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Identification of signaling molecules involved in mediating the anti-proliferative effects of gonadotropin… Kim, Ki Yon 2006

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IDENTIFICATION OF SIGNALING MOLECULES INVOLVED IN MEDIATING THE ANTIPROLIFERATIVE EFFECTS OF GONADOTROPIN RELEASING HORMONE-I AND -II ON OVARIAN CANCER CELL LINES b y KI YON KIM D . V . M . , K y u n g p o o k N a t i o n a l U n i v e r s i t y , 1 9 9 8 M . S c . , S e o u l N a t i o n a l U n i v e r s i t y , 2 0 0 0 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( R E P R O D U C T I V E & D E V E L O P M E N T A L S C I E N C E S ) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A D e c e m b e r , 2 0 0 6 © Ki Yon Kim, 2006 ABSTRACT In addition to its well-established role as a neuroendocrine regulator at the level of the pituitary gland, gonadotropin-releasing hormone (GnRH; GnRH-I) and a second form of GnRH have received consideration for therapeutic use in gynecological cancers. The objective of this thesis was to explore the signal transduction of GnRH-induced anti-proliferative effects in ovarian cancer cells. It has been hypothesized that the action of GnRH-II may be mediated by the GnRH-I receptor, which is expressed in OVCAR-3 and SKOV-3 ovarian cancer cells. GnRH-I and II induced the activation of ERK1/2 and anti-proliferative effect on on ovarian cancer cells and antide, a GnRH-I receptor antagonist, and transfection of short-interfering R N A (siRNA) of the GnRH-I receptor abolished GnRH-I and Il-induced anti-proliferation and extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) phosphorylation. In addition, the GnRH-induced ERK1/2 activation was mimicked by phorbol-12-myristate 13-acetate, a protein kinase C (PKC) activator, and pretreatment with GF109203X, an inhibitor of P K C , blocked GnRH-induced ERK1/2 activation and anti-proliferation. There is accumulating evidence that activation of mitogen-activated protein kinases (MAPKs) by GnRH-I is important for cell proliferation, differentiation and apoptosis. In this study, the role of GnRH-II in activating M A P K s was investigated in ovarian cancer cells. ERK1/2 and p38 M A P K were activated following GnRH-II treatment. The activation of ERK1/2 by GnRH-II led to the phosphorylation of Elk-1, which was not blocked by PD98059, an inhibitor of M A P K / E R K kinase (MEK). In addition, the transcription factor, AP-1 was activated by GnRH-II and attenuated in the presence of SB203580, an inhibitor of p38 M A P K . Moreover, PD98059 and SB203580 11 reversed the GnRH-II-induced anti-proliferation. Treatments with GnRH-I or II resulted in the induction of apoptosis in ovarian cancer cells. Moreover, GnRH-induced apoptosis was blocked by SB203580, but not by PD98059. Treatment with genistein, a protein tyrosine kinase (PTK) inhibitor, reversed GnRH-induced anti-proliferation in ovarian cancer cells. In summary, having demonstrated the functional involvement of the signaling pathways described above in GnRH-I and Il-induced anti-proliferation and apoptosis, our studies support the hypothesis that GnRH-I and II elicit anti-proliferative effects in ovarian cancer cells via GnRH-I receptor, ERK1/2, p38, PKC and PTK. in TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES v i i i LIST OF ABBREVIATIONS L . x i 1. INTRODUCTION 1 1.1 O v a r i a n c a n c e r 1 1 .2 . G n R H - I a n d G n R H - I I 2 1 . 2 . 1 P h y s i o l o g i c a l r o l e s 2 1 . 2 . 2 R o l e s o f G n R H - I a n d G n R H - I I i n c a n c e r c e l l s 4 1 . 3 . G n R H r e c e p t o r s 5 1 . 3 . 1 G n R H - I r e c e p t o r 5 1 . 3 . 2 G n R H - I I r e c e p t o r . . . 9 1 . 4 . G T P b i n d i n g p r o t e i n s a n d G n R H s i g n a l i n g 1 2 1 . 5 . P r o t e i n k i n a s e C a n d G n R H s i g n a l i n g 1 3 1 .6 . M A P K s a n d G n R H s i g n a l i n g 1 4 < 1 . 7 T r a n s c r i p t i o n f a c t o r s i n v o l v e d i n t h e G n R H r e s p o n s e 2 0 1 .8 I n t e r a c t i o n b e t w e e n t h e G n R H a n d E G F s i g n a l i n g p a t h w a y s 2 1 1 .9 G n R H a n d t h e i n d u c t i o n o f A p o p t o s i s 2 2 1 . 1 0 C l i n i c a l a p p l i c a t i o n s o f G n R H i n c a n c e r t r e a t m e n t 2 4 1 .11 H y p o t h e s i s 2 4 iv 1 . 1 2 S p e c i f i c O b j e c t i v e s 2 5 2. MATERIALS AND METHODS 2 6 2 . 1 . M a t e r i a l s 2 6 2 . 2 C e l l c u l t u r e 2 6 2 . 3 l l m m u n o b l o t a s s a y 2 7 2 . 4 In vitro M A P K a s s a y 3 0 2 . 5 T h y m i d i n e i n c o r p o r a t i o n a s s a y 3 0 2 . 6 D N A f r a g m e n t a t i o n a s s a y 3 2 2 . 7 T r a n s i e n t t r a n s f e c t i o n a s s a y 3 3 2 . 8 M T T a s s a y 3 4 2 . 9 In vitro t r a n s f e c t i o n w i t h s i R N A 3 4 2 . 1 0 F l o w c y t o m e t r y ; 3 5 2 . 1 1 T U N E L a s s a y 3 6 2 . 1 2 S t a t i s t i c a l a n a l y s i s 3 7 3. RESULTS 3 8 3 . 1 R o l e s o f t h e G n R H - I r e c e p t o r a n d p r o t e i n k i n a s e C p a t h w a y i n G n R H s i g n a l i n g 3 8 3 . 1 . 1 E f f e c t o f a G n R H - I a n t a g o n i s t o n G n R H - I a n d - I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n 3 8 3 . 1 . 2 G n R H - I a n d I I a c t i v a t e E R K 1 / 2 i n a P K C - d e p e n d e n t m a n n e r 3 9 3 . 1 . 3 T h e a n t i - p r o l i f e r a t i v e e f f e c t s o f G n R H - I a n d II a r e m e d i a t e d b y t h e G n R H - I r e c e p t o r 3 9 3 . 1 . 4 E f f e c t o f a P K C i n h i b i t o r o n t h e a n t i - p r o l i f e r a t i v e a c t i o n s o f G n R H - I a n d II 4 0 3 . 2 R o l e o f E R K 1 / 2 i n m e d i a t i n g t h e G n R H - I I - i n d u c e d i n h i b i t i o n o f o v a r i a n c a n c e r c e l l p r o l i f e r a t i o n 4 5 3 . 2 . 1 E f f e c t o f G n R H - I I o n E R K 1 / 2 a c t i v a t i o n 4 5 3 . 2 . 2 J N K / S A P K 1 a c t i v a t i o n b y G n R H - I I i n o v a r i a n c a n c e r c e l l s 4 6 3 . 2 . 3 G n R H - I I i n d u c e d a c t i v a t i o n o f E R K 1 / 2 d o e s n o t m e d i a t e t h e a c t i v a t i o n o f E l k - 1 4 7 3 . 2 . 4 G n R H - I I i n h i b i t s t h e p r o l i f e r a t i o n o f o v a r i a n c a n c e r c e l l s 4 7 3 . 3 R o l e o f p 3 8 M A P K i n G n R H - I I s i g n a l i n g i n o v a r i a n c a n c e r c e l l s 5 7 3 . 3 . 1 p 3 8 M A P K a c t i v a t i o n b y G n R H - I I i n o v a r i a n c a n c e r c e l l s 5 7 3 . 3 . 2 A c t i v a t i o n o f A P - 1 b y G n R H - I I r e q u i r e s p 3 8 M A P K a c t i v i t y 5 7 3 . 3 . 3 A n t i p r o l i f e r a t i v e e f f e c t o f G n R H - I I o n o v a r i a n c a n c e r c e l l s 5 8 3 . 3 . 4 I n d u c t i o n o f a p o p t o s i s b y G n R H - I I 5 9 3 . 4 I n v e s t i g a t i o n o f t h e r o l e s o f o t h e r s i g n a l i n g c o m p o n e n t s , a n d o f t h e d i f f e r e n t i a l r o l e s o f M A P K s , i n G n R H s i g n a l i n g 6 6 3 . 4 . 1 R o l e s o f G a s a n d G o d i n G n R H - i n d u c e d M A P K a c t i v a t i o n a n d i n m e d i a t i n g G n R H - i n d u c e d i n h i b i t i o n o f p r o l i f e r a t i o n 6 6 3 . 4 . 2 R o l e o f M A P K a c t i v a t i o n i n G n R H - i n d u c e d a p o p t o s i s i n o v a r i a n c a n c e r c e l l s 6 7 3 . 4 . 3 G n R H i n d u c e s the k i n e t i c a l l y d i s t i n c t a c t i v a t i o n o f M A P K s 6 7 3 . 4 . 4 I n v o l v e m e n t t h e E G F s i g n a l i n g p a t h w a y a n d p r o t e i n t y r o s i n e vi k i n a s e s i n G n R H s i g n a l i n g 6 8 4. DISCUSSION ..: 7 9 4 . 1 R o l e s o f t h e G n R H - I r e c e p t o r a n d P K C i n G n R H s i g n a l i n g a n d a n t i -p r o l i f e r a t i v e e f f e c t s 7 9 4 . 2 R o l e o f E R K 1 / 2 i n G n R H - I I s i g n a l i n g a n d t h e i n h i b i t i o n o f o v a r i a n c a n c e r c e l l p r o l i f e r a i o n 8 5 4 . 3 R o l e o f p 3 8 M A P K i n G n R H - I I s i g n a l i n g i n o v a r i a n c a n c e r c e l l s 8 9 4 . 4 I n v e s t i g a t i o n o f G T P b i n d i n g p r o t e i n s a n d P T K i n G n R H s i g n a l i n g 9 3 4 . 5 D i f f e r e n t i a l r o l e s o f E R K l / 2 a n d p 3 8 M A P K i n G n R H s i g n a l i n g 9 6 5. SUMMARY AND FUTURE STUDIES 9 9 1. S u m m a r y 9 9 2 . F u t u r e S t u d i e s 1 0 3 6. BIBLIOGRAPHY 1 0 6 vii LIST OF FIGURES Figure 1. The hypothalamic-pituitary-gonadal axis 3 Figure 2. Schematic representation of the human GnRH-I and GnRH-II genes 6 Figure 3 . Two-dimensional representation of the human GnRH-I receptor and monkey GnRH-II receptor Morphology of OSE in culture 8 Figure 4. Expression of GnRH-I receptor in OVCAR-3, SKOV-3 and aT-3 cells 11 Figure 5. Schematic generic overview of the sequence of events of ERK1/2, p38, and JNK MAPK pathways . 18 Figure 6 . Schematic representation of GnRH signaling in pituitary (aT3), extra-pituitary and prostate cancer (DU145) cell lines 19 Figure 7 . Effect of GnRH-I and II on ERK1/2 activation and growth inhibition in the presence or absence of Antide in OVCAR-3 and SKOV-3 cells 42 Figure 8. Effect of a PKC inhibitor on GnRH-I- and -II-induced ERK1/2 activation and growth inhibition in OVCAR-3 and SKOV-3 cells 43 Figure 9. Effect of GnRH-I receptor siRNA transfection on GnRH-I and II-induced growth inhibition in OVCAR-3 and SKOV-3 cells 44 Figure 10. Effect of GnRH-II on ERK1/2 in ovarian adenocarcinomas and IOSE cell lines 49 Figure 11. Effect of GnRH-II on the activation of JNK/SAPK1 in OVCAR-3 cells 51 Figure 12. Effect of GnRH-II on ERK1/2 activation in the presence or absence of PD98059 in OVCAR-3 cells 52 viii Figure 13. Effect of GnRH-II in the presence or absence of PD98059 on Elk-1 phosphorylation in OVCAR-3 cells 54 Figure 14. Effect of PD98059 on GnRH-II-induced growth inhibition in OVCAR-3 cells 55 Figure 15. Effect of GnRH-II on cell viability in OVCAR-3 cells 56 Figure 16. Time dependent effect of GnRH-II on p38 activation in OVCAR-3 cells 60 Figure 17. The effect of SB203580 pretreatment on GnRH-II-induced p38 activation in OVCAR-3 cells 61 Figure 18. The effect of GnRH-II and SB203580 on AP-1 activation in OVCAR-3 cells '. 62 Figure 19. The effect of GnRH-II on ovarian cancer cell proliferation in OVCAR-3 cells 63 Figure 20. The effect of p38 MAPK activation by GnRH-II in OVCAR-3 cells 64 Figure 21. The effect of GnRH-II in the induction of apoptosis in OVCAR-3 cells 65 Figure 22. Effect of 8-bromo-cAMP and H-89 on GnRH-induced ERK1/2 activation and anti-proliferation in OVCAR-3 cells 71 Figure 23. Effect of mastoparan and PTX on GnRH-induced ERK1/2 activation and anti-proliferation in OVCAR-3 cells 72 Figure 24. Effect of MAPK inhibitors on GnRH-induced apoptosis in OVCAR-3 cells ...73 Figure 25. Time-dependent apoptotic effects of GnRH-I and GnRH-II in SKOV-3 cells 74 ix Figure 26. Effect of GnRH-I or II on ERK1/2 and p38 activation in OVCAR-3 cells.... 