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Progesterone signaling in ovarian epithelial tumors

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Progesterone signaling in ovarian epithelial tumors

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Content PROGESTERONE SIGNALING IN OVARIAN EPITHELIAL TUMORS Copyright 2002 by Hong Zhou A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements of the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) August 2002 Hong Zhou Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3094406 UMI UMI Microform 3094406 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA The G raduate School U n iversity Park LOS ANGELES, CALIFO RNIA 900894695 This dissertation,, w ritte n b y Hong Zhou U n der th e d irectio n o f hex. . . . D issertatio n C om m i ttee, an d approved b y a ll its m em bers, has been p resented to an d accepted b y The G raduate School , in p a rtia l fu lfillm e n t o f requirem ents fo r th e degree o f D O C TO R O F P H I L O S O P H Y D ean o f G raduate S tudies Augufit-J6.., 2002 D1SSER TA T fO N C O M M IT T E E C hairperson Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To my parents Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS First, I would like to thank my mentor, Dr. Louis Dubeau. His insight contributed greatly to this work and his support and guidance make my study toward a Ph.D. degree successful. I thank all my other committee members Drs. Pradip Roy-Burman, Axel Schonthal, Michael Stallcup, and Hide Tsukamoto for their valuable advice, which significantly improved my thesis. I am also grateful to Ms. Martina Blumenthal and Sylvina Villalobos-Campos in Dr. Schonthal’s lab for their hard work and technical assistance on cell cycle study. My fellow students, postdocs, clinical fellows, and technicians in Dr. Dubeau’s lab provided important help during this work and also made this lab a fun place to be. Finally, I thank my family and my friends for their love and support and being there all the way. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF FIGURES vi ABSTRACT vii PART I BACKGROUND 1 Chapter 1 Ovarian cancer and reproductive factors 2 I. Risk factors for the development of ovarian epithelial 3 cancer II. Current theories on ovarian carcinogenesis 11 III. Role of reproductive hormones and their receptors in 13 ovarian cancer Chapter 2 Proliferation, cell cycle control, and cancer 19 I. Cell cycle machinery 20 II. Cell cycle control 22 III. Abnormalities of cell cycle regulators in cancer 25 IV. Hormonal regulation on cell growth regulators in 27 ovarian cancer V. Therapeutic targeting at deregulated cell cycle 28 VI. Rationale and Hypothesis 28 PART II PROGESTERONE SIGNALING IN OVARIAN EPITHELIAL 31 TUMORS Chapter 3 Effect of Progesterone on cell cycle activity in ovarian 32 epithelial tumors Abstract 33 Introduction 34 Materials and methods 36 Results 42 Discussion 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4 Role of the progesterone receptor in progesterone- 55 mediated ceil proliferation and angiogenic activity in ovarian epithelial tumors Abstract 56 Introduction 58 Materials and methods 63 Results 67 Discussion 82 PART III EPILOGUE 87 Chapter 5 Summary and future direction 88 Summary 89 Future directions 95 BIBLIOGRAPHY 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 3.1 Effects of reproductive hormones on thymidine incorporation Figure 3.2 Effects of P4 on proteins involved in cell cycle regulation Figure 3.3 Effect of P4 on MAP kinase phosphorylation Figure 3.4 Effect of P4 and RU486 on AP-1-mediated gene expression Figure 4.1a Effect of progesterone on DNA synthesis in SKOV-3 ovarian carcinoma cells Figure 4.1b Effect of progesterone on VEGF secretion in ovarian epithelial tumors Figure 4.2 Immunoprecipitation and Western blot analysis of progesterone receptor expression in cultures of benign and malignant ovarian tumor cells Figure 4.3 Effects of progesterone receptor agonists/antagonists on thymidine incorporation Figure 4.4a Effect of R5020 and RU486 on VEGF secretion Figure 4.4b Effect of ZK98299 on progesterone-induced VEGF secretion Figure 4.5 Effect of dexamethasone on DNA synthesis and VEGF secretion Figure 4.6 Down-regulation of progesterone receptors expression by antisense oligonucleotides Figure 4.7 Effect of antisense oligonucleotides against progesterone receptor mRNA on growth inhibition by progesterone Figure 4.8 Effect of antisense olionucleotides against progesterone receptor mRNA on progesterone-induced VEGF secretion page 46 47 49 50 72 73 74 75 76 77 78 79 80 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Epidemiological studies demonstrate that pregnancy and oral contraceptive use reduce the risk of ovarian cancer. We hypothesized that progesterone, the pregnancy hormone and the major component of oral contraceptives, may play an important role in protection against ovarian cancer. We examined the effect of progesterone on cell proliferation in benign and malignant ovarian epithelial tumor cells cultured in vitro. Progesterone significantly inhibited thymidine incorporation in these cell lines, down- regulated cyclin B1 expression and CDK1 kinase activity, and up-regulated p21 and p27 expression. DNA profile analysis confirmed cell cycle arrest in the G2 phase after progesterone treatment. MAP kinase phosphorylation was significantly reduced after progesterone treatment. AP-1 mediated gene expression was induced by progesterone and inhibited by RU486. We conclude that progesterone inhibits cell proliferation in ovarian epithelial tumors, at least in part, by down-regulating the activity of the cyclin B1/CDK1 complex. MAP kinase pathway may be involved in progesterone-mediated growth inhibition. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We further studied the role of the progesterone receptor in the actions of progesterone on growth and angiogenesis in ovarian epithelial tumor cells. Of the two isoforms of the progesterone receptor, PR-A is predominant in ovarian epithelial tumor cells, while PR-B expression is much lower. Progesterone inhibited cell proliferation in all the cell lines tested, but increased VEGF secretion only in certain cell lines. Both progesterone receptor agonists (R5020, ORG2058) and antagonists (RU486, ZK98299) acted like progesterone that inhibited thymidine incorporation in ovarian epithelial tumor cell lines. Although type II progesterone receptor antagonist RU486 acted like progesterone on VEGF secretion, type I progesterone receptor antagonist ZK98299 inhibited this effect of progesterone. Antisense oligonucleotides, which abolished expression of both PR-A and PR-B, blocked the effect of progesterone on VEGF secretion but not on cell proliferation. The effects of progesterone on cell proliferation and angiogenesis could not be reproduced by ligands for the mineralocorticoid and glucocorticoid receptors. These results suggest that the effect of progesterone on VEGF secretion is mediated via the progesterone receptor, while the inhibitory effect of progesterone on cell proliferation does not seem to involve this receptor. viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PARTI BACKGROUND Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1 Ovarian Cancer and Reproductive Factors Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ovarian cancer is the fourth most common malignancy and the fifth leading cause of cancer deaths among North American women (American Cancer Society 1997). More women die of ovarian cancer than from cancer arising in all other female reproductive organs combined (Holschneider and Berek 2000). It was estimated that more than 23,000 women in the United States were diagnosed with ovarian cancer in 2000 (Greenlee and others 2000). The majority was at advanced stages at the time of diagnosis, making a cure difficult to achieve. Although new chemotherapeutic agents are widely used in the treatment, the overall mortality from epithelial ovarian cancer has improved little over the past 20 years. The overall 5-year survival rate remains unchanged at about 30% (American Cancer Society 1997, Risch 1998, Wingo 1995). I. Risk factors for the development of ovarian epithelial cancer Although the cause of ovarian cancer is still unknown, some women are at higher risk than others. Risk factors of developing ovarian cancer include increasing age, North American or Northern European descent, nulliparity, and a family history of breast cancer or ovarian cancer. The latter is often 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. associated with a germ line mutation in the BRCA1 or BRCA2 genes. In the following brief comments are made about possible risk factors. 1- Age Ovarian cancer is a disease of increasing age. Ovarian cancer before the age of 40 is rare. It is mostly a disease of perimenopausal and postmenopausal women. The incidence rises dramatically through the fifth decade and then slowly reaches the maximum in the 80- to 84-year-old age group (Edmondson and Monaghan 2001). Although most cases are currently diagnosed in women younger than this age group, the elderly population will rise over the next two to three decades, therefore the total number of cases of ovarian cancer can be expected to increase. 2. Race Incidence of ovarian cancer varies among different ethnic groups and geographic regions, with a high frequency in Northern Europe and the United States, and a low incidence in Japan and some developing countries. Rates are significantly lower in Sub-Saharan Africa than in Europe and the United States (Parkin and others 1997). This difference was suggested to be due to racial variance other than other environmental factors. A lower incidence of 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. epithelial ovarian cancer among black women in the United States was first reported in 1977 (Weiss and others 1997). A similarly lower incidence was later found among Japanese, Chinese, and Hispanic women (Weiss and Peterson 1978). A study of immigrants to Britain from Africa and the Caribbean also showed that these women remain at lower risk of developing ovarian cancer than native white women, at least during the first generation (Grulich and others 1992). Although reproductive factors may account for some of the differences in these studies, it is thought the rate of ovarian cancer is lower in black women compared to white women even after correcting for such factors (John and others 1993). 3. Parity Pregnancy induces anovulation and inhibits pituitary release of gonadotropins (Parlow and others 1970, Jeppsson 1977). Epidemiological studies have consistently shown that increasing parity has dramatic effects on the reduction of ovarian cancer risk. Whittemore summarized the results of 12 US case-control studies and showed that parous women had ovarian cancer risk 40-60% lower than nulliparous women. Furthermore, risk decreases with increasing parity among parous women. Their studies suggested that the first term pregnancy has the greatest protection, which can significantly reduce ovarian cancer risk by 40%, while each following 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pregnancy after the first has smaller risk reduction, about 14% for every pregnancy. Among parous women, ovarian cancer risk even decreases with increasing number of incomplete pregnancies (miscarriages, abortions, and ectopic pregnancies), although the risk reduction is smaller in magnitude than with full term pregnancies, possibly due to the shorter duration of the anovulatory period (Whittemore and others 1992). 4. Breast-feeding Breast-feeding suppresses ovulation (Perez and others 1972) and reduces LH level, but increases FSH secretion (Reyes and others 1972). Separating the effects from pregnancy, breast-feeding showed a small protective effect on ovarian cancer development in epidemiological studies (Whittemore and others 1992). Parous women who ever breast-fed a child have 20-25% lower risk compared to those who never did so. The risk also decreases with increasing duration of breast-feeding. However, a month of breast-feeding within 6 months of delivery reduces risk more than does a month of subsequent breast-feeding, which is consistent with the observation that suppression of ovulation by breast-feeding is most effective in the first few months after delivery and becomes weaker afterwards (Reyes and others 1972). 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. Early menarche and late menopause It has been suggested that it takes longer to establish regular ovulatory cycles for women with later menarche than those who begin menstruating before their teens (Wallace and others 1978). Thus, women with early menarche may have higher ovarian cancer risk since they start ovulation earlier. Early menarche, together with late menopause, which results in a long menstrual life, may increase the risk of development of ovarian cancer. However, epidemiological studies failed to show any significant effect of age at menarche or at menopause on the risk of ovarian cancer (Whittemore and others 1992, Hankinson and others 1995). This suggests that length of menstrual life does not affect the risk of development of ovarian cancer. 6. Infertility and fertility drugs Increased ovarian cancer risk among nulliparous women suggests a possible association between ovarian cancer risk and infertility. There is a trend between infertility and the risk of developing epithelial ovarian cancer (Whittemore and others 1992, Mosgaard and others 1997, Risch and others 1994). Infertility was more commonly seen among ovarian cancer patients than controls. Infertile nulliparous women have an increased risk of epithelial ovarian cancer compared with nulliparous fertile women. However, parous 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. women who subsequently develop secondary infertility appear to have no greater risk than other parous women. In 1992, Whittemore et al reported that infertile women who had used fertility drugs had 2.8 times the risk of invasive ovarian cancer and 4.0 times for ovarian tumor of low malignant potential (LMP). Another study showed that prolonged use of 12 cycles or more of clomiphene citrate, a non-steroidal antiestrogen that induces ovulation by increasing FSH and LH secretion, significantly increased the risk of both LMP tumors and invasive carcinomas (Rossing and others 1994). A nationwide case-control study in Israel demonstrated that the use of ovulation induction agents, especially human menopausal gonadotropin, might increase the risk of ovarian cancer (Shushan and others 1996). However, other studies were unable to show a significant effect of fertility drugs on risk for ovarian cancer (Franceschi and others 1994, Venn and others 1995, Ness and others 2002). Whether an association exists between fertility drugs and ovarian cancer is still unresolved. 