The epidemiology, histopathology and clinical course of OSE-derived ovarian carcinomas differ profoundly from those of the mesotheliomas which arise in extraovarian mesothelium, e.g. a responsiveness to asbestos exposure, and lack of Mullerian phenotypes. This difference reflects, among other factors, the different developmental histories of these two components of the pelvic peritoneum, while may include inductive signals emanating from the ovary and acting on the developing OSE (Blaustein and Lee, 1979; Nicosia et al, 1997).  2.3. Regulation by hormones, growth factors and cytokines Ovarian carcinomas also secrete and have receptors for agents with growth-regulatory capabilities. The potential roles of peptide hormones, sex steroids and growth factors in ovarian cancer are described below.  2.3.1. Gonadotropin-releasing hormone (GnRH) GnRH acts as a key hormone in the regulation of pituitary gonadal axis (Conn, 1994). In addition to its well-documented role in gonadotropin biosynthesis and secretion in the pituitary, an autocrine/paracrine role for GnRH has also been suggested in tumors of the ovary and  20  endometrium (Emons et al, 1992; 1993a; 1993b; Gallagner et al, 1991). This concept is based on the detection of binding sites for GnRH, as well as the expression of GnRH and its receptor gene transcripts in these tumors. Especially noteworthy is the finding that GnRH and its receptor are expressed in normal and neoplastic OSE cells (Kang et al, 2000). GnRH receptors were detected in about 80% of human ovarian epithelial tumors and in numerous ovarian cancer cell lines such as EFO-21, EFO-27, and OV-1063 (Emons et al, 1997; Miyazaki et al, 1997). In vivo, long acting GnRH agonists are thought to act by desensitizing or down-regulating the GnRH receptors in the pituitary, resulting in a subsequent decline in gonadotropins which serve as tumor growth factors. The suppression of endogenous L H and F S H secretion by GnRHagonist treatment results in growth inhibition of heterotransplanted ovarian cancers in animal models (Peterson et al, 1994). In vitro, GnRH and its analogs have been shown to inhibit the growth of a number of GnRH receptor-bearing ovarian cancer cell lines (Emons et al, 1992; 1993a). To improve the therapeutic efficiency of GnRH analogs against cancer cells and to reduce cytotoxicity against normal cells, targeted chemotherapy based on the GnRH receptor has been developed recently (Schally and Nagy, 1999). The exact mechanism underlying the growth inhibitory effect of GnRH analogs remains to be elucidated. At the ovarian GnRH receptor level, the putative endogenous ligand may stimulate the proliferation of the cells through the receptor, which might be down-regulated by continuous treatment with a potent GnRH agonist. The finding that continuous treatment with GnRH agonists, which is thought to induce receptor down-regulation, inhibited ovarian cancer cell growth, and that this effect was abolished by co-treatment with a specific GnRH antagonist, corroborated this view (Kang et al, 2000; Thomson et al, 1991). Alternatively, the ovarian GnRH receptor might mediate direct antiproliferative effects of GnRH analogs. However, this  21  notion is not corroborated by the observation that both antagonistic and agonistic analogs have been reported to induce growth inhibition of ovarian cancer cells (Yano et al, 1994). Recently, it has been suggested that the well established GnRH receptor signaling mechanism mediated by phospholipase C (PLC) and protein kinase C (PKC) is likely not involved in the antiproliferative effects of GnRH in tumor cells (Emons et al, 1998). It has been reported that analogues of GnRH reverse the growth stimulatory effect of EGF and insulin-like growth factor (IGF) in cancer cells including carcinomas of the ovary (Emons et ah, 1996; Hershkovitz et al., 1993; Marelli et al, 1999), possibly by down regulating their receptor numbers and/or mRNA levels. In addition, it has been demonstrated that GnRH analogs reduce cell proliferation by increasing the portion of cells in the resting phase, Gn-Gi (Thomson et al, 1991) and inducing cell death or apoptosis (Motomura, 1998; Sridaran et al., 1998). Treatment of ovarian cancer cells with GnRH analogues may induce apoptosis mediated by the Fas ligand-Fas system, which has been shown to trigger apoptosis in a variety of cell types (Nagata and Golstein, 1995). Recently, it has been demonstrated that a GnRH analogue may modulate ovarian cancer cell growth by inhibiting telomerase activity without altering the R N A component of telomerase expression (Ohta et al., 1998).  2.3.2. Gonadotropins The involvement of gonadotropins in ovarian epithelial cancer development is supported by several observations. A number of epidemiological studies have demonstrated an increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during menopause or infertility therapy (Risch, 1998; Shushan et al, 1996; Whittemore et al, 1992). Clinically, administration of human menopausal gonadotropin (hMG) for ovulation induction may increase  22  the risk of epithelial ovarian tumors (Shushan et al, 1996). Reduced risk of ovarian cancer is associated with multiple pregnancy, breast feeding and oral contraceptive use which results in lower level and reduced exposure to gonadotropins (Rao and Slotman, 1991; Risch, 1998; Shoham, 1994; Whittemore et al, 1992). Receptors for FSH and L H / C G were demonstrated to be present in normal OSE and ovarian tumors (Konishi et al, 1999; Mandai et al, 1997; Zheng et al, 1996; 2000). As in normal OSE cells, F S H and L H / C G stimulated the growth of some ovarian cancer cells in a dose- and time-dependent manner in vitro (Kurbacher et al, 1995; Wimalasena et al, 1992). Elevated levels of gonadotropins may promote the growth of human ovarian carcinoma by induction of tumor angiogenesis in vivo (Schiffenbauer et al, 1997). Despite these observations, the roles that elevated levels and prolonged exposure to gonadotropins play in ovarian tumorigenesis remain to be elucidated. For instance, in other reports, increased risk of ovarian cancer development has not been demonstrated in women undergoing ovulation induction for in vitro fertilization (Franceschi et al, 1994; Venn et al, 1995). The mechanism by which gonadotropins increase ovarian cancer cell growth is unclear. It has been shown that h C G induced estradiol production in a dose dependent manner, whereas FSH had no such effect in primary cultures of epithelial ovarian cancer cells (Wimalasena et al, 1991). The combined treatment of hCG with estradiol may regulate the growth response of epithelial ovarian cancer cells through the IGF-1 and EGF pathways (Wimalasena et al, 1993). Human C G treatment has been demonstrated to suppress cisplatin-induced apoptosis by 58% in the ovarian carcinoma cell line, OVCAR-3 (Kuroda et al, 1998), suggesting that gonadotropins may play a role in preventing apoptosis. Taken together, gonadotropins may be a contributing factor in ovarian tumorigenesis, presumably by enhancing cell proliferation and/or inhibiting apoptosis.  23  2.3.3. Sex steroids Both epidemiological and experimental observations have implicated sex steroids in the pathogenesis and growth regulation of carcinomas arising from the ovary (Chien et al., 1994; Galtier-Dercure et al, 1992; Langon et al, 1994). A number of studies have suggested that the c  risk of developing ovarian cancer increase with the usage and duration of hormone replacement therapy (Garg et al, 1998; Rodriguez et al, 1995). Estrogens taken as oral contraceptives during premenopausal years are protective, but when used in postmenopausal years as hormone replacement therapy, may increase the risk of ovarian cancer (Clinton and Hua, 1997; Garg et al, 1998; Rao and Slotman, 1991; Risch, 1998; Rodriguez et al, 1995). Breast-feeding, which appears to offer protection, is associated with reduced serum concentrations of estradiol (Liu et al, 1983). In addition to estrogens, other ovarian steroids such as androstenedione, testosterone and progestins have also been implicated as risk factors for ovarian cancer (Rao and Slotman, 1991; Risch, 1998). In patients with ovarian cancer, elevated plasma levels of 17P-estradiol, estrone,  progesterone,  20a-hydroxyprogesterone,  dehydroepiandrosterone  sulfate,  androstenedione, and testosterone have been observed and shown to be correlated with tumor volume (Backstrom et al, 1983; Mahlck et al, 1985; 1986a; 1986b; 1988). Elevated levels of sex steroid hormones are thought to be produced by ovarian tumor cells. This notion is supported by the increased levels of sex steroids in the ovarian vein draining the tumor-bearing ovary, as compared with the contralateral ovarian vein and the peripheral blood (Aiman et al, 1986; Heinonen et al, 1986; Kitayama and Nakano, 1990). Exogenous estrogen stimulated the growth of several ER-positive ovarian carcinoma cell lines in vitro (Chien et al, 1994; Galtier-Dercure et al, 1992; Langon et al, 1994).  24  The classical estrogen receptor (ER), now referred to as E R a , and the progesterone receptor (PR) were found in <50% of ovarian tumors, whereas the androgen receptor (AR) was detected in the majority of cases reported (> 80%) (Rao and Slotman, 1991; Risch, 1998). In malignant epithelial ovarian tumors, the concentration of ER is generally higher, while the concentration of PR is generally lower in malignant lesions as compared to that of benign tumors or normal ovaries (Toppila et al., 1986; Vierikko et al, 1983; Willcocks et al, 1983). Also, the presence of a second isoform of estrogen receptor (ER(3) has been reported in normal and malignant ovarian cells in primary cultures or ovarian cancer cell lines (Brandenberger et al, 1998; Lau et al, 1999). Nevertheless, the relationship between receptor content and prognostic factors such as histology, stage and grade is unclear. Several authors found no correlation between estrogen receptor content and histological type or grade of differentiation (Anderl et al, 1988; Harding et al, 1999; Nestok et al, 1988). Others reported that endometrioid tumors express more frequently PR, while serous tumors were more frequently found to be ER positive (Harding et al, 1999; Nestok et al, 1988). Some investigators observed that E R positivity was correlated with poor differentiation (Teufel et al, 1983), whereas others found that well differentiated tumors express ER (Iversen et al, 1986) or both ER and PR more frequently (Creasman et al, 1981). PR status was found to be of significant prognostic value in advanced epithelial ovarian cancer cells (Hempling et al, 1998). However, in other studies, no clinical significance of ER and PR status in epithelial ovarian carcinomas was reported when correlated with age, parity, race, smoking, surgical stage, histologic type, histologic grade, progression-free interval, or patient survival (Masoo et al, 1989). Also, no correlation between the presence of A R and tumor histology was found (Kuhnel et al, 1987; Slotman et al, 1989). The apparent discrepancy of these observations may be explained by differences in the assay methods, the criteria for positivity for steroid  25  receptors, and/or heterogeneity of tumor cell populations with respect to steroid receptor contents (Slotman et al, 1989). The E R a mRNA mutation with a 32-bp deletion in exon 1 was found in SKOV-3 cell line, which is insensitive to E2 with respect to cell proliferation and induction of gene expression (Lau et al,  1999). This may provide an explanation for the lack of  responsiveness and resistance to E2 in some ovarian cancers. The exact mechanism of action of steroid hormones in ovarian cancer remains unclear. Induction of c-myc oncoprotein has been shown to mediate the mitogenic response to growth stimuli (Chien et al, 1994). Depending on the levels of ER, up-regulation of c-myc protein by estrogen has been shown to mediate estrogen-induced ovarian cancer cell growth. It has been demonstrated that estrogen interacts with other growth factors in the normal ovary and ovarian cancer cells. In the ovarian cancer cell line, PE01, the estrogen-mediated growth stimulatory effects were reversed by an EGF receptor-targeted antibody (Simpson et al, 1998). In addition, estrogen induced a significant increase in T G F - a protein concentration in media and estrogen regulated EGF receptor expression in those cells. These results suggest that estrogen may act through increasing production of TGF- a and regulation of the EGF receptor. Estrogen produced a concentration-related potentiation in the growth response to IGF-1 and EGF under conditions where the growth responses to EGF and IGF-1 were submaximal (Wimalasena et al, 1993). Estrogen has been shown to exert its enhancement of EGF- and IGF-1-mediated growth through increased binding affinity for EGF receptor and IGF-1 receptor number (Wimalasena et al, 1993). In other studies, estrogen caused a marked decrease in insulin-like growth factor binding protein-3 (IGFBP-3) mRNA, but increased IGFBP-5 m R N A levels, suggesting that IGFBP expression can be regulated in estrogen-responsive ovarian cancer cells by E2 (Krywicki et al, 1993).  26  Germline mutations in the BRCA1 gene are associated with increased cancer risk in breast, ovary and prostate, but not in other tissues. The obvious implication, that BRCA1 mutations therefore affect neoplastic transformation in conjunction with hormonal factors, is supported by recent reports which showed that estrogen and prolactin stimulate proliferation of ovarian and breast carcinoma cells and concurrently upregulate BRCA1 m R N A and protein (Fan et al., 1999). This demonstrates that in breast and prostate cancer cells, BRCA1 inhibits signaling by ligand-activated estrogen receptor ER-oc and blocks its transcriptional activation function. Together, these data suggest that BRCA1 functions as a negative feedback inhibitor of growth induced by estrogen and prolactin. It is important to note that some ovarian carcinoma cells proliferate in response to estrogen (Kang et al., 2001; Nash et al., 1989) while normal OSE cells do not (Kang et al., 2001; Karlan et al., 1995).  2.3.4. Activin/Inhibin Activin and inhibin are members of the TGF-R superfamily (Mathews, 1994; Vale et al., 1988; Woodruff, 1998).  Activin is a dimeric protein composed of two R subunits, RA-RA  (activin A), (3B-RB (activin B), or RA-RB (activin A B ) (Vale et al., 1988). Inhibin is composed of an a and one of two R subunits, a-RA (inhibin A ) or a-(3B (inhibin B). The main function of these gonadal peptides is to regulate FSH secretion from the anterior pituitary gland (Ling et al., 1986; Vale et al, 1986). However, since activin and inhibin are produced in the ovary (Eramaa et al., 1993), it has been hypothesized that they may act via an autocrine/paracrine mechanism to regulate ovarian function (Eramaa et al, 1993; Tuuri et al., 1996). Activin mediates its cellular effects through heterodimeric complexes of type I and II activin serine/threonine kinase receptors (Eramaa et al, 1995), which are expressed in normal and neoplastic OSE cells.  27  It has been demonstrated that recombinant activin has no mitogenic effect on normal OSE that also expresses activin receptors (Welt et al, 1997). Interestingly, activin may function to support cell survival and stimulate the proliferation of epithelial ovarian carcinoma cell lines, including O V C A R - 3 , CaOV-3, CaOV-4, and SW-626 (Di Simone et al, 1996, Fukuda et al, 1998), whereas follistatin, an activin binding protein, inhibits this action (Fukuda et al, 1998; Welt et al, 1997). Most primary epithelial ovarian tumors synthesize and secrete activin in vitro and serum levels of activin are frequently elevated in women with epithelial ovarian cancer (Welt et al, 1997). These findings suggested that, : 1) pA subunit mRNA is expressed, 2) activin is secreted more frequently than inhibin, 3) P A subunit mRNA expression is greater in neoplastic and normal epithelium after culture. Thus, activin may act as an autocrine/paracrine regulator of epithelial ovarian tumors, but its exact role in tumorigenesis has yet to be defined. Inhibin a subunit which was expressed in 47% cases of normal OSE, was not found in the epithelial component of ovarian cystadenomas, tumors of low malignant potential (LMP), or carcinomas. pA subunit was expressed in 93% cases of OSE, in the epithelial component of all cystadenomas, in 81% cases of L M P tumors and in 72% cases of carcinomas. These observations suggest that an imbalanced expression of inhibin and activin subunits in OSE may represent an early event that leads to epithelial proliferation (Zheng et al, 1998). Serum inhibin levels are elevated in most postmenopausal women with mucinous cystadenocarcinomas and mucinous borderline cystic types of epithelial ovarian tumors (Healy et al, 1993), whereas immunoreactive inhibin is undetectable or present at low levels in normal postmenopausal subjects. The a-inhibin has been proposed to be a serum marker for epithelial ovarian cancer in postmenopausal women (Lambert-Messerlian et al, 1997). Ovarian neoplasms may produce a variety of peptides related to the inhibin. It has been shown that inhibin B is  28 .  detected in more ovarian cancers than inhibin A (Robertson et al., 1999). The majority of granulosa cell tumors appear to secrete significant amounts of dimeric inhibin-A, whereas mucinous tumors secrete predominantly other forms of inhibin, presumably related to the asubunit (Burger et al., 1996; 1998). Serous tumors may also secrete inhibin-related peptides but not dimeric inhibin-A (Burger et al., 1996). The expression of inhibin subunit genes in granulosa cell tumors and in mucinous or serous epithelial ovarian tumors revealed that these tumors are the source of the increased immunoreactive inhibin observed in the serum of patients with ovarian tumors (Fuller et al, 1999). On the contrary, it has also been reported that ovarian carcinomatous epithelial cells do not secrete inhibin and that serum inhibin levels detected in patients with epithelial ovarian carcinoma may reflect an ovarian stromal response to the ovarian carcinoma (Ala-Fossi et al, 1999). Thus, the role of inhibin in ovarian cancer remains to be elucidated.  2.3.5. Growth factors Trends in the expression and response to growth regulators include the secretion of, and responses to, factors found in the normal OSE (Berchuck et al, 1993) as well as factors that may be typical of ovarian malignancies (Mills et al, 1992). The former includes growth inhibition by TGF-R (Berchuck et al, 1992) and growth stimulation by basic fibroblast growth factor (bFGF) (Di Blasio et al, 1993), EGF and T G F - a (Kohler et al, 1992).  2.3.5.1. Transforming growth factor-^ TGF-R is a multifunctional peptide that is involved in cell growth regulation, tissue remodeling, immune suppression and other crucial cellular functions via both autocrine and  29  paracrine mechanisms (Roberts and Sporn, 1990). Three mammalian TGF-P isoforms (TGF-pl, TGF-p2, and TGF-p3) that are encoded by different genes have been identified (Massague et al, 1992). The peptides share extensive homology in amino acid sequence (70-80%), and exist as homodimeric chains of between 111 and 113 amino acids, with molecular weights of 25 kDa. Three types of receptors for TGF-p (TpRI, TpRII, TpRIII) that belong to the family of serine/threonine kinase membrane receptors have been identified (Massague, 1992; Wrana et al, 1994). TGF-p binds to a type II TGF-p receptor (TpRII), which recruits and phosphorylates a type I TGF-p receptor (TpRI) (Bassing, 1994; Wrana et al, 1992; 1994). TpRIII, also known as betaglycan, has no known signaling motif (Wrana et al, 1992; 1994), and appears to bind and present TGF-p to TpRII (Carcamo et al, 1995; Lopez-Casilla et al, 1991; Wang et al, 1991). The expression of TGF-p has been demonstrated in ovarian tumors, suggesting an autocrine and/or paracrine role of TGF-p (Bartlett et al, 1992; 1997; Marth et al, 1990). TGF-p inhibited the proliferation of monolayers of normal human ovarian epithelial cells by 40-70% (Berchuck et al, 1992) and by 95% in primary epithelial ovarian cancer cell cultures obtained directly from ascites (Ffurteau et al, 1994). TGF-P inhibited colony formation of 7 of 9 fresh ovarian cancers in soft agar (Daniels et al, 1989). In contrast, epithelial ovarian cancer cell lines are found to be relatively resistant to the growth inhibition of exogenous TGF-P treatment (Berchuck et al, 1990; 1992). These data suggest that TGF-P may act as a growth inhibitor that prevents inappropriate proliferation of normal OSE cells, while loss of this autocrine inhibitory pathway may lead to cancer development in vivo and/or immortalization of cell in vitro. Several possible mechanisms have been proposed to explain the loss of responsiveness to TGF-p in primary culture of ovarian carcinomas and/or ovarian cancer lines. Some cells may  30  become resistant to the effects of endogenous TGF-R because they cannot produce and/or activate secreted latent TGF-R. In this regard, it has been shown that normal ovarian epithelial cells can produce and activate T G F - p l and 2, whereas production or activation does not occur in several ovarian cancer cell lines (Berchuck et al, 1992). Like in other cells, defective ligandbinding to the cell surface caused by absence of TpRII or expression of a truncated form or splice variant of TRRII may account for the resistance to activated TGF-R in ovarian cancer cells (Inagaki et al, 1993; Kadin et al, 1994; Kalkhoven et al, 1995 Park et al, 1994; Wrana, 1992). It is also possible that alterations in signal transduction pathways may account for the development of resistance to TGF-R during the transformation process. In this regard, the binding of TGF-R to its cell surface receptors has been shown to down-regulate c-myc, a DNA-binding protein whose expression is induced by growth factors that stimulate proliferation (Pietenpol et al, 1990). The loss of TGF-P responsiveness has been associatedAwith the inability of TGF-R to down-regulate c-myc in some, but not all, cases of ovarian tumors (Fynan and Reiss, 1993). It has been suggested that inactivation of the p53 or Rb tumor suppressing gene products due to deletion, mutation, or binding of viral oncoproteins may be responsible for the loss of TGF-R responsiveness (Reiss et al, 1993). However, in most ovarian cancers, it is thought that mutation and overexpression of p53 frequently occur, but this may not lead to the development of resistance to TGF-P (Hurteau et al, 1994; Jacobs et al, 1992; Kohler et al, 1993). The molecular mechanisms that mediate the growth inhibitory effect of TGF-p are poorly understood (Massague et al, 1992). Binding of TGF-P to its receptors initiates a cascade of molecular  events  that  are  thought  to  decrease  activity of cyclin-dependent  kinase  (CIPlAVA.Fl/p21), resulting in arrest of cell cycle from G l into S phase of D N A synthesis in  31  normal and neoplastic ovarian cells (Massague et al, 1992). In addition to the cell cycle inhibition, it has been shown that TGF-P can induce apoptosis in both normal and malignant cells under certain circumstances (Havrilesky et al, 1995; Selvakumuran et al, 1994). It is reported that malignant ovarian cells are more susceptible to apoptosis in response to TGF-P than their normal nontransformed counterparts (Havrilesky et al, 1995).  2.3.5.2. Epidermal growth factor and transforming growth factor-a The EGF receptor (also known as c-erbBl/HERl) is a membrane tyrosine kinase which forms homodimers after binding to either EGF or T G F - a (Ullrich and Schlessinger, 1990). Homodimerization activates tyrosine kinase activity and autophosphorylates several tyrosine moieties in the cytoplasmic domain of the receptor, thereby transmitting the growth stimulatory signal to the nucleus (Ullrich and Schlessinger, 1990). EGF receptors were detected in 33% to 75% of ovarian tumors using ligand binding, immunohistochemistry, or Northern blot analysis (Battagila et al, 1989; Bauknecht et al, 1988; 1993; Berchuck et al, 1991; Henzen-Logmans, 1992; Kohler et al, 1992; Morishige et al, 1991; Owens et al, 1991). The level of EGF receptor has been demonstrated to be higher in malignant ovarian tumors than in benign tumors or the normal ovary (Berns et al, 1992; Owens et al, 1993), implicating its prognostic importance. The contribution of a TGF-a/EGF receptor autocrine loop to the growth of epithelial ovarian cancer cells is corroborated by several studies. T G F - a levels in the normal ovary increase after menopause, i.e. at the peak incidence of ovarian neoplasms (Owens et al, 1991; Owens and Leake, 1992). Exogenous treatment with T G F - a promotes the growth of several ovarian cancer cell lines in vitro and enhances direct clonogenic growth of ovarian tumor cells (Crew et al, 1992; Kurachi et al, 1991; Zhou and Leung, 1992). Co-expression of EGF receptor with TGF-  32  a, but not EGF, in primary ovarian tumors was reported (Morishige et al, 1991). Neutralizing antibodies against either T G F - a or the EGF receptor induced growth inhibition in primary ovarian cancer cell cultures (Jindal et al, 1994; Morishige et al, 1991). The amplification and/or over-expression of the c-erbB-2 (HER2/neu) oncogene product (pl85  cerbB2  ), frequently observed in different types of tumors, was seen in 30-70% of human  ovarian cancers (Haldane et al, 1990; Slamon et al, 1989), but in only 5-10% of normal ovarian cells (Hung et al, 1992). At the mRNA level, c-erbB-2 has extensive homology with EGF receptor, c-erbB-3 and c-erbB-4 (Plowman et al, 1993). Immunohistochemically, increased expression of c-erbB-3 and c-erbB-4 proteins has been demonstrated in malignant ovarian tumors as compared to benign ones (Simpson et al, 1995). In spite of marked sequence homology between the EGF receptor and HER2, EGF and T G F - a do not bind to HER2 (Ullrich and Schlessinger, 1990). It has been demonstrated that HER2 can be transactivated by EGF through heterodimerization with EGF receptors (Goldman et al,  1990) or by heregulin through  heterodimerization with HER-3 or HER-4 receptors (Peles et al, 1993). In addition to cell proliferation, EGFR and pl85  CCTbB  " activation has been shown to play an important role in cell 2  motility (Wiechen et al, 1999), which is mediated in vitro by several polypeptide growth factors, including H G F and EGF (Christen et al, 1990; MacCawley et al, 1998). In this regard, overproduction of proteinases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) families has previously been reported in ovarian cancer cells and tissues (Stack et al, 1998). In  vitro,  EGF-dependent  stimulation of migration, and  induction of matrix  metalloproteinase (MMP)-9 (gelatinase B) were observed in two ovarian cancer cell lines (OVEA6 and OVCA429) (Young et al, 1996). These findings suggest that the E G F - or the  33  pl85  cerbB  " -dependent enhancement of cell motility may contribute to peritoneal spread and 2  invasion of tumor cells, resulting in tumor metastasis. Treatment of an ovarian cancer cell line with a human-mouse chimeric anti-EGF receptor monoclonal antibody (mAb) or an anti-HER2 mAb resulted in growth inhibition (Ye Dingwei et al., 1998). Concurrent treatment with two mAbs resulted in augmentation of inhibition. TGF-ocstimulated growth of ovarian cancer cell lines was completely inhibited by treatment with an EGF receptor-specific tyrosine kinase inhibitor, ZM252868, suggesting that blocking of receptor activation may have therapeutic value (Simpson et al., 1999). The use of antisense molecules that are designed to specifically block encoded genetic information from sense D N A have been developed for targeting the c-erbB-2 oncogene. It has been shown that the c-erbB-2 antisense oligonucleotide to reduce pl85  ccri,B  " levels results in a growth inhibition of an ovarian cancer cell 2  line (Wiechen K , Dietel M 1995 Wu et al., 1996). Single-chain immunoglobulin (scFv) molecules that retain antigen-binding specificity but lack other functional domains have been designed to modulate the expression levels of oncogenes, the intracellular mobilization and function of oncoproteins. Introduction of anti-erbB-2-scFV resulted in down-regulation of cell surface erbB-2 gene expression and marked inhibition of cellular proliferation (Deshane et al., 1994).  