75 Figure 27. Effects of AG1478 and Genistein on GnRH-induced ERK1/2 activation 76 Figure 28. Effects of GnRH on EGF receptor activation 77 Figure 29. Effects of AG1478 and Genistein on GnRH-induced anti-proliferation ........ 78 Figure 30. Proposed intracellular signaling cascade of GnRH-I and II in ovarian cancer cells 102 x LIST OF ABBREVIATIONS ANOVA Analysis of variance C Celcius Ca 2 + Calcium cAMP Cyclic adenosine monophosphate cDNA Complementary deoxyribonucleic acid cGMP Cyclic guanosine monophosphate Ci Curie Cpm Counts per minute DNA Deoxyribonucleic acid EDTA Ethylene diaminetetraacetic acid EGF Epidermal growth factor EGF-R EGF receptor ELISA Enzyme-linked immunosorbant assay ER Endoplasmic reticulum ERG Early response gene E R K 1 / 2 Extracellular signal-regulated kinase 1 /2 Fas L Fas ligand FBS Fetal bovine serum FSH Follicle-stimulating hormone FSH-R FSH receptor G Acceleration of gravity GDP Guanosine diphosphate GnRH Gonadotropin-releasing hormone GnRH a Gonadotropin-releasing hormone agonist GnRH-II Gonadotropin-releasing hormone-II GnRHR Gonadotropin-releasing hormone receptor G-protein GTP-binding protein GPCR G-protein coupled receptors GTP Guanosine triphosphate H Hour HBSS Hank's balanced salt solution HCG Human chorionic gonadotropin XI IOSE Immortalized ovarian surface epithelium IP Inositol phosphate IP3 Inositol 1, 4, 5-triphosphate IU International unit JNK/SAPK c-jun terminal kinase/stress-activated protein kinases LH Luteinizing hormone LPA Lysophosphatidic acid p. Micro MAPK Mitogen-activated protein kinase MAPKKs (=MEK) MAPK kinases MEK1/2 MAPK/ERK kinase 1/2 Min Minutes MMP Matrix metalloproteinases mRNA Messenger ribonucleic acid Mw Molecular weight n (as in nM) Nano OSE Ovarian surface epithelium p (as in pM) Pico PAGE Polyacrylamide gel electrophoresis PBS Phosphatase buffered saline PCR Polymerase chain reaction PI Phosphatidylinositol PIP Phosphatidylinositol 4-phosphate P1P2 Phosphatidylinositol 4, 5-phosphate PIP3 Phosphatidylinositol 3,4,5-triphosphate PI3K Phosphatidyl inositol 3 kinase PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C PMSF Phenylmethylsulfonyl fluoride rpm Revoultions per min sec Seconds SD Standard deviation SDS Sodium dodecyl sulphate x i i TCF Ternary complex factor TE Tris-EDTA TEMED N, N, N \ N'-tetramethylethlenediamine TGF-P Transforming growth factor-13 Tris Tris(hydroxy methyl) aminomethane UV Ultraviolet xiii I. INTRODUCTION 1.1 Ovarian cancer Ovarian cancer is a significant cause of cancer-related death in Western women, following breast, lung, and colorectal cancer (Schally et al. 2001). Because of the absence of symptoms in early-stages and location in the pelvis, ovarian cancer is difficult to detect in early stages (Herbst 1994) and has a high fatality and low five-year survival rate compared with other gynecologic cancers. Although the biological causes of ovarian cancer remain unknown, a few possibilities including endocrine factors that induce ovarian tumors have been suggested (Shoham 1994; Risch 1998). Hormonal factors have been implicated in the etiology of ovarian cancer, according to the incessant ovulation theory (Shoham 1994; Risch 1998). Women who have frequent ovulation in their twenties and have a family history of ovarian cancer may contribute to high risk of ovarian cancer, suggesting that ovarian cancer is essentially a hereditary genetic disease (Godwin et al. 1992; Negri et al. 2003; Purdie et al. 2003). Hormonal factors have also been considered to be an important determinant causing ovarian cancer. On the other hand, taking oral contraceptives and multiparity might reduce the risk of ovarian cancer, also suggesting that hormonal factors are important in ovarian cancer development (Risch 1998). 1 1.2 GnRH-I and GnRH-II 1.2.1 Physiological roles Gonadotropin-releasing hormone (GnRH) is a central regulator of the mammalian reproductive system. It is well documented that GnRH is secreted from the hypothalamus and intermittently binds to its receptor in the anterior pituitary to stimulate the synthesis and release of gonadotropins such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary (Fig. 1). Since the amino acid sequence of GnRH was reported in the 1970s (Matsuo et al. 1971; Burgus et al. 1972), the expression of GnRH and GnRH receptors are considered to be important for gonadal steroidogenesis and for the maintenance of pregnancy and efforts have focused on its molecular biology. Exogenously administered GnRH agonists action desensitizes, and down-regulates GnRH receptors in the pituitary and decreases LH and FSH production, which induces the subsequent decrease in the circulating sex steroid levels (Neill 2002). In the ovary, stimulatory effects of GnRH include augmentation of steroidogenic and ovulatory processes but GnRH has an inhibitory effect on ovarian function to reduce gonadotropin receptor biosynthesis, steroidogenesis, and follicular development. Three structural variants of GnRH were determined in non-mammalian vertebrates. These GnRH variants have almost similar amino acid sequences but different functions in the regulation of reproduction (Sherwood et al. 1993; Sealfon et al. 1997). One of these 2 Hypothalamus GnRH Pituitary FSH ) I C L H Gonads i Sex steroids Figure 1. The hypothalamic-pituitary-gonadal axis. Secretion of GnRH occurs in a pulsatile fashion and control synthesis and release of FSH and LH to regulate function of ovaries and testes. GnRH variants is GnRH-II (also called chicken GnRH-II), which is totally conserved in structure from fish to mammals (Fig. 2). GnRH-II was sequenced and its gene expression was identified in human (Neill 2002). Although the normal physiologic function of GnRH-II is poorly understood, it has been observed that GnRH-II induced the secretion of human chorionic gonadotropin (hCG) in cytotrophoblastic cells; however, GnRH-II had a lesser effect than GnRH-I (Islami et al. 2001). Chicken GnRH-II is less likely to regulate the gonadotropin secretion than chicken GnRH-I in hens (Sharp et al. 1990). Interestingly, the expression of GnRH-II was detected at higher levels (up to 30 times than GnRH-I) in kidney, bone marrow, and prostate (White et al. 1998). 1.2.2 Roles of GnRH-I and GnRH-II in cancer cells In addition to its important role in the reproductive system, GnRH-I and its analogs are known to have therapeutic value in the treatment of reproductive disorders such as infertility, polycystic ovarian disease, and precocious puberty (Homburg 2003; Heger et al. 2006). GnRH is also clinically valuable in the treatment of prostate, breast, and ovarian cancers (Harrison et al. 2004). GnRH-I showed inhibitory effects on human mammary, ovarian, endometrial, and prostatic tumor growth, and has been implicated as an anti-proliferative regulator of gynecologic cancers (Santen et al. 1990; Savino et al. 1992; Schally 1999; Schally et al. 2001). The general principle of GnRH therapy is to suppress the function of the hypothalamus-pituitary-gonadal axis, called 'chemical castration' (Florio et al. 2002). Moreover, the detection of GnRH-I and its receptor in breast, placenta, ovary, and prostate tissues implies that GnRH-I may have direct effects at peripheral targets 4 (Kleinman et al. 1994; Motomura 1998; Serially et al. 2001; Kim et al. 2004a). In ovarian cancer cells, it has been reported that GnRH-I induces apoptosis and has an autocrine/paracrine action (Motomura 1998; Kang et al. 2000b), suggesting that treatment with GnRH-I is important for the direct suppression of proliferation and the induction of apoptosis in gynecologic cancers. In addition to GnRH-I, the expression and potential anti-proliferative effect of GnRH-II indicate that GnRH-II, similar to GnRH-I, may have a growth-regulatory effect in normal and neoplastic ovarian surface epithelial cells (Choi et al. 2001b). Furthermore, a recent study demonstrated that GnRH-II has an anti-proliferative effect in gynecologic tumors and may exert a stronger anti-proliferative effect than GnRH-I in ovarian cancer cells (Grundker et al. 2002; Kim et al. 2004a), suggesting that GnRH-II could be considered as a novel target for anti-proliferative therapeutic approaches. However, in contrast to GnRH-I, the biological mechanism of GnRH-II remains obscure. 1.3 GnRH receptors 1.3.1 GnRH-I receptor The structure of mammalian GnRH-I receptor was determined by sequencing of a cDNA isolated from an immortalized murine gonadotroph cell line (aT3-l) (Reinhart et al. 1992; Tsutsumi et al. 1992). The GnRH-I receptor is a member of the G protein-coupled receptor (GPCR) family (Kraus et al. 2001) and a member of the 7 transmembrane receptor 5 Human GnRH-I mRNA : 5.1 kb pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH. Human GnRH-II mRNA: 2.1 kb • 5'-Untranslated region 100 bp Exon 1 Exon 2 Signal sequence GnRH Exon 3 GAP Exon 4 3'-Untranslated region pTyr-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- N H 2 GAP : Gonadotropin releasing hormone associated protein Figure 2. Schematic representation of the human GnRH-I and GnRH-II genes. Only exonic regions are drawn to scale. (Adapted from White et al., 1998) 6 superfamily that transduce an extracellular signal into an intracellular signal (G protein activation). The signaling of GnRH-I might then be passed onto the nucleus eliciting protein phosphorylation or dephosphorylation and eventually transcriptional activation or inactivation. The signal transduction pathway following the binding of GnRH-I to GnRH-I receptor has been extensively studied (Millar et al. 2004). Intracellular communication of the mammalian GnRH-I receptor is unique because it lacks the common carboxyl-terminal cytoplasmic domain and possesses a relatively short intracellular third loop among GPCRs (Reinhart et al. 1992) (Fig. 3). Understanding of the GnRH receptor structure can lay the foundation for the design of a new generation of GnRH analogs for the treatment of cancer and reproductive disorders. In pituitary cells, GnRH-I receptors have been identified (Clayton et al. 1979; Bourne et al. 1980; Clayton and Catt 1980; Clayton et al. 1980; Naor et al. 1980; Wormald et al. 1985; Pal et al. 1992; Weil et al. 1992; Schulz et al. 1993). In extrapituitary tissues, the gene of GnRH and its receptors are expressed in gonads (Currie et al. 1981; Iwashita et al. 1986), and placenta (Miller et al. 1985; Emons et al. 1992) but not in liver and spleen (Kakar et al. 1992). In neoplastic tissues, GnRH-I receptors have been identified in human epithelial ovarian carcinoma (Pahwa et al. 1989; Emons et al. 1992; Irmer et al. 1995), human breast cancer tissue (Miller et al. 1985; Fekete et al. 1989), endometrial cancer (Furui et al. 2002), and prostate tumors (Qayum et al. 1990; Hong et al. 1998). GnRH-I and its receptor have been detected in approximately 80% of ovarian cancer biopsy specimens as well as normal ovarian epithelial cells and immortalized ovarian surface epithelium cells (Irmer et al. 1995; Choi et al. 2001b). OVCAR-3 and 7 Human GnRH I Receptor 328 aa c o o Intracellular Monkey GnRH II 379 aa Receptor C O O H Figure 3. Two-dimensional representation of the human GnRH-I receptor and monkey GnRH-II receptor. Note that the absence of a carboxy-terminal cytoplasmic tail in the mammalian GnRH-I receptor is the most unique feature among GnRH receptor among G protein coupled receptors. 