7. Oral contraceptives B Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Oral contraceptives induce anovulation and suppress gonadotropin secretion (Lauritzen 1968). There is a decreased incidence of ovarian cancer with oral contraceptive use during the reproductive years (Whittemore and others 1992). Women who used oral contraceptives have 30% lower risk for ovarian cancer than those who never used. The risk also decreases with increasing years of oral contraceptive use. The results of the UK-based Oxford Family Planning Association study (Vessey and Painter 1995) showed that oral contraceptive use for over 8 years reduced the risk of ovarian cancer by 60%. This finding was confirmed by an Australian study, which demonstrated a risk reduction by 70% for 10 years of use (Purdie and others 1995). Furthermore, the reduced risk among past oral contraceptive users persists for at least 15 years after cessation of use (Whittemore and others 1992). 8. Hormone replacement therapy Studies on hormone replacement therapy (HRT) and risk of epithelial ovarian cancer have produced conflicting results, but most data do not support a strong association between HRT and risk of ovarian cancer. However, current studies do not exclude the possibility that estrogens alone could stimulate ovarian cancer growth in a small fraction of patients. Ever users of estrogen replacement therapy (ERT) may be at increased risk of epithelial ovarian cancer (Riman and others 2002). A recent study has reported that 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mortality from ovarian cancer is increased in women who have used ERT for over 10 years and that mortality falls with increasing time from last use of estrogen replacement. Women who had not used estrogen for 15 years were at no increased risk of death from ovarian cancer compared with women who had never used estrogen (Rodriguez and others 2001). 9. Family History Family history of ovarian cancer is the most significant known risk factor for ovarian cancer, although only a small percentage of epithelial ovarian cancers have been attributed to family history. Approximately 90% of familial ovarian cancers are due to mutations in the BRCA1 and BRCA2 genes (Boyd 2001). The protein products of these genes are involved in processes of DNA repair and recombination, cell cycle control, and transcription (Venkitaraman 2002). Other cases are caused by mutations including the hereditary nonpolyposis colon cancer (HNPCC) gene (Marra and Boland 1995). Inherited mutations in BRCA1 and BRCA2 are responsible for 5-10% of epithelial ovarian cancers (Reedy and others 2001). The lifetime risk of developing ovarian cancer as a carrier of a mutation of the BRCA genes has been estimated at approximately 15-60% (Easton and others 1995). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II. Current theories on ovarian carcinogenesis Only about 5-13% epithelial ovarian cancers are due to inherited familial predisposition. The majority of ovarian cancers are sporadic, which makes it most difficult to identify the etiology of these ovarian cancers. Two major theories on ovarian cancer pathogenesis are “incessant ovulation” (Fathalla 1971) and “excessive gonadotropin stimulation” (Cramer and Welch 1983). 1. Incessant ovulation The theory of “incessant ovulation” hypothesizes that monthly ovulation causes repetitive disruption and repair of the ovarian surface epithelium, which may result in a higher probability of spontaneous mutations and thus increase the risk of ovarian cancer development (Fathalla 1971). According to this theory, the number of ovulation in a woman’s lifetime, called ovulatory age, is related to the risk of developing ovarian cancer. Epidemiological studies support this theory since pregnancy or oral contraceptive use interrupts ovulation and reduces the risk of ovarian cancer. However, this theory cannot explain why infertility, usually due to hypo- or anovulation, is associated with increased risk of ovarian cancer (Mosgaard and others 1997, Venn and others 1995, Ness and others 2002). In contrast, early menarche and late menopause, which elongate women’s ovulatory age, have no 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significant effects on ovarian cancer risk (Mosgaard and others 1997, Franceschi 1994, Tortolero-Luna and Mitchell 1995). It cannot explain the lower rates in Japan and some developing countries compared to that in the United States. Besides, this theory is based on the hypothesis that the surface epithelium is the origin of ovarian epithelial tumors. This hypothesis is based on weak histological arguments and has recently been questioned (Dubeau 1999, Rodriguez and Dubeau 2001, Karseladze 2001). 2. Excessive gonadotropin stimulation The “excessive gonadotropin stimulation” theory hypothesizes that gonadotropins (follicle-stimulating hormone [FSH] and luteinizing hormone [LH]) promote the development of ovarian cancer by stimulating the ovarian epithelium proliferation (Cramer and Welch 1983). Pregnancy or oral contraceptive use can suppress endogenous levels of gonadotropins, therefore reducing the risk of ovarian cancer. However, since serum levels of FSH and LH are elevated in the years around menopause and thereafter, it is expected that there should be a significant increase in ovarian cancer incidence after age 70 years due to 25-30 year latency after exposure. In fact, epidemiological studies showed the mean age of incidence of ovarian cancer is in late 50s and there is only a slow increase in the incidence of ovarian cancer after age 70 years (Wingo and others 1995, Pike 1987). 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 1 1 . Role of reproductive hormones and their receptors in ovarian cancer Neither “incessant ovulation” nor “excessive gonadotropin stimulation” can fully account for the development of ovarian cancer, suggesting other mechanisms exist. The underlying hypothesis for this dissertation is that the hormonal fluctuations that take place during the normal menstrual cycle, when repeated continuously, can lead to the development of ovarian cancer. Reproductive hormones, including steroid hormones (estrogen and progesterone), and gonadotropins (FSH and LH), closely regulate development, growth, and function of the ovary. Brief comments will be made about each of these hormones. 1. Steroid hormones Sex steroids (estrogen and progesterone) typically act through their individual intracellular receptors. Binding to the receptor induces conformational changes in receptor structure, phosphorylation, and dimerization of the receptor. The hormone-receptor complex then binds to a specific DNA site (hormone responsive element) in the promoter region of a target gene and leads to initiation of transcription. 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Steroid hormones can stimulate or inhibit cell proliferation, thus modulate tumor progression. Sex steroid-related tumors in women are represented by breast cancer and endometrial cancer (Jordan and Murphy 1990, Horwitz and Clarke 1998, Jordan and Morrow 1999), and a possible relationship exists between sex steroids and ovarian cancer. Estrogen Estradiol, which is at a low concentration during menstruation, gradually increases and reaches its maximum in the follicular phase of the menstrual cycle. It is produced by granulosa cells under stimulation of FSH. It promotes granulosa cell proliferation and follicular growth and growth of endometrium. Epidemiological studies performed in the 1960’s showed that the average serum estradiol level in the premenopausal period was lower in Asian women than in U.S. women, and that this difference was consistent with a lower incidence rate of ovarian cancer in Asian women (Wu and Pike 1995). Another study on postmenopausal women indicated that estrogen replacement therapy increased the risk of ovarian cancer, by 40% after 6-10 years of use this therapy and by 70% for more than 10 years use of this therapy (Easton and others 1995). In vitro studies, estrogen was found to 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stimulate cell growth in ovarian cancer cells, while the antiestrogen tamoxifen was shown to inhibit cell proliferation (Nash and others 1989, Langdon and others 1988, Langdon and others 1990, Geisinger and others 1990, Persson 2000). Progesterone Progesterone is produced by the corpus luteum after ovulation and is therefore predominant only during the luteal phase. When corpus luteum regresses, progesterone levels fall. This hormone stimulates secretory and vascular activity of the endometrium, preparing the endometrium to receive the egg. If the egg is fertilized, progesterone secretion continues, preventing release of additional eggs from the ovaries. This hormone is also a major component in oral contraceptives. Numerous epidemiological studies confirmed that pregnancy and use of oral contraceptives have protective effect on ovarian cancer development. As the major hormone in pregnancy and the major component in oral contraceptives, progesterone may play an important role in this protective effect. 2. Gonadotropins 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gonadotropins, consisting of follicle stimulating hormone (FSH) and luteinizing hormone (LH), are glycopeptide hormones synthesized and secreted by the pituitary grand. These hormones are heterodimers composed of a common a-subunit and a specific p-subunit. While the p- subunit determines the different biological properties of each gonadotropin, the a-subunit is important for receptor binding and activation. The genes encoding the p-subunits of FSH and LH are structurally similar, each composed of 3 exons, located on chromosomes 19 and 11, respectively (Talmadge and others 1984, Watkins and others 1987). Both hormones exert a major effect on ovarian function through interaction with specific seven transmembrane domain glycoprotein receptors (G protein receptors). Binding of gonadotropins changes the conformation of these receptors and leads to interaction with G proteins, which activates second messengers. FSH is predominant in the follicular phase of the menstrual cycle. It stimulates ovary to produce estradiol during follicular phase and promotes granulosa cell division and differentiation (Simoni and others 1997). At the end of the follicular phase, a rapid surge in LH concentration triggers ovulation. LH also stimulates theca cell androgen production, granulosa cell luteinization, and progesterone production during luteal phase. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Postmenopausal women, who have a higher incidence of developing ovarian cancer, also show high circulating levels of gonadotropins. It was reported that FSH and hCG stimulated DMA synthesis and cell proliferation in a number of cultured ovarian cancer cell lines (Ohtani and others 1992, Simon and others 1979, Simon and others 1983). FSH and LH can also stimulate VEGF expression (Wang and others 2002). Elevated levels of gonadotropins (FSH and LH) may in some cases promote peritoneal metastatic dissemination of ovarian cancer by increasing cell adhesion (Schiffenbauer and others 2002). 3. Hormone receptors in ovarian cancer Hormonal actions are usually mediated via specific hormone receptors. Estrogen receptor (ER) and progesterone receptor (PR) are present in both normal ovaries and ovarian tumors. About 60% of ovarian epithelial tumors express ER, 50% express PR (Rao and Slotman 1991). FSH and LH receptors were also found in some benign and malignant ovarian epithelial tumors (Kammerman and others 1981, Rajaniemi and others 1981). These findings suggest that at least some types of ovarian tumor cells may respond to hormonal manipulations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The possibility of using the hormone receptor status as a prognostic factor for ovarian cancer has been widely studied. However, the results are not conclusive. Some studies are even contradictory. One study by Bizzi et al (Bizzi and others 1988) showed that ER positivity was associated with better survival. However, two other studies could not demonstrate correlation between ER status and survival (Slotman and others 1989, Slotman and others 1990). Teufel et al reported an association between presence of ER and poor differentiation of ovarian neoplasm (Teufel and others 1983). Others showed that well differentiated tumors more likely to express ER or both ER and PR (Ford and others 1983, Quinn and others 1982). Slotman et al suggested that ovarian cancer patients with higher PR levels have significantly longer survival than those with lower PR values, independent on age, stage, or tumor histology (Slotman and others 1989). In conclusion, risk factors and protective factors for ovarian cancer have been clearly identified. Epidemiological data and current theories (“incessant ovulation” and “excessive gonadotropin stimulation”) of ovarian cancer pathogenesis relate reproductive hormones to ovarian cancer. It is in that context and to better understand the role of reproductive hormones in ovarian tumorigenesis that the work presented in this dissertation was undertaken. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Proliferation, Cell Cycle Control, and Cancer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Uncontrolled cell proliferation is one the hallmarks of cancer cells (Albanes and Winick 1988, Cohen and Ellwein 1990, Moolgavkar 19933, Sherr 1996). Since mammalian cell proliferation is exquisitely controlled by a highly ordered process, called the cell cycle, it is not surprising to find deregulation of cell cycle regulators in cancer cells (Sherr 1996, Hahn and others 1999). Positive regulators of cell cycle progression, such as cyclin D1, cyclin E, CDK4, cdc25 are frequently overexpressed and activated in human cancer cells, while negative regulators, such as p53, ARE, Rb, p16, p27 (McDonald and el-Deiry 2000), Rb and p53 family members (Corn and others 1999), BRCA1, ATM (Zhou and Elledge 2000), and CHK (Bell and others 1999) are usually inactivated in cancer cells. Understanding the normal cell cycle regulation and the abnormality of cell cycle regulators in cancer will help us study molecular basis of tumor progression and develop better therapeutics for cancer treatment. I. Cell cycle machinery The cell cycle is composed of four continuous phases: G0/G 1 (quiescent state/gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis). Progression from one phase of the cell cycle to the next is driven by the active forms of cyclin-dependent kinases (CDKs). The CDKs are a conserved family of serine/threonine protein kinases. They are inactive as monomers and need 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to bind to specific cyclins, their regulatory subunits, to be activated. Different cyclin/CDK complexes are formed and activated in each phase to propel cell cycle progression. The CDKs also have to be phosphorylated on the threonine residue located in their T-loop to have proper catalytic activity. This phosphorylation is carried out by the cyclin H/CDK7 complex, a serine/threonine kinase that is also known as CDK-activating kinase (CAK). The phosphate is removed by the CDK-associated protein phosphatase (KAP) after the associated cyclin has been degraded (Morgan 1995). Entry into the cell cycle is induced by a variety of mitogenic signals, including hormones, growth factors, and cytokines. Cells respond to these stimuli during the early first gap (G1) phase of the cell cycle by the immediate rise in cyclin D expression, which associates with and activates CDK4 and CDK6 to start progression through G1 phase (Pines 1993a, Pines 1993b, Norbury and Nurse 1992). In late G1 phase, cyclin E complexes with CDK2 to promote G1/S transition. Cyclin A/CDK2 activity is required for S phase progression and entry into G2 phase (Knoblich and others 1994). Cyclin A also complexes with CDK1 to promote G2 phase progression. During G2 and M phase, cyclin B forms a complex with CDK1 to mediate G2/M transition (Sherr 1996, Knoblich and Lehner 1993, King and others 1994, McDonald and el-Deiry 2001). 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Active cyclin/CDK complexes drive the cell cycle progression, phosphorylating their substrates that carry out a series of changes in the cell to accomplish a cell cycle. The primary substrates of CDK4/6 and CDK2 in G1 phase progression are retinoblastoma protein family members, including Rb, p107, and p130. Rb is a tumor suppressor protein. It is active when unphosphorylated or hypo-phosphorylated. Active Rb inhibits growth by binding to and inhibiting transcription factors, such as the E2F family. In early G1 phase, Rb is phosphorylated by the active cyclin D/CDK4 complex. In late G1 phase Rb is subsequently phosphorylated by the cyclin E/CDK2 complex. Hyper-phosphorylated Rb releases E2F. Free E2F, in turn, activates transcription of cyclin A, cyclin E, CDK1, leading to progression into S phase (Harbour and others 1999, Harbour and Dean 2000). Thus, the Rb family members play a central role in the restriction point at G1/S transition. II. Cell cycle control The CDKs are central players in the cell cycle. The activity of cyclin/CDK complexes is tightly regulated throughout the cell cycle. The activity of the cyclin/CDK complex is regulated by its associated cyclin, phosphorylation of the CDK itself, and CDK inhibitors. 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Activation of CDKs requires both binding to appropriate cyclins and phosphorylation of a conserved threonine by the CDK-activating kinase (Desai and others 1995, Ducommun and others 1991). Cyclin levels regularly fluctuate during the cell cycle (Evans and others 1983), which regulates CDK activity, while CDK levels remain in constant excess throughout the cell cycle. Activating phosphorylation of the CDK by the CAK stabilizes the cyclin/CDK complex and increases its catalytic activity (Russo and others 1996). However, inhibitory phosphorylation on one or two residues at the N- terminal ATP-binding region of CDK reduces the kinase activity and inactivates the cyclin/CDK complex (Lew and Kornbluth 1996). These residues are a conserved tyrosine in many CDKs phosphorylated by Wee1/Mik1, and an adjacent threonine residue phosphorylated by Myt1. This inhibitory phosphorylation can be reversed by members of the cdc25 family of phosphatase, such as cdc25, phosphatases B and C (Hunter 1997), which removes the phosphate and activate the cyclin/CDK complex. An active cyclin/CDK complex can be inactivated by binding to CDK inhibitors (CKIs). There are at least two families of CDK inhibitors with distinct properties in sequence homology and specificity of binding with CDKs (Sherr and Roberts 1999). The first is the INK4 family, consisting of p15 (INK4B), p16 (INK4A), p18 (INK4C), and p19 (INK4D). These proteins all 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. contain four ankrin repeats in structure. They specifically bind to and inhibit CDK4 and CDK6 in G1 phase, but not CDK2 or CDK1, causing cell arrest in G1 phase. The second is the CIP/KIP family, including p21 (CIP1/WAF1/SD1), p27 (KIP1), and p57 (KIP2). They share homology at the N-terminal CDK inhibitory domain. They are universal CDK inhibitors that recognize all cyclin/CDK complexes and inhibit their kinase activity (McDonald and el-Deiry 2001, Sherr and Roberts 1995). However, they preferentially bind to G1 cyclin/CDK complexes since overexpression of these inhibitors leads to cell arrest in G1. The p21 protein is not only a CDK inhibitor but also a downstream target of p53. It is induced by p53 and binds to and inhibits G1 cyclin/CDK complexes, causing growth arrest in G1 phase in response to DMA damage (Harper and others 1993). It is also a critical determinant of G2/M transition by binding to G2 cyclin/CDK complexes (el-Deiry and others 1993, Dulic and others 1998). Expression of p21 can be regulated by p53 as well as p53-independent pathways (Medema and others 1998, Blagosklonny and others 1996, Li and others 1996, Michieli and others 1994). Overexpression of p21 has been reported to inhibit growth of mammary carcinoma (Shibata and others 2001), lung cancer (Joshi and others 1998), and prostate cancer (Eastham and others 1995). 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. III. Abnormalities of cell cycle regulators in cancer The cell cycle is such a delicately controlled process that any abnormal expression or function of components in the cell cycle machinery may lead to deregulated growth, a hallmark of cancer. In fact, cancer cells often show alterations in the components of the cell cycle machinery, including overexpression of cyclins and CDKs, as well as loss of CKI and tumor suppressors such as p53 and Rb. Positive regulators of the cell cycle are often up regulated in cancer cells. Overexpression of cyclin D1 has been reported in a variety of cancers, including those of the breast, esophagus and the B cell lymphoma (Bartkova and others 1995). Cyclin E overproduction has been found in a number of cancer types, such as breast, lung, colon, and ovarian cancers (Keyomarsi and others 1994, Leach and others 1993). Elevated levels of cyclin A have been found in hematologic malignancies (Paterlini and others 1993, Malumbres and Barbacid 2001). Amplification of CDK4 has been reported in breast, lung, and cervical cancers (Cheung and others 2001). The positive regulator of the cell cycle cdc25 phosphatase was found to be able to cooperate with Ras to transform cells, and injections of these transformed cells into nude mice result in tumor formation. Overexpression of cdc25B was found in 32% of primary breast cancers (Galaktionov and others 1995). 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Negative regulators of the cell cycle are usually inactivated or down regulated in cancer cells. Tumor suppressors p53 and Rb mutations are commonly found in human cancers. In addition, p53 and Rb are not only mutated in numerous types of cancer but also targeted and inactivated by DNA tumor virus proteins such as HPV E7, SV40 large T antigen, and adenovirus E1A (McDonald and el-Deiry 2000). Tumor suppressor BRCA1, causing G2 arrest in respond to DNA damage, is mutated in some breast and ovarian cancers (Venkitaraman 2002). CDK inhibitors are frequently deleted in a variety of tumors and cancer cell lines, such as p15 and p16 (Koduru and others 1995). Genetic analysis showed the locus surrounding the tumor suppressor protein, p16, is often lost by deletion in melanomas (Marx 1994). These deletions also eliminate the CKIs p15 and p14. Further studies have demonstrated point mutations in p16 as well as methylation in p16 promoter region (Hall and Peters 1996, Merlo and others 1995). Inactivation of the CKI, p27, has also been reported through different mechanisms, such as loss of expression, increase in degradation, and protein mislocalization (Tan and others 1997, Loda and others 1997, Singh and others 1998). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. Hormonal regulation on cell growth regulators in ovarian cancer Components of the ceil cycle machinery are regulated by extracellular signals, such as hormones, growth factors, etc. The c-myc proto-oncogene is needed for the initiation of cell proliferation. A rapid rise in the expression of this gene has been observed in many quiescent cells after stimulation with growth factors. It was also found that estrogen activated c-myc expression in ovarian and breast cancer cells in vitro (Chien and others 1994, Dubik and others 1987). The above data provided evidence for some of the earliest effects of hormonal agents on the transcription of genes thought to be necessary for cell proliferation. On the other hand, the BRCA1 gene, which is a tumor suppressor gene that inhibits growth of breast and ovarian cancers (Holt and others 1996), is mutated in 5-10% of ovarian cancers. Treatment with estrogen and progesterone up-regulated the BRCA1 mRNA and protein levels, suggesting the role of hormonal regulation of genes involved in growth regulation (Gudas and others 1995). Recent study showed that retinoid suppressed ovarian cancer cell growth by increasing p27 expression and decreasing CDK2, 4 and 6 activity (Zhang and others 2001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V. Therapeutic targeting at deregulated cell cycles Although cancers are diverse, uncontrolled proliferation is one of the characteristics of all cancer cells. This provides a target for therapeutic intervention for all cancers. This task can be achieved through a variety of paths, which depends on the molecular properties of individual tumors that cause their uncontrolled growth. Most anti-cancer drugs currently in use are inhibitors of DNA synthesis or cell division. They are not targeted at specific cause of deregulated proliferation. With the further understanding of the cell cycle regulation, drugs targeted at specific abnormal cell cycle regulators have been designed. Among these drugs, small molecule cyclin-dependent kinase inhibitors have been extensively studied and are currently being tested for clinical use, for example, Flavopiridol (Senderowicz and Sausville 2000). Other drugs of this kind being studied include drugs aimed at ATM and CHK1 (Shapiro and Harper 1999). VI. Rationale and Hypothesis Epidemiological studies have shown that reproductive hormones play important roles in ovarian cancer. Since the incessant ovulation hypothesis Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was proposed by Fathalla in 1971, epidemiological studies consistently confirmed the protective effect of increasing parity and use of oral contraceptives (see Chapter 1). As the major hormone in pregnancy and major component in oral contraceptives, progesterone plays a key role in epithelial ovarian tumors. However, the underlying mechanism of progesterone-mediated actions on ovarian cancer development is still unclear. We examined the effect of reproductive hormones on cell proliferation in cultured cystadenomas. The results showed that 3 of the 4 reproductive hormones involved in menstrual cycle regulation, including estradiol, FSH, and LH, stimulated cell proliferation while the fourth hormone, progesterone, was growth inhibitory. Given the potential therapeutic implications of such growth inhibitory activity, we sought to further investigate the mechanisms responsible for mediating the growth-inhibitory effects of progesterone in ovarian epithelial tumors. We hypothesize that progesterone directly influences the cell proliferation in ovarian epithelial tumor cells through cell cycle regulation. To test this hypothesis, we investigated the effects of progesterone on cell proliferation, 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell cycle regulation, and signal transduction pathway in various benign (cystadenomas) and malignant (carcinomas) ovarian tumors cultured in vitro. We further studied the role of progesterone receptor in progesterone- mediated growth inhibition and angiogenic activity. We examined benign tumors in addition to malignant ones because ovarian cystadenomas, which are made up of the same cell type as carcinomas, are better differentiated, implying that any effect from reproductive hormones may be more accentuated in these tumors. In addition, although not as serious as ovarian carcinomas, ovarian cystadenomas constitute a frequent health problem in women in their reproductive ages. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PART 1 1 PROGESTERONE SIGNALING IN OVARIAN EPITHELIAL TUMORS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 * Effect of Progesterone on Cell Cycle Activity in Ovarian Epithelial Tumors * This chapter is adapted from Zhou H et al. Effect of reproductive hormones on ovarian epithelial tumors: I. Effect on cell cycle activity. Cancer Biology and Therapy 1 (3): 298-304, 2002. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT We examined the effects of the 4 major female reproductive hormones, estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH) on thymidine incorporation in benign and malignant ovarian epithelial tumors cultured in vitro. Treatment of these tumors with E2, FSH and LH resulted in increased thymidine incorporation while treatment with P4 inhibited growth as well as thymidine incorporation. P4 down regulated cyclin B1 expression and cdkl activity, up-regulated the p21 protein, and inhibited MAP kinase phosphorylation. Expression of a reporter gene downstream to an AP-1 responsive element in a plasmid construct transfected into ovarian epithelial tumor cells was induced by P4 and inhibited by RU486. We conclude that P4 inhibits cell cycle activity in ovarian epithelial tumors, in part via down-regulation of the cyclin B1/CDK1 complex. MAP kinase signal transduction pathway may be involved in the inhibitory effect of progesterone on cell proliferation. This inhibitory effect of progesterone may have therapeutic utility against ovarian epithelial tumors. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Ovarian carcinoma is the fourth most common cause of cancer death among women in the United States, following lung, breast, and colorectal cancers (American Cancer Society 1997). It is the most lethal gynecological malignancy. There is strong epidemiological evidence for an association between ovulatory activity and risk of ovarian tumorigenesis (Whittemore and others 1992). Women with uninterrupted menstrual cycles throughout their reproductive years are at highest risk. Interruption of menstruation for only a few years can have a marked protective effect. This effect is independent of whether such interruption is achieved through pregnancy or oral contraceptives, although there is evidence of a first pregnancy being more protective than subsequent ones (Whittemore and others 1992). For example, use of oral contraceptives for 5 years results in an approximately 40% decrease in ovarian cancer risk, which is similar to the protective effect of 5 pregnancies after the first (Pike 1987). These epidemiological observations raise the possibility that reproductive hormones, which control ovulatory activity, may influence the biological behavior of ovarian epithelial tumors. A corollary to this statement is that 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. these hormones may constitute potentially useful tools in the clinical management of affected patients. We therefore sought to investigate the effect of reproductive hormones on cell cycle activity in benign (cystadenomas) and malignant (carcinomas) ovarian tumors cultured in vitro. We examined benign tumors in addition to malignant ones because ovarian cystadenomas, which are the same cell type as carcinomas, are better differentiated, implying that any effect from reproductive hormones may be more accentuated in these tumors. In addition, although not as serious as ovarian carcinomas, ovarian cystadenomas constitute a frequent health problem in women of reproductive age. The results showed that 3 of the 4 main hormones involved in menstrual cycle regulation, estradiol (E2), follicle stimulating hormone (FSH), and luteinizing hormone (LH), stimulated thymidine incorporation in the tumors examined while the fourth hormone, progesterone (P4), was growth inhibitory. Given the potential therapeutic implications of such growth inhibitory activity, we further investigated the mechanisms responsible for mediating the growth-inhibitory effects of P4 in ovarian epithelial tumors. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Source of reagents E2 and P4 were purchased from Sigma Chemical Company (St Louis, MO). They were both dissolved in absolute ethanol at stock concentration of 10 mM. RU486 (mifepristone) was obtained from Sigma Chemical Company. FSH and LH were obtained from the National Hormone and Pituitary Program Awards Management System. Cell lines and tissue culture ML5 and ML10 cells were derived from human ovarian cystadenomas and transfected with SV40 large T antigen to increase their longevity in vitro (Luo and others 1997). MCV 50 cells were derived from a subclone of ML10 that became spontaneously immortalized in culture. HOC-7 ovarian carcinoma cells were obtained from Dr. R. Buick, University of Toronto (Buick and others 1985). OVCAR-3 cells were purchased from the American Type Culture Collection (ATCC #HTB161). ML5, ML10, and MCV50 were cultured in MEM while HOC-7 and OVCAR-3 cells were cultured in RPMI 1640 (Life 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Technologies, Grand Island, NY). All tissue culture media were supplemented with 10% fetal bovine serum. 3H-thym«dine incorporation measurements Cells cultured in 24-well tissue culture plates (Corning Costar Corp., Cambridge, MA) were allowed to grow until 60-70% confluence, at which time the serum was switched to 2% charcoal filtered fetal bovine serum. After 2 days, the serum concentration was further lowered to 0.4%. One pCi/ml [3H]thymidine (ICN Pharmaceuticals, Irvine, CA) was added to each well and the cells were treated either with the desired hormonal agent or with vehicle only. The tissue culture dishes were washed twice with ice-cold phosphate- buffered saline (PBS) after completion of the appropriate treatment period, followed by treatment with ice-cold 5% trichloroacetic acid and solubilization of the resulting precipitate in 0.2 ml of 1 N NaOH. The amount of radioactivity was measured by liquid scintillation counting after neutralization with 0.2 ml of 1 M acetic acid. Western blot analyses Cell monolayers were detached from plastic tissue culture dishes by rubbing with a rubber “policeman” and stored at -70°C. After thawing, the cells were 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lysed in 20 mM Tris-CI (pH 8.0), 125 mM NaCI, 0.5% NP-40, 20 mM NaF, 0.2 mM Na3P04, 2 mM EDTA, 35 p,g/ml PMSF (Sigma Chemical Company), 0.7 pg/ml pepstatin A (Sigma), and 0.5 |ig/ml leupeptin (Sigma). Protein concentrations were determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Samples containing 50 p,g protein were electrophoresed on polyacrylamide and transferred onto nitrocellulose membranes (Biorad Laboratories, Richmond, CA). After incubating for 1 hr in 0.1% Tween-20, 50 mM Tris Base, 150 mM NaCI, pH 7.5, 5% non-fat dry milk (Biorad Laboratories, Richmond, CA), the membranes were exposed to the primary antibody at 4°C overnight. Monoclonal antibody against cyclin B1 was obtained from Pharmingen (La Jolla, CA). Monoclonal antibodies against cyclin D1, D2, D3 and E, and A as well as against p21 and p27 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for phosphorylated MAP kinase and total MAP kinase were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Monoclonal antibody against p-actin was purchased from Sigma Chemical Company. Anti-mouse antibody was from Promega (Madison, Wl). The secondary antibodies were coupled to horseradish peroxidase and were detected by the ECL western blotting detection reagents (Amersham Life Science, Buckinghamshire, England). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cyclin-dependent kinase assay The activity of cyciin-dependent kinases was analyzed as described by Schonthal et al (Schonthal and Feramisco 1993). A total amount of 100 (ig of cellular protein extract was immunoprecipitated with CDK1 or CDK2 antibody. The immunocomplex, bound to protein A-agarose, was incubated in kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCh, 1 mM dithiothreithol) in the presence of 10 jxCi [y-32 P]ATP (100 mM total ATP) and 2 jig histone H1 protein which served as substrate. The total reaction volume was 25 jil. The radiolabelled product was electrophoresed on 15% polyacrylamide gels stained with Coomassie blue (to control for the amount of histone H1 protein added) and analyzed using a phosphor-imager. Transient transfection and luciferase assay ML5 cells were plated onto 6-well plastic plates in MEM supplemented with 10% fetal bovine serum. The serum concentration was gradually lowered to 0.4% as explained for thymidine incorporation measurements (see above). The cells were transfected with 2 micrograms of a plasmid vector containing an AP-1 response element upstream of a luciferase reporter gene (obtained from Dr. Peter Kushner, University of California at San Francisco) using a Superfect kit (Qiagen Sciences, Germantown, MD). Luciferase activity was 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. measured in extracts from cells treated and untreated with reagents of interest using the Luciferase Assay System (Promega, Madison, Wl). Flow cytometry Cells of interest were resuspended in PBS containing 10 pg/ml propidium iodide and 100 pg/ml RNAse A. After incubation at 37°C for 30 min, the cells were analyzed using an Epics Profile Instrument (Coulter Electronics, Hialeah, FL). The laser excitation wavelength was 488 nm. The intensity of fluorescence emission over 630 nm was measured. The percentage of cells at different phases of the cell cycle was calculated using MultiCycle software from Phoenix Flow Systems (San Diego, CA). Statistical analyses All statistical analyses were performed using the Statistical Analysis System package program (SAS Institute, Carey, NC). All statistical significance levels quoted are 2-sided. Comparisons of thymidine incorporation was done by using the ratio of the levels of incorporation with the hormones to the levels of incorporation with the vehicle for each repeat of the experiment as the statistic of interest. Significance levels were calculated using Student’s t-test with the logarithms of these values. For comparisons of thymidine 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incorporation with different concentrations of compounds, the above method was used with one of the combinations playing the role of “vehicle”. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Effects of reproductive hormones on DMA synthesis in cultured ovarian cystadenoma cells We first measured the effects of E2 and P4, 2 steroid hormones important for controlling menstrual cycle activity, on the rate of thymidine incorporation in MLS ovarian cystadenoma cells (Figure 3.1a). Treatment with E2 at 0.1 pM stimulated DNA synthesis by 43% (p < 0.004). There was no further increase when the E2 concentration was increased to 1 pM. Treatment with P4 at 0.1, 1 and 10 pM showed increasing inhibitory effects (-11%, -28%, and -78% respectively, p = 0.02, 0.0009 and < 0.0001 respectively). The concentrations of E2 and P4 used in these studies were close to levels measured in the ovarian vein of pre-menopausal women (Lucisano and others 1978, McNatty and others 1976). We next examined the effect of gonadotropin hormones on thymidine incorporation in ML5 cells (Figure 3.1b). FSH at 0.04, 0.4 and 4 III/ ml increased DNA synthesis by 51%, 46% and 45% (p = 0.007, 0.0005 and 0.0006). LH at 1, 10 and 100 IU/L increased DNA synthesis by 42%, 99% and 104% respectively (p = 0.01, 0.07 and 0.04). The lowest concentration of 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FSH used, 0.04 IU/L, approached the highest circulating levels of this hormone during the normal menstrual cycles. The normal physiological concentration of circulating FSH increases 10 to 20 folds after menopause, reaching levels that fall between the 2 highest concentrations used in our thymidine incorporation studies. The normal physiological levels of circulating LH during the reproductive years, which vary between 5 and 20 IU/L, are likewise within the range of concentrations examined in our thymidine incorporation assays. The inhibitory effect of P4 on thymidine incorporation was reproduced in another cystadenoma cell strain (ML10) as well as in 3 ovarian carcinoma cell lines (Figure 3.1c). In each case, this effect was statistically significant at concentrations of 10 (iM (not shown). Differences in growth rates based on growth curve analyses of progesterone-treated and -untreated MLS cells are shown in Figure 3.1d. Effects of P4 on the expression of proteins involved in cell cycle regulation An important mechanism of regulation of progression through the cell cycle is the precise timing of expression of various cyclins (Grana and Reddy 1995). Western blot analyses of cells treated with 10 |iM P4 showed down- 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulation of cyclin B1, a cyclin important for progression through the G2/M phase of the cell cycle (Figure 3.2a) In contrast, P4 had much smaller effects on the expression of cyclins A, E, D1, D2, and D3 (not shown). The effect on cyclin B1 was accompanied by an up-regulation of p21 (Figure 3.2b), a universal cyclin-dependent kinase inhibitor involved in inhibition of CDK1 and CDK2 (Morgan 1995). Another member of this family of cyclin-dependent kinase inhibitors, p27 (Grana and Reddy 1995, Morgan 1995), was also slightly up regulated by P4 (Figure 3.2b). Up-regulation of p21 may have been mediated via a p53-independent pathway in ML5 cystadenoma cells because these cells express SV40 large T antigen, which binds to and inactivates p53 (DeCaprio and others 1989, Linzer and Levine 1979, Vogelstein and Kinzler 1992). The effect of P4 on down-regulation of cyclin B1, if biologically significant, should be accompanied by a reduction of CDK1 activity because such activity, which is essential for entry into mitosis, is dependent on interactions between this protein kinase and cyclin B1 (Morgan 1995). Indeed, incorporation of 32P into a histone H1 substrate by CDK1 activity was substantially lower in P4-treated cystadenoma cells than in controls. The intensity of the signal indicative of the amount of radioactive phosphate incorporated into the histone H1 substrate in protein extracts from cells treated with P4 for 24 and 48 hours was 79 and 12 respectively (Figure 3.2c). 