2.3.5.3. Hepatocyte growth factor The HGF/Met system is considered to be a principal paracrine mediator of normal mesenchymal-epithelial interaction (Rosen et al., 1994), and is also involved in the growth and spread of tumors (Jeffers, 1996). The Met/HGF receptor was overexpressed in a significant proportion of well-differentiated ovarian carcinomas (Di Renzo et al., 1994; Huntsman et al.,  34  1999; Moghul et al, 1994). Although little is known about the regulation of H G F and Met expression in ovarian tumors, the level of Met may be regulated by gonadotropin, steroids, certain cytokines and growth factors in vivo and in various cell lines (Hess et al., 1999; Moghul et al., 1994, Parrott and Skinner, 1998). HGF itself has been shown to autoregulate c-met mRNA levels (Boccaccio et al, 1994; Moghul et al., 1994). High levels of H G F are found in cystic fluids or ascites of ovarian cancer patients compared to the peritoneal fluid of normal women (Sowter et al., 1999). Recombinant HGF increased migration and proliferation of ovarian cancer cell lines that express high levels of Met protein (Corps et al, 1997; Ueoka et al, 2000). Thus, high levels of Met expression in ovarian cancer cells may facilitate HGF-mediated tumor growth and dissemination (Corps et al., 1997).  2.3.5.4. Insulin-like growth factors Insulin-like growth factor (IGF) affects the growth and differentiation in normal and neoplastic cells (Cullen et al., 1991; Daughaday, 1990; LeRoith et al., 1995). IGF receptor-I (IGF-RI) mRNA was detected in ovarian cancer cell lines and primary or metastatic ovarian cancer tissues, suggesting a role of the IGF system in neoplastic ovarian cells (Beck et al., 1994; van Dam et al., 1994; Yee et al., 1991). Expression of IGF-I, its receptor, and IGF-binding proteins (IGFBPs) in epithelial ovarian cancer cells and its mitogenic effect on these cells in vitro implicate a role for IGF-I in the regulation of human ovarian cancer (Conover et al., 1998; Karasik et al., 1994; Yee et al., 1991). IGF-II is also expressed in both normal ovary and ovarian cancer and expression level of IGF-II is elevated in ovarian cancer (Yun et al., 1996). The treatment of OVCAR-3 cells with hCG suppressed cisplatin-induced apoptosis via up-regulation of IGF-1 expression, suggesting that LH/hCG may influence the chemosensitivity of ovarian  35  cancer cells through an apoptotic-inhibitory signal (Kuroda et al., 1998). In addition, the transformed ovarian mesothelial cells, overexpressed with IGF-RI, are resistant to apoptosis as a result of down-regulation of Fas expression (Coppola et al, 1999). These results support the notion that the IGF system plays a role in tumor growth and apoptosis in ovarian cancer. IGFBPs appear to bind to IGF and deliver them to target organs. There are a limited number of studies implicating the involvement of IGFBPs in ovarian cancer. IGFBP-2, the major binding protein in benign and malignant ovarian cancers, is highly expressed in malignant as compared to benign neoplasms (Flyvbjerg et al., 1997; Kanety et ah, 1996), suggesting that IGFBP-2 may serve as a marker for ovarian cancer. Further, IGFBP-2 correlated positively with the serum tumor marker, C A 125. In contrast, the serum IGFBP-3 level was decreased in patients with ovarian cancer as shown by RIA and Western ligand blotting (Flyvbjerg et al., 1997). Treatment with estradiol induced a marked decrease in IGFBP-3, but enhanced IGFBP-5 levels, indicating that IGFBP expression is differentially regulated by estradiol in estrogen-responsive ovarian cancer (Krywicki et al., 1993). Considering that IGFs, mediated by IGF-Rs, induce cell growth and mitogenesis in ovarian cancer, antisense or antibody therapy against IGFs and/or IGF-Rs can be considered as a potential management strategy of ovarian cancer patients. Treatment of cells with anti-sense IGFI receptor oligonucleotides markedly inhibited cell proliferation (Muller et al., 1998; Resnicoff et al., 1993). Further, antisense oligonucleotide to IGF-II induced apoptosis in human ovarian cancer cells, suggesting that IGF-II may also be a potential target in the therapeutic treatment of ovarian cancer (Yin et al., 1998).  36  2.3.5.5. Vascular endothelial growth factor Angiogenesis is a critical phenomenon in the growth, progression, and metastasis of solid tumors. Vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF) is a 34- to 50-kDa dimeric, disulfide-linked glycoprotein synthesized by normal and neoplastic cells (Berse et al., 1992; Connolly et al., 1989). Through binding to the specific membrane tyrosine kinase receptors that are expressed in vascular endothelial cells (Neufeld et al., 1994), V E G F has been shown to be an important regulator of tumor angiogenesis. Abundant levels of VPF have been identified in the malignant effusions of ovarian tumors (Abu-Jawdeh et al., 1996; Olson et al., 1994; Yeo et al., 1993), indicating that VPF may be an important mediator of ascites formation and tumor metastasis observed in the neoplastic ovary. The expression of V E G F mRNA and protein (Abu-Jawdeh et al., 1996; Boocock et al., 1995; Olson et al., 1994) has been demonstrated in ovarian carcinoma, suggesting that neoplastic OSE is one source of V E G F production. In vitro, the conditioned medium from VEGF-positive ovarian cancer cell lines has been shown to stimulate D N A synthesis of vascular endothelium (Olson et al., 1994). In vivo, treatment of mice carrying tumor engraftment with a function-blocking V E G F antibody (A4.6.1) specific for human V E G F significantly inhibited subcutaneous SKOV-3 tumor growth as compared with controls (Mesiano et al., 1998). In mice bearing intraperitoneal tumors, ascites production and intraperitoneal carcinomatosis were completely inhibited by the treatment with a V E G F antibody (Mesiano et al., 1998). These results suggest that neutralization of V E G F activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer. Angiogenesis has been correlated with prognosis in patients with ovarian cancer. Higher positive immunostaining for V E G F and serum V E G F levels were observed in women with ovarian carcinoma than in tumors of low malignant potential (LMP) arid benign cystadenoma (Yamaoto  37  et al, 1997). High V E G F expression in epithelial ovarian carcinomas was found to be associated with poor overall survival (Hartenbach et al, 1997). Serum V E G F levels decreased after surgical removal of tumor in ovarian cancer patients, suggesting that serum V E G F could be used as a marker for monitoring tumor progression and ascites formation (Gadducci et al., 1999; Oehler and Caffier, 1999; Tempfer et al, 1998; Yamaoto et al, 1997).  2.3.5.6. Other growth factors Platelet-derived growth factor (PDGF) is a dimeric protein composed of two related A - and B - chain polypeptides encoded by separate genes. Two distinct receptors for PDGF have been found according to affinity (PDGF-Ra and PDGF-Rp). A functional role of PDGF via autocrine growth stimulation has been suggested. Expression of PDGF and P D G F - R a in ovarian tumor cells is related to progression of malignant ovarian tumors, suggesting an independent role for P D G F - R a as a prognostic factor (Henriksen et al., 1993). However, a contradictory report showed that many ovarian carcinomas lose the PDGF receptors, while PDGF stimulates growth of normal OSE cells, which have both a and [3 receptors (Dabrow et al., 1998). The loss of P D G F - R a and PDGF-R(3 may be indicative of independence from hormonal influences to cell growth. Platelet-derived endothelial cell growth factor  (PD-ECGF)  is associated  with  angiogenesis and the progression of human ovarian cancer. The levels of PD-ECGF and its mRNA were higher in ovarian cancers than in normal ovaries, suggesting that PD-ECGF might be related to advanced stages of ovarian cancers associated with neovascularization (Fujimoto et al., 1998). Thus, prevention of angiogenic activity by PD-ECGF may have a potential role in ovarian tumor therapeutics (Reynolds et al., 1994).  38  Basic FGF (bFGF) and other members of the FGF family share several biological properties that have the potential to mediate neoplastic cell growth. It has been shown that ovarian cancer cell lines produce and respond to bFGF and other members of the F G F family (Crickard et al, 1994). The bFGF and its receptor are also expressed in epithelial ovarian tumors (Di Blasio et al., 1995). In advanced primary ovarian tumors, the levels of bFGF mRNA and protein were significantly higher regardless of histological types (Fujimoto et al, 1997), indicating that this growth factor may contribute to growth, invasion and metastasis with neovascularization. It is hypothesized that bFGF may induce a fibroblastic response, which causes tumors with a high bFGF to be less aggressive than those with less stromal tissues (Obermair et al., 1998).  2.3.6. Cytokines While the secretion of cytokines is a normal OSE function (Ziltener et al., 1993), their recruitment into autocrine loops may be important during neoplastic progression. Cytokines produced by and growth stimulatory for ovarian carcinomas include M-CSF (Kacinski et al., 1990), GM-CSF (Cimoli et al, 1991), IL-1 and IL-6 (Malik and Balkwill, 1991; Scambia et al, 1994) and T N F - a (Balkwill, 1992; Bast et al, 1993; Marth et al, 1996; Wu et al, 1992; 1993). High levels of M-CSF and IL-6 in blood and ascitic fluid correlate with a poor prognosis in ovarian cancer, as does overexpression of the M-CSF receptor fins (Kacinski et al, 1990). Interestingly, fins is expressed by many ovarian cancers but not by benign ovarian tumors (Kacinski et al, 1990) or normal OSE (Berchuck et al, 1993). Thus, M-CSF, when secreted by normal OSE, acts in a paracrine manner but becomes an autocrine regulatory factor with malignant progression. GM-CSF is a regulatory glycoprotein that stimulates the production of granulocytes and macrophages. Recombinant human G M - C S F stimulates colony formation in  39  human ovarian cancer cell lines, IGROV-1, Kill A, ME-180, Pa-1 and A2780 (Cimoli et al, 1991). IL-1 and IL-6 enhance tumor cell motility and metastasis (Malik and Balkwill, 1991) and cause changes in gene expression including the induction of T N F - a which is mitogenic for OSE cells but growth inhibitory for ovarian cancer cells (Marth et al,  1996). Stimulation of  proliferation by IL-1 (3 could be partially blocked by an antibody against T N F - a or by soluble T N F - a receptor (Wu et al, 1993). Thus, T N F - a may function as an autocrine/paracrine growth factor in normal and malignant ovarian epithelial cells. Epithelial ovarian cancer cells, produce IL-6, a multifunctional cytokine with diverse biological effects, in both ovarian cancer cell lines and primary ovarian tumor cultures (Watson et al, 1990). IL-6 may be a useful tumor marker in some patients with epithelial ovarian cancer, as it correlates with tumor burden, clinical disease status, and survival (Berek et al, 1991). Inhibition of IL-6 gene expression by exposure to IL-6 antisense oligonucleotides resulted in greatly decreased cellular proliferation (Watson et al, 1993). However, the addition of exogenous IL-6 failed to restore the proliferation of the antisense-treated cells and antibodies to IL-6 did not consistently inhibit cell growth (Watson et al, 1990), suggesting that IL-6 is not an autocrine growth factor for these established ovarian tumor cell lines. As the majority of epithelial ovarian cancers produce IL-6, the direct specific inhibition of IL-6 gene expression may be of potential therapeutic value (Watson et al, 1993). Many of these agents are produced normally by various ovarian cell types and by cells of the immune system that reside in the ovary. Factors from these sources may contribute to the metaplastic and neoplastic changes in the OSE. Interferon-y (IFN-y) is known to modulate many cellular functions. A clinical relevance of IFN-y has been suggested because IFN-y has an antiproliferative activity on the majority of the  40  established human ovarian carcinoma cell lines (Mobus et al, 1993). It has been shown that IFNy decreases constitutive tyrosine phosphorylation of erbB-2 and inhibits erbB-2 kinase activity in an ovarian cancer cell line, SKOV3 cells, which over-expresses erbB-2 (Mishra and Hamburger, 1994). The elevated expression of tumor-associated antigens and major histocompatibility complex (MHC) antigens by IFN-y may improve immunogenicity of ovarian tumor cells and explain the therapeutic effects observed in IFN therapy of ovarian cancer (Mobus et al, 1993).  2.3.7. Lysophosphatidic acid A potent growth stimulatory factor from ascites of ovarian cancer patients has been purified and characterized as ovarian cancer activating factor (OCAF), which plays a role in ovarian tumorigenesis both in vitro and in vivo (Xu et al, 1995, Fang et al, 2000). In addition, this purified O C A F induced proliferation of ovarian cancer cells. O C A F is composed of various species of lysophosphatidic acid (LP A), including LP As with polyunsaturated fatty acyl chains (linoleic, arachidonic, and docosahexaenoic acids) (Xu et al,  1995).  L P A is a bioactive  phospholipid with mitogenic and growth factor-like activities that acts via specific cell-surface receptors present in many normal and transformed cell types. L P A has been implicated as a growth factor present in ascites of ovarian cancer patients (Westermann et al, 1998).  As reviewed above, multiple factors including peptide hormones, sex steroids, growth factors and cytokines have been implicated as stimulatory or inhibitory growth regulators in ovarian cancer. These regulators appear to exert their actions through specific receptors in an endocrine, paracrine or autocrine manner. A better understanding of the potential cross-talk between these  41  regulatory pathways in normal and neoplastic OSE cells will be a necessary first step in the understanding of ovarian tumorigenesis.  2.5. Apoptosis and bcl-2 gene family 2.5.1. Apoptosis and cancer Until recently, cancer was considered as a disease of abnormal cell proliferation. However, developments within the cell death field over the past ten years have provided a new prospective on how cell proliferation is normally maintained at equilibrium and unveiled how abnormalities in cell death regulation can contribute to the progression and development of malignancy. Apoptosis, which is documented as programmed cell death (PCD), plays a pivotal role in the normal development and homeostasis of all multicellular organisms. Deregulation of this process, resulting in either too much or too little cell death, may cause developmental defects and a wide range of disease status. The purpose of this process is to remove unwanted host cells. Apoptosis occurs in three situations: 1) for development and homeostasis, 2) as a defense mechanism, and 3) in aging (Vaux and Strasser, 1996). Morphologically, this phenomenon is characterized by chromatin condensation, membrane blebbing and loss of cell volume (Chao DT, Korsmeyer SJ 1998; Minn et al, 1998). In general, single cell surrounded by viable neighbors may be susceptible to apoptosis. Apoptosis is a genetically programmed mode of cell death that is regulated by genes, including oncogenes and oncosuppressor genes, which may be mutated, deleted or abnormally expressed in neoplasms, thus altering tumor cell susceptibility to apoptosis (Martin, 1997).  42  2.4.2. Bcl-2 family The 131 somatic cells undergoing programmed cell death among 1090 somatic cells are generated during the development of the nematode Caenorhabditis elegans. Genetic analysis revealed that three genes, CED-3, and CED-4 and CED-9 control all 131 of these developmental programmed cell death (Ffengartner and Horvitz, 1994). A mammalian homolog of CED-9 was identified as bcl-2 at the inter-chromosomal breakpoint of the t(14;18), the molecular hallmark of follicular B cell lymphoma (Bakhshi et al, 1985; Tsujimoto et al, 1985). The discovery that bcl2 plays a role in preventing apoptosis instead of promoting proliferation established a new concept of oncogenes (Hockenbery et al, 1990; Korsmeyer, 1992; Vaux et al, 1988). The identification of multiple bcl-2 homologs which form homodimers or heterodimers suggests that these molecules function at least in part through protein-protein interactions (Chao and Korsmeyer, 1998). The first pro-apoptotic homolog, bax, is a 21-kDa protein that shares homology with bcl-2 in conserved regions including BH-1 (bcl-2 homology region-1) and BH-2 (Fig. 4). It has been demonstrated that bax heterodimerizes with bcl-2 and homodimerizes with bax itself (Oltvai et al, 1993). When bax was overexpressed in cells, apoptotic death in response to a death signal was accelerated, however, when bcl-2 was overexpressed, it heterodimerized with bax and the death signal was repressed (Oltvai et al, 1993). Thus, the ratio of bcl-2 to bax is critical in determining susceptibility to apoptosis. Bax is widely expressed in tissues, including a number of sites in which cells die during normal maturation (Krajewski et al, 1994; Oltvai et al, 1993). The family has further expanded to include the death antagonists bcl-2, bcl-XL, bcl-W, Mcl-1 as well as the pro-apoptotic molecules bax, bcl-Xs, B A K and B A D (Fig. 4). The bcl-X gene gives rise to two mRNA species by alternative splicing, producing two protein products: bcl-X , L  43  Anti-apoptotic Bcl-2  Bcl-X  L  Loop  BH4  N  Bcl-W  Loop  BH4  N —1  N  _  _  1  BH4  BH3  TM  BH 1  BH  BH  BH 2  TM  1  BH  BH  TM  BH3  1 Loop  2  2  1  L 1 1 1 • •  Mcl-1  N  BH3  Loop  "I  BH 1  BH  BH  BH  TM  2  Pro-apoptotic  Bax  N  Bak  N  Bcl-Xs  N  Bad  N  Loop  Loop  BH3  BH3  1  2  BH  BH 2  1  BH4  Loop  BH3  Loop  BH3  TM  j" TlVf j  —c  Figure 4. Summary o f anti-apoptotic and pro-apoptotic bcl-2 family members. Bcl-2 homology regions (BH1 - B H 4 ) are indicated with shared boxes. TM: transmembrane domain  44  endowed with death repressor activity, and a shorter variant, bcl-Xs, which functions as a dominant inhibitor of bcl-2, thus inducing apoptosis (Boise et al, 1993). Mutational analysis of bcl-2 and bcl-X  L  identified key residues within BH1 and BH2 domains required for both  heterodimerization with bax and repression of cell death (Yin et al, 1994). The overall ratio of the death agonists to antagonists determines the susceptibility to a death signal. Conserved domains BH1, BH2 and BH3 participate in the formation of various dimer pairs as well as the regulation of cell death (Sedlak et al., 1995). Another mitochondrial component implicated in apoptotic cell death is the release of cytochrome-c from the inter membrane space. Release of cytochrome c into the cytosol is a strong activator of caspases (Kluck et al., 1997; Yang et al., 1997). When bcl-2 prevented apoptosis, cytochrome c was not released. It has been suggested that cytochrome c release may precede a fall in mitochondrial membrane potential. The CED-3 was cloned and demonstrated to be homologous to a previously cloned mammalian gene, interleukin-lR (IL-lR) converting enzyme (ICE) (Miura et al., 1993). The ICE was initially identified as a cysteine protease that converts the 31-kDa pro form of IL-1 R into a 17.5-kDa mature form. It was confirmed that ICE plays a critical role in programmed cell death when ICE was transiently introduced into a mammalian cell line and found to induce apoptosis. Many more ICE-like proteases have been cloned in mammalian cells, and these proteases have been renamed caspases (Faucheu et al., 1995; Kamens et al., 1995; Kumar et al, 1994). Transfection of many of these caspases into mammalian cells induces apoptosis, and elimination of these caspases in mice by gene targeting causes defects in apoptosis (Kuida et al., 1995; 1996). A l l of these caspases are synthesized in a pro form and activated by proteolytic cleavage (Kumar, 1995). The poly(ADP)-ribose polymerase (Lazebnik et al,  1994), nuclear lamins  (Takahashi et al, 1996), fodrin (Cryns et al, 1996), p21-activated kinases 2 (PAK2) (Rudel and  45  Stimulatory pathway Inhibitory pathway Pro-caspases cytochrome-C  Active caspases (caspase-3/-9)  • Cell death  Figure 5. Activation of apoptotic pathway through homodimerization (bax/bax) of pro-apoptotic  bcl-2 gene family, release of cytochrome-c, and activation of caspases.  46  Bokoch, 1997), and D N A fragmentation factor (DFF) (Liu et al, 1997) have been suggested as the targets for caspases. Thus, it has been proposed that cleavage of these substrates induces programmed cell death (Fig. 5) and is responsible for at least some of the morphological changes associated with apoptosis. Further, the studies have been extended to investigate the critical role of caspases in programmed cell death on multiple mammalian cell death receptors such as TNF receptor family, including TNF receptor-1 and Fas/APO-1 (Yuan, 1997). The cytoplasmic domain of these receptors contains an amino acid region known as a death domain. In addition, these death receptors play a role in apoptosis through a direct link to the downstream pathway of caspases, which is responsible for inducing apoptosis (Boldin et al, 1996).  2.4.3. Regulation of apoptosis and bcl-2 gene family The bcl-2 family constitutes a critical intracellular checkpoint of apoptosis within a common cell death pathway and these apoptotic proteins are widely accepted as regulators of cell death (Chao and Korsmeyer, 1998; Minn et al, 1998). Understanding the regulation of apoptosis in cancer cells has recently become an area of intense research. Overexpression of bcl-2 has been demonstrated to provide protection against a wide variety of death-inducing signals such as growth factor deprivation, loss of cell attachment to E C M proteins, oncogenes, tumor suppressor genes, cytotoxic T cells, radiation and essentially all available chemotherapeutic drugs (Reed, 1994). In contrast, overexpression of bax was reported to accelerate apoptosis by inhibiting the death repressor activity of bcl-2, probably by forming bcl-2-bax complexes or by competing with other bcl-2 targets (Oltvai et al; 1993). In addition, physiological and pathological stimuli, such as steroids (Bu et al, 1997; Perillo et al, 2000; Wang and Phang, 1995) or peptide hormones (Imai et al, 1998; Kuroda et al, 1998), growth factors (Havrilesky et al, 1995; Lafon et al,  Al  1996), cytokines (Gooch, 1998), radiation (Filippovich et al, 1997) and anticancer drugs (Judson et al, 1999'strobel et al, 1996; Zaffaroni et al, 1998) have been demonstrated to regulate apoptotic pathways in ovarian or breast cancer cells, suggesting that these factors may play a role in regulating pro- or anti-apoptotic genes in these cells. Bcl-2 inhibits programmed cell death triggered by several stimuli. It has been demonstrated that bcl-2 may inhibit apoptosis induced by glucocorticoids (Alnemri et al, 1992) and by anticancer drugs, such as 5-fluorodeoxyuridine or taxol (Fisher et al, 1993) in leukemic cells. The activity of bcl-2 has been shown to block gamma-radiation induced cell death (Sentman et al, 1991). The mechanism of apoptosis prevention has not been elucidated, but it has been suggested that bcl-2 may function in an antioxidant pathway which inhibits lipid peroxidation (Ffockenbery et al, 1993) and/or mitochondrial electron and metabolite transport (Hockenbery et al, 1990). Its main effect is to prolong cell survival by avoidance of apoptosis.  2.5. Mitogen-Activated Protein Kinases The mitogen-activated protein kinases (MAPKs) are a group of serine/threonine kinases that are activated in response to a diverse array of extracellular stimuli, and mediate signal transduction from the cell surface to the nucleus (Davis, 1994). Extracellular signal-regulated kinases (ERK1 and ERK2), c-jun terminal kinase/stress-activated protein kinases (JNK/SAPK), and p38 are three of the best characterized M A P K family members (Cobb and Goldsmith, 1995; Fanger, 1999) (Fig. 6). Other M A P K family members, ERK3, 4 and 5, four p38-like kinases, and p57 M A P K have been cloned, but a biological role for them has not been elucidated yet (Fanger, 1999). It is well known that the M A P K cascade is activated via two distinct classes of cell surface receptors, receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs).  48  The signals transmitted through this cascade lead to activation of a set of molecules that regulate cell growth, division and differentiation. The most extensively studied members of the cascade are the ERK1 (p44 M A P K ) and ERK2 (p42 M A P K ) . ERK1 and/or ERK2 are activated by mitogenic stimuli, whereas J N K / S A P K and p38 are activated in response to stress such as heat shock, osmotic shock, cytokines, protein synthesis inhibitors, antioxidants, ultra-violet, and DNA-damaging agents (Garrington and Johnson, 1999; Robinson and Cobb, 1997). M A P K family members are directly regulated by kinases known as M A P K kinases (MAPKKs), which activate the M A P K s by phosphorylation of tyrosine and threonine residues (Fanger, 1999; Robinson and Cobb, 1997). Currently, seven different M A P K K s have been cloned and characterized (Fanger, 1997). The first M A P K K s cloned were M A P K / E R K kinase 1 and 2 ( M E K 1/2), which specifically activate ERKs. M K K 3 and 6 specifically activate p38, whereas M K K 5 stimulates the phosphorylation of ERK5. The M K K 4 and 7 are known to activate JNK. The M A P K K s are activated by a rapidly expanding group of kinases called M A P K K kinases ( M A P K K K s ) , which activate the M A P K K s by phosphorylation of serine and threonine residues (Fanger, 1999; Robinson and Cobb, 1997). These include Raf-1, A-Raf, B-raf, M A P K / E R K kinase 1-4 (MEKK1-4), apoptosis-stimulating kinase-1 (ASK-1), and mixed lineage kinse-3 (MLK-3).  