8 SKOV-3 cells, ovarian adenocarcinoma cell lines, express the GnRH-I receptor protein (Fig. 4). Both cell lines were derived from serous carcinoma patient's ascitis, which accounts for most part of ovarian cancer patients. The each shape of OVCAR-3 and SKOV-3 cells implies that OVCAR-3 is more likely undifferentiated than SKOV-3 cell because OVCAR-3 has cuboidal shape which takes after normal ovarian surface epithelia cells but SKOV-3 cells has enlongated shape. Although it has been known that GnRH stimulates the release of the gonadotropins, FSH and LH, the functions of GnRH-I in other tissues are not well understood. Nonetheless, the presence of GnRH-I and its receptor indicate that it may have a functional role in the reproductive system. How GnRH recognizes and activates its receptor is considered important for elucidating the regulation of its reproductive function and treatment in cancer therapy. 1.3.2 GnRH-II receptor In most vertebrates, several structural variants of GnRH exist, suggesting that additional receptors for GnRH in mammalian may exist in some tissues and organisms. Indeed, three distinct types of GnRH receptor were determined in the bullfrog (Wang et al. 2001) and a novel chicken pituitary GnRH receptor has been cloned (Sun et al. 2001). Neill et al. observed that GnRH-II receptor gene is present in humans (Neill et al. 2001). In mammals, GnRH-II receptors have been found to be more widely expressed than GnRH-I receptors, suggesting that GnRH-II may have additional functions (Millar et al. 2001). GnRH-II receptors were identified in mammals and found more widely than GnRH-I receptors in the body including the brain (Millar et al. 1999; Kang et al. 2000b; Millar et al. 9 2001) , suggesting that GnRH-II may have more functions than GnRH-I. Nevertheless, to date, direct evidence demonstrating the existence of full-length, functional GnRH-II receptor RNA transcript in human tissues is lacking. Although a full length GnRH-II receptor has not been cloned or sequenced, Grundker et al. suggested the anti-proliferative effect of GnRH-II in endometrial and ovarian cancer cells (Grundker et al. 2002) . As to the role of GnRH-I and II receptors, there are discrepancies between previous reports. It has been suggested that the signal transduction pathways coupled to the GnRH-II receptor may be different from those triggered by activation of the GnRH-I receptor (Millar et al. 2001; Neill et al. 2001). Enomoto et al. showed that GnRH-II receptor is necessary to mediate the effect of GnRH-II (Enomoto et al. 2004) and Grundker et al. reported that the anti-proliferative effect induced by GnRH-II is not mediated through GnRH-I receptor (Grundker et al. 2004). However, a human GnRH-II receptor protein has not been identified since the human GnRH-II receptor transcript has a frame-shift resulting in a premature stop codon (Morgan et al. 2003) and the mechanisms of anti-proliferative action of GnRH-I and GnRH-II is not clearly understood. Thus, the issue of whether this transcript encodes a functional receptor protein in human tissues and the potential roles of the GnRH-II receptor in mediating the effects of GnRH-I and II remains obscure. In addition, a recent study showed that GnRH-II receptor inhibits the expression of GnRH-I receptor, indicating that GnRH-I receptor may be a common receptor that mediates the effects of both GnRH-I and GnRH-II in ovarian cancer cell lines (Pawson et al. 2005). Therefore, the exact mechanism mediated by these treatments needs to be elucidated. This will provide the basic knowledge for novel approaches in cancer therapy. 10 G I R • ( 6 0 k d ) A c t i n C O S - 1 O V C A R - 3 S K O V - 3 a T 3 F i g u r e 4 . E x p r e s s i o n o f G n R H - I r e c e p t o r ( G I R ) i n C O S - 1 , O V C A R - 3 , S K O V - 3 a n d a T - 3 c e l l s . T o d e t e r m i n e t h e e x p r e s s i o n o f G n R H - I r e c e p t o r i n o v a r i a n c a n c e r c e l l l i n e s , G n R H - I r e c e p t o r w a s e x a m i n e d u s i n g a n t i b o d i e s t a r g e t i n g G n R H - I . T h e p r o t e i n e x p r e s s i o n o f G n R H - I r e c e p t o r w a s i n v e s t i g a t e d b y W e s t e r n b l o t a n a l y s i s . 11 1.4 GTP binding proteins and GnRH signaling The signaling of GnRH is transferred to cytoplasmic signaling pathway to induce the cellular biological effect. After GnRH-I binds to cell membrane receptors, GnRH receptors become internalized and undergo lysosomal degradation and/ or undergo receptor recycling (Hazum and Conn 1988). Binding of GnRH to the GnRH receptor leads to conformational changes in the receptor. As stated, the GnRH-I receptor belongs to the GPCRs family and GnRH-I transmits extracellular signals into the intracellular milieu via heterotrimeric (a, p, and y subunits) GTP-binding proteins (G-proteins) (Birnbaumer 1992). The a-subunit has a binding site for GTP or GDP and carries the GTPase activity. The p, and y subunits exist as a complex and are active in this form. Although the Py subunit-complex is involved in signal transmission, the majority of G proteins-related signaling occurs via the a-subunit. Ga-proteins can be divided into four families based on amino acid sequence, G a s , Gaj, G a q and G, 2 (Downes and Gautam 1999). A characteristic of the members of the Gas subfamily is that they are inhibited by cholera toxin. Cholera toxin catalyzes the ADP-ribosylation of an Arg201 in Gas, which interferes with its function and inactivates the GTPase activity leading to the blockage of the intrinsic deactivation mechanism of the Gas-protein. Members of the G„j subfamily have inhibitory effects on adenylyl cyclase and are sensitive to pertussis toxin which catalyzes the ADP-ribosylation and subsequent inhibition of the Gai-subunit. Mastoparan isolated from wasp venoms is able to stimulate the GDP-GTP exchange of G i / o type G-proteins (Hirai et al. 1979; Higashijima et al. 1990; Tanaka et al. 1998). The next signal protein in the reaction sequence of G protein is phospholipase C (Zhu and Birnbaumer 1996; Drissi et al. 1998; 1 2 Kuhn and Gudermann 1999; Hubbard and Hepler 2006). Members of the G a q subfamily are not modified by cholera toxin or pertussis toxin (PTX). The functions of the G12 subfamily have been implicated as the activation of c-Jun N-terminal protein kinase, reorganization of the cytoskeleton, and stimulation of Na +/H + exchange (Gohla et al. 1999; Lin et al. 1999; Nagao et al. 1999). G a; is involved in GnRH signaling in ovarian carcinoma and uterine leiomyosarcomas (Imai et al. 1996). The anti-proliferative effect was mediated through the PTX-sensitive Gaj protein in these cell lines (Grundker et al. 2001b). However, it has also been reported that the G-proteins involved in GnRH-I signaling differ depending on cell type (Grundker et al. 2001b; Kraus et al. 2001; Fang et al. 2002) and it is assumed that the individual subtypes of G proteins have specific roles in different cell compartments, cells and tissues. The mechanisms involved in the activation of G-protein signaling pathways by GnRH have yet to be fully elucidated. 1.5 Protein kinase C and GnRH signaling The GPCRs can be coupled to the G a q / n protein that activates phospholipase CP, leading to the activation of protein kinase C (PKC) and various downstream signal transduction cascades, including the mitogen-activated protein kinase (MAPK) pathways (Harris et al. 1997b). In addition, the activation of the PKC pathway has been well documented in response to GnRH-I stimulation. GnRH-I induces the activation of ERK1/2 through PKC, which may participate in gonadotropin release or synthesis in pituitary cells (Shacham et al. 2001). A diversity of GPCRs can activate extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the activation of PKC is one of the important signaling 1 3 pathways in the activation of ERK1/2 by GnRH in pituitary cells (Andrews and Conn 1986; Zheng et al. 1994). The G-protein involved in GnRH-I signaling pathway in the pituitary gland cells is not Gaj but might be Gq/n, which activates phospholipase C (PLCP) to mediate inositol 1,4,5-triphosphate (EP3), and diacylglycerol (DAG) production (Hsieh and Martin 1992; Anderson et al. 1993). IP3 releases calcium from intracellular stores (Stojilkovic et al. 1994; Tse et al. 1997) and DAG stimulates the PKC pathway in the pituitary gonadotrophs. Activated PKC might be involved in the Ca 2 + influx and might up-regulate GnRH-I receptors (Naor 1990). The activation of PKC with an increase of Ca 2 + concentration in cytoplasm is important for mediating GnRH-I action for gonadotropin secretion in the pituitary gland. In granulosa cells, PKC is known as a major component in activating ERK signaling from GnRH-I receptor (Kang et al. 2001b). Activation of PKC appears to be an important second messenger mediating GnRH-I-induced ERK activation in pituitary cells (Harris et al. 1997a) and ovarian cancer cells (Chamson-Reig et al. 2003). However, unlike pituitary cells, it was reported that ERK activation of GnRH-I might be mediated by GpY complex, neither by G a subunit, PKC pathway, nor extracellular Ca 2 + . In addition, the activation of ERK is involved in the anti-proliferative effect of GnRH-I in the Caov-3, human ovarian cancer cell line (Kimura et al. 1999). 