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This is substantially lower than the corresponding values of 101 and 126 obtained from extracts of untreated cells kept in culture over the same period (Figure 3.2c). A 4-fold reduction in cdkl activity was also observed in HOC-7 cells treated with P4 (data not shown). The fact that this ovarian carcinoma cell line does not express SV40 large T antigen and harbors no known p53 mutation suggests that the effect of P4 on cdki activity was not a consequence of SV40 large T antigen expression in our cystadenoma cell lines. The effect of P4 on CDK2 activity, a cyclin-dependent kinase that interacts with cyclin A and cyclin E and regulates entry and progression through the S phase of the cell cycle, was comparatively much smaller (Figure 3.2c). We conclude that progesterone inhibits cell cycle progression in benign and malignant ovarian epithelial tumors by down-regulating the activity of the cyclin B1/CDK1 complex. Given that this complex acts at the G2/M phase of the cell cycle, we analyzed HOC-7, OVCAR-3, and MCV50 cells by flow cytometry to determine whether treatment with P4 resulted in an arrest at this phase. Similar experiments could not be performed with MLS cells because of their complex DNA profile, which precluded accurate determination of the proportion of cells at the various phases of the cell cycle. The results showed that progesterone treatment resulted in an increase in the proportion of cells in the G2 phase, with a concomitant decrease in the number of cells in the S phase. The changes in the G2/S ratios are shown in figure 4d for those 3 cell lines. The increase in this ratio ranged from about 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3H-thymidine Incorporation (°/bof 50% in OVCAR-3 cells to over 200% in MCV50 cells. These results support the conclusion that the growth inhibitory effect of P4 is due, at least in part, to an arrest at the G2 phase of the cell cycle. P= .004 -D 1 0 0 - M , ------r- 1 .02 .0009<.0001 •40 • 2 0 - 0.1 Estradiol ftiM) 0.1 1 10 Progesterone <HM ) P =.007 .0005.0006 .01 .07 .04 0.04 0.4 1 10 100 FSH (tU/mt) LH (IU/L) 0ML1O 8 MCV50 HHOC-7 BOVCAR-3 1 C f ■ O : 0.1 1 10 Progesterone Concentration (pM) 4 6 Days 10 Figure 3.1 Effects of reproductive hormones on thymidine incorporation. ML5 ovarian cystadenoma cells (a,b) or MLS and ML10 ovarian cystadenoma as well as MCV50, HOC-7, and OVCAR-3 ovarian carcinoma cells (c) were adapted to low serum concentrations and treated for 24 hours with either vehicle only or with the indicated amounts of hormone. Each value is an average from 3 to 6 experiments done in parallel, (d) shows a growth curve analysis of ML5 cells cultured in 35 mm dishes and either treated (triangles) or untreated (circles) with 10 pM P4 at the indicated time points using a Coulter counter (Coulter Electronics, Hialeah, FL). Each point represents the average of 3 dishes. The error bars indicate one standard error. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nene i*or# 18 § 24 24 Cyclin 8 1 non® non® 10jiM 8 49 m CyelU i 1 *■* * m « * ■ » - < K » \# V $ I W W W cyclin 11 p21 m non# IQjAt 9 24 48 24 48 12 .*« < £&)&»&✓ -Af^V V .-V T O S O It 91 57 Pr&issterofte Tim® (hours) ■ d *P4tt fetfki * Signal intensity *p-H| # * fcdR2) Signal intsmfty #=H1 ® 1 6 1 H O C -7 0VC»3 M C V 5 0 Figure 3.2 Effects of P4 on proteins involved in cell cycle regulation. ML5 cells were plated onto 100-mm plastic dishes in MEM supplemented with 10% fetal bovine serum. The serum concentration was gradually lowered to 0.4%. (a) Cells were treated with either vehicle only (control) or with 10 pM P4 for 24 hours or 48 hours. Cell extracts were electrophoresed on 10% polyacrylamide and analyzed by western blotting using antibodies for cyclin B1 and p-actin. (b) A total amount of 100 pg of cellular protein extracts was immunoprecipitated with anti-CDK1 or -CDK2 antibody. Each immunoprecipitate was assayed for kinase activity using histone H1 protein as substrate. The reaction products were electrophoresed on polyacrylamide gels and quantitated by radioactive phospho- imaging. The figure shows the phospho-imager signals corresponding to either CDK1 or CDK2 activity for each experimental condition as well as a numerical value of the signal intensities. The bottom portion shows the histone H1 substrate visualized through Coomassie blue staining of the polyacrylamide gel. (c) Western blot analysis of cyclin B1, p21, and p27 protein expression in MLS cells treated with the indicated concentrations of P4 over 48 hours, (d): Cells treated for 48 hours with 30 pM progesterone or with vehicle only were trypsinized, centrifuged at 4000g for 5 minutes, resuspended in 0.2 ml PBS, and fixed rapidly in 2 ml 70% ice-cold ethanol. The cells were washed and resuspended 1 ml of PBS. After addition of 10 pg/ml propidium iodide, 100 pg/ml RNAse A, the cell preparations were incubated at 37°C for 30 min and analyzed by flow cytometry. The ordinate indicates the percent increases in the ratio of cells in the G2 versus S phases following P4 treatment. The error bars indicate standard errors. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Effect of P4 on MAP kinase signal transduction pathway Mitogen-activated protein kinase (MAP kinase) is a key signal transducing protein which transmits signals involved in cell proliferation. To further study the signal transduction pathway responsible for the inhibitory effect of progesterone on cell growth, the effect of progesterone on MAP kinase phosphorylation was examined. Treatment with 10 pM P4 for 5, 15, and 30 min did not significantly alter MAP kinase phosphorylation, however, MAP kinase phosphorylation was markedly inhibited after P4 treatment for 24 or 48 hours (Figure 3.3), suggesting MAP kinase may be involved in the action of progesterone on growth inhibition. Role of AP-1 in mediating the effect of P4 Progesterone receptors mediate hormone signaling by binding to their response elements in the promoter regions of responsive genes, resulting in alterations in gene transcription. Another potential mechanism is via protein- protein interactions between the progesterone receptor and AP-1, which lead to hormone-dependent regulation of gene transcription through AP-1 response elements. It is well established that AP-1 can induce expression of genes involved in cell proliferation upon stimulation by extracellular signals (Pfahl 1993). We tested the hypothesis that the effect of P4 on cell 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. proliferation in ovarian epithelial tumors could be mediated by AP-1. ML5 cystadenoma cells were transfected with a plasmid construct containing an AP-1 response element upstream to a luciferase reporter gene. Treatment of the transfected cells with P4 had a minor effect on AP-1 promoter activity, as evidenced by a small increase in luciferase activity (Figure 3.4). In contrast, RU486, which has a similar effect as P4 on cell proliferation (shown in Chapter 4), inhibited AP-1 promoter activity (Figure 5; p = 0.02). The inhibitory effect of RU486 could be counteracted by the addition of equimolar amounts of P4 (Figure 3.4; p = 0.03 for differences between RU486 and RU486 plus P4). We conclude that the effect of P4 on cell proliferation in ovarian epithelial tumors probably does not involve interactions with the AP-1 pathway. a b P4 (jiM) 0 0 0 0 10 10 10 0 0 10 10 P4 QiM) Ti me (mirt) 0 5 15 30 5 15 30 24 48 24 48 Ti me (hr) p-ERK -m m f. m m m . Bf ». i p-ERK total ERK total ERK Figure 3.3 Effect of P4 on MAP kinase phosphorylation. MLS cell lysates were collected after (a) acute and (b) chronic exposure to progesterone. Western blot analysis was used to examine MAP kinase phosphorylation in these cells using specific antibodies recognizing phosphorylated ERK and total ERK. 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 - i 5 - Control Progesterone RU486 (10 pM) Progesterone (10 pM) (10 pM) + RU486 (10 pM f) Figure 3.4 Effect of P4 and RU486 on AP-1-mediated gene expression. MLS cells transfected with a vector containing an AP-1-responsive promoter upstream of a luciferase reporter gene were treated with P4 or RU486, or combination of both. Control cells received the same volume of vehicle only. Cell lysates were prepared after 24 hours and assayed for luciferase activity. The error bars indicate standard errors. Lysates from mock-transfected cells consistently generated light units below 150. These background values were not subtracted. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The results of our experiments clearly show that each of the 4 major hormones involved in menstrual cycle regulation can independently influence the growth of benign and malignant ovarian epithelial tumors, at least in a tissue culture environment. Treatment of such cells with P4 or progesterone receptor agonists/antagonists was growth inhibitory whereas treatment with E2, FSH, or LH was growth stimulatory. The cyclin B1/CDK1 complex is essential for progression of cells through the G2/M phase of the cell cycle (Grana and Reddy 1995). The level of cyclin B1 protein was significantly reduced in P4-treated ovarian epithelial tumor cells suggesting that the growth inhibitory effect of this hormone is due, at least in part, to an arrest at this phase of the cell cycle. This conclusion is further supported by the fact that the ratio of the percentage of cells in the G2 phase over that in the S phase was increased after treatment with P4. The observed concomitant decrease in kinase activity can readily be explained by the fact that cyclin B1 is essential for the enzymatic activity of the cyclin B1/CDK1 complex. Another effect of P4 in our cell culture system was to up regulate p21, a universal inhibitor of CDK activity. This inhibitor is known to 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. have two independent effects on CDK activity. One is at the posttranslational level where it binds to and inhibits the activity of cyclin-dependent kinases (Harper and Elledge 1996). The other acts at the transcriptional level (Innocente and othres 1999, Taylor and othres 1999). Although we have not formally investigated transcriptional activity of cyclin B1, it is reasonable to assume that regulation of cyclin B transcription by p21 in these cells is similar to what has been demonstrated in several other cell types (Innocente and othres 1999, Taylor and othres 1999). In addition, p21 appears to exert some posttranslational effects on other CDK complexes as well because we also saw reduced activity of another CDK, namely CDK2 (which associates with cyclin A and cyclin E). However, the effects on CDK2 activity were smaller. We conclude that the greatly reduced activity of cyclin B1/CDK1 complex is due to a combined effect of transcriptional and posttranslational regulation by p21 as well as, to a lesser extent, by p27. This cyclin-dependent kinase inhibitor was also up regulated by P4 in ML5 cystadenoma cells, although to a lesser extent than p21. MAP kinase plays a key role in transmitting extracellular signals into the cell and influencing cell proliferation. The finding that P4 treatment resulted in a marked decrease in MAP kinase phosphorylation suggests that the inhibitory effect of progesterone on cell proliferation may be mediated by the MAP kinase pathway. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It is unlikely that the effects of P4 on cell cycle down-regulation are mediated by AP-1 because the effects of P4 and RU486 on an AP-1 responsive promoter did not parallel those on the cell cycle. Our cell culture model did not include cell strains derived from the normal cells from which ovarian epithelial tumors originate. In fact, the issue of the tissue of origin of these tumors is still controversial (Dubeau 1999, Rodriguez and Dubeau 2001). However, it is fair to assume that our in vitro observations on ovarian epithelial tumors provide some indication of how the normal cells from which ovarian epithelial tumors originate respond to the fluctuating levels of the 4 reproductive hormones during the normal menstrual cycle. The results suggest that each follicular phase of the menstrual cycle, characterized by unopposed E2 and elevated FSH levels, is under the influence of a combination of hormones favoring growth stimulation. Such stimulation may be accentuated at the end of the follicular phase due to the rapid surge in LH concentration that triggers ovulation. Each luteal phase, in contrast, should be characterized by growth inhibition due to increased levels of P4. This scenario of growth stimulation followed by growth inhibition might contribute to the increased risk of tumor development in women with uninterrupted menstrual cycles. The protective role of either pregnancy or oral contraceptives could, in turn, be partly due to interruption 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of such scenario. Other contributing factors could be the decrease in intra- ovarian E2 concentration that results from oral contraceptive intake and the high levels of P4 that are present early during pregnancy. The importance of these mechanisms relative to the 2 prevailing theories for the association between ovulatory activity and ovarian cancer risk, namely the incessant ovulation theory (Fathalla 1971) and the excessive gonadotropin stimulation hypothesis (Stadel 1975), remains to be investigated. Our results suggest that P4 could provide an effective means of controlling the growth of at least some ovarian epithelial tumors in affected patients. The attractiveness of this idea is diminished by the fact that P4 is poorly tolerated due to undesirable side effects at the doses shown to be growth inhibitory in our studies. Equally undesirable side effects, however, are associated with most cancer chemotherapeutic agents. In addition, the finding that RU486 (shown in Chapter 4), a drug that should counteract several of the systemic effects of progesterone, is a progesterone receptor agonist with regard to its effect on cell cycle activity, suggests that specific P4 agonists/antagonists exist that retain the desirable effects of this hormone on tumor growth while lacking some of its undesirable side effects. Such drugs could prove as effective as P4 in the treatment of ovarian cancer while being better tolerated by the patients. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4* Role of the Progesterone Receptor in Progesterone- mediated Cell Proliferation and Angiogenic Activity in Ovarian Epithelial Tumors ‘ Parts of data in this chapter were published in Zhou H et al. Effect of reproductive hormones on ovarian epithelial tumors: I. Effect on cell cycle activity. Cancer Biology and Therapy 1 (3): 298-304, 2002 and Chen C et al. Effect of reproductive hormones on ovarian epithelial tumors: II. Effect on angiogenic activity. Cancer Biology and Therapy 1(3): 305-310, 2002. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Progesterone inhibits ovarian epithelia! tumor cell proliferation and, at the same time induces angiogenesis in some ovarian epithelial tumors by stimulating VEGF synthesis and secretion by the tumor cells. The mechanisms responsible for mediating these effects are investigated in this chapter. Since the concentration of progesterone needed to obtain both effects is much higher (10 pM) than the Kd value of the progesterone receptor, which is at nanomolar levels, we further investigated whether both of the effects of progesterone were mediated by this receptor. Both progesterone receptor agonists (R5020 and ORG2058) and antagonists (RU486 and ZK98299) act as agonists on the effect on the ovarian epithelial tumor cell growth. Although type II progesterone receptor antagonist RU486 had the same stimulatory effect as progesterone on VEGF secretion, ZK98299, a type I progesterone receptor antagonist, inhibited this effect of progesterone. Dexamethasone, a ligand for the glucocorticoid receptor and aldosterone, a ligand for the mineralocorticoid receptor, did not inhibit ovarian epithelial tumor cell growth or induce VEGF secretion, excluding the possibility that progesterone acted through this receptor. Antisense oligonucleotides complementary to the translation initiation site of the PR-A form of the progesterone receptor, which inhibited both the PR-A and PR-B 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. forms of the receptor, blocked the effect of progesterone on VEGF secretion but did not reverse the effect of progesterone on cell proliferation. Sense oligonucleotides for the same region of progesterone receptor mRNA had no effect on VEGF secretion or cell growth. Western blot analysis confirmed that there were decreased levels of PR-A and PR-B in cells treated with the antisense oligonucleotides, but not with the sense oligonucleotides. These results strongly suggest that the effect of progesterone on VEGF secretion in ovarian epithelial tumors is mediated via the progesterone receptor, while the effect of progesterone on cell proliferation may not be mediated via the progesterone receptor. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Progesterone is first isolated from the corpus luteum in 1934 (Allen and Wintersteiner 1934, Butenandt and others 1934, Hartman and Wettstein 1934, Slotta and others 1934). Since then extensive research has been done on progesterone and our knowledge about progesterone has been enriched rapidly. This steroid hormone plays a central role in the regulation of all aspects of female reproductive activity associated with establishment and maintenance of pregnancy. It acts at the levels of the hypothalamus, pituitary, ovary, and uterus to regulate cyclic gonadotropin production, ovulation, and uterine preparation for implantation (Conneely 2001). It has been implicated in the development of breast cancer (Horwitz and Clarke 1998). Epidemiological studies suggest that it may also have a protective effect in the development of ovarian cancer (Whittemore and others 1992, Pike 1987). Most of the effects of progesterone are thought to be mediated through interaction with specific intracellular receptors, progesterone receptors, which are members of the nuclear receptor superfamily of transcription factors (Milgrom and Baulieu 1970, Sherman and others 1970, Tsai and O’Malley 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1994). After progesterone enters cells, it binds to a progesterone receptor that induces conformational changes in the receptor structure, phosphorylation, and dimerization of the receptor. This is followed by binding to a specific DNA response element in the promoter region of a specific target gene and recruiting co-regulator proteins that interact with general transcriptional machinery to elaborate progesterone-triggered changes in promoter activity. In general, progesterone receptor agonists promote binding of co-activator proteins that promote transcription activity while binding of antagonists promotes interaction with co-repressor proteins that repress transcription (McKenna and others 1999, Giangrande and McDonnell 1999). The progesterone receptor is expressed as two distinct isoforms, PR-A and PR-B, which arise from a single gene (Conneely and others 1989, Kastner and others 1990). These two proteins are generated by alternate initiation sites of translation from two AUG codons located on a single mRNA transcript. The two isoforms of the human progesterone receptor are hPR-A, 94 kD, and hPR-B, 114 kD. They are originated from translational initiation at AUG2 (codon 165) and AUG1, respectively (Kastner and others 1990). PR-A and PR-B differ only in that the PR-B protein contains an additional fragment of amino acids at its N-terminus that is not contained in PR-A. This PR-B- specific region encodes a third transactivation function (AF3) that is absent 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from PR-A (Sartorius and others 1994, Wen and others 1994). It was recently shown that PR-B could bind to a subset of co-activators that are not available for PR-A (Giangrande and others 2000). When expressed individually in cultured cells, PR-A and PR-B display different transactivation characteristics that are specific both to cell type and target gene promoter context (Tora and others 1998, Meyer and others 1992, Vegeto and others 1993, Hovland and others 1988). Agonist-bound PR-B acts as a strong activator of transcription activity of several PR dependent promoters in a number of cell types in which PR-A is inactive. When both isoforms are co-expressed in cultured cells, the PR-A can repress the activity of PR-B. This repressor ability of PR-A also applies to other steroid receptors such as ERoc (Wen and others 1994, McDonnell and others 1994). The PR-A and PR-B isoforms also respond differently to progesterone receptor antagonists (Giangrande and McDonnell 1999). While antagonist bound PR-A is inactive, antagonist bound PR-B can be converted to a strongly active transcription factor by regulating intracellular phosphorylation pathways (Musgrove and others 1993, Beck and others 1993, Sartorius and others 1994). The ratios of the two progesterone receptor isoforms vary in reproductive tissues that may be related to development (Shyamala and others 1990), hormonal status (Duffy and others 1997), and carcinogenesis (Brandon and 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. others 1993, Graham and others 1997). Low hPR-B levels (high hPR-A: hPR-B ratio) have been detected in primary breast tumors and endometrial cancers. Numerous studies have showed that the majority of the physiological actions of progesterone are mediated through the progesterone receptor. The progesterone receptor is widely expressed in the different tissues of the body. Graham and Clarke reported that the target tissues of physiological action of progesterone include the uterus (mammalian endometrium and myometrium), the ovary (luteinizing granulose cells and corpus luteum and pre-ovulatory granulose cells), and other reproductive and non-reproductive tissues. Abolishing the progesterone receptor causes significant functional impairment in these tissues. Lydon et al showed that adult female mice with a null mutation of the progesterone receptor gene encoding both isoforms (PRKO) had remarkable abnormalities in all reproductive tissues. These defects included anovulation, uterine hyperplasia and inflammation, extremely limited mammary gland development and an inability to show sexual behavior. While most effects of progesterone are known mediated via the progesterone receptor, recent study showed other pathways exist. Grazzini et al recently 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reported that progesterone plays an essential role in establishing and maintaining pregnancy in mammals by directly binding to the oxytocin receptor and inhibiting its function, which is important in the maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peoptide hormone oxytocin. Although the binding affinity between progesterone and the oxytocin receptor is relatively low, it is compensated by the high concentration of progesterone during pregnancy. The oxytocin receptor is a G-protein-coupled receptor. These findings provided strong evidence for a direct interaction between a steroid hormone and a G-protein- coupled receptor and cross talk between the peptide- and steroid-hormone signaling pathways (Bouchard 1999). Our studies demonstrated that progesterone inhibits ovarian epithelial tumor cell proliferation but induces angiogenesis in some ovarian epithelial tumors by stimulating VEGF synthesis and secretion by the tumor cells. Since the concentration of progesterone needed to obtain this effect was much higher (10 pM) than the Kd value of the progesterone receptor, which is at nanomolar levels, we further investigated whether both of the effects of progesterone were mediated by this receptor or by other mechanisms. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Source of reagents Progesterone was purchased from Sigma Chemical Company (Saint Louis, MO). They were all dissolved in absolute ethanol at stock concentration of 10 mM. ORG2058 was obtained from Amersham Life Science (Amersham, UK). R5020 (promegestone) was obtained from NEN Life Science (Boston, MA). RU486 (mifepristone) was purchased from Sigma Chemical Company. ZK98299 (onapristone) was obtained from Siniwest (San Diego, CA). They were all dissolved in absolute ethanol at stock concentration of 10 mM. Quantikine human VEGF ELISA kit (Cat # DVE00) was purchased from R&D systems (Minneapolis, MN). Progesterone receptor antibody was purchased from Santa Cruz Biotechnololgy (Santa Cruz, CA). Cell lines and tissue culture ML5 cells were derived from a human ovarian cystadenoma and transfected with SV40 large T antigen to increase their longevity in vitro (Luo and others 1997). MCV 50 cells were derived from a subclone of ML10 human ovarian cystadenoma cells that became spontaneously immortalized in culture. HOC- 7 ovarian carcinoma cells were obtained from Dr. R. Buick, University of 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Toronto. SKOV-3 cells were purchased from American Type Culture Collection (ATCC). MLS and MCV50 were cultured in MEM while HOC-7 cells were cultured in RPMI 1640 (Life Technologies, Grand Island, NY). All tissue culture media were supplemented with 10% fetal bovine serum if not indicated. Cell cultures and measurement of VEGF by ELISA Cells were cultured in 6-well plates in medium with 10% fetal bovine serum until 50-60% confluence, then supplemented with 2% charcoal-stripped serum for 2 days, and switch to 0.4% serum the day before hormonal treatment. The cells were treated with hormones for 36 hours. The culture medium was changed 24 hours after initiation of the hormone treatment and conditioned medium was collected over the remaining 12 hours. The amount of VEGF in the conditioned medium was determined by ELISA. The VEGF concentration was normalized to the cell number. 3H-thymidine incorporation measurements Cells cultured in 24-well tissue culture plates (Corning Costar Corp., Cambridge, MA) were allowed to grow until 60-70% confluence, at which time the serum concentration was switched to 2% charcoal filtered fetal 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bovine serum. After 2 days, the serum concentration was further lowered to 0.4%. One pCi/ml [3H]thymidine (ICN Pharmaceuticals, Irvine, CA) was added to each well and the cells were treated either with the desired hormonal agent or with vehicle only. The tissue culture dishes were washed twice with ice-cold phosphate-buffered saline (PBS) after completion of the appropriate treatment period, followed by treatment with ice-cold 5% trichloroacetic acid and solubilization of the resulting precipitate in 0.2 ml of 1 N NaOH. The amount of radioactivity was measured by liquid scintillation counting after neutralization with 0.2 ml of 1 M acetic acid. Immunoprecipitation and western blot analyses of progesterone receptor Cells were cultured in 10 mm dishes and treated with 10 pM sense or antisense oligonucleotides against the progesterone receptor for 72 hours. The medium was changed and fresh oligonucleotides were added every 24 hours. Whole cell lysates were collected and immunoprecipitated with an antibody against progesterone receptor (purchased from Santa Cruz Biotechnology). The immunoprecipitates were analyzed by Western blotting. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Treatment with antisense oligonucleotides specific for progesterone receptor MCV50 cells were treated for 24 hours with either antisense or sense oligonucleotide sequences to a portion of the progesterone mRNA sequence that includes the initiation site of the PR-A form of this receptor. When indicated, the cells were then treated with a mixture of progesterone and oligonucleotides for an additional 24 or 36-hour period for different experiments. The oligonucleotide sequences antisense PR-A and sense PR- A were as described by Mani et al Antisense PR-A: 5’- GCTCATGAGCGGGGACAACA-35 ; Sense PR-A: 5’- TGTTGTCCCCGCTCATGAGC-3’. The oligonucleotide sequences of antisense PR-B and sense PR-B are: 5’-TCAGTCATGACGACTGGACT-3’ (antisense), 5’-AGTCCAGTCGTCATGACTGA-3’ (sense). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Effects of progesterone on DNA synthesis and VEGF secretion in cultured ovarian epithelial tumor cells We measured the effect of progesterone on the rate of thymidine incorporation and VEGF secretion in different cultured ovarian epithelial tumors cells. Treatment with progesterone at the concentration of 10 pM showed inhibitory effect on cell growth in all ovarian tumor cell lines tested (see Chapter 3 and Figure 4.1a). However, the effect of progesterone on VEGF secretion varied widely in different cell lines. Progesterone significantly increased VEGF secretion in MCV50, Hey, and A2780 ovarian epithelial tumor cells, decreased VEGF secretion in GT123 cells, did not show significant effect on CAOV-3, HOC-1, HOC-7 or OVCAR-3 cells (Figure 4.1b). Progesterone receptor expression in ovarian epithelial tumor cell lines To investigate whether the effects of progesterone on cell proliferation and VEGF secretion are mediated through the progesterone receptor, we first examined the expression of the progesterone receptor in ovarian epithelial 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tumor cells. Almost all cell lines examined expressed the PR-A isoform of the progesterone receptor. An ovarian carcinoma cell line, SKOV-3, expressed very little (figure 4.2). Much smaller amounts of the PR-B isoform were also present in most cell lines. No PR-B expression was detected in SKOV-3 cells (figure 4.2). Effects of progesterone receptor agonists and antagonists on DNA synthesis and angiogenic activity in ovarian epithelial tumor cells We next examined the effect of various progesterone receptor agonists and antagonists on thymidine incorporation and VEGF secretion in cultured ovarian epithelial tumor cells to further investigate the role of the progesterone receptor in mediating the growth inhibitory and angiogenic effects of progesterone. Inhibition of thymidine incorporation was observed after treatment with either R5020 or ORG2058, both progesterone receptor agonists (figure 4.3a) at the dose similar to that of progesterone. RU486 and ZK98299, which usually act as progesterone receptor antagonists, also showed inhibitory effects on thymidine incorporation and thus acted like progesterone receptor agonists with regard to their effects on DNA synthesis (figure 4.3a). All of these 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. compounds produced statistically highly significant inhibition at 10 |iM concentrations (p < 0.0001). These effects augmented those of P4 (figure 3b, p < 0.0001 for both RU486 and ZK98299). The responses of MCV50, HOC-7 and OVCAR-3 ovarian carcinoma cells to receptor agonists and antagonists were similar in nature and magnitude to those of MLS cystadenoma cells. Progesterone receptor agonist R5020 and type II progesterone receptor antagonist RU486 both increased VEGF secretion at the similar concentration as progesterone in MCV50 cells (figure 4.4a). However, progesterone receptor type I antagonist ZK98299 reversed the effect of progesterone on VEGF secretion (figure 4.4b). These data suggest that progesterone induces angiogenic activity via the progesterone receptor. Effect of other steroid receptor ligands on cell growth and angiogenic activity in ovarian epithelial tumor cells Progesterone has known affinity to glucocorticoid and mineralocorticoid receptors. To investigate the possibility of progesterone acting through these nuclear receptors, we then used ligands for these receptors to treat ovarian epithelial cells. Treatment with 1 to 10 jj,M Dexamethasone, a potent synthetic glucocorticoid receptor agonist, did not inhibit MLS cell growth 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Figure 4.5a). Treatment with 10 pM dexamethasone did not induce VEGF secretion in MCV50 cells, on the contrary, VEGF secretion was decreased by dexamethasone treatment (Figure 4.5b). Treatment with aldosterone, the natural ligand for mineralocorticoid receptor, did not show any significant effect on DNA synthesis or VEGF secretion in ovarian epithelial tumor cells (data not shown). These data suggest that the effects of progesterone on DNA synthesis and VEGF secretion in some ovarian epithelial tumor cells may not be due to interactions of progesterone hormone with other nuclear hormone receptors such as the glucocorticoid or mineralocorticoid receptors. Effect of antisense oligonucleotides against progesterone receptor on DNA synthesis and VEGF secretion To further study the role of the progesterone receptor in the actions of progesterone on DNA synthesis and VEGF secretion, we treated ovarian epithelial tumor cells with antisense oligonucleotides specific for the progesterone receptor and examined the consequences on progesterone responsiveness. T reatment with 10 jiM antisense oligonucleotides complementary to the translation initiation site of PR-A (Antisene PR-A) remarkably reduced the expression of both PR-A and PR-B isoforms of the progesterone receptor in MCV50 cells. Antisense oligonucleotides against to the translation initiation site of PR-B (Antisense PR-B) also significantly 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduced the expression of both PR-A and PR-B, with PR-B showing the greatest reduction (figure 4.6). We examined the effect of antisense oligonucleotides against the progesterone receptor on thymidine incorporation and VEGF secretion in cultured ovarian epithelial tumor cells to further investigate whether the progesterone receptor mediates the effects of progesterone on cell growth and angiogenic activity. Treatment with 10 pM antisense PR-A did not reverse progesterone-mediated inhibitory effects on DNA synthesis (Figure 4.7). Treatment with 5 and 10 pM antisense PR-A inhibited progesterone- induced VEGF secretion in MCV50 cells in a dose-dependent manner, however, 10 pM antisense PR-B did not reverse the effect of progesterone on VEGF secretion (figure 4.8). We conclude that progesterone-induced VEGF secretion is mediated via the PR-A isoform of the progesterone receptor, while the growth inhibitory effect of progesterone seems not to involve the progesterone receptor. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a 100000 S E Z * C L o c 80000 o is u O 60000 e- o u c 40000 a > E S E 20000 >. £ I 0 Control 0.1 1 Progesterone Concentration (jjM) 10 Figure 4.1a. Effect of progesterone on DNA synthesis in SKOV-3 ovarian carcinoma cells. SKOV-3 cells were treated with progesterone at the indicated concentrations or with vehicle (ethanol) only (control). Thymidine incorporation was measured after hormonal treatment for 24 hours. Each value is an average of six separate measurements. Error bars indicate standard errors. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. b 100 - □ Control ■ 10 ( jM P4 Figure 4.1b. Effect of progesterone on VEGF secretion in ovarian epithelial tumors. Cells were plated onto 6-well plastic tissue culture plates in appropriate medium. Serum concentration was gradually lowered to 0.4%. The cells in triplicates were treated for 36 hours with 10 pM progesterone or with vehicle (ethanol) only (controls). Conditioned medium was collected from each well over the last 12 hours of this 36-hour period and assayed for the presence of VEGF by ELISA. The units on the ordinate correspond to absorbance measurements from the ELISA plate reader and are proportional to VEGF concentration. The cell number was determined in each well using a Coulter Counter and the results were normalized for cell number. The ordinate shows pg of secreted VEGF per 100,000 cells. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HOC-7 OVCAR-3 MLS ML10 SKOV-3 T47D Figure 4.2. Immunoprecipitation and Western blot analysis of progesterone receptor expression in cultures of benign and malignant ovarian tumor cells. Total cell lysates were prepared and immunoprecipitated with an antibody specific for both A and B isoforms of the progesterone receptor. The antigen-antibody complex were collected with protein G- sepharose and electrophoresed on a polyacrylamide gel. T47D breast carcinoma cells, which are known to express both isoforms, were used as positive control. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a ■ 5 L. o m !— o 120 100 - 0E £ o t >. r . ■ * 1 * X C O ■ Progesterone QR5020 H OR02058 QRU486 HZKB8299 0.1 1 Concentration (|jM) o < 10 J = 2 0 iProgesterone (10 pk-l) a Agonist/antagonist (10 mM ) a Progesterone (10 jjtvf) + Agonist/antagonist (10 pM ) ZK98299 RU486 Figure 4.3. Effects of progesterone receptor agonists/antagonists on thymidine incorporation. Thymidine incorporation was measured in ML5 cystadenoma cells in the experiment described as above. The results are expressed as percent change in radioactive thymidine incorporation to compare cells treated with vehicle only. Each value is an average of six separate measurements. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a w u o o o o ' o C S > Q_ o L U > 10 0 - Control Progesterone (10 pM) R5020 (1 0 ijlM ) RU486 (10iiM) Figure 4.4a. Effect of R5020 and RU486 on VEGF secretion. MCV50 cells in triplicates were treated for 36 hours with 10 jxM R5020 or RU486 or with vehicle (ethanol) only (controls). Conditioned medium was collected from each well over the last 12 hours of this 36-hour period and assayed for the presence of VEGF by ELISA. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. b C D o o O o_ o ' o Q . 0 L U 30 25 2 0 1 5 1 0 - 5 - Control Progesterone ZK98299 Progesterone (10 p.M ) (10 nM) £ 10 (iM) + ZK98299 (10 fiM) Figure 4.4b. Effect of ZK98299 on progesterone-induced VEGF secretion. MCV50 cells in triplicates were treated for 36 hours with 10 pM progesterone or ZK98299 or both, or with vehicle (ethanol) only (controls). Conditioned medium was collected from each well over the last 12 hours of this 36-hour period and assayed for the presence of VEGF by ELISA. 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a o 80000 I - 60000 20000 Dexam ethasone Concentration (|iM) b Control Dexamethasone (10 jxM) Figure 4.5 Effect of dexamethasone on DNA synthesis and VEGF secretion, a. ML5 cells were untreated or treated with dexamethasone at the indicated concentrations for 24 hours thymidine incorporation was measured as described above. Each value is an average of six separate measurements, b. MCV50 cells in triplicates were treated for 36 hours with 10 pM dexamethasone or with vehicle (ethanol) only (controls). Conditioned medium was collected from each well over the last 12 hours of this 36-hour period and assayed for the presence of VEGF by ELISA. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Anti sense Sense Anti sen e Sense PR-A PR-A PR-B PR-B e-Heavy Chain Figure 4.6. Down-regulation of progesterone receptors expression by antisense oligonucleotides. MCV50 cells were treated with 10 pM sense or antisense oligonucleotides against the progesterone receptor for 72 hours. The medium was changed and fresh oligonucleotides were added every 24 hours. Whole cell lysates were collected and immunoprecipitated with an antibody against the progesterone receptor and then analyzed by Western blotting. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80000 i C l (J o a o < _ > c ® c 33 E >. x : 700 00 - 60000 50000 - 40000 - 30000 200 00 - 10000 - i iJ ■ Control P4 Antisense P4 (10 m -M ) + Sense PR-A P4(10|iM) + (10|iM) PR-A (10 pM) AntisensePR-A (10|iM) Sense PR-A ( % M ) (10 mM) Figure 4.7 Effect of antisense oligonucleotides against progesterone receptor mRNA on growth inhibition by progesterone. ML5 cells were first treated for 24 hours with either antisense or sense oligonucleotides against the progesterone receptor mRNA. When indicated, the cells were then treated with a mixture of progesterone and oligonucleotides for an additional 24 hours. Thymidine incorporation was measured as described above. Each value is an average of four separate measurements. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4.8 Effect of antisense olionucleotides against progesterone receptor mRNA on progesterone-induced VEGF secretion. MCV50 cells were treated for 24 hours with either antisense or sense oligonucleotides against the progesterone receptor mRNA. When indicated, the cells were then treated with a mixture of progesterone and oligonucleotides for an additional 36-hour period. Conditioned medium was collected over the last 12-hour period and assayed for VEGF amount by ELISA. 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Our results suggest that progesterone-induced VEGF secretion in ovarian epithelial tumor cells is mediated via the progesterone receptor. This conclusion is based on the fact that antisense oligonucleotides specific for progesterone receptor inhibited the effect of progesterone on VEGF secretion and that the progesterone receptor antagonist ZK98299 reversed the effect of progesterone on VEGF secretion. However, the growth inhibitory effect of progesterone in cultured ovarian epithelial tumors seems not to be mediated via interactions with the progesterone receptor. The treatment of antisense oligonucleotides for progesterone receptor did not show any effect on the inhibitory effect of progesterone on ovarian epithelial tumor cells. The response patterns to RU486 and ZK98299, which acted like agonists, were not typical. The possibility that the growth inhibitory effect was due to interactions with the glucocorticoid or mineralocorticoid receptors, which have known affinity for progesterone, is unlikely because treatment with natural ligands for these receptors, dexamethasone and aldosterone, did not result in growth inhibition. The possibility of interactions with other receptors such as the recently found PXR cannot be totally excluded. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Recent studies by Conneely et al demonstrated different functions of the PR- A and PR-B proteins in the physiological actions of progesterone using knock-out mice in which expression of the PR-A (PRAKO) or PR-B (PRBKO) isoform was selectively abolished. In PRAKO mice, the PR-B isoform functions in a tissue-specific manner to mediate a subset of the reproductive functions of progesterone receptors. Removal of PR-A isoform does not alter responses of the mammary gland or thymus to progesterone but causes significant defects in ovarian and uterine function. Surprisingly, the absence of PR-A in PRAKO uteri revealed an unexpected progesterone-dependent proliferative activity of PR-B in the epithelium and showed that the PR-A isoform is essential to reduce both progesterone (acting via PR-B) and estrogen-mediated proliferative responses in this tissue. The finding that the PR-A isoform is essential to inhibit estrogen-induced proliferation in the uterus is consistent with previous observations that agonist-bound PR-A is able to inhibit estrogen-dependent transcriptional activation in cell-based transactivation assays (McDonnell and others 1994). In addition, this inhibitory activity of PR-A was tissue specific and did not apply to the mammary gland where both PR-A and PR-B act as proliferative mediators of progesterone. In contrast to the reproductive defects observed in PRAKO mice, more recent studies using PRBKO mice have shown that ablation of PR-B isoform does not affect either ovarian, uterine, or thymic responses to 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. progesterone but leads to in reduced mammary ductal morphogenesis (Conneely and others 2002). Thus, PR-A is both necessary and sufficient for these progesterone-dependent reproductive responses while the PR-B isoform is required for normal proliferative responses of the mammary gland to progesterone. In the ovarian epithelial tumor cells used in our studies, both isoforms of the progesterone receptor are expressed in most cell lines. However, PR-A is the predominant form in all these cell lines, suggesting that PR-A plays a key role in these ovarian epithelial tumor cells. Progesterone induced-VEGF secretion was inhibited by antisense nucleotides specific for PRA but not by antisense nucleotides for PRB, indicating that the effect of progesterone on VEGF secretion in ovarian epithelial tumor cells is mediated by PR-A specifically. We showed that both progesterone receptor agonists and antagonists act like progesterone. The underlying mechanism remains to be studied. Similar effects were observed in the endometrium. Progesterone has antiproliferative effects in the endometrium. The same antiproliferative effects were also seen in rabbits, monkeys, and guinea pig under progesterone receptor antagonists, including type I progesterone receptor antagonist ZK98299 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Chwalisz and others 1991 and 1998, Slayden and others 1998, Slayden and Brenner 1994, Eiger and others 2000). The finding that the growth inhibitory effect of progesterone was dose- dependent at hormonal concentrations well above the reported Kd value for the progesterone receptor may reflect the fact that the ovary is a producer of progesterone. Cells within and even surrounding this organ are undoubtedly exposed to concentrations of this hormone that are orders of magnitude higher than circulating levels. Indeed, measurements of progesterone levels in the ovarian vein of fertile women showed concentrations similar to the maximal concentrations used in our studies (Lucisano and others 1978). The mechanism for this apparent resistance to progesterone is not clear. Our results suggest that progesterone acts on ovarian epithelial tumor cells through different pathways in terms of its effects on cell growth and angiogenic activity. The underlying mechanism remains to be studied. The finding that growth inhibitory effect of progesterone is not mediated via the classic nuclear progesterone receptor raises the possibility that it may be mediated through a membrane receptor. Some effects of estrogen, another 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. steroid hormone, have been reported via a membrane receptor (Nadal and others 2000). Our findings provide an effective mean of controlling the growth of ovarian epithelial tumors. The different effects of progesterone receptor antagonists on cell growth and angiogenic activity open up new applications in the treatment of ovarian cancer and other gynecological diseases. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PART ill EPILOGUE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5 Summary and Future Directions Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Summary Ovarian cancer is the most lethal malignancy in the female reproductive system. Despite the extensive use of new chemotherapeutics, its survival rate remains very low. Epidemiological studies persistently demonstrate the protective effect of increasing parity and use of oral contraceptives on the development of ovarian cancer. The incessant ovulation hypothesis and the excessive gonadotropin stimulation theory proposed by Fathalla in 1971 and Cramer in 1983, respectively, link the etiology of ovarian cancer to reproductive hormones. However, the molecular mechanism underlying the hormonal actions is still lacking. The goal of this dissertation is to study the mechanism of protective effects of progesterone, the pregnancy hormone and major component of oral contraceptives, in ovarian epithelial tumors. We examined the effect of progesterone on cell proliferation in benign (cystadenomas) and malignant (carcinomas) ovarian tumor cells cultured in vitro. We examined benign tumors in addition to malignant ones because ovarian cystadenomas, which are made up of the same cell type as carcinomas, are better differentiated, implying that any effect from reproductive hormones may be more accentuated in these tumors. In 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. addition, although not as serious as ovarian carcinomas, ovarian cystadenomas constitute a frequent health problem in women in their reproductive ages. Using [3H]thymidine incorporation assay, We showed that progesterone significantly inhibited DNA synthesis in various ovarian epithelial cell lines, in a dose-dependent manner. We further investigated the mechanism underlying the growth inhibitory effect of progesterone. We studied the effect of progesterone on cell cycle regulation. Western blot analyses showed that progesterone down regulated the expression of cyclin B1, a cyclin important for cell cycle progression through G2/M transition. In contrast, progesterone did not show significant effect on the expression of cyclins A, Ds, and E. The decreased expression of cyclin B1 was accompanied by an increased expression of p21, a universal inhibitor of cyclin/cycin-dependent kinase (CDK) complexes. Another member of this family of cyclin-dependent kinase inhibitors, p27, was also slightly up regulated by progesterone. Consistent with the down-regulation of cyclin B1, the kinase activity of the cyclin B1/CDK1 complex was substantially decreased by progesterone. Progesterone also showed a smaller inhibitory effect on the activity of CDK2, 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a cyclin-dependent kinase that interacts with cyclin A and cyclin E and regulates entry and progression through the S phase of the cell cycle. DNA profile analysis by flow cytometry showed that progesterone treatment resulted in an increase in the proportion of cells in the G2 phase, with a concomitant decrease in the number of cells in the S phase. We conclude that progesterone inhibits cell cycle progression in benign and malignant ovarian epithelial tumors, at least in part, by down-regulating the activity of the cyclin B1/CDK1 complex, up-regulating p21 expression, and arresting cells at the G2 phase of the cell cycle. We also investigated the signal transduction pathway that may be responsible for mediating the growth inhibitory effect of progesterone. Western blot analysis showed decreased expression of phosphorylated MAP kinase after progesterone treatment for 24 or 48 hours, suggesting MAP kinase pathway maybe involved in the growth inhibition by progesterone. We further studied the role of the progesterone receptor in mediating the effects of progesterone on growth inhibition and angiogenesis induction (ongoing study in our lab) in ovarian epithelial tumor cells since the concentration of progesterone needed to obtain both effects was much 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. higher (10 micromolar) than the Kd value of the progesterone receptor, which is at nanomolar levels. Western blot analysis showed varied progesterone receptor expression in ovarian epithelial tumor cell lines, low in SKOV-3 cells. Of the two isoforms of the progesterone receptor, PR-A isoform predominate in all the ovarian epithelial cell lines, while PR-B isoform expression is much lower, even not detected in SKOV-3 cells. Despite the varied expression levels of progesterone receptors in the ovarian epithelial cell lines, [3H]thymidine incorporation assay showed that progesterone inhibited cell proliferation in all the ovarian epithelial tumor cells examined. Both progesterone receptor agonists (R5020 and ORG2058) and antagonists (RU486 and ZK98299) acted the same as progesterone, significantly inhibiting cell proliferation at the same concentrations, in a dose- dependent manner. Progesterone induced angiogenesis in certain cell lines by increasing VEGF secretion shown by ELISA. Although type II progesterone receptor antagonist RU486 showed the same stimulatory effect as progesterone on VEGF secretion, ZK98299, a type I progesterone receptor antagonist, inhibited this effect of progesterone. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We also examined the possibility of progesterone acting through other steroid receptors, which have known affinity for progesterone. Neither dexamethasone, a potent synthetic ligand for the glucocorticoid receptor, nor aldosterone, a natural ligand for the mineralocorticoid receptor, could mimic progesterone on growth inhibition or VEGF secretion, which excluded the possibility that progesterone acted through these receptors. Antisense oligonucleotides complementary to the translation initiation site of the PR-A isoform of the progesterone receptor, which abolished expression of both PR-A and PR-B isoforms of the progesterone receptor confirmed by Western blot analysis, blocked the effect of progesterone on VEGF secretion but did not reverse the effect of progesterone on cell proliferation. Antisense oligonucleotides specific for the PR-B isoform of the progesterone receptor did not show significant effects on progesterone-induced VEGF secretion. Sense oligonucleotides for the same regions of the progesterone receptor mRNA had no effect on VEGF secretion or cell growth. These results suggest that the effect of progesterone on VEGF secretion in ovarian epithelial tumors is mediated via the progesterone receptor, PR-A 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. isoform, however, the current evidence suggests that inhibition of cell proliferation by progesterone may not be mediated by the progesterone receptor. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Future Directions 1. Finding the receptor that mediates the growth inhibitory effects of progesterone in ovarian epithelial tumor cells. There is no evidence showing that progesterone inhibits cell proliferation via the progesterone receptor, the glucocorticoid receptor, or the mineralocorticoid receptor, which raises the possibility of a role for other receptors, such as PXR, or unidentified membrane receptors or intracellular receptors. The concentration of progesterone or other progesterone receptor ligands needed to achieve growth inhibition in ovarian epithelial tumor cells is at micromolar level, far above 10 nM, which is the Kd for the progesterone receptor. We are therefore looking for a receptor that has a low affinity binding to progesterone. One of the possible candidates would be pregnane X receptor (PXR). PXR is a new member of the steroid receptor superfamily. It has been reported to mediate the genomic effects of steroid hormones, including 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. progesterone, pregnenolone, glucocorticoid, synthetic glucocorticoids (dexamethasone), antiglucocorticoids (RU486), and xenobiotics in the mouse, rat, and human. In contrast to other nuclear receptors that bind to one or few ligands with high affinity, PXR has the ability to bind to structurally varied ligands with low affinity (Kliewer and others 1998, Lehmann and others 1998, Bertilsson and others 1998, Schuetz and others 1998). Ligands usually bind to PXR at the concentrations above 10 jiM, which is close to the concentrations used in our studies. To investigate the possibility of progesterone acting through the PXR to inhibit cell proliferation in ovarian epithelial tumor cells, the expression of PXR should be examined by Western blotting. If it is present in the ovarian epithelial tumor cell lines, PXR antisense strategy or double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) can be used to abolish PXR so that the effect of progesterone on cell proliferation can be examined with no presence of PXR. If the inhibitory effect of progesterone on cell proliferation in ovarian epithelial tumor cells is not mediated by the PXR, or other known intracellular receptors, it may be via a new membrane receptor or intracellular receptor. Although there is no report on membrane receptors for progesterone yet, 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membrane receptors for estrogen have been identified (Nadal and others 2000). Binding assays using fluorescence- or radio-labeled progesterone can be used to identify possible receptors for progesterone. 2. Further studying the role of p21 in the effect of progesterone on cell cycle regulation. It is known that p21 not only is a CDK inhibitor, but also represses transcription of cyclin B1. Therefore, the effects of progesterone on down- regulation of cycin B1 expression and CDK1 and CDK2 activity may be all attributed to up-regulation of p21 expression. If this is the case in the progesterone treatment, using antisense strategies to diminish p21 should block the effects of progesterone on cylin B1 expression and CDK1 and CDK2 activity and cell proliferation. 3. Studying the synergistic effects of progesterone and anti- angiogenic agents on ovarian epithelial tumor growth in nude mice. Our finding that progesterone inhibits ovarian epithelial tumor cell proliferation provides evidence for the therapeutic potential of progesterone. 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Creator Zhou, Hong (author)

Core Title Progesterone signaling in ovarian epithelial tumors

Degree Doctor of Philosophy

Degree Program Pathobiology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,health sciences, oncology,health sciences, pathology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-550681

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Identifier 3094406.pdf (filename),usctheses-c16-550681 (legacy record id)

Legacy Identifier 3094406.pdf

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Document Type Dissertation

Rights Zhou, Hong

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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