The M A P K K K s may be activated by kinases known, as M A P K K K kinases  ( M A P K K K K s ) , one of which is p21-activated kinase (PAK). In addition, low molecular weight GTP-binding (LMWG) proteins regulate the activity of M A P K K K s and M A P K K K K s (Fanger, 1999). There are several different families of L M W G proteins, two of which include the Ras (NRas, K-Ras, and H-Ras) and Rho (Rac 1, 2 and 3, Cdc42 and Rho A , B and C) families. Activated M A P K s have been shown to phosphorylate a large number of both cytoplasmic and nuclear proteins, exerting their specific functions. For example, activated ERK1/2 phosphorylate  49  Growth factor, hormone, neurotransmitter  Stress/cytokine  Heat shock Osmotic shock UV  G-proteincoupled receptors  TNFa ILla,p  Tyrosine kinase receptors  r?  ^f^ PT C  MF.KK  M A P K T C M(s  I  \AAPVK  All  MAPKKK/ MEKK  T  MF.K 1 n  MAPK/Erk  MAPKK/ MEK  )  MAPK  Transcriptional factors C e l l proliferation, differentiation and survival  Stress response  Figure 6 . Signal-transduction pathways of receptors or stress-activated M A P K s .  50  ternary complex factor (TCF) proteins such as Elk-1 and SAP-1, which form transcriptional complexes with serum response factor (SRF) in the promoter region of early response genes (e.g. c-fos, egr-1, junB) and thereby regulate their expression (Wasylyk et al, 1998) (Fig. 6). Many of these nuclear proteins, as a result of their ability to modulate expression of other proteins, are potential candidates for critical factors involved in the cellular response to stimuli. As mentioned earlier, the M A P K cascade can be activated via both RTKs and GPCRs, which include F S H and GnRH receptors. In ovarian cancer cells, M A P K s were regulated by cisplatin (Persons et al, 1999), paclitaxel (Wang et al, 1999), endothelin-1 (Vecca et al, 2000) and GnRH (Kimura et al, 1999) suggesting that the M A P K signaling pathway plays an important role in the regulation of proliferation, survival and apoptosis in response to these external stimuli.  3. Rationale and Objectives The common epithelial ovarian tumors appear to arise from the ovarian surface epithelium (OSE), which is a simple squamous-to-cuboidal mesothelium covering the ovary, as mentioned earlier (Auersperg et al, 1998; Herbst, 1994; Nicosia et al, 1991). The exact mechanism of ovarian tumorigenesis is not known even though this disease accounts for the most common cancer among gynecological malignancies. The incessant ovulation theory was suggested, whereby repeated ovulation contributes to (pre)neoplastic change of OSE, suggesting that the wound healing process of ruptured OSE may play a role in the disease in women (Fathalla, 1971). Therefore, endocrine and autocrine factors including hormones and multiple growth factors were suggested to influence the occurrence of ovarian tumors during the menstrual cycle (Godwin et al, 1993; Hamilton, 1992; Piver et al, 1991; Rao and Slotman, 1991 Risch, 1998; Shoham, 1994; Westerman et al, 1997). Therefore, we tested the hypothesis that endocrine and autocrine  51  factors may play a role in the induction of proliferation and/or apoptosis related with bcl-2 and bax expression in normal and neoplastic OSE cells during the development of ovarian cancer.  Specific aim 1: To investigate the differential expression of activin/inhibin subunit and activin receptor mRNAs {EXPERIMENTA) The importance of the activin/inhibin system in regulating cell proliferation and possibly ovarian tumor development has been suggested recently (Di Simone et al., 1996; Fukuda et al., 1998; Welt et al, 1997). A study of six human epithelial ovarian cancer cell lines revealed the expression of PA and/or PB subunits as well as activin type II receptor. Several ovarian cancer cell lines produced activin in vitro and exogenous activin induced proliferation of these cells (Di Simone et al., 1996). However, the exact role of activin and its receptors has not been elucidated. Therefore, this study was designed to further examine the autocrine role of activin in normal and neoplastic OSE cells, via determining the expression of activin/inhibin subunits and activin type II receptors, and its effect on cellular proliferation.  Specific aim 2: To determine the effect of activin and TGF-P on the regulation of apoptosis {EXPERIMENT B) The TGF-p superfamily plays a critical role in the regulation of proliferation and apoptosis for ovarian tumorigenesis. The importance of activin, a member of the TGF~P superfamily, in regulating cell proliferation and possibly ovarian tumor development has been suggested (Di Simone et al., 1996; Fukuda et al., 1998; Welt et al., 1997). Therefore, activin appears to act as an autocrine/paracrine factor in epithelial ovarian tumors, but its role in tumorigenesis has yet to be defined (Welt et al, 1997). In addition, it has been demonstrated that TGF-P inhibits  52  proliferation but does not induce apoptosis in normal OSE cells. In contrast, TGF-R induces apoptosis in some ovarian tumors that are growth-inhibited by TGF-R (Havrilesky et al., 1995). This study was performed to investigate the role of activin and TGF-R in normal, early neoplastic and tumorigenic OSE cells. The expression of the activin/inhibin subunits and activin receptors was determined. In addition, the effects on cell number and induction of apoptosis by activin and TGF-R were also examined. Finally, the regulation of pro-apoptotic bax and antiapoptotic bcl-2 was investigated following treatment with activin and TGF-R.  Specific aim 3: To examine the role of E2 on the regulation of anti-apoptotic bcl-2 mRNA and protein (EXPERIMENT Q The actions of estrogen are mediated through interactions with its intracellular receptor, a member of the steroid/thyroid/retinoid receptor gene superfamily (Tsai and O'Malley, 1994). The classical estrogen receptor (ER, now referred to as ERa) was thought to be the only form of nuclear receptor able to bind estrogen, and mediate its hormonal effects in their target tissues. However, the cloning of a second form of estrogen receptor, now referred to as ERR, has prompted a reexamination of the estrogen signaling system (Mosselman et al., 1996). Recent studies have revealed different tissue distributions and expression levels of E R a and ERR in the ovary, suggesting different biological roles of E R a and ERR in this tissue (Kuiper et al., 1996; Mosselman et al., 1996; Sar and Welsch, 1999). In addition, the existence of E R a and ERR in normal OSE and ovarian cancers has been demonstrated (Brandenberger et al., 1998; Lau et al., 1999). While E2 is not a mitogen for normal OSE (Karlan et al., 1995), treatments with exogenous estrogen resulted in growth stimulation of several ER-positive ovarian carcinoma cell  53  lines in vitro (Chien et al, 1994; Galtier-Dercure et al, 1992; Langon et al, 1994). However, the role of estrogen in ovarian tumorigenesis and regulation of apoptosis by estrogen in neoplastic OSE cells remains uncertain. The present study was performed to investigate the role of E2 in the regulation of apoptosis in normal, early neoplastic, tumorigenic and late neoplastic OSE cells. The expression of E R a and ERP was determined in these OSE cell lines to investigate the effect of E2. Furthermore, cell proliferation and prevention of apoptosis by E2 were examined in immortalized OSE cell lines. Finally, to elucidate the mechanism of E2 in the prevention of apoptosis, the regulation of proapoptotic bax and anti-apoptotic bcl-2 was investigated following treatments with E2 and/or antiestrogen, tamoxifen.  Specific aim 4: To investigate the effect of FSH on activation of mitogen-activated protein kinase {EXPERIMENT D) In addition to its well-established function in reproductive physiology, follicle-stimulating hormone (FSH) has been implicated in ovarian cancer development (Konishi et al, 1999; Zheng et al, 2000). Epidemiological studies demonstrated an increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during postmenopause or infertility therapy (Risch, 1998 Shushan et al, 1996; Whittemore et al, 1992). Expression of F S H receptor (FSH-R), a G protein-coupled receptor, has been demonstrated in normal OSE (Zheng et al, 1996), ovarian inclusions and epithelial tumors (Zheng et'al, 2000), implicating a possible role of FSH in these cells.  In addition, treatment of F S H resulted in growth-stimulation of ovarian cancer cells  (Wimalasena et al, 1992; Zheng et al, 2000) in a dose- and time-dependent manner in vitro. Despite these findings, the precise molecular mechanism of FSH in terms of growth stimulation  54  and intracellular signaling in ovarian cancer remains unknown. A number of factors including cisplatin (Persons et al, 1999), paclitaxel (Wang et al, 1999), endothelin-1 (Vecca et al, 2000) and GnRH (Kimura et al, 1999) have been shown to regulate M A P K activity on ovarian cancer cells, suggesting a possible signaling pathway in the regulation of growth and differentiation. The effect of FSH on growth stimulation in ovarian cancer cells may be mediated via the M A P K signaling cascade. Little is known about the molecular events that mediate FSH actions in normal and neoplastic ovarian cells. Considering that FSH plays a role in these cells, the effect of FSH in normal OSE and IOSE cell lines was investigated. In the present study, experiments were designed to examine 1) the expression of FSH-R at the mRNA level in normal OSE and IOSE cell lines, 2) the proliferative effect of FSH in these cells, and 3) the effect of FSH on ERK1/2 activation.  Specific aim 5: To elucidate the expression and apoptotic role of the second form of gonadotropin-releasing hormone (GnRH-II) {EXPERIMENT E) In addition to its well-documented role in the regulation of gonadotropin synthesis and secretion in the reproductive hormone cascade, GnRH, a hypothalamic decapeptide, has been suggested to act as an autocrine/paracrine regulator in normal ovarian surface epithelium (Kang et al, 2000). GnRH and its synthetic analogs have a direct growth-inhibitory effect on ovarian tumors (Emons et al,  1993a; Yano et al, 1994). This concept is based on the detection of  binding sites for GnRH, as well as the expression of GnRH and its receptor gene transcripts in these tumors. Recently, a second form of GnRH with characteristics of chicken GnRH-II, now referred to as GnRH-II (pGln-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly) has been cloned in brain extracts from rhesus monkeys (Lescheid et al,  55  1997). In addition, GnRH-II has been  demonstrated to encode a different gene and is expressed at significantly higher levels outside the brain, including kidney (30-fold higher than in any brain region), bone marrow, and prostate, suggesting that GnRH-II may have multiple functions in various tissues (White et al, 1998). Interestingly, GnRH-II has been shown to bind the GnRH receptor (GnRH-R) up to 100 times more effectively than GnRH-I, suggesting GnRH-II may act via GnRH-R outside the brain (King and Millar, 1991). However, the role of GnRH-II in normal and neoplastic OSE cells remains to be elucidated. To examine the potential role of GnRH-II as a possible regulator, it has been investigated for the first time the expression of GnRH-II in normal and neoplastic OSE cells. In addition, to investigate the physiological significance, the direct receptor-mediated growth regulatory effect of GnRH-I and -II on neoplastic OSE cells was investigated for the first time in this study. Finally, to examine the regulatory effects of GnRH-I and -II on apoptosis, the induction of cell death was investigated following treatment with exogenous GnRH-I and -II.  56  II. MATERIALS AND METHODS  1. Materials Human recombinant activin A (rh-activin A), follistatin and follicle-stimulating hormone (FSH) were generously provided by Dr. A. F. Parlow in National Hormone & Pituitary Program of Harbor-UCLA Medical Center (Torrance, CA). Recombinant TGF-(3l, tamoxifen, staurosporin, 3-isobutyl-l-methylxanthine agonist) were purchased from Sigma-Aldrich  17(3-estradiol,  (IBMX), and (D-Ala )-GnRH (GnRH  Corp.  6  (Oakville, Canada). PD98059,  a  M A P K / E R K kinase (MEK) inhibitor, was purchased from New England Biolabs Inc. (Berverly, MA). GnRH-II was purchased from Peninsula Laboratories (Belmont, CA).  2. Cell cultures 2.1. Normal OSE cells Normal OSE cells were scraped from the ovarian surface during laparoscopics for nonmalignant disorders and cultured as previously described (Kruk et al., 1990) in medium 199:MCDB 105 (Sigma-Aldrich Corp., Oakville, Canada, respectively) containing 10% FBS, 100 U/ml penicillin G and 100 fig/ml streptomycin (Life Technologies, Inc., Burlington, Canada) in a humidified atmosphere of 5% C02-95% air and passaged with 0.06% trypsin (1:250)/0.01% E D T A in M g  2 +  /Ca  2+  - free HBSS when confluent. The human ovarian epithelial carcinoma cell  line, OVCAR-3 was cultured in medium 199:MCDB 105 containing 10% FBS, 100 U/ml penicillin G and 100 Hg/ml streptomycin in a humidified atmosphere of 5% C02-95% air.  57  2.2 Primary cultured ovarian tumors (PC-OVC) from surgical specimens in women Primary ovarian tumors and ascite fluid from ovarian cancer patients were obtained from Vancouver General Hospital and British Columbia Cancer Agency, respectively. Solid tissue was processed within 1 hr after surgery in medium 199 :MCDB 105 supplemented with 100 U/ml penicillin G and 100 fig/ml streptomycin as previously described (Crickard et al., 1983). Areas of fat, nontumor, and obviously necrotic tissue were removed from the tumor mass and minced with a scalpel into 2 mm pieces. The minced tissue was incubated in medium 199 :MCDB 105 3  containing 4mg/ml collagenase(type III) and 0.1 mg/ml bovine pancreatic DNase (Sigma Co.) for 1 hr in 37 C CO2 incubator. The suspension was mixed with a pipet, and the cells were passed through a fine mesh in the presence of mediuml99:MCDB 105 containing 20% FBS, 100 U/ml penicillin G, 100 ilg/ml streptomycin, and 2.5 u\g/ml Fungizone. The cell suspension was layered onto Ficoll paque (Amersham-Pharmacia Co.) and centrifuged to remove red blood cells. The cancer cells were placed on a 10 cm plastic culture dish with 10 ml of mediuml99:MCDB 105 supplemented with 20% FBS for 1 to 2 h. Fibroblasts and mesothelial cells tend to attach to the plastic substrate during this period, leaving a relatively pure preparation of tumor cells in suspension. The tumor cells could be preplated on plastic substrate with subsequent passages to eliminate any fibroblasts present. The suspended tumor cells were centriftiged at 500 x g for 10 min and resuspended in complete medium. The tumor cells were maintained on culture dishes in the presence of medium 199:MCDB 105 supplemented with 20% FBS, 100 U/ml penicillin G, and 100 ug/ml streptomycin. Ascites fluid was processed and maintained as previously described (Hurteau et al., 1994). Briefly, cancer cells were removed from the fluid by centrifugation. The cells were layered onto Ficoll paque and centrifuged to remove red blood cells. The cancer cells were placed in medium 199:MCDB 105 supplemented with 10% FBS for 24 h. The suspended  58  tumor cells were pelleted, resuspended and maintained on culture dishes in the presence of mediuml99:MCDB 105 supplemented with 20% FBS, 100 U/ml penicillin G, and 100 itg/ml streptomycin.  2.3. Immortalized ovarian surface epithelium cell lines (IOSE cell lines) As outlined in Table 1, a culture system with cells representing several stages in the neoplastic progression of OSE has been developed. The IOSE-29 cell line (referred to as IOSEMar in some previous publications) was generated by transfection with the immortalizing simian virus 40 early genes into normal human OSE at passage 5 (Maines-Bandiera et al., 1992). The IOSE-29EC cell line was made from IOSE-29 at passage 11 by transfecting the full length mouse E-cadherin cDNA under the control of the (3-actin promoter (Auersperg et al., 1999). The IOSE-29EC/T4 and IOSE-29EC/T5 were established from tumors that arose in IOSE-29ECinoculated SCID mice, and they formed tumors on mesenteries and omentum, invaded the liver and thigh musculature, and produced ascites (Ong et al., 2000). For monolayer culture, all cell lines were maintained on tissue culture dishes (Corning; Corning Laboratory Sciences Co.) in a 1:1 mixture of medium 199 / M C D B 105 medium supplemented with 10% FBS, 100 U/ml penicillin G and 100 lig/ml streptomycin.  2.4. Established epithelial ovarian cancer cell lines O V C A R - 3 , the epithelial ovarian cancer cell line derived from adenocarcinoma was kindly provided by Dr. T.C. Hamilton, Fox Chase Cancer Center, Philadelphia, P A (Chien et al., 1994). Epithelial ovarian cancer cell lines including CaOV-3, O V C A R - 3 and SKOV-3 cells were cultured under the conditions as described above.  59  TABLE 1. Development of early neoplastic, tumorigenic and late neoplastic OSE cells from normal human OSE  TRANSFECTION  CELL TYPE  NEOPLASTIC PROGRESSION  OSE SV40 Tag Immortalized OSE ('IOSE-29')  Extended life span Increased growth rate Genetic instability  Tumorigenic OSE (TOSE-29EC)  Epithelial differentiation Anchorage independence Tumorigenicity Invasiveness  E-cadherin  Tumor-derived OSE (TOSE-29EC7T4, T5')  60  Selected for tumorigenesis in SCID mice  3. Treatments To study the basal expression level of activin/inhibin subunits and activin type II receptors, 2 x 10 normal OSE and OVCAR-3 cells were plated and cultured on 35-mm culture dishes in the 5  above-mentioned medium. In a dose-response experiment, 2 x 10 OVCAR-3 cells were cultured 5  in 35 mm dish for 48 h and treated with rh-activin A (1, 10 and 100 ng/ml) for 24 h. To investigate the specificity of activin, the cells were treated with activin (10 ng/ml) plus follistatin (100 ng/ml), which is a specific activin binding protein. In a time-course experiment for activin, 2 x 10 OVCAR-3 cells were treated with 10 ng/ml rh-activin A for 3, 6, 12, and 24 h. 5  To study the regulation of pro-apoptotic bax and anti-apoptotic bcl-2 mRNA by 17P-estradiol (E2, Sigma-Aldrich Corp., Oakville, Canada), 2 x 10 IOSE-29EC cells were plated onto 35-mm 5  culture dishes. After a preincubation of 48 h, the cells were treated with E2 at concentrations of 10" , 10" and 10" M in phenol-red free medium 199 (Sigma-Aldrich Corp.) with 2 % 8  7  6  charcoal/dextran treated FBS (HyClone Laboratories Inc.) for 24 h. To confirm the specificity of E2, the cells were treated with E2 (10" M) plus tamoxifen (10~ M , Sigma-Aldrich Corp.) for 24 7  6  h. Control cultures were treated with vehicle (absolute ethyl alcohol). Furthermore, to investigate the regulation of bax and bcl-2 apoptotic proteins by E2, 2 x 10 IOSE-29EC cells were plated 5  onto 35-mm culture dishes and cultured for 48 h. Subsequently, the cells were treated with E2 (10" , 10" and 10" M) plus tamoxifen (10" M ) for 48 h. 8  7  6  6  4. RNA extraction and RT-PCR procedures Total R N A was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer's suggested procedure. R N A integrity was confirmed by agarose gel electrophoresis and ethidium bromide staining. The total R N A  61  concentration was determined from spectrophotometric analysis at A260/280- Complementary D N A (cDNA) was synthesized from 2.5 tig total R N A by reverse transcription at 37 C for 2 h using a first strand cDNA synthesis kit (Pharmacia Ltd., Uppsala, Sweden). The synthesized cDNA was used as template for polymerase chain reaction (PCR) amplification. The amplification was achieved using a thermal cycler ( D N A Thermal Cycler, Perkin-Elmer, Norwalk, CT). Total R N A (2.5 |ng) was reverse transcribed into first strand cDNA (Amersham Pharmacia Biotech, Oakville, Canada), following the manufacturer's procedure. Synthetic oligonucleotides used for PCR primers and PCR conditions are listed in Table 2 based on the published sequences. The P C R reactions were performed in 25 u.1 P C R mixture containing 1 X PCR buffer, 0.2 m M each dNTP, 1.6 m M M g C ^ , 50 pmol specific primers, each cDNA template, and 0.25 unit Taq polymerase. The P C R reaction was performed for two or three independent cDNA preparations of each R N A sample. Twelve microliters of PCR products were analyzed by agarose (1%) gel electrophoresis and visualized by ethidium bromide staining, and the sizes were estimated by comparison to D N A molecular weight markers.  5. Southern blot analysis Following electrophoresis, P C R products were transferred to nylon membranes (Hybond-N, Amersham Co.) and fixed by U V irradiation. The blotted membranes were prehybridized for 3 h at 42 C in prehybridization solution containing 50 % formamide, 5X SSC, 0.1 % N-lauroyl sarcosine, 0.02 % SDS, and 2 % blocking solution. The prehybridized membranes were hybridized overnight at 42 C with digoxigenin-labeled probes. The c D N A clones for a, PA, and PB subunits were subcloned from human granulosa cells and verified by sequencing.  62  TABLE 2. Oligonucleotide sequences of PCR primers for human Oligo  Sequence  Cycle No.  a subunit  sense antisense  5'-CCC A G T TTC A T C TTC C A C TAC-3' 5'-CCC A T A G G C A C A G A A G T G A A - 3 '  27  pA subunit  sense antisense  5'-AGG T C A A C A TCT GCT G T A AG-3' 5'-TTC TCT G G A C A A CTC T T G CT-3'  27  pB subunit  sense antisense  5'-TGT TGC A G G C A A C A G TTC TTC-3' 5'-GAA T G A C T G T A C T T A G C C CAC-3'  27  ActR-IA  sense antisense  5'-GAT G A G A A G T C A T G G TTC A G G - 3 ' 5'-TAT GTT T G G CCT T T G T T G ATC-3'  30  ActR-IB  sense antisense  5'-CTG G C T GTC C G T C A T G A T G C A - 3 ' 5' - C A A TTC GCT CTC A G A GTC T C C - 3 '  40  ActR-IIA  sense antisense  5'-ACC A G T GTT G A T G T G G A T CTT-3' 5'-TAC A G G T C C A T C T G C A G C AGT-3'  30  ActR-HB  sense antisense  5'-TTC T G C T G C TGT G A A G G C A A C - 3 ' 5'-GAG GTC GCT CCT C A G C A A TAC-3'  30  ERa  sense antisense  5'-ATG A C C A T G A C C CTC A A C A C C A A - 3 ' 5'-CTT G G C A G A TTC C A T A G C C A T A C - 3 '  30  ERP  sense antisense  5'-TAC A G C A T T C C C A G C A A T GTC A C - 3 ' 5 ' - G A A G T G A G C A T C C C T CTT T G A A C - 3 '  30  Bax-a  sense antisense  5'-ATG G A C G G G T C C G G G G A G C A G C-3' 5'-CCC C A G T T G A A G T T G C C G T C A G-3'  24  Bcl-2  sense antisense  5'-GGT G C C A C C TGT GGT C C A CCT G-3' 5'-CTT C A C TTG T G G C C C A G A T A G G-3'  30  FSH-R  sense antisense  5 ' - G A G A G C A A G G T G A C A G A G A T T C C-3' 5' -CCT TTT G G A G A G A A T G A A TCT T-3'  30  GnRH-II  sense antisense  5'-GCC C A C CTT G G A C C C T C A G A G - 3 ' 5'-CGG A G A A C C T C A C A C TTT A T T GG-3'  30  GnRH-R  sense antisense,  5'-GTA T G C T G G A G A GTT A C T C T G C A - 3 ' 5'-GGA T G A T G A A G A G G C A G C T G A A G - 3 '  33  p-actin  sense antisense  5'-GGA C C T G A C T G A C T A CCT C A T G A A - 3 ' 5'-TGA T C C A C A TCT GCT G G A A G G TGG-3'  15  GAPDH  sense antisense  5*-ATG TTC G T C A T G G G T G T G A A C C A - 3 ' 5'-TGG C A G GTT TTT C T A G A C G G C A G - 3 '  18  63  The cDNA clones for activin receptor IA and IB were provided by Dr. C. Peng (York Univ., Toronto, Canada). The cDNA clones for activin receptor IIA and IIB were kindly provided by Dr. W.Vale (The Salk Institute, La Jolla, CA) and Dr. C. Peng, respectively. The cDNA clones for bax and bcl-2 were subcloned from ovarian cancer cell line (OVCAR-3) and verified by sequence analysis. PCR products of ERa and ERp isolated from human granulosa cells were cloned into pCRII vector using the T A Cloning Kit (Invitrogen, San Diego, C A ) and were sequenced by the dideoxy nucleotide chain termination method using the T7 D N A polymerase sequencing kit (Amersham Pharmacia Biotech.). The cDNA clones for bax and bcl-2 were subcloned from ovarian cancer cell line (OVCAR-3) and verified by sequence analysis abovementioned. The cDNA clone for FSH-R was kindly provided by Dr. Minegishi (Gunma University School of Medicine, Gunma, Japan) (Minegish et al., 1991). The cDNA probes were labeled with digoxigenin cDNA labeling kit (Roche Molecular Biochemicals, Laval, Canada) for hybridization. The hybridized membranes were detected with luminescence method (Boehringer Mannheim Co.) and exposed to X-ray film for 1 to 10 min at room temperature. The specific bands were scanned and quantified using a computerized visual light densitometer (model 620, BioRad Laboratories, Richmond, CA).  6. Cloning and sequencing PCR products of a, pA, P B subunits, bax, bcl-2, GnRH-II were subsequently cloned using T A cloning kit (Invitrogen, San Diego, C A ) and sequenced to verify their identities using T7 sequencing kit (Amersham Pharmacia Biotech.). A l l sequence analysis was performed using both M13 universal forward and reverse primers. Double-strand D N A templates (2 ug in 32 ul) were denatured for 10 min by adding 8 ul of 2 M NaOH in reaction volume of 40 ul. Following  64  incubation, D N A was precipitated with a sodium acetate/ethanol (7 ul of 3 M sodium acetate, pH 4.8, 4 ul of distilled water, and 120 ul absolute ethanol) and collected by centrifugation (10, 000 x g, 20 min), washed with 70% ethanol, and dried under vacuum. The denatured D N A was resuspended in 10 ul of distilled water, combined with 2 ul of primer, and 2 ul of annealing buffer (1 M Tris-HCl, pH 7.6, 100 m M M g C l , 160 m M DTT). The reaction was incubated for 5 2  min at 65 C and then for 10 min at 37 C. After incubation, the reaction mixture was allowed to cool down to room temperature for 15 min. Following the annealing reaction, 3 ul of Labeling Mix, 5 uCi of [a- S]dATP (Amersham Pharmacia Biotech.), and 2 ul of diluted T7 D N A 35  polymerase (3 unit) were added and incubated for 5 min at room temperature. Four of 2.5 ul dNTP mix (840 ul M of A , C, G, and T mix) were prepared and prewarmed for 5 min before addition of 4.5 ul of the labeling reaction to each mix. After incubation for 5 min at 37 C, reactions were stopped with 5 ul of Stop Solution (0.3 % each bromophenol blue and xylene cyanol FF, 10 m M E D T A (pH 7.5), and 97.5% deionized formamide) and boiled for 5 min prior to loading (2.5 ul) onto a sequencing gel. Polyacrylamide 6%/7M urea sequencing gels were prepared and prewarmed at 45 W constant power for 1 h. The samples were loaded and run for 3-4 h at 45 W constant power. Subsequently, gels were dried at 80 C for 2 h using a gel dryer (Model 583, Biorad Laboratories, Richmond, CA) and exposed to autoradiography at -70 C for 8-24 h. Each P C R product was verified to be identical to the published sequence (GenBank, NIH) through the website (www.ncbi.nlm.nih.gov).  