1.6 MAPKs and GnRH signaling The family of MAPK consists of both mitogen-activated protein kinase and stress-activated protein kinase. Every MAPK has dual phosphorylation motif (Thr-Xaa-Tyr) and is activated by upstream kinases called MAPK kinases (MEKs or MKKs). MAPK family 1 4 members are directly regulated by kinases known as MAPK kinases (MAPKKs), which activate MAPKs by the phosphorylation of tyrosine and threonine residues (Cobb and Goldsmith 1995; Robinson and Cobb 1997) (Fig. 5). Although 12 different MAPKs have been identified in mammalian cells to date, ERK1/2, c-Jun N-terminal protein kinase/Stress activated protein kinase 1 (JNK/SAPK1) and p38/SAPK2 are three of the best-characterized MAPK moieties (Cobb and Goldsmith 1995). Biological roles of other MAPKs (ERK3, 4, and 5, four p38-like kinases, and p57 MAPK, etc) have not been elucidated. ERK1 (p44 MAPK) and ERK2 (p42 MAPK) are activated by mitogenic stimuli and represent a group of the most extensively studied members. In contrast, JNK/SAPKl and p38 MAPK are activated in response to stress such as heat shock, osmotic shock, cytokines, protein synthesis inhibitors, antioxidants, ultra-violet, and DNA-damaging agents (Cobb and Goldsmith 1995; Garrington and Johnson 1999; Kennedy and Davis 2003; Lin 2003). Although ERK is proposed to contribute to protecting against UV-induced damage, it has been reported that ERK1/2 is involved in cell cycle arrest and the inhibition of growth as well as cell survival and differentiation (Herskowitz 1995; Alblas et al. 1998; Yen et al. 1998). The activation of ERK1/2 by GnRH is relatively sustained in alpha T3-1 and HEK293 cells but it is transient in GT1-7 neurons (Shah et al. 2003b). It has been proposed that gene expression and other specific intracellular responses may vary according to the duration and the magnitude of MAPK activation in individual cell types (Shah et al. 2003a) and the effect of the activation of MAPK seems to vary in many cell types (Mansour et al. 1994; Alblas et al. 1998; Yen et al. 1998) 1 5 GnRH-I might activate diverse cytoplasmic proteins to transfer its signal into the nucleus, and MAPK is considered to be one of the important pathways in GnRH-I signaling pathway (Naor et al. 2000; Kraus et al. 2001). MAPK cascades are activated via two distinct classes of cell surface receptors, receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Signals transmitted through these cascades induce the activation of diverse molecules which regulate cell growth, survival and differentiation (Naor et al. 2000). In pituitary aT3-l cells, the GnRH receptor signals are mediated via all four major MAPK cascades including ERK1/2, INK, p38 MAPK and BMK1/ERK5 (Naor et al. 2000; Kraus et al. 2001). Four subtypes of Got (Gs, G q , Gj, and G12) might activate MAPK family and GpY dimer of G-protein also activates MAPK through PI3K (Jiang et al. 1998). G12/13 exerts its action by stimulating protein tyrosine kinases (PTKs) (Dikic and Blaukat 1999). Furthermore, GPCRs have cross talk with RTKs and PTK can convey GPCRs signals to regulate proliferation, migration, and adhesion, suggesting that the MAPK cascade is activated by RTKs and GPCRs (Cobb and Goldsmith 1995). Although it is difficult to define each of the mechanisms involved in the regulation of MAPKs in response to external stimuli, it is important to clarify the specific signaling pathways utilized by GnRH. Thus, it is important to study the MAPK pathway involved in GnRH-I and GnRH-II signaling pathway to elucidate the mechanism of anti-proliferative effect. GnRH-I revealed distinct differences in signaling pathways between cell types ( F i g . 6 ) . The signaling mechanism mediating the activation of MAPKs by GnRH-I also seems to be significantly different in cell types. In pituitary cells, it has been reported that GnRH-I activates ERK 1/2, JNK and p38 in a T 3 and L P T 2 cells (Reiss et al. 1997; L e v i et al. 16 1998a; Roberson et al. 1999; Liu et al. 2002). GnRH-I induces the activation of ERK1/2 through protein kinase C (PKC), which may participate in gonadotropin synthesis or release in pituitary cells. The role of the MAPK family in the anti-proliferative effect of GnRH in CaOV-3 has been demonstrated (Kimura et al. 1999). In our previous reports, we demonstrated that follicle-stimulating hormone (FSH) stimulated the activation of the ERK1/2 cascade and induced the phosphorylation of Elk-1 in neoplastic ovarian surface epithelial cells (Choi et al. 2002) and that the p38 MAPK pathway is involved in the anti-proliferative effect of GnRH-II in ovarian cancer cells (Kim et al. 2004a). The sequential transcriptional cascade, which is followed by GnRH-induced MAPK activation, should be characterized. Studies conducted in COS7 cells showed that the antiproliferative effect of GnRH-I agonist was mediated through Gj (Kraus et al. 2001). Furthermore, GnRH-I had no effect in the activation of JNK in this cell line. Following binding to its own receptors, the activation of PKC is involved in the activation of ERK 1/2 directly through Raf-1, Ras and dynamine, which are downstream of Src in aT3 cells. However, it was shown that the Gpy complex, receptor tyrosine kinase, and focal adhesion kinase (FAK) did not play a role in ERK1/2 activation (Sundaresan et al. 1996; Benard et al. 2001). In granulosa cells, PKC is a major component in the activation of ERK from GnRH-I receptor (Kang et al. 2001b). However, the effect of GnRH-II on MAPKs is pooly understood. 1 7 Stimulus MAPKKK I MAPKK MAPK Cellular responses Growth Factors, Mitogens Raf, B-Raf MEK1/2 MAPK/ERK i Growth, Differentiation, Development Stress, Inflammatory cytokines, Growth Factors MLKs, TAK ASK1 I MKK3/6 MEKK1,4 MLKs ASK1 MKK4/7 P38 MAPK SAPK/JNK \ / Inflammation, Apoptosis, Growth, Differentiation Figure 5. Schematic generic overview of the sequence of events of ERK1/2, p38, and INK MAPK pathways. A variety of stumuli activate MAPKKK. The activated MAPKKK activates MAPKKs. MAPKs are activated by the activated MAPKKs and control cellular responses such as cell growth and apoptosis by transcription factors and other regulatory factors. 1 8 aT3 GnRH-I Gaq 7 ;g3 Y Src PKC Ras I N K 1 M E K 1 ERK P38 G o n a d o t r o p i n s y n t h e s i s a n d s e c r e t i o n COS7 EGFR Ras v ERK (^PBK) t INK G r o w t h a r r e s t D U 1 4 5 EGFR Ras PI3K MLK3 ERK JNK A p o p t o s i s Figure 6 . S c h e m a t i c r e p r e s e n t a t i o n o f G n R H s i g n a l i n g i n p i t u i t a r y ( a T 3 ) , e x t r a - p i t u i t a r y ( C O S 7 ) a n d p r o s t a t e c a n c e r ( D U 1 4 5 ) c e l l l i n e s . ( A d a p t e d f r o m K r a u s e t a l . , 2 0 0 6 ) 1 9 1.7 Transcription factors involved in the GnRH response GnRH-I stimulates the activation of transcriptional factors such as Elk-1 by the activation of MAPK in aT3 cells (Roberson et al. 1995). GnRH-I also increases immediate early response gene (ERG) mRNA such as c-fos and c-jun in this cell line (Cesnjaj et al. 1995). The p38 MAPK might be involved in GnRH-I integration of c- fos promoter activity (Roberson et al. 1999). It has been reported that GnRH-I analog activates AP-1, a transcriptional factor in human endometrial cancer cells (Grundker et al. 2001a). In our previous reports, we demonstrated that follicle-stimulating hormone (FSH) stimulates the activation of the ERK1/2 cascade and phosphorylates Elk-1 in neoplastic ovarian surface epithelial cells (Choi et al. 2002). However, despite these observations, the exact mechanism of the signaling pathway between MAPK and transcriptional factors by GnRH-I and GnRH-II in ovarian cancer cells remains to be investigated. It has been demonstrated that MAPKs associate with transcription factors such as c-Myc, Elk-1, c-Jun, and ATF-2, (Wasylyk et al. 1998; Garrington and Johnson 1999; Johnson and Lapadat 2002). ERK, JNK and p38 activate Elk-1 that binds to serum response element (SRE) and enhances SRE-dependent c-fos expression (Cavigelli et al. 1995; Gille et al. 1995b; Gille et al. 1995c; Whitmarsh et al. 1995; Raingeaud et al. 1996). JNK phosphorylates c-jun and ATF-2 (Derijard et al. 1994; Gupta et al. 1995), and p38 activates ATF-2 (Raingeaud et al. 1995; Raingeaud et al. 1996). Both ATF-2 and c-jun form heterodimers, which bind to TPA-response elements (TREs) resulting in enhancing c-jun expression (van Dam et al. 1995). c-Fos and c-Jun form AP-1, which induce a cellular 20 response following binding to AP-1 response elements in gene promoter. It is suggested that AP-1 is involved in a protective function against the effects of UV-induced cell damage. JNK and p38 are involved in apoptosis via AP-1 (Xia et al. 1995; Verheij et al. 1996). MAPK contributes to the induction of AP-1 activity through activating transcription factor 2 (ATF-2) (Raingeaud et al. 1995). 1.8 Interaction between the GnRH and EGF signaling pathway The EGF receptor family (EGFR, also known as type I receptor tyrosine kinases or ErbB tyrosine kinase receptors) is the best studied growth factor receptor system. This family is comprised of four homologous receptors: the epidermal growth factor receptor (ErbB 1 /EGFr/HERl), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (Klapper et al. 2000; Jorissen et al. 2003). This receptor family regulates the intracellular effects of ligands such as EGF and transforming growth factor-a (TGFa) (Wells 1999; Yarden and Sliwkowski 2001). It has been observed that EGFRs are markedly overexpressed on a number of various epithelial cancers including ovarian cancers (Salomon et al. 1995). Therefore, it has been hypothesized that the binding of EGF to its receptor could be blocked to prevent receptor activation and thereby inhibit cell proliferation. They might be candidates for the development of novel therapeutics for ovarian cancer treatment (Maihle et al. 2002). The EGF signaling pathway is also considered to be an important mechanism for GnRH-I signaling because GnRH-I is known to reverse the effects of EGF by the activation of tyrosine phosphatases (Lee et al. 1991). Alpha T3-1 cells express EGF receptor and 2 1 respond to EGF stimulation with significant but transient ERK1/2 phosphorylation (Shah et al. 2003b). In addition to the anti-proliferative effect of GnRH-I by interfering with the EGF pathway mediating the mitogenic action in prostatic cancer cells (Dondi et al. 1996), the binding of GnRH-I agonists and antagonists to GnRH-I receptors inhibits the activation of MAPK and the expression of c-fos by EGF in gynecologic cancer cell lines (Grundker et al. 2000). Furthermore, GnRH-I inhibits MAPK activity induced by EGF in endometrial and ovarian cancer cell lines (Emons et al. 1998). Genistein, a protein tyrosine kinase (PTK) inhibitor, inhibits GnRH-induced FSH release and reduces GnRH-I-analog-stimulated MAPK activity in aT3-cells, suggesting that PTK signaling may be involved in GnRH action in the pituitary (Johnson et al. 1995; Reiss et al. 1997). However, the role of PTK remains to be determined in other tissues. 