7. Northern blot analysis Total RNAs (50- 100 ug) were denatured in 50 % formamide/2.2 M formaldehyde, incubated at 60 C for 15 min, and electrophoresed on 1% denaturing agarose gel (20 m M MOPS, 2.2 M  65  formaldehyde, 8 m M sodium acetate, 1 m M E D T A pH 8.0) at a constant 70 voltage for 3-4 h. A capillary transfer of R N A to nylon membrane was performed overnight in 10X SSC, as described in Southern blot analysis. The membrane was then irradiated by U V light for 5 min to cross-link the R N A to membrane. Radioactive labeled cDNA probe of FSH-R was prepared from Ramdon Labeling Kit (Life Technologies Inc.) according to the manufacturer's suggested procedure. D N A templates (100 ng) were denatured at 100 C for 5 min and placed on ice. Two ul each of following dNTP (dCTP, dGTP, and dTTP), 15 ul of random priming buffer, 50 uCi of [a- P]dATP (3000 Ci/mmol, Amersham Pharmacia Biotech.) and 3 unit of Klenow fragment 32  were mixed. After 3 h incubation period at 25 C, the reaction was stopped by adding 5 ul of Stop Buffer (0.2 m M N a E D T A , pH 7.5). The labeled D N A was purified using G-50 Sephadex 2  column (Amersham Pharmacia Biotech.). The membrane was pre-hybridized in standard hybridization solution (50% formamide, 5X SSPE, 5X Denhardt's, 0.5 % SDS, 100 ug/ml denatured herring sperm D N A ) for. 3 h at-42 C. The membrane was washed once with 2 X SSC/0.1 % SDS at room temperature and twice with 0.1 X SSC/0.1% SDS at 65 C for 15 min. After washing, the membrane was exposed to Kodak Omat x-ray film (Eastman Kodak Co.).  8. Immunoblot analysis The immortalized ovarian surface epithelium cells (IOSE-29, IOSE-29EC, IOSE-29EC/T4, and IOSE-29EC/T5) were seeded at a density of 2 x 10 cells in 35 mm culture dishes and 5  cultured in a humidified atmosphere of 5 % C02-95 % air at 37 C. Cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer (150 m M NaCl, 1 % Nonidet P-40, 0.5 % deoxycholate, 0.1 % SDS, 50 m M Tris (pH, 7.5), and 1 m M PMSF, 10 ug/ml leupeptin, 100 Ug/ml aprotinin). The extracts were placed on ice for 15 min and centrifuged to remove cellular  66  debris. Protein content of the supernatants was determined using a Bradford assay (Bio-Rad Laboratories) (Fig. 7). Twenty-five or 30 ixg of total protein was run on 10 % SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech.). The membranes were immunoblotted using rabbit polyclonal antibodies of activin receptor IA, IB, IIA, and IIB provided by Dr. W. Vale (The Salk Institute, La Jolla, C A ) or a mouse monoclonal antibody for E R a (Santa Cruz Biotech., Santa Crus, CA) and a goat polyclonal antibody for ERp (Santa Cruz Biotech.). To determine i f the in vitro treatments affected the expression of the genes involved in apoptosis, the membranes were immunoblotted using mouse monoclonal antibodies of bax (BD Pharmingen Inc., Mississauga, O N , Canada) and bcl-2 (SantaCruz Biotech.). The loaded amount of protein was normalized with actin protein in the same membrane. In addition, the membrane was immunoblotted using a mouse monoclonal antibody specific to the phosphorylated p44/p42 M A P K (P-MAPK, T h r / T y r ) (New England Biolabs 202  204  Inc.). Alternatively, a membrane was probed with a rabbit polyclonal antibody for p44/42 M A P K (New England Biolabs Inc.), which detects total M A P K (T-MAPK,  phosphorylation-state  independent) levels. After washing, the signals were detected with horseradish peroxidaseconjugated secondary antibody, and visualized using the E C L chemiluminescent system (Amersham Pharmacia Biotech.).  9. [ H1 thymidine incorporation assay 3  Normal OSE and O V C A R - 3 cells were plated in 24-well plates at l x l O cells/well in 0.5 ml 4  medium 199:MCDB 105 supplemented with 10 % FBS and antibiotics for 24 h.  After a  preincubation period, the cells were treated with rh-activin A (1, 10, and 100 ng/ml) and/or follistatin (100 ng/ml) in medium plus 2% FBS as previously described (Di Simone et al, 1996; Wang et al., 1996a). After 48 h, the medium was removed, and the cells were treated with the  67  Optical Densitiy (O.D) Figure 7. A standard curve for the protein assay. Cellular lysate (10 ]x\) was incubated with 25 | i l o f Reagent A and 200 (il o f Reagent B , for 15 min with gentle shaking at room temperature. A n absorbance o f the samples was measured using E L I S A reader at 630 n m wavelength.  68  same concentration of rh-activin A for an additional 48 h. After 96 h incubation, the medium was removed again, the cells were cultured in the presence of the same concentration of rh-activin A and 1 (iCi[ H]thymidine (5.0 Ci/mmol; Amersham Inc.) for another 24 h. Human recombinant 3  follistatin was used to block the effect of activin. For TGF-p treatment, the cells were incubated with 0.1, 1, 10 ng/ml TGF-P and replaced daily for 72 h. During the last 6 h, 1 ixCi[ H]thymidine 3  and  the same concentration of TGF-P were added to each well as previously described  (Havrilesky et al., 1995). On the day of treatment, the cells were incubated with increasing concentrations (10" ,10" or 10" M) of E2 in phenol-red free medium 199 with charcoal/dextran treated FBS for 2-6 days. On 6  the days indicated in the results, during the last 6 h of the incubations to be harvested, the medium was changed to include the same concentration of E2 and 1 |iCi[ H]thymidine. 3  Tamoxifen (10" M , Aldrich-Sigma Corp.), a specific E2 antagonist, was used to block the E2 6  effect. In addition, the cells were incubated with increasing concentrations (10, 100 and 1000 ng/ml) of F S H and 1 itCi[ H]thymidine in serum free media for 24 h. In this study, the cells were 3  treated with increasing concentrations (10" , 10" and 10" M ) of GnRH agonist, (D-Ala )-GnRH 9  8  7  6  or GnRH-II everyday for 6 days. To block the effect of the GnRH agonist, the cells were treated with.the (D-Ala )-GnRH (10" M ) or GnRH-II plus antide ( 1 0 M , Sigma-Aldrich Corp.) at 6  7  7  equimolar concentration for 6 days. After the culture medium was removed, to determine [ H]thymidine incorporation, the cells 3  were washed three times with PBS and precipitated with 0.5 ml 10 % trichloroacetic acid for 20 min at 4 C (Wang et al., 1996a). The precipitate was washed in methanol twice and solubilized in 0.5 ml 1 N sodium hydroxide. The incorporated radioactivity was measured in a 1217  69  Rackbeta liquid scintillation counter ( L K B Wallac, Turku, Finland). Each experiment was repeated three times.  10. DNAfluorometricassay In addition to the [ H]thymidine incorporation assay, the effect of E2 on the growth of IOSE3  29 and IOSE-29EC was determined by measuring the D N A content as previously described with some modifications in 24-well plates (Rago et al, 1990). The cells were treated with various concentrations (10" , 10" or 10" M) of E2 and/or tamoxifen (10~ M ) in phenol-red free medium 8  7  6  6  199 with charcoal/dextran treated FBS for 6 days. After treatment, the cells were washed with T N E buffer (10 m M Tris, 1 m M EDTA, 2 M NaCl, pH 7.4) three times and stored at -70 C. On the day of assay, 250 ul of distilled water was added in the wells and incubated for 1 h at room temperature (RT). The plates were frozen then for 1 h at -70C and thawed until reaching RT. The amount of D N A was measured using an automated microplate fluorescence reader (Model FL600, Bio-Tek Instruments Inc., Winooski, V A ) at an excitation wavelength of 350 nm and emission wavelength of 460 nm (sensitivity = 90) following adding the D N A fluorescent dye. The amount of D N A in the culture was calculated by inserting the fluorescence unit into a standard curve.  11. Quantification of apoptotic cells To quantify the induction of apoptosis, D N A fragmentation was measured using the cell death detection ELISA kit (Roche Molecular Biochemicals) as previously described (4). Briefly, 1 x 10 cells were plated in each well of 24-well plates. After treatment with E2 and tamoxifen for 6 4  days, the conditioned media were collected and stored. The cells were washed with PBS, and 0.1  70  ml lysis buffer was added. Following 15 min incubation on ice, apoptotic cells in cell lysates and conditioned media were assayed for D N A fragments according to the manufacturer's protocol. The same amount (lu.g) of cell lysate was used in each experiment. D N A fragmentation was measured at 405 nm.against the untreated control.  12. In vitro MAPK assay IOSE-29 and IOSE-29EC cells were serum starved for 4 h. The cells were then treated with FSH (100 ng/ml) in the presence or absence of PD98059 for 10 and 30 min, respectively and washed twice with ice-cold PBS and lysed in 1 x lysis buffer (20 m M Tris (pH 7.5), 150 m M NaCl, 1 m M EDTA, 1 m M EGTA, 1% Triton, 2.5 m M sodium pyrophosphate, 1 m M p glycerolphosphate, 1 m M NaaVO^ 1 u.g/ml leupeptin, 1 m M PMSF). The extracts were placed on ice for 15 min and centrifuged to remove cellular debris, and protein amount of supernatants was determined.  Cellular protein (200 ug) was immunoprecipitated with immobilized phospho-  p44/42 M A P K monoclonal antibody. In vitro M A P K assays were performed using the Elk-1 fusion protein as a substrate for activated M A P K , according to the manufacturer's suggested procedure (New England Biolabs Inc.).  13. RIA for intracellular cAMP To measure intracellular cAMP, IOSE cell lines and human granulosa luteal cells (2 x 10  5  cells) were plated onto 35 mm culture dishes and cultured for 4 days. The cells were then preincubated in serum-free  medium containing 0.1% B S A and 0.5 m M 3-isobutyl-l-  methylxanthine (IBMX, Sigma-Aldrich Corp.) for 30min, and treated with FSH for 0, 5, 10, 20,  71  or 60 min. Intracellular cAMP levels were measured using a [ H]-cAMP assay system 3  (Amersham Pharmacia Biotech), according to the manufacturer's suggested procedure.  14. Statistical analysis Data are shown as the means of two or three individual experiments with triplicate samples, and are presented as the mean ± SD. Expression levels of activin/inhibin subunits and type II activin receptors mRNA were analyzed by Student's / test using raw data before transformation to percentage and expressed as the percent change from the control value. For a multiple comparison test, data were analyzed by one-way A N O V A (Analysis of Variance) followed by Tukey's multiple comparison or Dunnett's test using raw data before transformation to percentage by the computer software PRISM GraphPad (Ver. 2, GraphPad Software Inc., San Diego, USA). A l l data were considered significantly different from each other at P<0.05 and expressed as the change from the control value in the Result.  72  III. RESULTS  1. EXPERIMENT  A  1.1. Expression of activin/inhibin subunits To investigate the relative importance of activin and inhibin in normal and neoplastic OSE, basal expression levels of a, pA and PB subunits in OSE and OVCAR-3 were examined. The mRNA levels of activin/inhibin subunits were quantitated by RT-PCR. The 382-bp, 377-bp and 424-bp P C R products were obtained and confirmed as a, PA and PB subunits of activin/inhibin using Southern blot hybridization, respectively, as previously described (Fukuda et al., 1998). The P C R products amplified were subcloned and sequenced, and found to be 100% identical to published sequences of activin/inhibin subunits in these cells (data not shown). As shown in Fig. 8, a significantly higher expression level of a subunit was observed in normal OSE cells when compared to OVCAR-3 cells (Fig. 8A). Also, PA subunit was significantly higher in normal OSE, when compared to OVCAR-3 cells (Fig. 8B). In contrast, a significantly higher mRNA level of pB subunit was detected in OVCAR-3 cells when compared to normal OSE cells (Fig. 8C).  1.2. Expression of activin receptors Basal expression levels of activin type II receptors (IIA and IIB) in normal and neoplastic OSE cells were investigated. The 456-bp and 699-bp PCR products were obtained and confirmed as activin receptor IIA and IIB using Southern blot hybridization, respectively, as previously described (Fukuda et al, 1998). The P C R products amplified were subcloned and sequenced, and found to be 100% identical to published sequences of activin type IIA and IIB receptors in  73  OVCAR-3  OSE  a subunit  382-bp  P actin  y^^*  ^ ^ ^ ^  OSE  ]^  524-bp  OVCAR-3 Cell Types  OVCAR-3  OSE  B  377-bp  R A subunit  P actin  524-bp  OSE  OVCAR-3 Cell Types  74  Figure  8.  Differential expression level o f activin/inhibin subunits i n normal O S E and O V C A R - 3  cells. The m R N A levels o f a (A), pA (B) and pB (C) subunits were quantitated by R T - P C R as described in the Materials and Methods. The 382-bp, 377-bp and 424-bp P C R products were obtained and confirmed as a , pA and PB subunits o f activin/inhibin using Southern blot hybridization, respectively. Values are standardized with p-actin expression and represented as the mean ± S D . a, P<0.05 vs basal expression level o f activin/inhibin subunits in O S E .  75  OSE  A  OVCAR-3  ActRIIA P actin  524-bp  o.o  OSE  OVCAR-3  Cell Types B  OSE  ActRIIB  •L  OVCAR-3  1  699-bp  P actin  524-bp 3n  OSE  OVCAR-3  Cell Types Figure 9. Differential expression level of type II activin receptors in normal O S E and O V C A R - 3 cells. The m R N A levels o f activin receptor IIA (A) and IIB (B) were quantitated by R T - P C R . The 456-bp and 699-bp P C R products were obtained and confirmed as activin receptor IIA and IIB using Southern blot hybridization, respectively. Values are standardized with P-actin expression and represented as the mean ± SD. a, P<0.05 vs basal expression level o f each activin receptor in O S E .  76  these cells (data not shown). As seen in Fig 9A, no difference of activin receptor IIA was observed in normal OSE and OVCAR-3 cells. Interestingly, a significantly higher level of activin receptor IIB mRNA was observed in OVCAR-3 cells when compared to normal OSE cells (Fig. 9B).  1.3. Effects of activin on cell proliferation The effect of activin on cellular proliferation was analyzed in normal and neoplastic OSE cells using a thymidine incorporation assay. As shown in Fig. 10, rh-activin A (1, 10, 8and 100 ng/ml) induced a significant increase in cell proliferation to 150 % of control in OVCAR-3 cells. This stimulatory effect of activin was attenuated by treatment of follistatin (100 ng/ml), which is a specific activin binding protein. However, no difference was observed following activin treatment in normal OSE cells (data not shown).  1.4. Effects of activin on activin/inhibin subunits and activin receptors To investigate whether the growth stimulatory effect of activin in neoplastic OSE is mediated via an autocrine mechanism, the effect of exogenous activin on the expression of activin/inhibin subunits and activin receptors in OVCAR-3 cells were investigated. Treatment of rh-activin A at concentrations of 1, 10, and 100 ng/ml for 24 h induced a significant increase in a subunit mRNA expression (Fig. 11). Interestingly, a dose-dependent increase in PA subunit mRNA was observed after activin treatments (Fig. 12A). In addition, this stimulatory effect of activin (10 ng/ml) on PA subunit mRNA expression was attenuated with treatment of follistatin (Fig. 12B). However, no difference in PB subunit was observed in OVCAR-3 cells following rh-activin A  77  200-1  Act Fol  0 0  1 0  10 0  100 0  10 100  0 100  Treatments of activin and/or follistatin (ng/ml)  Figure 10. Effects of rh-activin A on OVCAR-3 cell proliferation. Normal OSE and OVCAR-3 cells were cultured and treated with rh-activin A (1, 10, and 100 ng/ml) and/or follistatin (100 ng/ml) as described in the Materials and Methods. A [ HJthymidine incorporation assay was performed to quantify D N A synthesis following activin treatments. Values are the mean ± SD for three individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs 10 ng/ml activin treatment.  78  Activin (ng/ml) Control  1  Control  10  1  10  100  100  Activin Treatment (ng/ml)  Figure 11. Effect of rh-activin A on a subunit expression in O V C A R - 3 cells. Cells were treated with activin (1, 10 and 100 ng/ml) for 24 h as described in the Materials and Methods. To investigate the specificity of activin, the cells were treated with activin (10 ng/ml) plus follistatin (100 ng/ml) simultaneously. The expected size of PCR product for a subunit was obtained as 382-bp. Values are standardized with (3-actin expression and represented as the mean ± SD for three individual experiments, each with triplicate samples, a, P<0.05 vs untreated control.  79  Activin (ng/ml) 100  10  Control I  i  ir  377-bp  p A subunit P actin  L L . I  524-bp  I I .  Control  1  10  100  Activin Treatment (ng/ml)  Treatment o f activin and/or follistatin (ng/ml) activin 0 10 10 follistatin 0 0 100 i  \r~  II  1  P A subunit  P actin  Figure 12. Effect o f rh-activin A on p A subunit expression i n O V C A R - 3 cells. Cells were treated with activin ( 1 , 1 0 and 100 ng/ml) for 24 h as described i n the Materials and Methods (A). T o investigate the specificity o f activin, the cells were treated with activin (10 ng/ml) plus follistatin (100 ng/ml) simultaneously (B). The expected size o f P C R product for p A was obtained as 377-bp. Values are standardized with P-actin expression and represented as the mean ± SD for three individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs 1 ng/ml activin treatment.  80  Activin (ng/ml) 1  Control  100  10  424-bp  RB subunit  p  actin  [flBjHt  Control  1  XT  JXi X — X  10  100  JE^^I  524-bp  Activin Treatment (ng/ml)  Figure 13. Effect of rh-activin A on pB subunit expression in OVCAR-3 cells. Cells were treated with activin (1,10 and 100 ng/ml) for 24 h as described in the Materials and Methods. To investigate the specificity of activin, the cells were treated with activin (10 ng/ml) plus follistatin (100 ng/ml) simultaneously. The expected size of PCR product for pB was obtained as 424-bp. Values are standardized with p-actin expression and represented as the mean ± SD for three individual experiments, each with triplicate samples.  81  treatments (Fig. 13). Further, the levels of activin receptor IIA and IIB were determined after rhactivin A treatments. Treatments of OVCAR-3 cells with activin (1 - 100 ng/ml) did not affect activin receptor IIA and IIB mRNA levels in O V C A R - 3 cells (Fig. 14A and B). The effect of activin on activin/inhibin a and PA subunits was further investigated in a timecourse experiment (Fig. 15A and B). OVCAR-3 cells were treated with 10 ng/ml activin A for 3, 6, 12 and 24 h. Treatment of activin (10 ng/ml) for 24 h induced a significant increase in a subunit mRNA level as shown in Fig. 15A. The pA subunit mRNA level also significantly increased following activin treatment at same amount for 6, 12 and 24 h (Fig. 15B). Thus, exogenous rh-activin A increased the mRNA levels of a and PA subunit in a time-dependent manner.  82  ED  ID (Kinase Domain)  TD  Primers of activin receptor type lis:  0.0  sense  antisense  Control 1 10 100 Activin Treatment (ng/ml)  B  Control  1  10  100  Activin Treatment (ng/ml) Figure 14. Effects of rh-activin A on type II activin receptors mRNA expression in OVCAR-3 cells. Cells were treated with 1,10 and 100 ng/ml activin for 24 h as described in the Materials and Methods. Primer sets for activin receptor IIA and IIB were designed to amplify coding sequences of intracellular domain of type II activin receptors. The 456-bp and 699-bp of PCR products were observed for activin type IIA (A) and IIB (B) receptors as previously published. Values are standardized with P-actin expression and represented as the mean ± SD for two individual experiments, each with triplicate samples. E D , extracellular domain; TD, transmembrane domain; ID, intracellular domain of activin receptors.  83  Duration of Activin Treatment (Hours) B  Duration of Activin Treatment (Hours)  Figure 15. A time-dependent increase of a and RB subunit by rh-activin A in O V C A R - 3 . The effect of activin on activin/inhibin a (A) and RA (B) subunits was further investigated in a timecourse experiment.  The cells were treated with 10 ng/ml activin for 3, 6, 12 and 24 h as  described in the Materials and Methods. Values are relative to R-actin and plotted as the mean ± SD for three individual experiments, each with duplicate samples, a, P<0.05 vs untreated control.  84  2. EXPERIMENT B  2.1. Expression of activin/inhibin subunit mRNAs The mRNA levels of a, pA, and pB subunits in IOSE-29 (passages 13-16), IOSE-29EC (passages 15-17), IOSE-29EC/T4 (T4), and IOSE-29EC/T5 (T5) were investigated by RT-PCR and Southern blot analysis. The possibility of cross-contamination was rule out, because no P C R products were observed and detected in the negative control [TmA(-); without template in the PCR reaction] by ethidium bromide (data not shown) and Southern blot analysis (Fig. 16). Linear relationship between PCR products and amplification cycles was obtained in G A P D H , a, pA and PB subunits in all cell types (data not shown). Expected PCR products of G A P D H , a, PA and PB subunits were obtained at 3 73-bp, 382-bp, 377-bp and 424-bp respectively and confirmed by Southern blot analysis (Fig. 16) and sequence analysis (data not shown).  Semi-quantitative  analysis of the present study demonstrated that all types of activin/inhibin subunits are expressed in IOSE-29, IOSE-29EC, T4, and T5. Interestingly, pB subunit was less expressed in IOSE cell lines when compared in OVCAR-3 cells (Fig. 16).  2.2. Expression of activin receptor mRNAs The mRNA levels of activin receptor IA, IB, IIA, and IIB in IOSE-29 (passages 13-16), IOSE-29EC (passages 15-17), T4 and T5 were investigated by RT-PCR and Southern blot analysis (Fig. 17). The expected sizes of PCR products for activin receptors were obtained as 651-bp, 684-bp, 456-bp and 699-bp, respectively using sense and antisense primers located within the intracellular kinase domains of each activin receptor. The P C R of G A P D H was amplified to rule out the possibility of R N A degradation, and used to control the variation in mRNA concentrations in RT reaction. PCR products of the predicted sizes were obtained and  85  IOSE-29EC  IOSE-29  Tm(-) GAPDH  a subunit  T4  T5  OVCAR-3  I <- 373-bp  <r 382-bp  (  PA subunit  PB subunit  <r 377-bp  jjULjtKjk  ^kft  424-bp  Figure 16. The m R N A expressions of activin/inhibin subunits in IOSE cell lines. The mRNA levels of a, PA, and PB subunits in IOSE-29 (passages 13-16), IOSE-29EC (passages 15-17), IOSE-29EC/T4 (T4), and IOSE-29ECT5 (T5) were investigated by RT-PCR and Southern blot analysis as previously described in the Materials and Methods. The possibility of crosscontamination was ruled out, because no PCR products were observed and detected in the negative control [TmA(-); without template in the PCR reaction] by ethidium bromide (data not shown) and Southern blot analysis. The expected sizes of PCR product for G A P D H , a, PA and PB subunits were obtained as 373-bp, 382-bp, 377-bp and 424-bp, respectively.  86  IOSE-29  IOSE-29EC  T4  T5  GAPDH  OVCAR-3  l<r 373- bp  ED  TD  ID (Kinase Domain) 3'  5'  Primers:  sense  antisense  ActR-IA  651-bp  ActR-IB  <r 684-bp  ActR-IIA  <r 456-bp  ActR-IIB  <r 699-bp  Figure 17. The m R N A expressions o f activin receptors i n I O S E cell lines. The m R N A levels o f activin receptor I A , I B , IIA, and IIB in IOSE-29 (passages 13-16), I O S E - 2 9 E C (passages 15-17), T4, and T5 were investigated by R T - P C R and Southern blot analysis. The expected sizes o f P C R products for activin receptors were obtained as 651-bp, 684-bp, 456-bp and 699-bp, respectively using sense and antisense primers located within the intracellular kinase domains o f activin receptors. The P C R o f G A P D H was amplified to rule out the possibility o f R N A degradation, and used to control the variation in m R N A concentrations in R T reaction. E D , extracellular domain; T D , transmembrane domain; ID, intracellular domain o f activin receptors.  87  confirmed by Southern blot analysis using DIG-labeled probes (Fig. 17) and sequence analysis (data not shown). Semi-quantitative analysis of the present study demonstrated that all forms of activin receptors were observed in IOSE-29, IOSE-29EC, T4 and T5.  2.3. Expression of activin receptor proteins Immunoblot analysis was performed using the rabbit polyclonal antibodies against activin receptor IA (amino acid 474-494), IB (aa 493-505), IIA (aa 482-494), and IIB (aa 524-536) based on COOH terminal amino acids in IOSE cell lines. As shown in Fig. 18, activin receptor IA protein (60 kDa) was observed in all cell types. OVCAR-3 cell line was used for positive control of the expression of activin receptors (Fukuda et al., 1998). Similarly, activin receptor IB protein (55 kDa) was also observed in all cell types (Fig. 18). In addition, activin receptor IIA and IIB were clearly detected at 80 kDa and 60 kDa respectively in IOSE cell lines and O V C A R 3 cells (Fig. 18). Immunoblot analysis of the present study demonstrated that all forms of activin receptor protein were observed in IOSE-29, IOSE-29EC, T4 and T5.  2.4. Effects of activin on cell number To evaluate the role of recombinant human activin A (rh-activin A ) in IOSE cell lines, IOSE29 and IOSE29EC were treated with different concentrations (1, 10, and 100 ng/ml) of rh-activin A for 6 days. The proliferative index was measured by thymidine incorporation assay. Follistatin, which is activin-binding protein, was used to block the action of activin in cell proliferation study. As seen in Fig. 19, a dose-dependent decrease of cell number was observed following rhactivin A (1, 10, and 100 ng/ml) treatments in IOSE-29 (Fig. 19A, 100.0 ± 7.63 vs 83.7 ± 6.06, 67.9±4.10or59.9±9.06)andIOSE-29EC(Fig. 19B, 100.0 ± 5.89 vs 75.9 ±9.11,61.4 ± 8.11 or 52.9 ± 9.70) cells. Co-treatment with follistatin (lOOng/ml) with activin blocked the growth-  88  O V C A R - 3 IOSE-29 IOSE-29EC  ActR-IA  ActR-IB  WtmP  ActR-IIA  ActR-IIB  T4  T5  <r  60 kDa  <r  55 kDa  <r  80 kDa  <r  60 kDa  Figure 18. The expression of activin receptor proteins in IOSE cell lines. Immunoblot analysis was performed using the rabbit polyclonal antibodies against activin receptor IA, IB, IIA and IIB as previously described in the Materials and Methods. Activin receptor IA (60 kDa) and IB (55 kDa) were observed in all cell types. OVCAR-3 cell line was used for positive control of the expression of activin receptors (Fukuda et al., 1998). In addition, activin receptor IIA and IIB were clearly detected at 80 kDa and 60 kDa, respectively, in IOSE cell lines and O V C A R - 3 cells.  