1.9 GnRH and the induction of apoptosis Apoptosis is a natural cellular process which directs programmed cell death to achieve homeostasis. Apoptosis is accompanied by changes in cell morphology such as chromatin condensation, the degradation of DNA, cell shrinkage and fragmentation of the cell nucleus, which distinguish apoptosis from the another form of cell death known as necrotic cell death. Cancer is considered as a disease of abnormal cell growth (Chao and Korsmeyer 1998; Minn et al. 1998). It has been shown that treatment with GnRH-I induces apoptosis in pituitary and prostate cancer cells (Rose et al. 2004; Kraus et al. 2006). In addition, it has been reported that GnRH-I analogs induce apoptosis in GnRH receptor-bearing 22 gynecologic cancers (Kleinman et al. 1994; Thompson 1995; Motomura 1998; Kim et al. 1999a; Grundker et al. 2000; Kang et al. 2000a; Kraus et al. 2006). However, the effect of GnRH-I analogs on apoptosis is still controversial (Motomura 1998; Kim et al. 1999a; Grundker et al. 2000; Kim et al. 2004a) and it might be due to the different cell lines. It has been demonstrated that GnRH-I increased Fas ligand (FasL) expression in reproductive tract tumors and GnRH-I analogs had no effect on the cell growth in Fas-negative cells (Imai et al. 1998a; Imai et al. 1998b). This accounts for the anti-proliferative effect through the Fas/FasL complex. However, different cell types display various sensitivity for Fas-induced apoptosis (Schulze-Osthoff et al. 1998). Although GnRH-I also increased the expression of Fas/FasL in cultured uterine leiomyoma cells, the effect of GnRH on the expression of Fas/FasL is still controversial (Huang et al. 2002a; Huang et al. 2002b; Wang et al. 2002). The caspases, existing as procaspases in cells, are also important mediators for apoptosis as well as Fas. These procaspases are activated by the cell death signals such as Fas activation. The two main apoptotic signals are mediated by a death receptor (caspase-8) and mitochondria (caspase-9), and they initiate to activate downstream caspases. Caspase 12 in endoplasmic reticulum (ER) can be activated by ER stress such as disruption of Ca 2 + homeostasis (Mehmet 2000; Nakagawa et al. 2000). Caspase 8 can be activated by Fas or TNF receptor activation. Alternatively, caspase 9 can be activated by the release of cytochrome c from mitochondria by apopototic triggers such as mutagens or ionizing radiation. Caspases 8 and 9 activate downstream capases such as caspases 3, 6, and 7 (Salvesen and Dixit 1997; Mehmet 2000). However, the role of Fas and caspases in ovarian cancer apoptosis by GnRH-I and II is not known, which provide a basis for future studies and promising new therapeutic approaches for ovarian cancer. 2 3 1.10 Clinical application of GnRH in cancer treatment Despite continueing efforts for ovarian cancer therapy, the techniques for early diagnosis and treatment by surgery and chemotherapy are not very effective. In addition, the responses have been variable and the treatments are not particularly effective. A number of treatment strategies are still in development. Recent findings suggest that hormone treatment including gonadotropin-releasing hormone (GnRH) has potential benefits for patients with ovarian cancer that is unresponsive to chemotherapy (Rzepka-Gorska et al. 2003). GnRH analogs produced favorable results when conventional therapy was supplemented for late-stage ovarian cancer treatment (Zidan et al. 2002). Increasing our understanding of GnRH signaling pathways may improve the efficacy of chemotherapy in using these agonists in ovarian cancer treatment. 1.11 Hypothesis GnRH-induced MAPK activation plays a role in anti-proliferative effect and apoptosis in ovarian cancer cells and the activation of transcriptional factors related to cellular responses. The activation of PKC/PKA pathway is involved in the GnRH-induced anti-proliferative effect in ovarian cancer cells. 2 4 1.12 Specific Objectives 1. To examine the effect of GnRH-I and-II on anti-proliferation and the activation of MAPKs (ERK1/2, JNK, p38) and transcription factors in ovarian cancer cells 2. To investigate the involvement of the activation of PKC/PKA pathway in GnRH-induced anti-proliferative effect in ovarian cancer cells. 3. To investigate whether Gai and Gas subunits are involved in GnRH-induced MAPK (ERK1/2) activation. 4. To elucidate the involvement of Gai and Gas subunits in GnRH-induced anti-proliferation. 5. To elucidate the involvement of GnRH-I receptor expression in GnRH-I and II signal transduction 6. To investigate the involvement of ERK1/2 and p38 MAPK in the induction of apoptosis by GnRH. 2 5 II. MATERIALS AND METHODS 2.1. Materials GnRH-I agonist, (D-Trp6)-GnRH (Triptorelin), phorbol 12-myristate 13-acetate (TPA), H-89, pertussis toxin (PTX), and 8-bromo-cAMP were purchased from Sigma-Aldrich Corp (Oakville, Canada). A native GnRH-II and GnRH-II analog, D-Arg(6)-Azagly(10)-NH2, were purchased from Peninsula laboratories (Belmont, CA). PD98059, a MAPK/ERK kinase (MEK) inhibitor, was purchased from New England Biolabs Inc. (Beverly, MA) and was dissolved in dimethyl sulfoxide (DMSO). SB203580 (Sigma-Aldrich Corp., St. Louis, MO), a pyridinyl imidazole that suppresses the activation of p38 MAPK and MAPKAP kinase-2 by inhibition of their phosphorylation was purchased and dissolved in DMSO. GF109203X, inhibitor of protein kinase C (PKC), and genistein, a inhibitor of protein tyrosine kinase, were purchased from EMD Biosciences, Inc. Calbiochem (San Diego, CA). Caspase-3 inhibitor (Z-DEVD-frnk) was purchased from EMD Biosciences, Inc. Calbiochem (San Diego, CA). 2.2 Cell culture The human epithelial ovarian cancer cell line OVCAR-3, an ovarian adenocarcinoma derived from the ascites of ovarian cancer patient, was kindly provided by Dr. T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA) or purchased from 2 6 ATCC (American Type Cell Culture, Manassas, VA). SKOV-3, another human ovarian cancer cell line, was purchased from ATCC (American Type Cell Culture, Manassas, VA). The cells were cultured in medium 199:MCDB 105 (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10 % FBS (Hyclone, Logan, UT), 100 U/ml penicillin G and 100 ug/ml streptomycin (Life Technologies, Inc., Rockville, MD) at 37 C° in a humidified atmosphere of 5 % C02-95 % air as previously described (Kang et al. 2000b; Choi et al. 2001c). Then, the cells were passaged with 0.06 % trypsin (1:250)/0.01 % EDTA (Life Technologies, Inc.) in Mg 2 + /Ca 2 + - free HBSS when they were confluent. The non-tumorogenic SV40 Tag-immortalized OSE-derived cell line, and IOSE-80 post crisis (IOSE-80PC), were cultured in the above mentioned culture conditions and used in the present study. 2.3. Immunoblot assay Immunoblot analysis was performed to investigate the effect of GnRH-II on the activation of MAPK as previously described (Kang et al. 2001b; Choi et al. 2002; Kim et al. 2004a). To investigate the effect of GnRH-I and II on p38 MAPK, ERK1/2 and JNK1/2 activation, cells were seeded at a density of 2 x 105 cells in 35 mm dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ) and cultured for 2 days. Then, the cells were washed once with the medium, and serum starved for 6 h prior to GnRH-II treatment. The cells treated with GnRH-I or II were washed once with ice-cold PBS and lysed in 100 ul of in ice-cold RIPA buffer (150 mM NaCl, 1 % Nondiet P-40, 0.5 % deoxycholate, 0.1 % SDS, 50 mM Tris (pH7.5) 1 mM PMSF, 10 ug/ml leupeptin, 100 jig/ml aprotinin). 2 7 The extracts were placed on ice for 10 min, collected into 1.5 ml tubes, and centrifuged for 10 min at 14,000 rpm. The supernatants were moved to new tubes and the protein concentration of supernatants was determined using Bradford assay (Bio-rad Laboratories). Thirty five pg of total protein was mixed with 6x sample buffer (75 mM Tri-HCl of pH 6.8, 15 % SDS, 0.15 % bromophenol blue, 15 % glycerol, 37.2 % 2-mercapthoethanol) and boiled for 10 min. The sample mixture was run on 10 % SDS-PAGE gels (acrylamide: bisacrylamide =29:1) in lx gel running buffer (25 mM Tris/250 mM glycine, pH 8.3/0.1 % SDS) at 100 V for 2.5 h and electrotransferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech Inc., Oakville, ON) at 100 V for 1.5 h. The membrane was immunoblotted using a rabbit polyclonal antibody for phosphorylated p38 MAPK (Biosource International Inc., Camarillo, CA) with protein molecular marker (New England Biolabs, Inc., Ontario). After washing three times with TBS-T (0.1 % Tween 20 in TBS) for 15 min, the signals were detected with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc.) and visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech Inc.). Alternatively, the membrane was probed with a mouse monoclonal p38 MAPK antibody (Biosource International Inc.), which detects total p38 MAPK levels. The membrane was immunoblotted using a mouse monoclonal antibody for phosphorylated ERK1/2 (New England Biolabs, Inc., ON) and a rabbit polyclonal antibody for JNK/SAPK (Biosource International Inc., Camarillo, CA) (Akagi et al. 2000; Lee et al. 2001) with protein molecular marker (New England Biolabs, Inc., ON). Alternatively, the membrane was probed with pan ERK1/2 (New England Biolabs, Inc., ON) and pan JNK/SAPK1 antibodies (Biosource International Inc.) using a rabbit polyclonal antibody, which detect 2 8 total ERK1/2 and JNK/SAPK1 level, respectively. The activity of ERK1/2 and JNK/SAPK was represented as a ratio of phosphorylated-MAPKs (P-MAPKs) to total-MAPKs (T-MAPKs). The intensity of signals was quantitated by densitometry (BioDocAnalyze, Biometra, Germany). To investigate the direct effect on p38 MAPK, SB203580 (1 uM), the inhibitor of p38 MAPK, was added for 20 min (Saklatvala et al. 1996), and then GnRH-II was added in a time dependent manner at 100 nM (Millar et al. 2001). To investigate the direct effect on ERK1/2, the cells were pretreated with 10 uM PD98059 for 1 h and then treated with 100 nM GnRH-II. To investigate the involvement of the GnRH-I receptor in GnRH-I and Il-induced ERK1/2 activation, the cells were pre-treated with antide (100 nM) for 15 min and then treated with GnRH-I or II (100 nM) for 10 min. Immunoblot analysis was carried out as previously described above. To explore the role of PKC activation in GnRH-I or Il-induced ERK1/2 activation, cells were pretreated with the PKC specific inhibitor, GF109203X (3 uM) for 30 min, followed by treatment with GnRH-I or -II (100 nM). To block the PKA pathway, the cells were pre-treated with 1 uM H-89 for 1 h and then treated with GnRH-I or II (100 nM) for 10 min. Cells were treated with or without 50 ng/ml PTX a specific inhibitor of G t t i , for 16 h prior to GnRH-I or II treatment or 1 uM mastoparan for 10 min. To identify the expression of GnRH-I receptor, OVCAR-3, SKOV-3 and aT3-cells were cultured and proteins from these cells were electrotransferred to a nitrocellulose membrane as described above. The membrane was probed with a mouse monoclonal 2 9 GnRH-I receptor antibody (Lab Vision Corp. Fermont, CA). The membrane was immunoblotted using a Actin antibody (Santa Cruz, CA). To investigate the effect of GnRH on EGF receptor activation, the cells were treated with EGF (0.1 ng/ml) in the presence of GnRH-I and II for 30 min. and The membrane was immunoblotted using a rabbit polyclonal antibody for phosphorylated EGF receptor and a rabbit polyclonal antibody for EGF receptor (New England Biolabs, Inc., ON). Alternatively, the membrane was probed with EGF receptor (New England Biolabs, Inc., ON) using a rabbit polyclonal antibody, which detect total EGF receptor level, respectively. 2.4. In vitro M A P K Assay OVCAR-3 cells were seeded at a density of 4 x 105 cells in 60 mm dishes (Corning, Corning Laboratory Sciences Co. NY) and cultured for 2 days. After treatment with GnRH-II in the presence or absence of PD98059, protein extracts were prepared under the conditions described above. Cellular protein (200 pg) was immunoprecipitated with an immobilized phospho-ERKl/2 MAPK monoclonal antibody. The in vitro MAPK assay was performed using an Elk-1 fusion protein as a substrate for activated MAPK, according to the manufacturer's suggested procedure (New England Biolabs, Inc.). 2.5. Thymidine incorporation assay Cell proliferation was monitored using [3H]thymidine incorporation as previously described (Wang et al. 1996; Choi et al. 2001a; Choi et al. 2001b; Kang et al. 2001a). To 3 0 investigate the effect of p38 MAPK activation on GnRH-induced anti-proliferative effect, 2 x 10 4 cells were seeded in 24-well plates and cultured in 0.5 ml medium with 10% FBS and antibiotics for 24 h as prescribed above. After 24 h preincubation, cells were washed once with medium and serum starved overnight before treatment with GnRH-II. The medium was changed with 2% FBS as previously described (Di Simone et al. 1996; Wang et al. 1996) after 24 h. On the day of treatment, GnRH-II was diluted appropriately with medium and the cells were treated for 2, 4, and 6 days every 12 h. The medium was changed after 24 h incubation. In order to block the effect of p38 MAPK, the cells were pretreated with SB203580 (1 uM), followed by treatment with GnRH-II for 6 days. Control culture was treated with vehicle. To investigate if the activation of ERK1/2 is involved in the GnRH-II-induced anti-proliferative effect, 2 x 104 OVCAR-3 cells were plated in 24-well dishes in 0.5 ml medium as described above. After preincubation for 24 h, the medium was changed with 10% FBS and antibiotics and GnRH-II was appropriately diluted with medium and the cells were treated with a final concentration of 100 nM GnRH-I. Cells were treated for 4 days with medium changes every 24 h. In order to block the activation of ERK1/2, the cells were pretreated with 10 uM PD98059 for 1 h, followed by the addition of GnRH-II (100 nM final concentration) or vehicle. In order to block the activation of GnRH-I receptor or PKC, 4 x 104 OVCAR-3 or SKOV-3 cells were plated in 24-well dishes in 0.5 ml of medium as described above. After 24 h of incubation the cells were pretreated with antide (100 nM), a inhibitor of GnRH-I or GF109203X (0.03, 0.3 or 3 uM), a inhibitor of PKC for 15 min, followed by treatment with a final concentration of 100 nM GnRH-I (Triptorelin) or II agonist for 24 h. The cells 3 1 were transfected with siRNA targeting the GnRH-I receptor 24 h prior to treatment with GnRH-I or -II. To explore the role of PKA pathway and Gai protein, OVCAR-3 cells were pretreated with H-89 (1 pM) or PTX (50 ng/ml), followed by treatment with GnRH-I and II. In order to block EGF signal pathway and PTK activity, OVCAR-3 cells were pretreated with 1 nM AG1478 or 100 pM genistein, followed by treatment with GnRH-I (Triptorelin) and II agonist. Control cultures were treated with vehicle. Following treatment, the cells were then incubated with medium containing 1 pCi [ H]thymidine (0.5 Ci/mmol; Amersham Pharmacia Biotech Inc.) and collected after 6 h incubation. The cells were washed three times with PBS and precipitated with 0.5 ml 10 % trichloroacetic acid for 20 min at 4 C°. The precipitate was washed in methanol twice and solubilized in 0.5 ml 0.1 N sodium hydroxide. The radioactivity was measured in the Tri-Carb Liquid Scintillation Analyzer (Model 2100TR; Packard Instrument Com., Meriden, CT). 2.6. DNA fragmentation assay The amount of DNA fragmentation was measured using the cell death detection ELISA kit according to the manufacturer's instructions (Roche Applied Science, Laval, QC) to quantify the induction of apoptosis. The cells were placed in each well of 24-well plates at lx 104 concentrations (Choi et al. 2001b; Choi et al. 2001d). After treatment with GnRH-II (100 nM), the conditioned media were collected and cells were washed with PBS, and 0.1 ml lysis buffer was added. Following 15 min incubation on 3 2 ice, apoptotic cells in cell lysates and conditioned media were assayed for DNA fragments according to the manufacturer's protocol using ELISA kit. The same amount (1 ug) of cell lysate was used and the amount of DNA fragmentation was measured at 405 nm. The control was treated with vehicle. 2.7. Transient transfection assay AP-l-TA-Luc vector (Clonetech, Polo Alto, CA) was designed to monitor the induction of AP-1 signal transduction pathway and contained the firefly luciferase (luc) gene from Photinus pyralis. AP-l-TA-Luc contains an AP-1 response element, located upstream of the minimal TA promoter, the TATA box from the herpes simplex virus thymidine kinase promoter (PTA)- Located downstream of P T A is the firefly luciferase reporter gene (luc). The pTAL-Luc was used as a negative control to determine the background signals associated with the cell lysates. The enhancerless pTAL-Luc contains HSV-TK upstream of the Luciferase coding sequence. Transient transfection was performed using Lipofetamine™ 2000 transfection reagent (Invitrogen Life Technologies Carlsbad, CA), following the manufacturer's protocol. The OVCAR-3 cells were cotransfected 1 p,g plasmid with AP-1 per well. Five hours after transfection, the medium was changed to 10 % FBS/DMEM, and 24 h after transfection, GnRH-II (100 nM) was treated for 6 h before harvest. Cellular lysates were collected with 200 ul cell lysis buffer and immediately assayed for luciferase activity with the Luciferase Assay kit (Promega, Madison, WI) using a Lumat LB9507 luminometer (EG&G, Berthold, Germany). 6-glactosidase activity was also measured and normalized for 3 3 varying transfection efficiencies. AP-1 activity was calculated as luciferase activity/ B-glactosidase activity. A pTA-Basic vector was included as a control in the transfection experiments. 2.8. MTT assay Cell viability was estimated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] (Sigma-Aldrich Corp., St. Louis, MO) assay. OVCAR-3 cells were seeded onto 96-well dishes. The MTT colorimetric assay was performed to detect tumor cell viability after 96 h of incubation (Mohseni et al. 2004). The cells were incubated at 37 °C with 50 pi MTT solution [2 mg/ml in phosphate-buffered saline (PBS)] for 4 h. The supernatants were removed and the cells were solubilized in DMSO (200 pi) for 30 min. The optical density at 570 nm was determined using an ELISA reader (Fisher Scientific Ltd., Ottawa, ON). 2.9. In vitro transfection with siRNAs targeting the GnRH-I receptor GnRH-I receptor siRNAs (small interfering RNAs) were synthesized by Invitrogen (Burlington, ON), and the sequences were as follows: hGnRH-I-RS (5'-GCUCU CUGCG ACCUU UAAU-3'), and hGnRH-I-RA (5'-AUUAA AGGUC GCAGA GAGC-3'). In addition, a nonspecific scrambled siRNA was purchased from Invitrogen to be used as controls and the sequences were as follows: S (5'-GCUUC CGAGC CUUUC UAAU-3') and A (5'-AUUAG AAAGG CUCGG AAGC-3'). The siRNA transfection was performed 3 4 according to the manufacturer's suggested procedure (Invitrogen). In brief, 1 day before transfection, 2 x 104 cells per well of a 6-well plate were seeded in 2 ml of culture medium or 4x 104 cells per well of a 24-well plate were seeded in 500 ul. The cells were transfected with GnRH-I receptor (12.5 pM final concentration) or scrambled siRNA with 1 ul Lipofectamine 2000 reagent, following the manufacturer's protocol. Then, the cells were challenged with GnRH-I or -II for 24 h. To monitor the siRNA transfection efficiency, immunoblot analysis was performed using GnRH-I receptor antibody (Neomarkers, Fremont, CA). 2.10. Flow cytometry Apoptotic and necrotic cells expose phosphatidylserine (PS), which is normally present on the inner cell membrane leaflet to the outer leaflet, allowing Annexin V to bind to PS at the cell surface (Vermes et al. 1995). To investigate if GnRH-I and II induce apoptosis in ovarian cancer cells, 2 x 10 5 OVCAR-3 cells were seeded in 100 mm dishes and cultured in 10 ml medium with 10% FBS and antibiotics for 24 h as prescribed above, and GnRH-I (Triptorelin) and II agonist were diluted appropriately with medium and the cells were treated for 2, 4, and 6 days every 12 h. The cells were incubated with Annexin V-FITC and 7-amino actinomycin D (7AAD, BD-Biosciences, San Jose, CA) for a 10 min at room temperature. Untreated and treated cells were collected after 10 min incubation by trypsinization and centrifugation at 500 x g for 5-10 minutes at room temperature. Cells were washed and resuspended in ice cold PBS and pelleted by centrifugation. Binding buffer was then added to each sample at a concentration of approximate 1 x 106 cells/ml. 3 5 C e l l s were then move to 12 x 75 mm tube and Annexin V was added at a concentration of 1 x 106 cells/10 pi and 7AAD was added at a concentration o f 1 x 106 cells/20 pi, and incubated in the dark f o r 10 minutes at room temperature. Incubation with Annexin V and 7 AAD was performed according to the manufacturer's suggested procedure. Annexin V-FITC is detected as green fluorescence and 7 AAD is detected as red fluorescence. Samples were then analyzed within one hour by flow cytometry (FACScalibur, Becton Dickinson and Co., Mountain View, CA) with Cell Quest software (BD-Biosciences, San J o s e , CA) and evaluated based on the percentage o f the population o f cells staining low or high f o r Annexin V (apoptotic cells) and 7 AAD (necrotic cells). To investigate the role of p38 MAPK activation on GnRH-induced apoptosis, the cells were pretreated with SB203580 (1 pM), followed by treatment with GnRH-I and II f o r 2, 4, and 6 days prior to GnRH treatment to block the effect o f p38 MAPK; control culture was treated with vehicle. To investigate i f the activation of ERK1/2 is involved in the GnRH-induced apoptosis effect, cells were cultured as described above. GnRH-I and II were appropriately diluted with medium and the cells were treated with a final concentration o f 100 nM. Cells were treated f o r 2, and 6 days. In order to block the activation o f ERK1/2, the cells were pretreated with 10 pM PD98059 for 1 h, followed by the addition of GnRH or vehicle. 2.11. TUNEL assay To confirm the induction of apoptosis by GnRH, end-labeling o f exposed 3' OH ends o f DNA fragments was undertaken with TUNEL (TdT-mediated dUTP-X nick end 3 6 labeling) in situ cell death detection kit AP (Roche Diagnostics). Treated cells were fixed with 4% paraformaldehyde solution and incubated in a 0.1% Triton permeabilization solution on ice according to the manufacturer's instructions. Cell were then rinsed twice in phosphate-buffered saline (PBS) and reacted with 50 ul of the TUNEL reaction mixture for 60 min in a dark, humidified chamber at room temperature. Cells were then rinsed three times in PBS and incubated for a further 30 min with 50 ul of the Converter-POD (Roche Diagnostics, Castle Hill, Australia) followed by 10 min with DAB. This procedure ensures the detection of TUNEL labeled cells. 