89  IOSE-29  0  Activin Follistatin  0 0  Treatment with activin and/or follistatin (ng/ml)  B IOSE-29EC  0  Activin Follistatin  0 0  Treatment with activin and/or follistatin (ng/ml) Figure 19. Effect of activin on cell proliferation in IOSE cell lines. The IOSE-29 and IOSE29EC were treated with different concentrations (1, 10, and 100 ng/ml) of rh-activin A for 6 days. Proliferative index was measured using the thymidine incorporation assay as previously described in the Materials and Methods. A dose-dependent decrease was observed following rhactivin A (1, 10, and 100 ng/ml) treatments in IOSE-29 (A) and IOSE-29EC (B). Co-treatment with follistatin (lOOng/ml) with activin was demonstrated to block the growth-inhibitory of activin in both cell lines. Values are the mean ± SD for three individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs 1 ng/ml activin treatment; c, P<0.05 vs treatment with 100 ng/ml activin.  90  inhibitory effect of activin in both cell lines (Fig. 19). However, no significant difference was observed in follistatin treatment only.  2.5. Effects of TGF-R on cell proliferation To examine the role of TGF-R, normal OSE, IOSE-29 and IOSE-29EC cells were treated with different concentrations (0.1, 1 and 10 ng/ml) of TGF-R for 72 h. As seen in Fig. 20A, TGF-R (1 and 10 ng/ml) induced a significant decrease of normal OSE cell proliferation in a dosedependent manner (100.0 ± 15.62 vs 58.6 ± 11.78 or 43.3 ± 12.03). Also, a significant decrease was observed following TGF-R treatments (1-10 ng/ml) in IOSE-29 (Fig. 20B, 100.0 ± 5.03 vs 81.1 ± 7.59 or 69.8 ± 4.08). In addition, treatment with TGF-R resulted in a decrease of proliferative index in IOSE-29EC (Fig. 20C, 100.0 ± 11.70 vs 74.2 ± 5.63, 67.1 ± 7.05 or 55.0 ± 6.75).  2.6. Effects of activin and TGF-P on apoptosis To quantify the induction of apoptosis, IOSE-29EC cells were treated with rh-activin A for 6 days. As shown in Fig 21, treatments with 10 and 100 ng/ml activin increased D N A fragmentation in a dose-dependent manner (100.0 ± 8.06 vs 190.6 ± 13.58 or 221.3 ± 15.72). Cotreatment with follistatin (lOOng/ml) with activin attenuated the effect of activin. No significant difference was observed in follistatin treatment only in IOSE-29EC cells. Similarly, IOSE-29EC cells were treated with different concentrations of TGF-P for 72 h. Treatments with TGFP induced a significant increase of D N A fragmentation in a dose-dependent manner in IOSE29EC (Fig. 22, 100.0±5.20 vs 123.7 ±10.03, 191.3 ±16.94 or 201.9 ±25.06).  91  V  A  Normal OSE  E  5*  H  100  o o c o O  50  0  Con  0.1  10  Treatment with TGF-B (ng/ml)  Figure 20 (including next page). Effects of TGF-R on cell proliferation in IOSE cell lines. Normal OSE, IOSE-29 and IOSE-29EC cells were treated with increasing concentrations (0.1, 1 and 10 ng/ml) of TGF-R for 72 h.  Proliferative index was measured using the thymidine  incorporation assay as previously described in the Materials and Methods. Treatments with TGF-P (1 and 10 ng/ml) induced a significant decrease of growth in normal OSE (A), IOSE-29 (B) and IOSE-29EC (C) in a dose-dependent manner. Values are the mean ± SD for three individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs 0.1 ng/ml TGF-p treatment.  92  IOSE-29  Treatment with T G F - f i (ng/ml)  IOSE-29EC  Treatment with TGF-p (ng/ml)  93  IOSE-29EC  Activin 0 1 10 100 100 0 Follistatin 0 0 0 0 100 100 T r e a t m e n t s of activin a n d / o r follistatin (ng/ml)  Figure 21. Effect of activin in the induction of apoptosis. To quantify the induction of apoptosis, IOSE-29EC cells were treated with rh-activin A for 6 days. Attached and detached cells were collected and D N A fragmentation was measured by cell death detection ELISA as described in the Material  and Methods. Treatments with 10 and 100 ng/ml activin increased D N A  fragmentation in a dose-dependent manner. Co-treatment with follistatin (lOOng/ml) with activin attenuated the effect of activin. Values are the mean ± SD for two individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs treatment with 100 ng/ml activin.  94  IOSE-29EC  300-i CO  Treatment with TGF-p (ng/ml) Figure 22. Effect of TGF-p in the induction of apoptosis. To quantify the induction of apoptosis, IOSE-29EC cells were treated with TGF-R for 72 h. Attached and detached cells were collected and D N A fragmentation was measured by cell death detection ELISA as described in the Material and Methods. Treatments with TGF-R for 72 h induced D N A fragmentation of IOSE29EC in a dose-dependent manner. Values are the mean ± SD for two individual experiments, each with triplicate samples, a, P<0.05 vs untreated control; b, P<0.05 vs treatment with 0.1 ng/ml TGF-P treatment.  95  2.7. Expression of pro- and anti-apoptotic gene mRNAs The mRNA levels of bax and bcl-2 in IOSE-29 (passages 13-18), IOSE-29EC (passages 1318) were investigated by RT-PCR and Southern blot analysis (Fig. 23). Linear relationship between PCR products and amplification cycles was obtained in G A P D H , bax and bcl-2 in all cell types (data not shown). PCR products of G A P D H , bax and bcl-2 were obtained as 373-bp, 323-bp and 459bp, respectively and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 23). No difference was observed in the expression level of bax mRNA between IOSE-29 and IOSE-29EC cells. In contrast, the expression level of bcl-2 m R N A was higher in IOSE-29EC cells than IOSE-29 cells (Fig. 23).  2.8. Effects of activin and TGF-R on apoptotic proteins To investigate the mechanism of activin and TGF-R in the induction of apoptosis, the regulation of apoptotic bax and bcl-2 was examined by immunoblot analysis. The IOSE-29EC cells were treated with increasing doses of rh-activin A and TGF-R respectively for 24 h and immunoblot analysis was performed as described in the Material and Methods. Bax and bcl-2 protein were detected at 21 kDa and 26 kDa respectively. As seen in Fig 8A, treatments with 10 and 100 ng/ml activin had no effect on both bax and bcl-2 proteins in these cells. No significant difference of bax protein was observed in TGF-P treatments (Fig 8B). In contrast, treatments with 1 and 10 ng/ml TGF-p induced a significant decrease of bcl-2 protein up to 50% (Fig. 8B and C, 100.0 ±5.17 vs 58.2 ±7.35 or 54.0 +5.39). The loaded amount of proteins in treatment groups was normalized by actin protein (41 kDa).  96  IOSE-29  IOSE-29EC  GAPDH  373-bp  Bax <r 323-bp  AM  Bcl-2  wfffff  <- 459-bp  Figure 23. The expression o f bax and bcl-2 m R N A s in I O S E cell lines. The m R N A levels o f bax and bcl-2 i n IOSE-29 (passages 13-18), IOSE-29EC (passages 15-20) were investigated by R T P C R and Southern blot analysis. P C R products o f G A P D H , bax and bcl-2 were obtained as 373bp, 323-bp and 459-bp, respectively and confirmed by Southern blot analysis using DIG-labeled probes. N o difference was observed i n the expression level o f bax m R N A between IOSE-29 and I O S E - 2 9 E C cells. In contrast, the expression level o f bcl-2 m R N A was higher i n I O S E - 2 9 E C cells than IOSE-29 cells.  97  Treatment with activin (ng/ml) 0 10 100 Bax  <- 21 k D a  Bcl-2  <-26 k D a  Actin  <- 41 k D a  Figure 24. Effect o f activin on bax and bcl-2 proteins. I O S E - 2 9 E C cells were treated with various doses o f rh-activin for 24 h and immunoblot analysis was performed as described in the Material and Methods. Treatments with 10 and 100 ng/ml activin had no effect on both bax (21 kDa) and bcl-2 (26 kDa) proteins in these cells. The loaded amount o f protein in treatment groups was normalized by actin protein (41 kDa).  98  Treatment with T G F - p (ng/ml) 0 1 10  Bax  <- 21 k D a  Bcl-2 <- 26 k D a  Actin  /  B  <r 41 k D a  V  IOSE-29EC  ® 150 CD  c 'o o  Q.  iooH  CM  o CD > _CC  CD  DC  0  1  10  T r e a t m e n t with T G F - p (ng/ml) Figure 25. Effect o f T G F - p on bax and bcl-2 proteins. I O S E - 2 9 E C cells were treated with various doses o f T G F - P for 24 h and immunoblot analysis was performed as described i n the Material and Methods. N o significant difference o f bax protein was observed in T G F - p treatment. In contrast, treatments with 1 and 10 ng/ml T G F - P induced a significant decrease o f bcl-2 protein up to 50% ( A and B ) . The loaded amount o f protein i n treatment groups was normalized by actin protein (41 kDa). Values are the mean ± S D for two individual experiments, each with duplicate samples, a, P<0.05 vs untreated control.  99  3. EXPERIMENT C  3.1. Expression of ERa and ERP mRNAs The mRNA levels of E R a and ERp in IOSE-29 (passages 13-16), IOSE-29EC (passages 1517), IOSE-29EC/T4 (T4) and IOSE-29EC/T5 (T5) were investigated by RT-PCR and Southern blot analysis. The possibility of cross-contamination was ruled out, because no P C R products were observed and detected in the negative control [TmA(-); without template in the RT reaction] by ethidium bromide (data not shown) and Southern blot analysis (Fig. 26). A Linear relationship between P C R products and amplification cycles was obtained in all cell types (data not shown). Predicted PCR products of G A P D H , E R a and ERp were obtained as 373-bp, 540-bp and 279-bp respectively and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 26) and sequence analysis (data not shown). This result indicates that the mRNAs of E R a and ERp are expressed in IOSE-29, IOSE-29EC, IOSE-29EC/T4 and IOSE-29EC/T5.  3.2. Expression of ERa and ERp proteins To investigate the expression of E R a and ERp proteins in immortalized OSE cell lines, immunoblot analysis was performed using the mouse monoclonal antibody for E R a and a goat polyclonal antibody for ERp. As shown in Fig. 27 E R a protein (68 kDa) was observed in all cell types. OVCAR-3 cell line was used for positive control of the expression of ER expression. ERp protein was also observed as 55 kDa in immortalized OSE cell lines. Immunoblot analysis of the present study demonstrated that E R a and ERP proteins were observed in IOSE-29, IOSE-29EC, IOSE-29EC/T4 and IOSE-29EC/T5.  100  TmA(-)  IOSE-29  IOSE-29EC  T4  T5  OVCAR-3  GAPDH <r 373-bp  m mmmmm mmm m <- ^  P  Figure 26. The m R N A levels of E R a and ERp in IOSE cell lines. The m R N A levels of E R a and ERp in IOSE-29 (passages 13-16), IOSE-29EC (passages 15-17), IOSE-29EC/T4 (T4) and IOSE-29EC/T5 (T5) were investigated by RT-PCR and Southern blot analysis. Expected PCR products of G A P D H , E R a and ERP were obtained as 373-bp, 540-bp and 279-bp, respectively and confirmed by Southern blot analysis using DIG-labeled probes and sequence analysis (data not shown).  101  OVCAR-3  IOSE-29 IOSE-29EC  T4  T5  Figure 27. The protein levels of E R a and ER(i in IOSE cell lines. In parallel of mRNA expression of E R a and ERP, immunoblot analysis was carried out using specific antibodies for E R a and ER(3 in IOSE cell lines. OVCAR-3 cell line was used for positive control of the expression of ERs. E R a protein (68 kDa) was observed in all cell types. ERp protein was also observed as 55 kDa in IOSE cell lines.  102  3.3. Effects of E2 on cell proliferation To evaluate the role of E2 in normal and immortalized OSE cell lines, the cells were treated with increasing concentrations (10" , 10" and 10" M ) of E2 for 2-6 days. The [ H]thymidine 8  7  6  3  incorporation and D N A fluorometric assays were performed as previously described in the Materials and Methods. Tamoxifen (Txf, 10" M), which is an estrogen antagonist, was used to 6  block the action of E2 in the cell proliferation study. Treatment with E2 did not affect the growth of normal OSE (Fig. 28A), whereas E2 treatment for 2-6 days resulted in an increase of the growth in O V C A R - 3 cells in a time-dependent manner as positive control (Fig. 28B). Highest proliferative effect of E2 was observed at 10" M , whereas the effect of E2 was less increased at 7  10" M in OVCAR-3 cells (Fig. 28B). No difference was observed following E2 treatment for 6 6  days in IOSE-29 cells by thymidine incorporation (Fig. 29A) and fluorometric assay (Fig. 29B), In contrast, treatment with E2  (10" - 10" M) prior to day 6 had no effect on thymidine  incorporation, but it resulted in a significant increase on day 6 (Fig. 30A, 100.0 + 8.16 % vs 134.7 ± 5.37 %, 156.8 ± 12.23 % or 132.8 ± 6.85 %) in IOSE-29EC cells. Similarly, D N A content in culture also increased significantly on day 6 (Fig. 30B, 100.0 ±6.15 % vs 138.4 ± 4.08 %, 176.3 ± 22.02 % or 147.6 ± 24.00 %). Co-treatment with E2 (10" M ) plus tamoxifen ( 1 0 M ) 7  6  attenuated the effect of E2 (100.0 + 8.16 % vs 74.2 + 6.50 % in the thymidine and 100.0 ± 6.15 % vs 77.7 ± 7.94 % in the D N A content) in IOSE-29EC cells. The ratio of thymidine incorporation / D N A content per culture did not change following E2 and/or tamoxifen treatments in IOSE-29 (Fig. 29, Panel C) and IOSE-29EC cells (Fig. 30, Panel C), suggesting that E2 effect does not include stimulation of proliferation. Treatment with tamoxifen only also caused inhibitory effect in IOSE-29 (Figs. 29A and B) and IOSE-29EC cells (Figs. 30A and B). In addition, a significant increase of thymidine incorporation by E2 (10" M) was also observed in IOSE-29EC/T4 and  103  Duration o f E 2 treatment [day]  Figure 28. Effect of E2 on cell proliferation/apoptosis in normal OSE and OVCAR-3 cells. To evaluate the role of E2 in IOSE cell lines, the cells were treated with increasing concentrations (10~ , 10" and 10" M ) of E2 and/or tamoxifen (10~ M ) for 2-6 days as previously described in 8  7  6  6  the Materials and Methods. [ H]thymidine incorporation was analyzed following E2 treatment 3  for 2-6 days in normal OSE (A) and OVCAR-3 cells (B).  104  E2 Txf C  0 10" 10" 10" 10" 0 0 0 0 0 10" 10" Treatment with E2/tamoxifen (Txf) (M) 8  7  6  7  6  The ratio of thymidine/DNA content E2 (M) Txf (M)  0 0  10" 0  1.00  0.90  8  io0  7  1.08  10" 0  6  1.12  10 10"  -7 6  0.99  0 10"  6  0.96  Figure 29. Effect of E2 on cell proliferation/apoptosis in IOSE-29 cells. A [ H]thymidine 3  incorporation (A) and D N A fluorometic assays (B) were performed following E2/tamoxifen treatment for 6 days in IOSE-29. The ratio of thymidine incorporation / D N A content per culture did not change following E2 and/or tamoxifen treatments in IOSE-29 (Panel C). Data are shown as the means of three individual experiments with triplicate, and are presented as the mean ± S D . a, P<0.05 vs. untreated control.  105  CD  E2 Txf  C  0 10" 10" 10" 10" 0 0 0 0 0 10" 10 Treatm ent with E2/tamoxifen (Txf) (M) 8  7  6  7  6  The ratio of thymidine / D N A content E2 (M) Txf (M)  0 0  10" 0  1.00  0.97  8  10" 0  7  0.89  10" 0  10" 10"  6  7  6  0.96  0.90  0 10"  6  1.11  Figure 30. Effect of E2 on cell proliferation/apoptosis in IOSE-29EC cells. A [ H]thymidine 3  incorporation (A) and D N A fluorometic assays (B) were performed following E2/tamoxifen treatment for 6 days in IOSE-29EC. The ratio of thymidine incorporation / D N A content per culture did not change following E2 and/or tamoxifen treatments in IOSE-29EC (Panel C). Data are shown as the means of three individual experiments with triplicate, and are presented as the mean ± SD. a, P O . 0 5 vs. untreated control; b, P<0.05 vs. E2 (10" M ) treatment. 7  106  B  IOSE-29EC/T5  Txf  0  0  10°  10"  Treatment with E2/tamoxifen (Txf) (M)  Figure 31. Effect of E2 on cell proliferation/apoptosis in IOSE-29EC/T4 and /T5. A thymidine incorporation by E2/tamoxifen was also analyzed in IOSE-29EC/T4 (A) and IOSE-29EC/T5 (B) cells after 6-day treatment. Data are shown as the means of three individual experiments with triplicate, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. E2 (10" M) treatment. 7  107  IOSE-29EC/T5 cells, whereas co-treatment with tamoxifen attenuated the E2 effect (Figs. 31A and B).  3.4. Effects of E2 on apoptosis To examine the role of E2 in the prevention of apoptosis, D N A fragmentation was measured using the cell death detection ELISA. To quantify the induction of apoptosis, IOSE-29EC cells were treated with tamoxifen (10" M) and/or E2 (10~ , 10" and 10" M ) for 6 days. As shown in 6  8  7  6  Fig 32, treatment with tamoxifen resulted in a significant increase of D N A fragmentation in IOSE-29EC cells (100.0 ± 5.89 % vs 287.7 ± 11.26 %). Co-treatments with E2 (10 , 10" and 10" 8  6  7  M ) plus tamoxifen attenuated tamoxifen-induced apoptosis in a dose-dependent manner (Fig.  32, 287.7 ±11.26 % vs 219.8 ± 21.47 %, 175.8 ± 12.02 % or 174.9 ± 16.50 %).  3.5. Expression of pro- and anti-apoptotic gene mRNAs and proteins The mRNA levels of pro-apoptotic bax and anti-apoptotic bcl-2 in IOSE-29 and IOSE-29EC were investigated by RT-PCR and Southern blot analysis. Predicted P C R products of G A P D H , bax and bcl-2 were obtained as 373-bp, 323-bp and 459-bp respectively and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 33) and sequence analysis (data not shown). No difference was observed in the expression level of bax mRNA between IOSE-29 and IOSE-29EC cells. In contrast, the mRNA expression level of bcl-2 was higher in IOSE-29EC cells than IOSE-29 cells (Fig. 33A). In parallel of mRNA level, protein level of bcl-2 was investigated in these cell lines. As shown in Fig. 5B, the expression level of bcl-2 protein was higher in IOSE-29EC cells than IOSE-29 cells.  108  E2 Txf  0 0  0 10~  6  10 10"  10~  6  6  10"  6  T r e a t m e n t with E2/tamoxifen (Txf) (M)  Figure 32. Effect of E2 on apoptosis in IOSE-29EC cells. To examine the role of E2 in the prevention of apoptosis, D N A fragmentation was measured using the cell death detection ELISA. Data are shown as the means of three individual experiments with duplicate, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. tamoxifen (10' M ) 6  treatment; c, P<0.05 vs. E2 (10~ M) plus tamoxifen (10~ M) treatment. 8  6  109  Figure 33. Expression o f bcl-2 protein i n IOSE-29 and I O S E - 2 9 E C cells. The protein levels o f bax and bcl-2 in IOSE-29 and I O S E - 2 9 E C were investigated by immunoblot analysis. The protein amount was normalized by actin protein (41 kDa).  110  3.6. Effect of E2 on pro- and anti-apoptotic mRNAs To investigate the mechanism of E2 action in the prevention of apoptosis, the regulation of bax and bcl-2 was examined using RT-PCR in IOSE-29EC cells. The cells were treated with E2 and/or tamoxifen for 24 h. The expected sizes of PCR products for bax and bcl-2 were obtained as 323-bp and 459-bp respectively (Fig. 34). The mRNA expression of bax and bcl-2 was normalized with G A P D H (373-bp) to quantify the mRNA levels. Treatments with E2 (10" to 10" 8  6  M) up-regulated bcl-2 mRNA up to 2-fold in these cells (Figs. 34 and 35, 100.0± 7.19 % vs  172.9 ± 14.47 %, 190.9 ± 22.03 % or 171.8 ± 17.55 %). Co-treatment with tamoxifen (10" M) 6  plus E2 attenuated this E2 effect (190.9 ± 22.03 % vs 116.8 ± 10.92 %) as shown in Figs. 34 and 35. However, no significant difference in the m R N A level of bax was observed by E2 treatment (Fig. 34).  3.7. Effect of E2 on pro- and anti-apoptotic proteins To investigate the protein levels of bax and bcl-2 by E2, immunoblot analysis was performed following estrogen and/or tamoxifen treatments. The cells were treated with E2 and/or tamoxifen for 48 h as previously described in the Material and Methods. Specific signals for bax and bcl-2 protein were detected at 21 kDa and 26 kDa, respectively, as shown in Fig. 36. Consistent with the mRNA levels, treatments with E2 (10" to 10" M) significantly up-regulated bcl-2 protein 8  6  level in these cells (Figs. 36 and 37, 100.0 ± 9.92 % vs 162.05 ± 12.68 %, 166.7 ±19.61 % or 154.4 ± 20.86 %). Co-treatment with tamoxifen (10" M ) plus E2 attenuated this E2 effect (166.7 6  ± 19.61 % vs 109.9 ± 10.22 %) as shown in Fig. 37. In contrast, no significant difference in the protein level of bax was observed by E2 treatment in IOSE-29EC cells (Fig. 36).  Ill  Figure  34. Effect o f E 2 on bax and bcl-2 m R N A levels i n I O S E - 2 9 E C cells. To investigate the  mechanism o f E 2 i n the prevention o f apoptosis, the regulation o f bax and bcl-2 was examined using R T - P C R i n I O S E - 2 9 E C cells. The cells were treated with E 2 and/or tamoxifen (Txf) for 24 h. The expected sizes o f P C R products for bax and bcl-2 were obtained as 323-bp and 459-bp respectively. The m R N A expression levels o f bax and bcl-2 were normalized with G A P D H (393bp) to quantify the m R N A levels. 1, untreated control; 2, E 2 (10" M ) treatment; 3, E 2 (10" M ) 8  treatment; 4, E 2 (10" M ) treatment; 5, tamoxifen ( 1 0 6  tamoxifen (10~ M ) treatment. 6  112  6  7  M ) treatment; 6, E 2 (10~ M ) plus 7  0  E2 Txf  0 0  10" 0  10" 0  8  7  10" 0  6  0 10"  6  10" 10~  7  6  Treatment with E2/tamoxifen (Tmx) (M)  Figure 35. Effect of E2 on bcl-2 mRNA level in IOSE-29EC cells. A relative bcl-2 mRNA expression level was examined after treatment E2 and/or tamoxifen. Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. E2 (10" M ) treatment 7  113  Figure 36. Effect o f E2 on bax and bcl-2 proteins i n I O S E - 2 9 E C cells. In parallel o f m R N A level, protein level o f bax and bcl-2 was investigated b y immunoblot analysis. The cells were treated with E2 and/or tamoxifen (Txf) for 48 h as previously described i n the Materials and Methods. Specific signals for bax and bcl-2 protein were detected at 21 k D a and 26 k D a respectively. The protein amount i n the groups was normalized b y actin protein (41 kDa). 1, untreated control; 2, E 2 (10" M ) treatment; 3, E2 (10" M ) treatment; 4, E 2 (10~ M ) treatment; 5, 8  7  6  tamoxifen (Iff M ) treatment; 6, E 2 (10" M ) plus tamoxifen (10" M ) treatment. 6  7  6  114  E2  Txf  0 0  10" 0  10" 0  8  7  10" 0  6  0 10"  6  10" 10"  7  6  Treatment with E2/tamoxifen (Tmx) (M)  Figure 37. Effect of E2 on bcl-2 protein in IOSE-29EC cells. In parallel of mRNA level, protein level of bax and bcl-2 was investigated by immunoblot analysis. The cells were treated with E2 and/or tamoxifen (Txf) for 48 h as previously described in the Materials and Methods. The protein amount in the groups was normalized by actin protein (41 kDa). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P O . 0 5 vs. E2 ( 1 0 M) treatment. 7  115  4. EXPERIMENT D  4.1. Expression of FSH-R mRNA The mRNA expression of FSH-R in normal and neoplastic OSE cells was investigated by RTPCR, Southern blot and Northern blot analysis. A predicted PCR product of FSH-R was obtained as 369-bp and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 38) and sequence analysis (data not shown). The human granulosa luteal cells (hGLCs) were used for positive control. As demonstrated in Fig. 38, the FSH-R mRNAs are expressed in normal OSE and IOSE cell lines (IOSE-29, IOSE-29EC, IOSE-29EC/T4 and IOSE-29EC/T5). To confirm FSH-R mRNA expression by RT-PCR and Southern blot, Northern blot analysis was performed in IOSE-29, IOSE-29EC and ovarian cancer cell lines, including OVCAR-3 and SKOV-3 cells (Fig. 39). As seen in Fig. 39, the low levels of two transcripts (4.1 and 2.4-kb) of FSH-R mRNA were demonstrated in IOSE cell lines (IOSE-29 and IOSE-29EC) and two ovarian cancer cell lines (OVCAR-3 and SKOV-3). The predominant transcript was 4.1-kb in size as shown in Fig. 39 (Minegishi et al, 1997). The high level of FSH-R mRNA transcripts was observed in human granulosa-luteal cells as positive control.  4.2. Effects of FSH on proliferative index To evaluate the role of FSH in normal and immortalized OSE cell lines, the cells were treated with increasing concentrations (10, 100 and 1000 ng/ml) of human recombinant FSH for 24 h and a [ H]thymidine incorporation assay was performed as previously described (30). Treatments 3  with increasing doses of FSH (10, 100 or 1000 ng/ml) resulted in a significant growthstimulation in normal OSE (Fig. 40A, 100.0 ± 8.33 % vs. 135.1 ± 7.49, 137.1 ± 9.06 or 135.4 ± 13.90) and OVCAR-3 cells (Fig. 40B, 100.0 ± 9.38 % vs. 128.4 ± 7.21 or 128.9 ± 9.60).  116  hGLCs  Normal  IOSE  IOSE  IOSE  IOSE  Figure 38. Expression of FSH-R mRNA in normal OSE and IOSE cell lines. The mRNA expression of FSH-R in normal and neoplastic OSE cells was investigated by RT-PCR and Southern blot analysis. A predicted PCR product of FSH-R was obtained as 369-bp and confirmed by Southern blot analysis using DIG-labeled probes and sequence analysis (data not shown). The amplification of G A P D H (373-bp) was performed to rule out the possibility of R N A degradation, and was used to control the variation in m R N A concentration in the PCR reaction.  117  Figure 39. Expression o f F S H - R m R N A in IOSE and ovarian cancer cell lines. The m R N A expression o f F S H - R in neoplastic O S E cells was investigated by Northern blot analysis. Total R N A (50 fig) was prepared and resolved by formaldehyde denaturing agarose gel electrophoresis. The hybridization was performed using a radioactive labeled F S H - R probe. 1, IOSE-29; 2, IOSE-29EC; 3, O V C A R - 3 ; 4, S K O V - 3 ; 5, Human granulosa-luteal cells (positive control).  118  Normal OSE CD £Z TD  E  >> .c  175 150 125  hX 100 CO M—  O O  75  c  50  o  O  25  vo  0  0  10  100  1000  Treatment with F S H (ng/ml) B  OVCAR-3 ID  175  cz 150  T3 E  125  hX 100 CO O  75  O •*—»  c  o  O  50 25 0  0  10  100  1000  Treatment with F S H (ng/ml) Figure 40. Effects of FSH on cell proliferation in normal OSE and OVCAR-3 cells. The cells were treated with increasing concentrations (10, 100 and 1000 ng/ml) of human recombinant FSH for 24 h and a [ H]thymidine incorporation assay was performed as previously described in 3  the Materials and Methods. Treatments with increasing doses of F S H (10, 100 or 1000 ng/ml) resulted in a significant growth-stimulation in normal OSE (A) and OVCAR-3 cells (B). Data are shown as the means of three individual experiments with triplicate, and are presented as the mean ± S D . a, P<0.05 vs. untreated control.  119  IOSE-29 175-i <D C  TD  E s:  h-  150125-  X 100o  -t—' c  oo  7550250-  0  10  100  1000  Treatment with FSH (ng/ml) IOSE-29EC  B  0  10  100  1000  Treatment with FSH (ng/ml) Figure 41. Effects of FSH on cell proliferation in IOSE-29 and IOSE-29EC cells. The cells were treated with increasing concentrations (10, 100 and 1000 ng/ml) of human recombinant FSH for 24 h and a [ H]thymidine incorporation assay was performed as previously described in the 3  Materials and Methods. Treatments with increasing doses of F S H (10, 100 or 1000 ng/ml) resulted in a significant growth-stimulation in IOSE-29 (A) and IOSE-29EC cells (B). Data are shown as the means of three individual experiments with triplicate, and are presented as the mean ± SD. a, P<0.05 vs. untreated control.  120  In addition, treatments with same concentrations of F S H induced a significant growthstimulation of IOSE-29EC (Fig. 41A, 100.0 ± 9.08 % vs. 126.9 ± 10.12, 130.6 ± 1.1.23 or 129.9 ± 7.25) and IOSE-29EC cells (Fig. 41B, 100.0 ± 11.05 % vs.137.5 ± 8.21, 147.6 ±11.65 or 154.6 ± 12.45). This result indicates that both normal and neoplastic OSE cells are responsive to F S H treatments, which resulted in a growth-stimulation in these cells.  4.3. Expression of MAPKs in normal and neoplastic OSE cells The basal expression level of phosphorylated p44/p42 M A P K (P-MAPK) normalized by total M A P K (T-MAPK) was compared using the same amount of protein (30 ug) in normal OSE, IOSE cell.lines (IOSE-29 and IOSE-29EC) and ovarian cancer cell lines (CaOV-3, OVCAR-3 and SKOV-3). As shown in Fig. 42, normal OSE cells expressed a high level of P - M A P K . Interestingly, the basal level of P - M A P K in CaOV-3 cells was very low, when compared with other cell lines (Fig. 42).  121  4.4. Effects of FSH and/or PD98059 on MAPK activation To investigate the role of FSH on M A P K activation, the cells were pretreated with 50 j j M PD98059, a M A P K / E R K kinase  (MEK) inhibitor, for 30 min, followed by treatment with  increasing doses of FSH (10, 100 or 1000 ng/ml) for 10 min. As shown in Figs. 43A and B, treatments with FSH induced a significant increase in M A P K activation in IOSE-29 cells (100.0 + 9.94 % vs. 147.8 ± 9.31, 151.9 ± 10.62 or 156.7 ± 9.83). The stimulatory effect of FSH was completely reversed by pretreatment with PD98059 (157.3 ± 11.21 % vs.56.3 ± 5.18). Similarly, treatments with FSH resulted in a significant increase in M A P K activation in IOSE-29EC cells (Figs. 44A and B, 100.0 ± 7.54 % vs. 182.9 + 11.84, 183.4 ± 9.52 or 179.2 ± 9.00). This stimulatory effect of FSH was completely blocked by pretreatment with PD98059 (179.3 ± 12.04 % vs. 65.7 ± 4.98). Treatment with PD98059 alone resulted in a significant decrease of basal PM A P K in both IOSE-29 and IOSE-29EC cells (Figs. 43B and 44B).  122  Normal OSE  IOSE -29  IOSE -29EC  CaOV -3  OVCAR -3  SKOV -3  T-MAPK  -p44 -p42  P-MAPK  Figure 42. Basal expression level of P - M A P K and T - M A P K in normal and neoplastic OSE cells. The P - M A P K normalized by T - M A P K was compared after running the same amount of protein (30 ug) in normal OSE, IOSE cell lines (IOSE-29 and IOSE-29EC) and ovarian cancer cell lines (CaOV-3, OVCAR-3 and SKOV-3).  123  B  ^ 200n P  FSH 0 PD98059 0  10 100 1000 100 100 0 0 0 0 50  0 ng/ml 50 uM  Treatment with F S H and/or PD98059 Figure 43. Effects of FSH in the presence or absence of PD98059 on M A P K activation IOSE-29 cells. To investigate the role of FSH on M A P K , the cells were pretreated with 50 u M PD98059, a M A P K / E R K kinase (MEK) inhibitor, for 30 min, followed by treatment with increasing doses of FSH (10, 100 or 1000 ng/ml) for 10 min. The P - M A P K normalized by T - M A P K was analyzed in IOSE-29 (A and B). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. FSH (100 ng/ml) treatment; c, P<0.05 vs. PD98059 (50 uM) treatment. 1, untreated control; 2, FSH (10 ng/ml) treatment; 3, FSH (100 ng/ml) treatment; 4, FSH (1000 ng/ml) treatment; 5, FSH (100 ng/ml) treatment; 6, FSH (100 ng/ml) plus PD98059 (50 uM) treatment; 7, PD98059 (50 uM) treatment.  124  IOSE-29EC 1  2  3  4  5  6  7  B  FSH 0 PD98059 0  10 0  100 1000 100 100 0 0 0 50  0 ng/ml 50 [M  Treatment with F S H and/or P D 9 8 0 5 9 Figure 44. Effects o f F S H i n the presence or absence o f PD98059 on M A P K activation IOSE2 9 E C cells. To investigate the role o f F S H on M A P K , the cells were pretreated with 50 (iM PD98059, a M A P K / E R K kinase  ( M E K ) inhibitor, for 30 min, followed b y treatment with  increasing doses o f F S H (10, 100 or 1000 ng/ml) for 10 min. The P - M A P K normalized by TM A P K was analyzed i n I O S E - 2 9 E C ( A and B ) . Data are shown as the means o f three individual experiments, and are presented as the mean ± S D . a, P<0.05 vs. untreated control; b, P<0.05 vs. F S H (100 ng/ml) treatment; c, P<0.05 vs. PD98059 (50 |iM) treatment. 1, untreated control; 2, F S H (10 ng/ml) treatment; 3, F S H (100 ng/ml) treatment; 4, F S H (1000 ng/ml) treatment; 5, F S H (100 ng/ml) treatment; 6, F S H (100 ng/ml) plus PD98059 (50 (iM) treatment; 7, PD98059 (50 | i M ) treatment.  125  Time-dependent experiment was performed following treatment with F S H (100 ng/ml) and/or pretreatment with PD98059 (50 u M ) on M A P K activity. A s shown in Figs. 4 5 A and B , treatment with F S H induced a significant increase in P - M A P K at 5-10 m i n in IOSE-29 cells (100.0 ± 9 . 1 5 % vs. 132.6 ± 7.07 or 163.3 ± 9.22). The activated M A P K declined to control level after 20 min in these cells. In contrast, treatment with F S H significantly activated M A P K after 5 min and sustained for 60 m i n in I O S E - 2 9 E C cells (Figs. 4 6 A and B , 100.0 ± 9.35 % vs. 195.0 ± 10.70, 184.6 ± 14.47 or 190.8 ± 14.26). FSH-stimulated M A P K activation was completely abolished by pretreatment with PD98059 in both cell lines. In addition, treatment with PD98059 alone significantly decreased M A P K activity as well in both IOSE-29 and I O S E - 2 9 E C cells (Figs. 45B and 46B).  4.5. Effects of FSH and/or PKC inhibitor staurosporin on MAPK activation To assess whether the P K C signal transduction pathway is involved in M A P K activation in neoplastic O S E cells, the cells were treated with F S H (100 ng/ml) and/or pretreated with staurosporin (1 |J.M), a P K C inhibitor, in a time dependent manner. A s demonstrated in Figs. 47A and B , treatments with F S H resulted in a significant increase in M A P K activation at 5-10 min in IOSE-29 cells as expected. Similarly, treatments with F S H induced a significant increase of M A P K activation after 5 min and sustained for 20 min in I O S E - 2 9 E C cells (Figs. 4 8 A and B). FSH-stimulated M A P K activation was completely abolished by pretreatment with staurosporin for 30 min in both cell lines. Treatment with staurosporin alone resulted in a significant decrease of P - M A P K activation as well in both IOSE-29 and I O S E - 2 9 E C cells (Figs. 47B and 48B).  126  B  Time 0 FSH 0 PD98059 0  5 100 0  10 100 0  20 100 0  60 100 0  10 100 50  10 min 0 ng/ml 50 uM  Treatment with FSH and/or PD98059 Figure 45. Effects o f F S H in the presence or absence o f PD98059 on M A P K activation i n IOSE29 cells. A time-dependent experiment was performed following treatment with F S H (100 ng/ml) and/or pretreatment with PD98059 (50 uM) on M A P K activity. The P - M A P K normalized by T - M A P K was analyzed in IOSE-29 ( A and B ) . Data are shown as the means o f three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. F S H (100 ng/ml) treatment for 10 min. 1, untreated control; 2, F S H (100 ng/ml) treatment for 5 m i n ; 3, F S H (100 ng/ml) treatment for 10 min; 4, F S H (100 ng/ml) treatment for 20 m i n ; 5, F S H (100 ng/ml) treatment for 60 min; 6, F S H (100 ng/ml) plus PD98059 (50 u M ) treatment for 10 min; 7, PD98059 (50 u M ) treatment.  127  IOSE-29EC 1  2  3  4  5  6  7  B  FSH  0  PD98059 0  100  100  100  100  100  0  0  0  0  50  0  ng/ml  50 [iM  Treatment with FSH and/or PD98059 Figure 46. Effects of FSH in the presence or absence of PD98059 on M A P K activation in IOSE29EC cells. A time-dependent experiment was performed following treatment with FSH (100 ng/ml) and/or pretreatment with PD98059 (50 |iM) on M A P K activity. The P - M A P K normalized by T - M A P K was analyzed in IOSE-29EC (A and B). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. FSH (100 ng/ml) treatment for 10 min. 1, untreated control; 2, FSH (100 ng/ml) treatment for 5 min; 3, FSH (100 ng/ml) treatment for 10 min; 4, FSH (100 ng/ml) treatment for 20 min; 5, FSH (100 ng/ml) treatment for 60 min; 6, FSH (100 ng/ml) plus PD98059 (50 uM) treatment for 10 min; 7, PD98059 (50 |iM) treatment.  128  IOSE-29 1  Time FSH Stauro  2  0 0 0  3  4  10 100 0  5 100 0  5  20 100 0  6  10 100 1  10 0 1  min ng/ml  uM  Treatment with FSH and/or Staurosporin Figure 47. Effects o f F S H i n the presence or absence o f staurosporin on MAPK activation i n IOSE-29 cells. T o assess whether the PKC signal transduction pathway is involved i n MAPK activation i n neoplastic O S E cells, the cells were treated with F S H (100 ng/ml) and/or pretreated with staurosporin (1 uM), a PKC inhibitor, in a time dependent manner. The P-MAPK normalized by T-MAPK was analyzed in IOSE-29 (A and B). Data are shown as the means o f three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. F S H (100 ng/ml) treatment for 10 min; c, P<0.05 vs. staurosporin (1 uM) treatment. 1, untreated control; 2, F S H (100 ng/ml) treatment for 5 min; 3, F S H (100 ng/ml) treatment for 10 m i n ; 4, F S H (100 ng/ml) treatment for 20 m i n ; 5, F S H (100 ng/ml) plus staurosporin (1 uM) treatment for 10 min; 6, Staurosporin (1 uM) treatment.  129  IOSE-29EC 1  2  3  4  5  6  B  Time  0  5  10  20  10  10  min  FSH Stauro  0 0  100 0  100 0  100 0  100 1  0 1  ng/ml uM  Treatment with FSH and/or Staurosporin Figure 48. Effects of FSH in the presence or absence of staurosporin on M A P K activation in IOSE-29EC cells. To assess whether the P K C signal transduction pathway is involved in M A P K activation in neoplastic OSE cells, the cells were treated with FSH (100 ng/ml) and/or pretreated with staurosporin (1 uM), a P K C inhibitor, in a time dependent manner. The P - M A P K normalized by T - M A P K was analyzed in IOSE-29EC (A and B). Data are shown as the means of three individual experiments, and are presented as the mean ± S D . a, P<0.05 vs. untreated control; b, P<0.05 vs. FSH (100 ng/ml) treatment for 10 min; c, P<0.05 vs. staurosporin (1 uM) treatment. 1, untreated control; 2, FSH (100 ng/ml) treatment for 5 min; 3, FSH (100 ng/ml) treatment for 10 min; 4, FSH (100 ng/ml) treatment for 20 min; 5, F S H (100 ng/ml) plus staurosporin (1 uM) treatment for 10 min; 6, Staurosporin (1 uM) treatment.  130  4.6. Effects of FSH and/or PD98059, staurosporin on MAPK activation To examine the role of FSH on M A P K s in OVCAR-3 and SKOV-3 cells, the cells were pretreated with 50 u M PD98059 or 1 u M staurosporin for 30 min, followed by treatment with 100 ng/ml FSH for 10 min. As shown in Fig. 49A, treatment with F S H appeared to induce a significant increase in P - M A P K activation in O V C A R - 3 cells. In contrast, no difference was observed in SKOV-3 cells following FSH treatment (Fig. 49B). Pre-treatments of PD98059 or staurosporin resulted in a decrease of FSH-induced P - M A P K activation in O V C A R - 3 cells (Fig. 49A), whereas it appears that pre-treatments with PD98059 or staurosporin did not affect on M A P K activity in SKOV-3 cells (Fig. 49B).  4.7. Effect of FSH and/or PD98059 on Elk-1 phosphorylation The Ets family transcription factor, Elk-1 is a physiological substrate for p42 M A P K and p44 M A P K (Gille et al, 1995; Janknecht et al, 1993). To investigate whether the FSH-induced activation of M A P K leads to phosphorylation of Elk-1 in vitro, the cells were treated with FSH (100 ng/ml) for 10 min and/or PD98059 (50 uM) for 30 min. As shown in Fig. 50, treatment of FSH resulted in a significant increase in Elk-1 phosphorylation, whereas pretreatment with PD98059 significantly inhibited FSH-induced Elk-1 phosphorylation in both IOSE-29 (100.0 + 9.66 % vs. 173.5 ± 14.59 or 52.0 ± 6.27) and IOSE-29EC cells (100.0 ± 10.46 % vs. 196.3 ± 13.76 or 56.1 ±7.70).  131  OVCAR-3 1  2  3  4  5  6  T-MAPK  P-MAPK  SKOV-3 1  2  3  4  5  6  T-MAPK  P-MAPK  Figure 4 9 . Effects of FSH in the presence or absence of PD98059 or staurosporin on M A P K activation in ovarian cancer cell lines. To examine the role of FSH on M A P K s in O V C A R - 3 and SKOV-3 cells, the cells were pretreated with 50 u M PD98059 or 1 uJVl staurosporin for 30 min, followed by treatment with 100 ng/ml FSH for 10 min. The P - M A P K normalized by T - M A P K was analyzed in OVCAR-3 (A) and SKOV-3 cells (B). 1, untreated control; 2, FSH (100 ng/ml) treatment; 3, FSH (100 ng/ml) plus PD98059 (50 uM) treatment; 4, PD98059 (50 uJVl) treatment; 5, FSH (100 ng/ml) plus staurosporin (1 u.M) treatment; 6, Staurosporin (1 uM) treatment.  132  B  FSH PD98059  0 0  100 0  100 50  ng/ml uM  Treatment with FSH and/or PD98059 Figure 50. Effect o f F S H i n the presence or absence o f PD98059 on Elk-1 phosphorylation. To investigate whether the FSH-induced activation o f M A P K leads to phosphorylation o f Elk-1 in vitro, the cells were treated with F S H (100 ng/ml) for 10 m i n and/or PD98059 (50 u M ) for 30 min. The phosphorylation o f Elk-1 was investigated following F S H and/or PD98059 treatment in IOSE-29 and I O S E - 2 9 E C cells ( A and B ) . Data are shown as the means o f three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. F S H (100 ng/ml) treatment for 10 min. 1, untreated control; 2, F S H (100 ng/ml) treatment for 10 min; 3, F S H (100 ng/ml) plus PD98059 (50 u M ) treatment.  133  4.8. Effects of MAPK and PKC inhibitors on FSH-stimulated cell growth To evaluate the effect of M A P K and P K C inhibitors on FSH-stimulated cell growth, the cells were pretreated with PD98059 (50 uM) or staurosporin (1 uM) for 30 min and then treated with FSH (100 ng/ml) for 24 h in IOSE-29 and IOSE-29EC cells. A [ H]thymidine incorporation 3  assay was performed as previously described in the Materials and Methods. Treatment with FSH (100 ng/ml) resulted in a significant growth-stimulation in these cells as expected (Fig. 51). As seen in Fig. 51, pre-treatments with PD98059 and staurosporin attenuated completely F S H stimulated cell growth in both IOSE-29 and IOSE-29EC cells.  4.9. Effects of FSH on intracellular cAMP accumulation To investigate whether FSH modulates intracellular cAMP levels, the cells were treated with FSH (100 ng/ml), and intracellular c A M P levels were measured. Treatment of FSH did not affect basal intracellular c A M P levels in IOSE-29 and IOSE-29EC cells (Fig. 52), whereas FSH treatment induced a significant increase of intracellular cAMP in human granulosa luteal cells (hGLCs).  134  FSH" PD98059 Staurospo  0 0 0  100 0 0  100 50 -  100 1  (ng/ml) (uM) (uM)  Treatments with FSH and/or PD98059 or Staurosporin Figure 51. Effects of M A P K and P K C inhibitors on FSH-stimulated cell growth. To evaluate the effect of M A P K and P K C inhibitors on FSH-stimulated cell growth, the cells were pretreated with PD98059 (50 \M) or staurosporin (1 |iM) for 30 min and then treated with F S H (100 ng/ml) for 24 h in IOSE-29 and IOSE-29EC cells. A [ H]thymidine incorporation assay was 3  performed as previously described in the Materials and Methods. Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control; b, P<0.05 vs. FSH (100 ng/ml) treatment.  135  mmm h G L C s ^ IOSE-29 IOSE-29EC  60 min Time of F S H treatment (100ng/ml) Figure 52. Effect of FSH on intracellular c A M P accumulation. To investigate whether F S H modulates intracellular cAMP levels, the cells were treated with F S H (100 ng/ml), and intracellular c A M P levels were measured in IOSE-29, IOSE-29EC and human granulose luteal cells (hGLCs). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P<0.05 vs. untreated control.  136  5. EXPERIMENT E  5.1. Expression of GnRH-II mRNA The mRNA expression of GnRH-II in normal and neoplastic OSE cells was investigated as previously described. A predicted PCR product of GnRH-II was obtained as 327-bp using specific primers and confirmed by Southern blot analysis using DIG-labeled probes in normal OSE and IOSE cell lines (Fig. 53A) and sequence analysis (data not shown). Similarly, a PCR product of GnRH-II was also detected in primary cultured ovarian cancers from the patients (PCOVC) and ovarian cancer cell lines (CaOV-3, O V C A R - 3 , and SKOV-3) as shown in Fig. 53B. These results indicate that GnRH-II mRNAs are expressed in both normal and neoplastic OSE cells.  5.2. Expression of GnRH-R mRNA The mRNA expression of GnRH-R in IOSE cells was investigated by RT-PCR and Southern blot analysis. A predicted PCR product of GnRH-R was obtained as 347-bp  and confirmed by  Southern blot analysis using DIG-labeled probes (Fig. 54) and sequence analysis (data not shown). OVCAR-3 cells were used for positive control. As demonstrated in Fig. 54, the GnRH-R mRNAs are expressed in IOSE cell lines (IOSE-29, IOSE-29EC, IOSE-29EC/T4 and IOSE29EC/T5).  5.3. Effects of GnRH-I and -II on proliferative index To evaluate the role of GnRH-I and -II in IOSE cell lines, the cells were treated with increasing concentrations (10" - 10" M ) of GnRH-I and GnRH-II for 6 days and a [ H]thymidine 9  7  3  137  Tm(-)  Normal O S E  GAPDH  I O S E cell lines 1 2 3 4  ••••  GnRH-n  -373-bp  327-bp  B  Ovarian cancer cell lines 1 2 3  PC-OVC  GAPDH  4fe 4fe  W w f l l  GnRH-R  -373-bp  327-bp  Figure 53. Expression o f GnRH-II m R N A in normal and neoplastic O S E cells. The m R N A expression o f G n R H - I I in normal and neoplastic O S E cells was investigated as previously described. A predicted P C R product o f GnRH-II was obtained as 327-bp using specific primers and confirmed by Southern blot analysis using DIG-labeled probes i n normal O S E and I O S E cell lines (1: IOSE-29; 2: I O S E - 2 9 E C ; 3: IOSE-29EC/T4; 4: I O S E - 2 9 E C / T 5 ) . Similarly, a P C R product o f GnRH-II was also detected in primary cultured ovarian cancers from the patients (PCO V C ) and ovarian cancer cell lines (1: C a O V - 3 ; 2: O V C A R - 3 ; 3: S K O V - 3 ) .  138  Tm(-)  IOSE -29  IOSE IOSE IOSE OVCAR - 2 9 E C -29EC/T4 - 2 9 E C / T 5 -3  GAPDH  GnRH-R  Figure 54. Expression o f G n R H - R m R N A in IOSE cell lines and O V C A R - 3 cells. The m R N A expression o f G n R H - R in IOSE cells was investigated by R T - P C R and Southern blot analysis. A predicted P C R product o f G n R H - R was obtained as 347-bp and confirmed by Southern blot analysis using DIG-labeled probes. O V C A R - 3 cells were used for positive control.  139  incorporation assay was performed as previously described (Kang et al., 2000). Treatments with increasing doses of GnRH-I resulted in a significant growth-inhibition in both IOSE-29 and IOSE-29EC cells (Fig. 55). Co-treatment with antide plus GnRH-I completely blocked the growth-inhibitory effect of GnRH-I in both cell lines. In addition, treatments with same concentrations of GnRH-II also induced a significant growth-inhibition of IOSE-29 and IOSE29EC cells (Fig. 56). Similarly, co-treatment with antide plus GnRH-II completely blocked the growth-inhibitory effect of GnRH-II in both cell lines. These results indicate that neoplastic OSE cells are responsive to GnRH-I and -II treatments, which resulted in a growth-inhibition through GnRH-R in these cells.  5.4. Effects of GnRH-I and -II on apoptosis To examine the role of GnRH-I and -II in the induction of apoptosis, D N A fragmentation was measured by cell death detection ELISA. To quantify the induction of apoptosis, IOSE-29 and IOSE-29EC cells were treated with GnRH-I and -II for 6 days. As shown in Fig 57A, treatments with GnRH-I (D-Ala-GnRH, 10" M) increased D N A fragmentation in both cell lines. Co7  treatment with antide (10~ M) with GnRH-I blocked completely GnRH-I effect on the induction 7  of D N A fragmentation. In addition, treatments with GnRH-II (10" M ) resulted in an increase of D N A fragmentation in IOSE-29 and IOSE-29EC cells (Fig. 57B). Similarly, co-treatment with antide (10~ M ) with GnRH-II blocked completely GnRH-II effect on the induction of D N A 7  fragmentation.  140  GnRHa  0  10"  10"  10  Antide  0  0  0  0  9  8  -7  1CV  0  10"  10"  7  7  7  Treatments with G n R H a and/or antide (M) Figure 55. Effects of GnRH-I on proliferative index in IOSE cell lines. To evaluate the role of GnRH-I in IOSE cell lines, the cells were treated with increasing concentrations ( 1 0 - 10" M ) 9  7  of GnRH-I for 6 days and a [ HJthymidine incorporation assay was performed as previously described (Kang et al., 2000). Treatments with increasing doses of GnRH-I resulted in a significant growth-inhibition in both IOSE-29 and IOSE-29EC cells. Co-treatment with antide plus GnRH-I completely blocked the growth-inhibitory effect of GnRH-I in both cell lines.  IOSE-29  GnRH-II Antide  0 0  10~ 0  9  10" 0  8  10" 0  7  10" 10" 7  7  0 10"  7  Treatments with GnRH-II and/or antide(M) Figure 56. Effects of GnRH-II on proliferative index in IOSE cell lines. To evaluate the role of GnRH-II in IOSE cell lines, the cells were treated with increasing concentrations (10~ - 10" M) 9  7  of GnRH-II for 6 days and a [ H]thymidine incorporation assay was performed as previously described (Kang et ah, 2000). Treatments with increasing doses of GnRH-II resulted in a significant growth-inhibition in both IOSE-29 and IOSE-29EC cells. Co-treatment with antide plus GnRH-II completely blocked the growth-inhibitory effect of GnRH-II in both cell lines.  142  mmm IOSE-29 VZZm IOSE-29EC  0  0  GnRHa Antide  10"  7  0  0  10-  7  v7  10"  Treatments with GnRHa and/or antide (M) B  mmm IOSE-29 vzzm IOSE-29EC  200-  SD. 0)  of apopl  o o 150'•5 100-  O r—  \_  o  50-  O  0  GnRH-II Antide  Treatment with GnRH-II and/or antide (M) Figure 57. Effects of GnRH-I and -II on apoptosis. To examine the role of GnRH-I and -II in the induction of apoptosis, D N A fragmentation was measured by cell death detection ELISA. To quantify the induction of apoptosis, IOSE-29 and IOSE-29EC cells were treated with GnRH-I and -II for 6 days. Treatments with GnRH-I (D-Ala-GnRH, 10" M ) and GnRH-II (10" M ) 7  7  resulted in an increase of D N A fragmentation in IOSE-29 and IOSE-29EC cells. Co-treatment with antide (10" M ) with GnRH-I and -II blocked completely GnRH-I and -II effect on the 7  induction of D N A fragmentation.  143  5.5. Effects of GnRH-I and -II on the regulation of bax and bcl-2 proteins , To investigate the mechanism of GnRH-I and -II in the induction of apoptosis, the regulation of apoptotic bax and bcl-2 was examined by immunoblot analysis. The IOSE-29 and IOSE29EC cells were treated with increasing doses (10  9  to 10" M) of GnRH-I and GnRH-II 7  respectively for 24 h and immunoblot analysis was performed as described in the Material and Methods. Bax and bcl-2 protein were detected at 21 kDa and 26 kDa respectively. As seen in Fig 58, treatments with GnRH-I had no effect on both bax and bcl-2 proteins in both cell lines. In addition, no significant difference of bax and bcl-2 proteins was observed in GnRH-II treatments (Fig. 58). The loaded amount of proteins in treatment groups was normalized by actin protein (41 kDa, data not shown).  144  Treatment with G i l (M) Control lfT If/ 1CT 9  8  7  Bax IOSE-29  <r 21 k D a  IOSE-29EC  <r 21 k D a  Bcl-2 IOSE-29  <r 26 k D a  IOSE-29EC  <r 26 k D a  Figure 58. Effects o f G n R H - I and - I I on the regulation o f bax and bcl-2 proteins. T o investigate the mechanism o f G n R H - I and -II in the induction o f apoptosis, the regulation o f apoptotic bax and bcl-2 was examined by immunoblot analysis.  The IOSE-29 and I O S E - 2 9 E C cells were  treated with increasing doses (10' to 10" M ) o f G n R H - I and G n R H - I I respectively for 24 h and 9  7  immunoblot analysis was performed as described in the Material and Methods. B a x and bcl-2 protein were detected at 21 k D a and 26 k D a respectively. The loaded amount o f proteins in treatment groups was normalized by actin protein (41 k D a , data not shown).  