2.12. Statistical analysis Data were subjected to ANOVA, and differences were determined by Tukey's multiple comparison test. Each experiment was repeated three times with duplicate or triplicate. Data are shown as means of three individual experiments and presented as the mean ± S.D. Expression level of MAPK are shown as fold changes compared with control levels. In DNA fragmentation assay, values are expressed as the percentage of apoptosis compared with untreated control value and are the mean ± S.D. of three individual experiments. [3H]thymidine incorporation assay was presented as the percentage of growth compared with control level. The data from three separate experiments are presented as the mean ± S.D. P<0.05 was considered statistically significant. 3 7 III. RESULTS 3.1 Roles of the GnRH-I receptor and protein kinase C pathway in GnRH signaling 3.1.1 Effect of a GnRH-I antagonist on GnRH-I and II-induced ERK1/2 activation To examine the effects of GnRH-I and II on ERK1/2 activation in OVCAR-3 and SKOV-3cells, ovarian adenocarcinoma, were treated with GnRH-I or II (100 nM) for 10 min and the amount of ERK1/2 phosphorylation was measured. The phospho-specific antibody to ERK1/2 was employed to estimate alterations in ERK1/2 activity induced by GnRH-I and II treatment. It was found that GnRH-I and II induced a four-fold increase in phosphorylation of ERK1/2 (Fig. 7A). To determine the role of the GnRH-I receptor in ERK1/2 activation, cells were pretreated with antide, a GnRH-I receptor antagonist, (100 nM) for 15 min, followed by GnRH-I or II for 10 min. Results showed that pretreatment with antide significantly attenuated GnRH-I and II-induced ERK1/2 activation, whereas antide alone did not alter the phosphorylation of ERK 1/2 as seen in Fig. 7A. As discussed in more detailed below, transfection with GnRH-I receptor siRNA blocked the activation of ERK1/2 induced by GnRH-I or II, and scrambled RNA had no a significant effect on GnRH-I or-II-induced activation of ERK1/2 (Fig. 9B). 3 8 3.1.2 GnRH-I and II activate ERK1/2 in a PKC-dependent manner To determine the role of the protein kinase C (PKC) pathway in GnRH-I and -Il-induced ERK1/2 activation, cells were pretreated with GF109203X, a specific PKC a, p and y isoform inhibitor (Alessi 1997), followed by GnRH-I or II. As shown in F i g . 8A, activation of ERK1/2 by GnRH-I and -II was blocked by GF109203X, indicating that GnRH-induced ERK1/2 activation is PKC-dependent. Treatment with a PKC-activating phorbol ester (TPA) induced the phosphorylation of ERK 1/2. However, pretreatment with GF109203X for 15 min completely abolished the activation of ERK1/2 by TPA. 3.1.3 The anti-proliferative effects of GnRH-I and II are mediated by the GnRH-I receptor To determine the role of GnRH-I or II in ovarian cancer cells, OVCAR-3 and SKOV-3 cells were treated with GnRH-I or II (100 nM) for 24 hours (h). Cell proliferation was measured by a thymidine incorporation assay. Antide, which is a GnRH-I receptor antagonist, was used to block the action of GnRH-I or II in cell proliferation. As seen in F i g . 7B, decreased cell proliferation was observed following treatment with GnRH-I (83.0 ± 3.5 % in OVCAR-3 cells and 75.7 ± 4.2 % in SKOV-3 cells) and GnRH-II (84.3 ± 1.5% in OVCAR-3 cells and 65.0 ± 6.6% in SKOV-3 cells). These results are consistent with the finding that GnRH-I and II have a growth inhibitory effect on normal and neoplastic ovarian surface epithelium cells (Choi et al. 2001b; Schally et al. 2001). Co-treatment with antide reversed the anti-proliferative effects of GnRH-I and II in SKOV-3 and OVCAR-3 3 9 cells (Fig. 7B), suggesting that GnRH-II-induced anti-proliferation may be mediated by the GnRH-I receptor. To confirm the role of the GnRH-I receptor on the effects of GnRH-I or GnRH-II, OVCAR-3 and SKOV-3 cells were transfected with siRNAs targeting the GnRH-I receptor. The efficiency of siRNA-mediated down-regulation of the GnRH-I receptor was monitored by immunoblot analysis which found that treatment with GnRH-I receptor siRNA resulted in a significant decrease in GnRH-I receptor expression at the protein level (Fig. 9 A ) . In addition, siRNA treatment completely reversed the anti-proliferative effects of GnRH-I or -II (Fig. 9 C ) . Therefore, these two observations indicate that the GnRH-I receptor is required for both GnRH-I and -II-induced growth inhibition in these ovarian cancer cells. 3.1.4 Effect of a PKC inhibitor on the anti-proliferative actions of GnRH-I and II To demonstrate the relevance of the PKC pathway in the proliferation of ovarian cancer cells, SKOV-3 and OVCAR-3 cells were treated with GnRH-I, GnRH-II (100 nM) or vehicle for 24 h in the absence or presence of the PKC inhibitor, GF109203X (0.03 or 0.3 pM). As seen in Fig. 8B, treatment with GnRH-I or II resulted in a significant decrease of proliferation in these cells (76.6 % ~ 79.7 %). In the presence of 0.03 pM GF109203X, the anti-proliferative effects of GnRH-I or -II were reduced on SKOV-3 cells (GnRH-I: 84.8 ± 2.6% and GnRH-II: 94.6 ± 6.7%) while the inhibitory effects of GnRH-I and II on OVCAR-3 cell proliferation were blocked by GF109203X at both concentrations tested. In SKOV-3 cells, no significant difference (P>0.05) was seen in cell proliferation for samples 4 0 treated with GnRH alone compared to samples treated with GnRH and GF109203X (0.3 pM). Similarly, no significant difference (P>0.05) was seen in cell proliferation for samples treated with GnRH-I versus samples treated with GnRH-I and GF109203X (0.03 uM) Moreover, co-treatment with GF109203X and GnRH did not show an additive effect in inhibiting proliferation compared to GF109203X or GnRH alone. This indicates that GnRH-induced signaling leading to the inhibition of proliferation may interact with the signaling pathways affected by GF109203X, and thus that the PKC pathway may mediate the anti-proliferative effects of GnRH-I and II in these cells. It is of interest that cell proliferation was dose-dependently decreased at both concentrations of GF109203X in the absence of GnRH-I or -II: 92 ± 8.8 % at 0.03 uM and 68.5 ± 2.2 % at 0.3 uM in SKOV-3 cells, and that the inhibitory effect on cell proliferation of GF109203X alone was not observed at both concentrations of GF109203X on the OVCAR-3 cells. This discrepancy might result from differences in ovarian cell line membrane permeability to GF109203X. 4 1 A S K O V - 3 O V C A R - 3 T - E R K 1 / 2 P - E R K 1 / 2 T - E R K 1 / 2 P - E R K 1 / 2 6.00 s.oo 4.00 3.00 2.00 1 .OO O.OO Control G n R H - I G n R H - I I Antide Antide + Antide+ G n R H - I G n R H - I I m SKOV-3 O OVCAR-3 b wn i n . I B Control G n R H - I G n R H - I I Antide Antide + Antide+ G n R H - I GnRH-II • S K O V - 3 • O V C A R - 3 G n R H - I G n R H - I I Antide Antide + Antide-t-G n R H - I GnRH-II F i g u r e 7 . E f f e c t o f G n R H - I a n d I I o n E R K 1 / 2 a c t i v a t i o n a n d g r o w t h i n h i b i t i o n i n t h e p r e s e n c e o r a b s e n c e o f a n t i d e . ( A ) O V C A R - 3 a n d S K O V - 3 c e l l s w e r e p r e t r e a t e d w i t h a n t i d e ( 1 0 0 n M ) f o r 1 5 m i n p r i o r t o t r e a t m e n t w i t h G n R H - I o r I I ( 1 0 0 n M ) f o r 1 0 m i n . T h e T - E R K 1 / 2 a n d P - E R K 1 / 2 l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y a n d P - E R K 1 / 2 l e v e l s a r e e x p r e s s e d a s a f o l d c h a n g e r e l a t i v e t o b a s a l l e v e l . D a t a w e r e a n a l y z e d b y A N O V A f o l l o w e d b y T u k e y ' s m u l t i p l e c o m p a r i s o n t e s t . V a l u e s i n t h e f i g u r e a r e p r e s e n t e d a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , P O . 0 5 vs. c o n t r o l ; b , P O . 0 5 vs. t r e a t m e n t o f G n R H - I o r G n R H - I I a l o n e . (B) T h e c e l l s w e r e t r e a t e d w i t h G n R H - I o r I I ( 1 0 0 n M ) i n t h e p r e s e n c e o r a b s e n c e o f a n t i d e ( 1 0 0 n M ) . A [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y w a s p e r f o r m e d t o q u a n t i f y S - p h a s e g r o w i n g c e l l s . T h e d a t a f r o m t h r e e i n d i v i d u a l e x p e r i m e n t s a r e p r e s e n t e d a s t h e m e a n ± S . D . . a , P < 0 . 0 5 vs. c o n t r o l . 4 2 S K O V - 3 O V C A R - 3 T - E R K 1 / 2 P-ERK1/2 | T - E R K 1 / 2 g P-ERK1/2 GI G i l G F G F + G I GF+II T P A G F + T P A mm SKOV-3 CD O-VC:AR.-3 J G fcl GI G i l G F GF+GI GF+II • GF109203X BGF109203X+GnRH-I • GF109203X + GnRH-II OGF109203X 0 GF109203X +GnRH-I • GF109203X +GnRH-II 0.03 0.3 GF109203X (|iM) GI G i l 0.03 0.3 GF109203X (uM) GI G i l F i g u r e 8 . E f f e c t o f a P K C i n h i b i t o r o n G n R H - I - a n d - I l - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d g r o w t h i n h i b i t i o n . ( A ) S K O V - 3 o r O V C A R - 3 c e l l s w e r e p r e t r e a t e d w i t h G F 1 0 9 2 0 3 X ( G F , 3 u M ) f o r 3 0 m i n , f o l l o w e d b y s t i m u l a t i o n w i t h G n R H - I ( G I ) , - I I ( G i l ) ( 1 0 0 n M ) a n d T P A ( 1 6 0 n M ) f o r 1 0 m i n . C o n t r o l c e l l s w e r e t r e a t e d w i t h v e h i c l e . T - E R K 1 / 2 a n d P - E R K 1 / 2 l e v e l s w e r e a n a l y z e d b y a n i m m u n o b l o t a s s a y . V a l u e s i n t h i s f i g u r e a r e p r e s e n t e d a s t h e m e a n + S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , P O . 0 5 v s . c o n t r o l ; b , P O . 0 5 v s . t r e a t m e n t w i t h G n R H - I o r II a l o n e ; c , P < 0 . 0 5 v s . T P A a l o n e . ( B ) T h e c e l l s w e r e t r e a t e d w i t h G n R H - I o r - I I ( 1 0 0 n M ) f o r 2 4 h i n t h e p r e s e n c e o r a b s e n c e o f G F 1 0 9 2 0 3 X ( 0 . 0 3 o r 0 . 3 u M ) . A [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y w a s p e r f o r m e d t o q u a n t i f y S - p h a s e g r o w i n g c e l l s . D a t a a r e s h o w n a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , P O . 0 5 v s . c o n t r o l ( 1 0 0 % ) . 43 A S K O V - 3 O V C A R - 3 c C GI G i l h G I R h G I R h G I R sc + GI sc + G i l + GI + G i l Figure 9. Effect of GnRH-I receptor siRNA transfection on GnRH-I and II-induced growth inhibition. (A) The cells were transfected with human GnRH-I receptor siRNA (hGIR) or scrambled siRNA (sc) for 1 day and GnRH-I receptor levels were monitored by immunoblot assay. (B) The activation of E R K 1/2 induced by GnRH-I and II was monitored after hGIR transfection. (C) To evaluate whether hGIR blocks GnRH-I and II- induced anti-proliferation, cells were transfected with hGIR and treated with GnRH-I and II (100 nM) for 24 h. Cell growth was assessed by thymidine incorporation assay and data are presented as the mean ± S.D. of three individual experiments, a, PO.05 vs. control; b, PO.05 vs. treatment with GnRH-I or GnRH-II alone; b, PO.05 vs. treatment with GnRH-I or II alone. 