145  IV. DISCUSSION  1. Experiment A Normal OSE and OVCAR-3 cells have been demonstrated to express all forms of inhibin subunits and activin receptors at the mRNA and protein levels (Fukuda et al, 1998; Ito et al., 2000, Welt et al, 1997). In addition, OVCAR-3 cells expressed the Smad-4 mRNAs and Smad-2, which are proteins specific for TGF-R signaling pathway (Ito et al, 2000). The present study clearly demonstrates that activin/inhibin subunits and activin receptor IIB are differentially expressed in normal and neoplastic OSE cells. The a and pA subunit were highly expressed in normal OSE when compared to OVCAR-3 cells. In contrast, the mRNA level of pB subunit was greatly expressed in O V C A R - 3 cells when compared to normal OSE cells. These results indicate that inhibin may be predominantly expressed in this ovarian compartment. In contrast, O V C A R 3 cells expressed a low level of the a subunit, but a high level of PB subunit, when compared to normal OSE cells, suggesting that activin may be predominantly expressed in this cell line. These results are in an agreement with the recent finding that inhibin a subunit expression was found in 47% of OSE, but was not found in the epithelial component of ovarian cystadenomas, tumors of low malignant potential (LMP), or carcinomas by immunohistochemisty and RT-PCR. PA subunit was expressed in 93% of OSE, in the epithelial component of all cystadenomas, in 81% of L M P tumors and in 72% of carcinomas, suggesting that imbalanced expression of inhibin and activin subunits in OSE may represent an early event that leads to epithelial proliferation (Zheng et al, 1998). The p subunits may be dominantly expressed in neoplastic OSE cells because most epithelial ovarian tumors (96%) synthesize and secrete activin in vitro (Welt et al, 1997). The detection of pA subunit alone in the absence of a subunit suggests the  146  synthesis of activin with little or no inhibin expression in epithelial ovarian cancer (Zheng et al., 1998). The switch from an inhibin dominant to an activin dominant status in neoplastic OSE may play a role in epithelial ovarian tumorigenesis. Although no difference was observed in the basal mRNA level of activin receptor IIA, OVCAR-3 cells expressed a higher level of activin receptor IIB, indicating that this cell line may be more responsive to activin treatment than normal OSE cells. Our observation that OVCAR-3 expressed low level of a subunit appears to be at variance somewhat with another report (Di Simone et al., 1996) that a subunit was not expressed in six other ovarian cancer cell lines. The apparent discrepancy between our results and the study by Di Simone et al. may be due to 1) different cell lines used, 2) the different specificity of primers used in the experiments, 3) the different sensitivity of Southern blot analysis using [ P]ATP32  labeled oligonucleotide probes or digoxigenine-labeled cDNA probes, 4)  different culture  conditions. Most primary epithelial ovarian tumors (96%) synthesize and secrete activin in vitro and serum levels of activin are frequently elevated in women with epithelial ovarian cancer (Welt et al., 1997). In the present study, activin stimulated cell proliferation in a neoplastic OSE cell line, O V C A R - 3 , but not in normal OSE cells, whereas the stimulatory effect of activin was attenuated by follistatin treatment. These results indicate that activin is mediated via its receptors, by stimulating the proliferative index of O V C A R - 3 . We have previously shown that OVCAR-3 cells expressed activin receptors, supporting the notion that activin may act in an autocrine manner (Fukuda et al., 1998). Treatments with activin (1-100 ng/ml) resulted in an increase, whereas follistatin treatment (1-100 ng/ml) resulted in a decrease in cellular proliferation of CaOV-3, CaOV-4, and SW-626 ovarian cancer cell lines (Di Simone et al., 1996). The proliferative index of normal OSE cells in the present study was unaffected following activin  147  treatment, even though these cells also express activin receptors (Welt et al, 1997). Therefore, it is hypothesized that activin may stimulate the proliferation of ovarian tumors following the transformation of normal OSE to neoplastic OSE cells. To determine i f the growth stimulatory effect of activin A is mediated via an autocrine mechanism in neoplastic OSE, the expression of activin/inhibin subunits and activin receptors by rh-activin A was examined in OVCAR-3 cells. Treatment with activin resulted in a significant increase in RA subunit mRNA levels in a dose- and time-dependent manner. In addition, this stimulatory effect of activin on RA subunit mRNA expression was attenuated following treatment with follistatin, suggesting that exogenous activin A may enhance activin A expression through its receptors. Thus, activin A may enhance cellular proliferation via an autocrine pathway in ovarian carcinoma cells. Similarly, activin A induced an increase in the a subunit mRNA in O V C A R - 3 , implicating an increase in inhibin expression (LaPolt et al, 1989). In addition, biologically active follistatin, capable of binding human activin, has been demonstrated to secrete in an ovarian teratocarcinoma cell line (PA-1) (Wang et al, 1996). Treatment with activin induced up to a 12-fold increase in RA subunit expression and up to a 2-fold increase in a subunit. These results indicate that exogenous activin A may induce more activin A than inhibin A production in OVCAR-3 cells, further suggesting an autocrine role of activin in ovarian cancer growth. Although the RB subunit is highly expressed in O V C A R - 3 cells, it appears that the primary targets for activin treatment are a and RA subunit but not RB subunit in O V C A R - 3 cells. Our results on the effects of activin on activin/inhibin subunit m R N A levels in human neoplastic OSE (i.e., OVCAR-3) cells are in an agreement with the previous findings in the rat ovary. In rat granulosa cells, activin induced the expression of both a and RA subunit mRNAs, but no difference in RB subunit level was observed in these cells (LaPolt et al, 1989). In contrast,  148  activin A induced only PB subunit mRNA in a dose- and time-dependent manner without affecting basal expression levels of the a and PA subunit mRNAs in human granulosa cells (Eramaa et al, 1995). Together, these results indicate that activin/inhibin subunits may be differentially regulated by activin in an autocrine manner. In this regard, activin/inhibin subunits may be differentially regulated by other endocrine and autocrine factors as well. TGF-p has been found to regulate PB subunit mRNA level in cultured human granulosa-luteal cells, whereas gonadotropins potently induce the a- and pA subunit mRNAs, suggesting that distinct components of the activin/inhibin system are regulated (Eramaa and Ritvos, 1996). In addition, estrogen may affect expression of the activin/inhibin subunits in the rat granulosa cells, and regulate the production of inhibin and activin differentially (Tate et al., 1996). The activation of protein kinase-A and -C by 8-bromo-cAMP and 12-(9-tetradecanoylphorbol 13-acetate (TPA), respectively, results in imbalanced expression of activin/inhibin subunits in human granulosaluteal (Tuuri et al, 1996) and placental cells (Li et al., 1994) in vitro. Multiple activin receptors are present in the human ovary and placenta, suggesting activin may play a role in these tissues (Sidis et al., 1998; Peng et al., 1999). Normal and neoplastic OSE cells have been demonstrated to express all known subtypes of activin receptors, further supporting a role for activin in ovarian neoplastic progression. (Welt et al., 1997). Activin may act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-a and p53-deficient mice (Shikone et al, 1994). In addition, treatment with anti-activin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth via its receptors. However, activin-responsive testicular tumor cells, derived from a p53 " / a-inhibin" " mouse testicular tumor, respond to activin treatment by _/  7  inhibition of cell proliferation. Furthermore, in the presence of exogenous activin, m R N A levels  149  of activin type IIA receptor and activin/inhibin RA subunit were significantly suppressed, suggesting that the inhibition of cellular proliferation in testicular tumor cells can be induced by down-regulation of activin receptor mRNA levels (Di Simone et al, 1998). However, treatment with activin, which stimulated cell proliferation, did not alter the mRNA levels of activin receptor IIA and IIB in OVCAR-3 cells. In summary, the present study demonstrates that (1) activin/inhibin are differentially expressed in normal and neoplastic OSE (i.e., OVCAR-3) cells, (2) activin induces cell proliferation in O V C A R - 3 cells, but not in normal OSE cells, and (3) activin induces an upregulation of a and RA subunits in OVCAR-3 cells. Taken together, these results support the hypothesis that activin may be an autocrine regulator of neoplastic OSE progression.  2. Experiment B Immortalized neoplastic OSE cells from human normal OSE have been generated by transfection with SV40-large T antigen and E-cadherin (Auersperg et al, 1999; Maines-Bandiera et al, 1992). These IOSE-29EC cells were found to be anchorage independent and formed transplantable, invasive subcutaneous and intraperitoneal adenocarcinmas in injected SCID mice (Ong et al, 2000). Therefore, two additional cell lines, designated IOSE-29EC/T4 and IOSE29EC/T5 were established from tumors that arose in IOSE-29EC-inoculated SCID mice. These cell lines may represent early neoplastic (IOSE-29), tumorigenic (IOSE-29EC) and late neoplastic (IOSE-29EC/T4 and T5) transformation stages of OSE. The characteristics of IOSE29EC resemble those of ovarian cancer (Auersperg et al, 1999; Ong et al, 2000). Thus, these cell lines may be a useful in vitro model to study the progression of ovarian tumorigenesis.  150  Activin and TGF-R belong to the TGF-R superfamily, which are known to have either growthpromoting and/or growth-inhibiting activities depending on the particular cell type (Zheng et al, 1998). Activin is an important regulator that mediates hormonogenesis, cellular homeostasis (cell growth and apoptosis) and differentiation (Mathews 1994). The actions of activin can be regulated by follistatin, which binds activin with high affinity and neutralizes activin actions (Woodruff, 1998). Members of this superfamily are prime candidates for the regulation of cell proliferation during morphological changes in the ovary, as well as transformation of these tissues (Mather et al., 1997). In epithelial ovarian cancer, Smad-2, specific signaling pathway of the TGF-R family, was regulated by activin treatment (Schneyer et al., 1994). The present study clearly demonstrated that mRNAs of activin/inhibin subunits are expressed in the IOSE cell lines. Both mRNAs and proteins for activin receptors were expressed in these cell lines, suggesting that activin may have an autocrine role in neoplastic OSE cells. Only RB subunit was expressed at lower levels in IOSE cell lines. However, no difference was observed in the expression levels of activin receptors when compared to OVCAR-3 cells. Activin/inhibin subunits are differentially expressed in normal OSE, epithelial ovarian cystadenomas, low malignant potential (LMP) tumors and ovarian carcinomas, suggesting that an imbalanced expression of activin/inhibin subunits in OSE may represent an early event that leads to epithelial proliferation (Wang et al., 1996). Our recent findings have demonstrated that activin/inhibin subunits and activin receptor IIB were differentially expressed in normal and neoplastic OSE cells in the EXPERIMENT  A. Furthermore, treatment with activin stimulated the growth of  O V C A R - 3 cells but not normal OSE. Thus, the differential expression and production of activin/inhibin subunits, activin receptors and follistatin suggest that activin may be involved in neoplastic OSE cell proliferation (Di Simone et ah, 1996). Continuous treatments of activin (1-  151  100 ng/ml) for 6 days resulted in a significant decrease in cell proliferation in both IOSE-29 and IOSE-29EC cell lines. These growth-inhibitory effects of activin were attenuated following cotreatment with follistatin (100 ng/ml), a specific binding protein of activin. These findings were unexpected because activin has been thought to be a growth-stimulatory factor in some ovarian cancer cell lines (Di Simone et al., 1996; Fukuda et al., 1998). Furthermore, most primary epithelial ovarian tumors (96%) synthesize and secrete activin in vitro and serum levels of activin are frequently elevated in women with epithelial ovarian cancer (Welt et al., 1997). In contrast, treatment or overexpression of activin resulted in a decrease in cell proliferation, which was blocked by follistatin, in human ovarian teratocarcinoma-derived cell line (Delbaere et al., 1999; McPherson et al., 1997). Similarly, it has been demonstrated that activin induced growth inhibition in prostate cancer cell lines (McPherson et al, 1997; Wang et al., 1996b). However, no difference was observed in the proliferative index of normal OSE even though all forms of activin receptors are expressed in these cells (Di Simone et al., 1996). The mechanism by which activin suppressed a growth in the IOSE cell lines remains uncertain. Treatments with TGF-P (0.1-10 ng/ml) induced a significant decrease in the proliferative index of normal and neoplastic OSE cells in a dose-dependent manner. The expressions of TGFP isoforms and its receptors have been demonstrated in ovarian tumors, suggesting an autocrine and/or paracrine role of TGF-p (Bartlett et al, 1992; 1997; Marth et al, 1990). TGF-p inhibited the proliferation of monolayers of normal human ovarian epithelial cells by 40-70% (Berchuck et al., 1992) and by 95% in primary epithelial ovarian cancer cell cultures obtained directly from ascites (Hurteau et al., 1994). In contrast, immortalized epithelial ovarian cancer cell lines were found to be relatively resistant to the growth inhibition by exogenous TGF-p treatment (Berchuck et al., 1990; 1992). These data suggest that TGF-P may act as a growth inhibitor that  152  prevents inappropriate proliferation of normal OSE cells, while loss of this autocrine growthinhibitory pathway may lead to cancer development in vivo and/or immortalization of cell in vitro. The results in this study confirm that TGF-R is a prime inhibitory regulator of cell proliferation in both normal and neoplastic ovarian cells and show that it effectively inhibits cell proliferation in early neoplastic and tumorigenic transformation stages. Increase in proliferation and/or decrease in apoptosis play critical roles in tumorigenesis. Treatments with increasing concentrations of activin and TGF-R resulted in an increase in D N A fragmentation of IOSE-29EC cells in a dose-dependent manner. The effect of activin on induction of apoptosis was attenuated following 100 ng/ml follistatin treatment. The previous reports have demonstrated that activin has been shown to induce apoptosis in B-cell lymphoma (Koseki et al., 1995; 1998), hepatoma (Chen et al., 2000) and androgen-dependent prostate cancer cells (Wang et al., 1996b). The exact mechanism by which TGF-R induced growthinhibition in ovarian tumor cells remains unknown. However, previous studies suggested that binding of TGF-P to its receptors initiates a cascade of molecular events that are thought to decrease activity of cyclin-dependent kinase, resulting in arrest of cell cycle from G l into S phase of D N A synthesis in normal and neoplastic ovarian cells (Massague, 1992). In addition to the cell cycle inhibition, TGF-p induced apoptosis in epithelial ovarian cancer but not in normal OSE, suggesting that neoplastic cells are more susceptible to apoptosis than their normal counterparts (Havrilesky et al., 1995, Lafon et al., 1996). The present study indicates that both exogenous activin and TGF-p induced apoptosis in neoplastic OSE cells that were growthinhibited in vitro. It is hypothesized that growth inhibition by activin or TGF-P may be derived from induction of apoptosis in this model, suggesting that apoptosis may be also one of important phenomena in growth-inhibited ovarian cancer cells.  153  The bcl-2 gene family has been widely reviewed as regulators of cell death (reviewed in Chao and Korsmeyer, 1998; Minn et al, 1998). Among pro- and anti-apoptotic genes in the bcl-2 family, bax and bcl-2 genes are dominant regulators of apoptosis. The ratio of bcl-2 to bax is important in determining susceptibility to apoptosis (Chao and Korsmeyer, 1998). It has been shown that steroid hormones and growth factors may regulate pro- or anti-apoptotic genes in ovarian and breast cancer cells (Lafon et al, 1996; Lapointe et al, 1999; Wang and Phang, 1995). The present study has demonstrated that bax and bcl-2 mRNAs are expressed in IOSE cell lines. No difference was observed in the expression level of bax m R N A between IOSE-29 and IOSE-29EC cells. In contrast, the expression level of bcl-2 mRNA is higher in IOSE-29EC cells than IOSE-29 cells, suggesting that IOSE-29EC cells may be more resistant to apoptosis. Relatively high expression levels of bcl-2 in IOSE-29EC cells suggest that this cell line is more resistant to serum deprivation than IOSE-29 cells (data not shown). To examine the exact mechanism by which activin and TGF-P regulates apoptosis in neoplastic OSE cells, the regulation of pro-apoptotic bax and anti-apoptotic bcl-2 protein was investigated following treatments of activin and TGF-P, respectively. Treatments of TGF-P (1 and 10 ng/ml) resulted in a significant decrease in bcl-2 protein (up to 50%), whereas no difference was observed in bax protein level. These findings are in agreement with a previous report where TGF-p 1 downregulated the endogenous expression of anti-apoptotic bcl-2 gene (Lafon et al, 1996). Thus, down-regulated bcl-2 may elicit apoptosis in IOSE-29EC cells, suggesting that anti-apoptotic bcl-2 appears to be a dominant regulator of apoptosis in these cells. However, no difference was observed in bax and bcl-2 protein expression following treatments of increasing doses of activin. It has been reported that the expression of the pro-apoptotic bax was unchanged after activin treatment in B-cell lymphoma (Koseki et al,  154  1995), however, over-expression of bcl-2  suppressed activin-induced apoptosis. Thus, different pro- and/or anti-apoptotic genes or possibly another apoptotic pathway may be related with activin-induced apoptosis in our culture system (Chao and Korsmeyer, 1998; Koseki et al., 1998). In conclusion, the present study indicates that both activin and TGF-R induced growth inhibition and apoptosis in experimentally produced early neoplastic (IOSE-29) and tumorigenic (IOSE-29EC) OSE cells. Furthermore, anti-apoptotic bcl-2 protein was down-regulated by TGFR, whereas no difference was observed in bax protein by activin or TGF-P and in bcl-2 protein by activin. These results suggest that activin and TGF-p may play a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic transformation stages of ovarian cancer.  3. Experiment C The present studies demonstrated that both mRNAs and proteins of E R a and ERp are expressed in IOSE cell lines by semi-quantitative RT-PCR and immunoblot analysis. No difference in the expression levels of ERs was observed among early neoplastic OSE, tumorigenic OSE, late neoplastic OSE and OVCAR-3 cells. In the previous studies, it has been demonstrated that human normal OSE cells express both mRNAs and proteins of E R a and ERp (Brandenberger et al., 1998; Kang et al., 2001; Lau et al., 1999). In addition, the expression levels of E R a were enhanced when compared to those in normal ovaries, whereas ERp levels were significantly decreased in ovarian tumors, suggesting that E R a and ERP mRNAs are differentially expressed in normal and neoplastic OSE cells (Brandenberger et al., 1998; Pujol et al., 1998). These results suggest that overexpression of E R a relative to ERP m R N A may be a marker of ovarian tumorigenesis. Consistent with mRNA levels, E R protein is also highly  155  expressed in ovarian carcinomas when compared to normal or benign ovarian tumors (Tropila et al, 1986). The open reading frame predicted from the ERP cDNAs encodes a protein of molecular weight of approximately 54 kDa, which contrasts with the size of ERa (approximately 67 kDa) detected by Western blotting (Green et al, 1986; Kuiper et al, 1996). ERa and ERp can homodimerize (a/a or p/P) or heterodimerize (a/P) upon binding to the ERE (Cowley et al, 1997). Thus, it is hypothesized that differential expression of E R a and ERp in ovarian tumors may alter a responsiveness of estrogen or anti-estrogen treatment. Recently, a mutation involving a 32-bp deletion in exon 1 of E R a transcripts was detected in SKOV-3 cell line, which is not responsive to estrogen treatment even though this cell line is ER-positive (Lau et al, 1999). In addition to its well-documented role in reproductive organs, it has been suggested that estrogen, especially 17P-estradiol  (E2), may be associated with ovarian tumorigenesis.  Treatments with exogenous estrogen resulted in a growth stimulation of several ER-positive ovarian carcinoma cell lines in vitro (Chien et al, 1994; Galtier-Dercure et al, 1992; Langon et al, 1994). Some cultures of human epithelial ovarian cancer cells have been demonstrated to produce E2 and progesterone (Wimalasena et al, 1991). The present studies demonstrated that o  c  exogenous E2 (10" - 10" M ) resulted in an increased thymidine incorporation and D N A content in IOSE-29EC cells but not in IOSE-29 cells. The effect of E2 was attenuated by the estrogen antagonist tamoxifen (10" M), suggesting that the effect of E2 is mediated through specific 6  receptors. As there was no stimulatory effect on thymidine incorporation prior to day 6, and since the ratio of thymidine incorporation / D N A content per culture did not change, E2 does not stimulate proliferation. The growth of ER-positive ovarian tumors that are responsive to E2 is also attenuated by antiestrogen, such as tamoxifen and the pure antiestrogen ICI 164,384  156  (Clinton and Hua, 1997; Langdon et al, 1990). It is not yet known which ERs (a, (3 or both) are blocked by tamoxifen treatment. In the present study, E2 does not appear to be mitogenic for IOSE-29EC cells even though E2 resulted in a significant increase in thymidine incorporation after 6-day treatment, because the increase in thymidine incorporation was paralleled by an increase in D N A content per culture. In addition, no difference in proliferative index was obtained after E2 treatment for 1 or 2 days (data not shown). These observations suggest that the increase in thymidine incorporation and D N A content may be due to reduced apoptosis. This increase in thymidine incorporation could reflect the stimulation of proliferation by E2 (i.e. an increase in the proportion of dividing cells per total cell number) or it could be the result of an unchanged rate of proliferation in cell populations that had increased in size because apoptosis was inhibited. The observation that a significant increase in thymidine incorporation was only observed on day 6 of E2 treatment supports the latter possibility. To define the basis for the increase in thymidine incorporation more definitely, total D N A determination was carried out on the cell populations. These determinations showed an increase in D N A content that paralleled the changes in thymidine incorporation, i.e. the ratio of thymidine incorporation over total cell number did not change. Therefore, it appears that the increase in cell number on day 6 was the result of suppression of apoptosis rather than enhanced proliferation. Further, in the work presented here, treatment with tamoxifen (10" M ) only resulted in a growth-inhibitory effect in both IOSE-29 and IOSE-29EC 6  cells, regardless of E2 treatment. Clinically relevant concentrations of tamoxifen (10 —10" M) 7  5  have been shown to inhibit the growth of the ER-negative ovarian cancer cell line, A2780, and to induce apoptosis (Ercoli et ah, 1998). This estrogen-independent role of tamoxifen in ERnegative ovarian and breast cancer cells have been well documented (Ercoli et al., 1998; Kang.ef  157  al, 1996; Markman et al, 1996), suggesting that tamoxifen ( 1 0 M ) may have dual functions, 6  antagonizing the effects of estrogen by blocking E R and inhibition of growth through an estrogen-independent manner, as demonstrated in this experiment. In addition, tamoxifen may have estrogenic and antiestrogenic effects even in the same tissue. It has been demonstrated tamoxifen induces progesterone receptors in breast tissue (agonistic effect), but inhibits the growth of breast cancer (antagonistic effect). This cell-specific effect of tamoxifen in the same tissue can be explained by the tripartite theory (ligand, receptor, and effector) based on the celland promoter specific action of steroid hormones (Katzenellenbogen et al, 1996), indicating that the biocharacter of ligand (i.e., agonist-antagonist balance) is determined principally through this receptor-effector coupling. This molecular explanation of the agonistic and antagonistic effects of the ligand-binded estrogen receptor is further supported by other groups (Brzozowski et al., 1997; Parker, 1998). In the present study, our results confirm those of others (Karlan et al, 1995), which indicated that E2 does not affect the growth of normal OSE. The role of ER's in OSE and IOSE-29 remains to be further elucidated, but our results suggest that the introduction of E-cadherin resulted in an altered ER-elicited effect and responsiveness to E2 or tamoxifen resembling that of ovarian cancer lines. Dysregulation of proliferation and/or cell death plays a critical role in tumorigenesis. The present study demonstrates that tamoxifen (10" M) can induce apoptosis in IOSE-29EC cells, 6  whereas E2 can attenuate the effect of tamoxifen on these cells. Only IOSE-29EC cells were used for further studies of apoptosis because this cell line expressed both ERs and responded to E2/tamoxifen treatments. Co-treatment with E2 (10" to 10~ M) plus tamoxifen attenuated 8  6  tamoxifen-induced apoptosis in a dose-dependent manner. Among the pro- and anti-apoptotic genes in the bcl-2 family, bax and bcl-2 genes are dominant regulators for apoptosis. The ratio of  158  bcl-2 to bax is important in determining susceptibility to apoptosis (Chao and Korsmeyer, 1998). As an alternative pathway for apoptosis, Fas-Fas ligand system (CD95/CD95 ligand) mediates apoptosis in several tissues and tumors. Fas ligand is a type II integral membrane protein that bears homology to T N F - a , and both can be released from the surface as soluble cytokines (Herr et al., 1999). In addition, TNF-related apoptosis-inducing ligand (TRAIL) is another member of the TNF family of cytokines and mediates rapid apoptosis in transformed cell lines of various origin (Nagata, 1997). Among members of the TNF receptor superfamily, the intracellular "death domain" is highly conserved. The Fas associating protein with death domain (FADD), a major adaptor molecule, binds either directly or indirectly to the death domains of Fas, TNF and TRAIL receptors to tranduce the apoptosis signal to caspases (Nagata, 1997). The present study has demonstrated that bax and bcl-2 are expressed at both mRNA and protein levels in neoplastic OSE cells. No difference was observed in the expression level of bax mRNA between IOSE-29 and IOSE-29EC cells. Interestingly, the expression level of bcl-2 mRNA and protein is higher in IOSE-29EC cells than IOSE-29 cells, suggesting that IOSE29EC cells may be more resistant to apoptosis. In addition, treatments with E2 resulted in a significant increase in bcl-2 m R N A (up to 2-fold), whereas the effect of E2 was attenuated with tamoxifen treatment, suggesting that the up-regulation of bcl-2 m R N A by E2 is mediated through specific estrogen receptors. These findings are in agreement with a previous report where estrogen up-regulated anti-apoptotic bcl-2 gene, while bax levels remain unaffected by E2 in breast cancer cells (Leung and Wang, 1999; Perillo et al., 2000; Teixeira et al., 1995; Wang and Phang, 1995). The up-regulation of bcl-2 by E2 in this series of experiments indirectly suggests that E2 affects the survival of IOSE-29EC cells through bcl-2 which is known to be a dominant regulator of apoptosis in other tissues (Leung and Wang, 1999; Perillo et al, 2000;  159  Teixeira et al, 1995; Wang and Phang, 1995). It has been shown that estrogen down-regulated pro-apoptotic bak and anti-apoptotic b c l - X mRNA and protein in a dose-dependent manner, L  suggesting different members of bcl-2 family may be regulated via different pathways by estrogen (Leung and Wang, 1999). In parallel with the mRNA level, E2 induced a significant upregulation of bcl-2 protein level (up to 1.7-fold), whereas no difference was observed in bax mRNA level. This induction of bcl-2 protein by E2 was attenuated with tamoxifen treatment (10" 6  M). Thus, the mechanism by which estrogen regulates the apoptotic pathway may be related to  up-regulation of the bcl-2 gene. Recently, it has been demonstrated that the bcl-2 major promoter does not contain cis-acting elements, which are directly involved in transcriptional control by E2, and that E2 induces bcl-2 expression via two estrogen-responsive elements located within its coding region (Perillo et al, 2000). In conclusion, the present study indicates that early neoplastic (IOSE-29), tumorigenic (IOSE29EC) and late neoplastic (IOSE-29EC/T4 and T5) OSE cell lines, which were generated from normal OSE, express both E R a and E R p at the mRNA and protein levels. E2 has been demonstrated to prevent tamoxifen induced-apoptosis through ERs. The mechanism of action of E2 may be associated with up-regulation of bcl-2 gene at the mRNA and protein levels. These results suggest that estrogen may play a role in the prevention of apoptosis in tumorigenic OSE cells for ovarian tumorigenesis.  4. Experiment D In addition to its well-documented role in ovarian physiology, F S H , one of the pituitary glycoprotein hormones, has been suggested to play a role in ovarian cancer development (Konishi et al, 1999; Zheng et al, 2000). A n increased occurrence of ovarian cancer with  160  exposure to high levels of gonadotropins during postmenopause or infertility therapy has been suggested by epidemiological studies (Risch, 1998;. Shushan et al, 1996; Whittemore et al., 1992). Little information is available regarding the expression of FSH-R and exact role of FSH in normal and neoplastic OSE cells. FSH-R, a G-protein coupled receptor, is expressed in normal OSE (Zheng et ah, 1996), ovarian inclusions and epithelial tumors (Zheng et al., 2000). In addition, treatment with F S H resulted in growth-stimulation of rabbit OSE (Osterholzer et al., 1985) and ovarian cancer cells (Wimalasena et al., 1992; Zheng et ah, 2000) in a dose- and timedependent manner in vitro. In agreement with these reports, the present study demonstrated that FSH-R mRNA was expressed and that treatment of FSH induced a growth-stimulation in both normal and neoplastic OSE cells. It has been shown that FSH-R expression was decreased with increasing tumor  grade in epithelial inclusions, cystadenomas,  borderline tumors  and  carcinomas, suggesting that constitutive expression of FSH-R may represent a cellular differentiation marker for epithelial ovarian tumors (Zheng et al., 2000). However, in the present study, it appears that no difference was observed in the expression level of FSH-R in normal, early neoplastic (IOSE-29), tumorigenic (IOSE-29EC) and late tumorigenic OSE (IOSE29EC/T4 and /T5) cells by RT-PCR and Southern blot analysis. A recent report demonstrated that elevated level of gonadotropins stimulated growth of ovarian carcinoma by induction of tumor angiogenesis, and the F S H effect was connected with the expression of vascular endothelial growth factor (VEGF), which is an angiogenic factor presumably involved in ovarian tumorigenesis (Schiffenbauer et al, 1997), implying that gonadotropins may facilitate the growth of existing microtumors by enhancing blood supply. Protein phosphorylation is a critical regulatory response to cellular stimulation and differentiation. M A P K cascade is known to regulate acute cellular responses and to control  161  transcriptional events through phosphorylation of target enzymes and transcriptional factors (Biesen et al, 1996; Cobb and Goldsmith, 1995; Davis, 1994). Activation of E R K is induced by phosphorylation of both threonine and tyrosine residues of the enzymes as a result of stimulation of Ras, E R K kinase kinase, M E K kinase and M E K (Cobb and Goldsmith, 1995; Davis, 1994; Seger and Krebs, 1995). The M A P K s have been shown to mediate a diverse range of regulatory molecules such as F S H (Das et al, 1996), prostaglandin F2oc (Chen et al, 1998), TGF-oc (Sasanami et al, 1999), or EGF (McClellan et al, 1999) in the ovarian cells. In addition, treatment with GnRH analog (GnRHa) resulted in a sustained (24 h) activation of E R K , while PD98059, which binds M E K , blocked GnRHa-induced growth inhibition as well as hypophosphorylation of pRB in CaOV-3 cells (Kimura et al, 1999). As the mechanism of FSH action in ovarian tumors is not clear, we investigated the possible regulatory action of FSH on M A P K activation and its role in neoplastic OSE cells. In the present study, F S H stimulated M A P K activation in both IOSE-29 and IOSE-29EC cells, whereas the stimulatory effect of FSH was reversed completely by pretreatment with PD98059, an E R K inhibitor, suggesting that the growth stimulatory effect of FSH may be mediated by the M A P K pathways in neoplastic OSE cells. In previous reports, exogenous EGF activated ERK1/2, increased and sustained levels of cjun mRNA, but had no effect on JNK1 activation in IOSE-29 cells (McClellan et al, 1999). Similarly, EGF has been demonstrated to induce activation of E R K and cellular proliferation was partially inhibited by PD98059 in a prostate cancer cell line (Price et al, 1999). Additionally, it has been shown that EGF-induced cell proliferation, MMP-9 induction and invasion through reconstituted basement membrane were significantly reduced when breast epithelial cells were exposed to M E K inhibitor (PD 98059) or M A P K inhibitors (Apigenin or M A P K antisense phosphorothioate oligodeoxynucleotides). These results suggest that interference with M A P K  162  activity may affect the growth and invasiveness of tumors in which the signaling cascade is activated (Reddy et al, 1999). Whether interference of the M A P K cascade with E R K or M A P K inhibitors may induce a growth-inhibition in normal and neoplastic OSE cells warrants future investigation. In a time-dependent study, treatment with F S H induced a significant increase in M A P K activation at 5-10 min in IOSE-29 cells. The activated M A P K declined to control level after 20 min in these cells. In contrast, treatment with FSH significantly induced M A P K activation after 5 min and the activity was sustained for 60 min in IOSE-29EC cells. It appears that cellular responses to M A P K may be influenced by the duration of its activation. Sustained activation of M A P K is associated with cellular differentiation by nerve growth factor (NGF) in PC 12 cells, whereas transient activation of M A P K by epidermal growth factor (EGF) leads to cellular proliferation (Heasley and Johnson, 1992; Nguyen et al., 1993). Thus, a rapid activation of M A P K by FSH in IOSE-29 and IOSE-29EC cells is related with a growth-stimulation in the present study. However, the cause of sustained response following FSH treatment in IOSE-29EC cells has yet to be elucidated. EGF stimulated an early rise in E R K activity at 4 min followed by a rapid decline in normal breast epithelium, whereas a sustained (1 h) elevation of E R K activity was observed in the tumor cells (Xing and Imagawa, 1999), suggesting the time course of E R K activity may be different between normal and neoplastic cells. In addition, PD98059 inhibited EGF-stimulated proliferation and E R K activity in a parallel, dose-dependent manner, indicating that E R K activation is at least permissive for the proliferative response to EGF (Xing and Imagawa, 1999). FSH-R belongs to a superfamily of G-protein coupled receptors, which interact with intracellular signaling system via 7-transmembrane domains (Simoni et al., 1997). Transient  163  increase of c-fos, c-myc expression and M A P K activation were demonstrated in granulosa cell cultures in response to FSH (Cameron et al, 1996; Das et al, 1996; Pennybacker and Herman, 1991). The actions were mediated by either cAMP-dependent or independent pathway. The M A P K pathway has been shown to mediate the cAMP-independent FSH induced growth promotion (Babu et al., 2000). In the present study, staurosporin, a P K C inhibitor, was employed to investigate whether FSH-activated M A P K is mediated by c A M P independent and P K C dependent pathway (Watson et al, 1988; Melner, 1996). The FSH-induced activation of M A P K was completely blocked by pretreatment with staurosporin (1 uM) for 30 min in both cell lines, suggesting that FSH acts via a P K C pathway in neoplastic OSE cells. As demonstrated in this study, FSH did not stimulate basal c A M P level, suggesting that the P K A pathway is not involved in the FSH-induced M A P K activation in neoplastic OSE cells. This is in contrast to human granulosa-luteal cells where FSH stimulates c A M P accumulation and M A P K activation occurs via a PKA-dependent pathway. In other ovarian cancer cell lines examined, it appears that FSH induced a significant increase in M A P K activation in OVCAR-3 cells, not in SKOV-3 cells. Pretreatment with PD98059 or staurosporin resulted in a decrease in FSH-induced M A P K activation in OVCAR-3 cells, whereas pre-treatment with PD98059 or staurosporin did not affect on FSHinduced M A P K activity in SKOV-3 cells. Several studies have shown that M A P K s phosphorylate ternary complex factor (TCF) proteins such as Elk-1 and SAP-1 (Gille et al, 1995; Janknecht et al, 1993, Treisman, 1994). The activated TCF protein regulates the expression of c-fos and other co-regulated genes through their actions on the serum response element. Therefore, the ability of F S H to activate a downstream effector of the M A P K pathway was examined using immunoprecipitation. The • present study demonstrated that treatment with FSH resulted in substantial phospohorylation of  164  Elk-1 fusion protein in vitro. These results confirmed that FSH action is mediated by the M A P K pathway, as treatment with PD98059 completely reversed the effect of F S H on Elk-1 phosphorylation. Taken together, these results suggest that FSH-stimulated M A P K activation resulted in phosphorylation of Elk-1, the Ets family of transcription factors, which possibly mediates cellular functions in response to FSH in neoplastic OSE cells. In conclusion, we demonstrated that FSH-R was expressed and F S H induced growthstimulation in both normal and neoplastic OSE cells. In addition, F S H activated the M A P K cascade presumably through a PKC-dependent pathway. Activated M A P K phosphorylated Elk-1 in neoplastic OSE cells. These results suggest that M A P K cascade may be involved in cellular function such as growth stimulation in response to FSH in OSE cells.  5. Experiment E Epithelial ovarian tumors are the most common cause of death from gynecological malignancies and appear to arise from OSE, which is a simple  squamous-to-cuboidal  mesothelium covering the ovary based on histopathological observations (Auersperg et al., 1995). The exact mechanism of ovarian tumorigenesis has not been elucidated, but repeated ovulation and process of healing ruptured OSE have been suggested to contribute to neoplastic transformation of OSE (Godwin et al, 1993). Considering the fundamental role of OSE cells in ovarian tumorigenesis, the growth regulation of normal and neoplastic OSE cells by intraovarian regulators may play an important role in ovarian cancer development. Recent cloning of a second form of GnRH has been demonstrated in the brain (Lescheid et al, 1997; Urbanski et al., 1999) and expressed at significantly higher levels outside the brain (White et al., 1998). Therefore, it is tempting to investigate the role of GnRH-II in normal and neoplastic OSE cells, which will  165  contribute to a better understanding of normal ovarian physiology and the role of OSE in ovarian tumorigenesis. In the present study, we demonstrated for the first time that GnRH-II is expressed in normal and neoplastic OSE cells, suggesting that GnRH-II exerts its actions in an autocrine/paracrine manner. Sequence analysis indicated that GnRH-II in these cells have a nucleotide sequence identical to that of other tissues (White et al, 1998). The presence of GnRH-II in normal OSE as wells as neoplastic OSE indicates that the local regulatory system based on GnRH-II in normal OSE is a normal component of the cells. In a previous report, it has been demonstrated that GnRH-II is expressed and down-regulates FSH/LH receptors in human granulose luteal cell (Kang et al, 2001), suggesting that GnRH-II may have biological effects similar to those of GnRH-I. The present study demonstrated that GnRH-R is expressed in these cell lines, suggesting that the actions of GnRH-I and -II are a receptor-mediated event. GnRH-II binds GnRH-R up to 100 times more effectively than GnRH-I, suggesting GnRH-II may act through GnRH-R outside the brain (King and Millar, 1991). One interesting finding in the present study was the demonstration that treatments (10~ - 10" ) with GnRH-I and -II resulted in a growth9  7  inhibitory effect in IOSE-29 and IOSE-29EC cells. It has been demonstrated previously that GnRH has antiproliferative effects and regulates its own receptor mRNA, suggesting that GnRHI may act as an autocrine regulator in normal OSE cells (Kang et al., 2000). In addition, GnRH analogs have been proved to be efficient in treating GnRH-R bearing tumors, including carcinomas of the ovary (Emons et al., 1993a; Yano et al., 1994). These results indicate that GnRH-II may play a role in growth-inhibition similar to GnRH-I in normal and neoplastic OSE cells. The mechanism of growth-inhibitory effect of GnRH-II remains to be elucidated. It has been demonstrated GnRH analogs reduce cell proliferation by increasing the portion of cells in  166  the resting phase, Go-Gi (Thomson et al, 1991) and by inducing cell death or apoptosis (Motomura, 1998; Sridaran et al,  1998). Treatment of ovarian cancer cells with GnRH  analogues may induce apoptosis mediated by the Fas ligand-Fas system, which has been shown to trigger apoptosis in a variety of cell types (Nagata and Golstein, 1995).  In this study,  treatment with GnRH-II (10" M) resulted in an induction of apoptosis in IOSE-29 and IOSE7  29EC cells by D N A fragmentation assay. In addition, co-treatment with antide (10" M ) and 7  GnRH-II completely blocked the effect of GnRH-II on the induction of D N A fragmentation, suggesting that GnRH-II may trigger a cellular signaling pathway via GnRH-R. However, no significant change in bax and bcl-2 proteins was observed following GnRH-II (10"' M ) treatment, suggesting that another pathway may be involved in terms of the induction of apoptosis by GnRH-II in neoplastic OSE cells. More studies of the effect of GnRH-II on apoptosis in normal and neoplastic OSE cells need to be extended. In conclusion, we demonstrated that GnRH-II is expressed in normal and neoplastic OSE cells. In addition GnRH-II induces a growth-inhibition and apoptosis in neoplastic OSE cells. These results suggest that GnRH-II may be an integral regulator in normal OSE physiology and play a role in a growth-inhibition in neoplastic OSE cells.  167  V. SUMMARY AND FUTURE STUDIES  1. Summary The exact mechanism of ovarian tumorigenesis is not well known even though this disease is the most frequent cause of cancer death in gynecological malignancies. Repeated ovulation contributes to neoplastic transformation of OSE, indicating that the process of healing ruptured OSE may contribute to the disease. Therefore, it has been hypothesized that endocrine and autocrine factors may influence the occurrence of ovarian tumors in women. Recently, nontumorigenic and tumorigenic immortalized OSE (IOSE) cells were generated from normal OSE directly by transfection with simian virus 40 (SV40)-large T antigen (IOSE-29) and subsequent E-cadherin (IOSE-29EC). These IOSE-29EC cells were found to be anchorage independent and formed transplantable, invasive subcutaneous and intraperitoneal adenocarcinomas in SCID mice. Thus, two additional cell lines, designated IOSE-29EC/T4 and IOSE-29EC/T5 were established from tumors that arose in IOSE-29EC-inoculated SCID mice. This study was performed to investigate the effects of activin, TGF-P, estradiol (E2), Follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone-II (GnRH-II) in the growth-stimulation or -inhibition and regulation of apoptosis in normal and neoplastic OSE cells using IOSE cell lines. This study indicates that a and (3A subunits were highly expressed in normal OSE compared to OVCAR-3 cells, whereas (3B subunit was highly expressed in OVCAR-3 cells, compared to normal OSE cells. In addition, activin receptor IIB mRNA levels were significantly higher in OVCAR-3 compared to normal OSE cells. At concentrations of 1, 10 and 100 ng/ml, rh-activin A stimulated the growth of OVCAR-3 cells, but not of normal OSE. Treatment with follistatin, binding protein of activin, attenuates the stimulatory effect of activin. Treatments with activin  168  increased the a and p A subunit mRNA levels in a dose- and time-dependent manner. But, no difference was observed in levels of p B subunit, or in activin receptor type IIA and IIB mRNAs following activin treatments in OVCAR-3 cells. These results suggest that different levels of activin/inhibin and activin receptor isoforms are expressed in normal and neoplastic OSE cells. In addition, the altered expression of the activin/inhibin subunits, as well as the cell proliferative effect of activin observed in OVCAR-3 but not in normal OSE cells, indicate that activin may act as an autocrine regulator of neoplastic OSE progression. In IOSE cell lines, activin/inhibin subunits and activin receptors were expressed at both the mRNA and protein levels in these cells. Treatments with activin (1-100 ng/ml) resulted in a significant decrease in cellular proliferation in both IOSE-29 and IOSE-29EC cells, though we have shown that it had no effect in normal OSE. This inhibitory effect was attenuated by cotreatment with follistatin. In addition, treatment with TGF-p (0.1-10 ng/ml) also significantly decreased the proliferation of normal, IOSE-29 and IOSE-29EC in a dose-dependent manner. Treatments with both activin and TGF-p resulted in an increase in D N A fragmentation in IOSE29EC cells in a dose-dependent manner. This apoptotic effect of activin was attenuated with cotreatment by follistatin. Furthermore, treatment with TGF-P (1 and 10 ng/ml) resulted in a significant decrease in bcl-2 protein (up to 50%) in IOSE-29EC, whereas no difference was observed in bax protein levels. Therefore, down-regulation of bcl-2 by TGF-P may eventually induce apoptosis in IOSE-29EC cells. No difference was observed in bax and bcl-2 protein expression following treatment with activin. In conclusion, the present study indicates that activin and TGF-p inhibited growth and induced apoptosis in early neoplastic (IOSE-29) and tumorigenic OSE (IOSE-29EC) cells. In addition, anti-apoptotic bcl-2 protein was downregulated by TGF-P, whereas no difference was observed in bax protein by activin or TGF-P  169  treatment and in bcl-2 protein by activin. These results suggest that activin and TGF-p may play 1  a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic stage of ovarian cancer. Both mRNAs and proteins of estrogen receptor (ER) a and P were expressed in IOSE cell lines. No difference was observed in normal OSE and IOSE-29 cells, whereas treatment with 17P-estradiol (E2, 10" - 10" M) resulted in an increased thymidine incorporation and D N A 8  6  content per culture in IOSE-29EC cells. This effect of E2 was attenuated with tamoxifen treatment (10~ M), an estrogen antagonist, suggesting that the effect of E2 is mediated through 6  specific ERs. There was no stimulatory effect on thymidine incorporation prior to day 6, but after 6 days of E2 treatment, thymidine incorporation was significantly increased. Since the ratio of thymidine incorporation / D N A content per culture did not change, this E2 effect does not appear to indicate stimulation of proliferation, but, rather, inhibition of apoptosis. Treatment with tamoxifen (10~ M ) induced apoptosis up to 3-fold in IOSE-29EC cells, whereas co-treatment 6  with E2 (10 to 10" M ) plus tamoxifen attenuated tamoxifen-induced apoptosis in a dose8  6  dependent manner. Both mRNA and protein levels of pro-apoptotic bax and anti-apoptotic bcl-2 were expressed in IOSE cell lines. Treatments with E2 resulted in a significant increase in bcl-2 mRNA and protein levels (2 and 1.7-fold respectively), whereas no difference was observed in bax mRNA level. Thus, E2 may enhance survival of IOSE-29EC by up-regulating bcl-2. Antiapoptotic bcl-2 may be a dominant regulator in the apoptotic pathway in these cells. In conclusion, the present study indicates that early neoplastic (IOSE-29), tumorigenic (IOSE29EC) and late neoplastic (IOSE-29EC/T4 and T5) OSE cells expressed both E R a and ERp at the mRNA and protein levels. In addition, E2 prevented tamoxifen induced-apoptosis through ERs. The mechanism of E2 action may be associated with up-regulation of bcl-2 gene at mRNA  170  and protein levels. These results suggest that estrogen may play a role in ovarian tumorigenesis by preventing apoptosis in tumorigenic OSE cells. In the present study, FSH-R mRNA was expressed in normal and neoplastic OSE cells. F S H exerted a growth-stimulatory effect in both normal and neoplastic OSE cells. Treatment with FSH activated M A P K in immortalized OSE cell lines (IOSE-29 and IOSE-29EC), whereas the stimulatory effect by F S H was completely abolished in the presence of PD98059, a M E K inhibitor, suggesting that the growth stimulatory effect of FSH may be mediated by M A P K activation in neoplastic OSE cells. In a time-dependent study, F S H significantly increased M A P K activity at 5-10 min in IOSE-29 cells. The activated M A P K declined to control levels after 20 min in these cells. In contrast, treatment with FSH significantly activated P - M A P K after 5 min and the activation was sustained for 60 min in IOSE-29EC cells. FSH-stimulated M A P K activity was completely blocked by pretreatment with staurosporin (1 uM), a protein kinase C (PKC) inhibitor, for 30 min in both cell lines. Treatment with F S H did not affect basal intracellular c A M P levels in IOSE-29 and IOSE-29EC cells, suggesting that the P K A pathway is not involved with FSH-induced M A P K activation in neoplastic OSE cells. A M A P kinase assay revealed that FSH resulted in substantial phosphorylation of Elk-1, confirming that FSH action is mediated by activation of M A P K . In conclusion, it has been demonstrated that FSH-R was expressed and F S H induced a growth-stimulatory effect in normal and neoplastic OSE cells. In addition, FSH stimulated the activation of M A P K cascade presumably through a PKC-dependent pathway. Activated M A P K phosphorylated Elk-1 in neoplastic OSE cells. These results suggest that the M A P K cascade may be involved in cellular function such as growth stimulation in response to FSH in neoplastic OSE cells. GnRH-II mRNA was expressed in normal OSE, immortalized OSE (IOSE), ovarian tumors  171  from patients and ovarian cancer cell lines, suggesting that the actions of GnRH-II are exerted in an autocrine/paracrine manner in these cells. Treatment with increasing doses (10~ - 10" M) of 9  7  GnRH-I and -II resulted in a growth-inhibition of IOSE-29 and IOSE-29EC cells. In addition, treatment with GnRH-I (D-Ala-GnRH, 10" M) increased D N A fragmentation in both cell lines. 7  Co-treatment of antide ( 1 0 M) and GnRH-I completely blocked the effect of GnRH-I on the 7  induction of D N A fragmentation. Furthermore, treatment with GnRH-II ( 1 0 M) resulted in an 7  increase in D N A fragmentation in IOSE-29 and IOSE-29EC cells. Similarly, co-treatment of antide (10" M) and GnRH-II completely blocked the effect of GnRH-II on the induction of D N A 7  fragmentation. However, treatment with GnRH-I had no effect on both bax and bcl-2 proteins in both cell lines. In addition, no significant difference in bax and bcl-2 proteins was observed following GnRH-II treatments. These results suggest that GnRH-II may be an integral regulator similar to GnRH-I, in normal OSE physiology and play a role in the induction of growthinhibition and apoptosis, via mechanisms other than bax and bcl-2, in neoplastic OSE cells. Taken together, the above-mentioned studies have investigated the roles of activin, TGFP, estradiol, FSH and GnRH-II as endocrine and autocrine regulators of proliferation and apoptosis in normal and neoplastic OSE cells. The proposed intracellular signaling cascades of activin, TGF-P, estradiol, FSH and GnRH-II have been suggested based on the conclusions in these studies (Fig. 59). These findings strongly suggest that these regulators may play a role in ovarian tumorigenesis in terms of the regulation of cell growth and/or cell death.  172  Figure 59. Proposed intracellular signaling cascades o f activin, T G F - p \ E2, F S H and GnRH-II neoplastic O S E cells.  173  2. Future Studies  2.1. Intracellular signaling pathways of activin and TGF-R in normal and neoplastic OSE  cells Future studies are required to elucidate the intracellular signaling pathways involved in the growth-stimulatory or -inhibitory effects of activin and TGF-R in normal and neoplastic OSE cells. It has been demonstrated that OVCAR-3 cells express Smad-4 and Smad-2, proteins specific for the TGF-R superfamily signaling pathway (Ito et al., 2000). Although no detectable change was induced in Smad-4 mRNA in OVCAR-3 cells, Smad-2 m R N A levels were increased following treatment with activin A (50 ng/ml) for 48 h. Therefore, it is tempting to identify whether these specific signaling pathways are activated following activin or TGF-R treatment in normal and immortalized OSE cells in vitro. Future experiments will provide a better understanding of the mechanisms involved in activin or TGF-P action in epithelial ovarian carcinoma.  2.2. Apoptotic pathways by activin and TGF-P in normal and neoplastic OSE cells In the present study, treatment with activin and TGF-P induced growth inhibition and apoptosis in early neoplastic and tumorigenic OSE cells. The anti-apoptotic bcl-2 protein was down-regulated by TGF-P treatment, but not by activin treatment. No difference was observed in bax protein levels following treatment with either TGF-p or activin. Thus, different pro- and/or anti-apoptotic genes or possibly other apoptotic pathways may be related with activin-induced apoptosis in our culture system (Chao and Korsmeyer, 1998; Koseki et al, 1998). The involvement of other pro- and anti-apoptotic genes such as bcl-XL, bcl-W, bcl-Xs, bad, and bak  174  should be evaluated to explain the molecular mechanism of activin-induced apoptosis in neoplastic OSE cells.  2.3. Apoptotic pathways by Estradiol in tumorigenic OSE cells In the present study, treatment with E2 prevented tamoxifen-induced apoptosis through ERs in tumorigenic OSE cells. The mechanism of action of E2 may be associated with up-regulation of bcl-2 gene at the m R N A and protein levels. Thus, future experiments are required to investigate the effect of E2 on downstream pathways of apoptosis such as caspase-3, caspase-9 and/or poly(ADP)-ribose polymerase in these cells. In addition, future experiments should clarify whether  the bcl-2 major  promoter  contains  cis-acting elements  directly involved in  transcriptional control by E2, and whether E2 induces bcl-2 expression via estrogen-responsive element(s) located within its coding region.  2.4. Involvement of PKC pathway in activating MAPK by FSH in neoplastic OSE cells M A P K pathway has been shown to mediate the cAMP-independent FSH activation of growth promotion in granulosa tumors (Babu et al, 2000). In the present study, treatment with F S H activated the M A P K (ERK-1/-2) cascade and phosphorylated Elk-1 in neoplastic OSE cells presumably through a PKC-dependent pathway. Involvement of P K C pathway in FSH-induced M A P K activation needs to be extensively examined using IOSE cell lines.  In addition,  activation of other transriptional factors including c-fos and c-myc by FSH treatment should be determined in these cells.  175  2.5. Apoptotic pathways by GnRH-II in normal and neoplastic OSE cells In the present study, GnRH-II induces a growth-inhibitory and apoptotic effect in neoplastic OSE cells. However, no significant change in the levels of bax and bcl-2 proteins was observed following GnRH-II treatment, suggesting that another pathway may be involved in the induction of apoptosis by GnRH-II in neoplastic OSE cells. 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