44 3.2 Role of ERK1/2 in mediating the GnRH-II-induced inhibition of ovarian cancer cell proliferation 3.2.1 Effect of GnRH-II on ERK1/2 activation To investigate the activation of ERK1/2 in ovarian cancer cell lines and immortalized OSE-derived cell lines (IOSE), the cells were treated with GnRH-II (100 nM) in a time-dependent manner. The phosphorylation of ERK1/2 was examined using antibodies targeting phosphorylated-ERKl/2 (P-ERK1/2) and total-ERKl/2 (T-ERK1/2, activated plus inactivated forms). Treatment with GnRH-II (100 nM) activated ERK1/2 in OVCAR-3, SKOV-3, IOSE-80PC, and IOSE-80 cell lines in a time-dependent manner (Fig. 10A). These results indicate that GnRH-II activates ERK 1/2 in both ovarian carcinomas and IOSE cell lines. The regulatory pattern of ERK1/2 activity in these experiments seems to differ among cell types. GnRH-II stimulated the phosphorylation of ERK1/2 at 10 and 20 min in OVCAR-3 cells, whereas GnRH-II significantly activated p-ERKl/2 in differing time-dependent manners in other cell lines. Moreover, a biphasic effect of GnRH-II on P-ERK1/2 activity was observed in IOSE-80 and IOSE-80PC cells, suggesting that the mechanism of action of GnRH-II in inducing the activation of ERK1/2 may differ among cell types. To quantify the GnRH-II-induced ERK 1/2 activity, three independent immunoblot experiments were performed in OVCAR-3 cells and the levels T-ERK1/2 and P-ERKl/2 were analyzed. As seen in Fig. 10B, GnRH-II activated ERK1/2 significantly in OVCAR-3 cells and a maximal level of ERK1/2 activation was observed at 10 min. To further elucidate the direct effect of GnRH-II on the activation of ERK1/2 through 45 MEK1/2, OVCAR-3 cells were pretreated with the MEK inhibitor PD98059 (10 pM), followed by treatment with 100 nM GnRH-II for 10 min. As seen in Fig. 11 A, pretreatment with PD98059 attenuated GnRH-II-induced phosphorylation of ERK1/2, while ERK1/2 activity was at a similar low level in control cells (no GnRH-II treatment) or cells treated with DMSO (vehicle) or PD98059 alone. Phosphorylation levels of ERK1/2 are expressed as a relative fold change in untreated and treated GnRH-II groups compared to their relative control groups (Fig. 11B). The comparison of these shows a significant difference (P<0.05) between GnRH-II and GnRH-II with PD98059. 3.2.2 JNK/SAPK1 activation by GnRH-II in ovarian cancer cells To determine the activation of JNK/SAPK1 by GnRH-II, we treated OVCAR-3 cells with 100 nM GnRH-II in a time-dependent manner. In contrast with the activation of ERK1/2, no difference was observed in P-JNK/SAPK1 levels following treatment with GnRH-II (Fig. 12A). In addition, GnRH-II had no effect on the activation of JNK/SAPK1 in SKOV-3, IOSE-80, and IOSE-80PC cells (Fig. 12B). The functionality of the phospho-JNK/SAPK1 MAPK antibody was verified on TE671 (human brain tumor) cells in previous report (Yeung et al. 2005) and found that GnRH-II phosphorylates JNK/SAPK1 MAPK in this cell line after 60 min treatment. It is of interest to note that the basal level of P-JNK/SAPK1 in this cells were high, suggesting that JNK/SAPK1 is already activated in these cells but there is no good explanation for this observation. 46 3.2.3 GnRH-II induced activation of ERK1/2 does not mediate the activation of Elk-1 To investigate whether GnRH-II-induced activation of ERK1/2 leads to phosphorylation of Elk-1 in vitro as a down-stream target of MAPK, the cells were treated with GnRH-II (100 nM) for 10 min in the presence or absence of 10 uM PD98059 for 1 h. As shown in Fig. 13A, treatment with GnRH-II resulted in an increase in Elk-1 phosphorylation. Treatment with DMSO (vehicle) had little effect on Elk-1 phosphorylation, and PD98059 alone reduced Elk-1 phosphorylation compared to that observed in untreated control samples. Despite this inhibitory effect of PD98059 alone on 'basal' Elk-1 phosphorylation, the effect of GnRH-II also appeared to be reduced by pre-treatment with PD98059. To verify this, the experiment was repeated two more times. Results as in Fig. 13 A were quantified and analyzed for statistical significance. Phosphorylation levels of Elk 1/2 are expressed as a relative fold change in untreated and treated GnRH-II groups compared to their relative control groups (Fig. 13B). There was no significant difference (P>0.05) in the fold-increase in Elk-1 pshosphorylation between samples treated with GnRH-II or with GnRH-II with PD98059. 3.2.4 GnRH-II inhibits the proliferation of ovarian cancer cells To determine the role of GnRH-II in ovarian cancer, we further examined the effect of GnRH-II on proliferation by thymidine incorporation and MTT assays in OVCAR-3 cells. The cells were treated for 4 days with 100 nM GnRH-II following 24 h culture. As 4 7 illustrated in Fig. 14, treatment with GnRH-II (100 nM) inhibited cell proliferation in OVCAR-3 cells as assessed by thymidine incorporation assay. To confirm the anti-proliferative effect of GnRH-II, cellular viability was measured by MTT assay. Treatment with GnRH-II (100 nM) resulted in a significant reduction in cell viability after 4-days of treatment (Fig. 15). Furthermore, to elucidate the relevance of ERK1/2 activation in the proliferation of ovarian cancer cells, we challenged OVCAR-3 cells with 10 uM PD98059. Pretreatment with PD98059 (10 uM) completely abolished the anti-proliferative effect induced by GnRH-II (Fig. 14), suggesting that the MEK/MAPK pathway mediates the anti-proliferative effect of GnRH-II in ovarian cancer cells. 48 A OVCAR-3 T-ERK1/2 P-ERK1/2 SKOV-3 T-ERK1/2 P-ERK1/2 IOSE-80PC T-ERK1/2 P-ERK1/2 IOSE-80 T-ERK1/2 P-ERK1/2 1 1 1 i i n i i n i i i i i r i W i i i l i i i t ^jj^wns^^ ^ S ' ^ ^ ^ j i J i i . MiBftiiffiiiiliBfcf- • : J^HIfl^ H^ih%i' • M i f f WimM^^- • - ^ ^ g a a t ^ ^ ^ f e . ' r"'  • • • ' ' • ...... ... ... .. ... mmrni » t Control 2 10 20 30 60 90 120 (min) Treatment with GnRH-II (100 nM) 49 B W 0.0 Control 2 5 10 20 30 60 90 120 Treatment with GnRH-II (min) Figure 10. Effect of GnRH-II on ERK1/2 in ovarian adenocarcinomas and IOSE cell lines. (A) OVCAR-3, SKOV-3, IOSE-80PC and IOSE-80 cells were treated with GnRH-II (100 nM) in a time-dependent manner. The total- (T-ERK1/2) and phosphorylated-ERKl/2 (P-ERK1/2) levels were analyzed by immunoblot assay. (B) EKR.1/2 level of OVCAR-3 cells is expressed as a relative fold change to basal level. Data were analyzed by ANOVA followed by Tukey's multiple comparison test. Values are represented as the mean ± S.D. of three individual experiments, a, P<0.05 vs. control. 50 Control G n R H - I I D M S O PD98059 G n R H - I I + PD98059 B 3 © I § I i i 8 2 o PD98059 + PD98059 G n R H - I I treatment F i g u r e 1 1 . E f f e c t o f G n R H - I I o n E R K 1 / 2 a c t i v a t i o n i n t h e p r e s e n c e o r a b s e n c e o f P D 9 8 0 5 9 . ( A ) O V C A R - 3 c e l l s w e r e p r e t r e a t e d w i t h 1 0 u M P D 9 8 0 5 9 f o r 1 h , f o l l o w e d b y t r e a t m e n t w i t h 1 0 0 n M G n R H - I I f o r 1 0 m i n . A c o n t r o l w a s t r e a t e d w i t h v e h i c l e . T h e t o t a l - E R K l / 2 a n d p h o s p h o r y l a t e d - E R K l / 2 l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . E R K 1 /2 l e v e l i s e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e t o b a s a l l e v e l . V a l u e s a r e r e p r e s e n t e d a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s . ( B ) G n R H - I I - i n d u c e d E R K 1 / 2 p h o s p h o r y l a t i o n l e v e l i s e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e , a , G n R H - I I ( P O . 0 5 ) vs. c o n t r o l . ; b , G n R H - I I ( P O . 0 5 ) vs. G n R H - I I - t r e a t e d g r o u p w i t h P D 9 8 0 5 9 . 51 A Control 5 10 20 30 60 120 180 240 (min) Control 2 5 10 20 30 60 90 120 Treatment with GnRH-II (min) 52 B S K O V - 3 IOSE-80 IOSE-: Control 5 10 20 30 60 (min) T r e a t m e n t w i t h G n R H - I I (100 n M ) F i g u r e 12. E f f e c t o f G n R H - I I o n t h e a c t i v a t i o n o f J N K / S A R K 1 i n o v a r i a n a d e n o c a r c i n o m a s a n d I O S E c e l l l i n e s . ( A ) J N K 7 S A P K 1 l e v e l o f O V C A R - 3 c e l l s i s e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e t o b a s a l l e v e l . D a t a w e r e a n a l y z e d b y A N O V A f o l l o w e d b y T u k e y ' s m u l t i p l e c o m p a r i s o n t e s t . V a l u e s a r e r e p r e s e n t e d a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , P < 0 . 0 5 vs. c o n t r o l . ( B ) S K O V - 3 , I O S E - 8 0 P C a n d I O S E - 8 0 c e l l s w e r e t r e a t e d w i t h G n R H - I I ( 1 0 0 n M ) i n a t i m e - d e p e n d e n t m a n n e r . T h e t o t a l - ( T - J N K / S A P K 1 ) a n d p h o s p h o r y l a t e d - J N K / S A P K l ( P - J N K / S A P K 1 ) l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . 53 Control GnRH-II D M S O PD98059 GnRH-II + P D 98059 B 3 i i S S 2 s 0» f i t l 1 - PD98059 + PD98059 G n R H - I I treatment F i g u r e 1 3 . E f f e c t o f G n R H - I I i n t h e a b s e n c e o r p r e s e n c e o f P D 9 8 0 5 9 o n E l k - 1 p h o s p h o r y l a t i o n . ( A ) T o e v a l u a t e w h e t h e r G n R H - I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n l e a d s t o t h e a c t i v a t i o n o f E l k - 1 in vitro, O V C A R - 3 c e l l s w e r e p r e t r e a t e d w i t h 1 0 p M P D 9 8 0 5 9 f o r 1 h , f o l l o w e d b y t r e a t m e n t w i t h G n R H - I I ( 1 0 0 n M ) f o r 1 0 m i n . C e l l u l a r p r o t e i n ( 2 0 0 p g ) w a s i m m u n o p r e c i p i t a t e d w i t h a n i m m o b i l i z e d p h o s p h o - E R K l / 2 M A P K m o n o c l o n a l a n t i b o d y . T h e in vitro M A P K a s s a y w a s p e r f o r m e d u s i n g a n E l k - 1 f u s i o n p r o t e i n a s a s u b s t r a t e f o r a c t i v a t e d M A P K . T h e p h o s p h o r y l a t i o n s t a t e o f E l k - 1 w a s a n a l y z e d b y i m m u n o b l o t a s s a y u s i n g a s p e c i f i c a n t i b o d y f o r p h o s p h o - E l k - 1 . A c o n t r o l w a s t r e a t e d w i t h v e h i c l e . V a l u e s r e p r e s e n t t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s ( n = 3 ) . ( B ) P h o s p h o r y l a t i o n l e v e l s o f E l k - 1 a r e e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e i n u n t r e a t e d a n d t r e a t e d G n R H - I I g r o u p s c o m p a r e d t o t h e i r r e l a t i v e c o n t r o l g r o u p s ( P > 0 . 0 5 ) . 5 4 Control GnRH-II DMSO PD98059 GnRH-II + PD98059 Treatment F i g u r e 1 4 . Effect of PD98059 on GnRH-II-induced growth inhibition. OVCAR-3 cells were treated with GnRH-II (100 nM) in the presence or absence of the MEK inhibitor PD98059 (10 uM). A [3H]thymidine incorporation assay was performed to quantify s-phase growing cells. After 24 h of culture, the medium was changed and 20 ul of GnRH-II (100 nM) was added. The radioactivity of thymidine incorporated cells was measured by beta-counter. Data are shown as the means of three individual experiments, and are presented as the mean ± S.D.. a, PO.05 vs. control. 5 5 o e o U U 100 80 60 £ 40 = 20 0 Control GnRH-II Treatment with GnRH-II (100 nM) Figure 15. Effect of GnRH-II on cell viability. The cells were seeded into 96-well plates at a density of 104 cells/well. The cells were treated with GnRH (100 nM) for 4 days and cell viability was measured by MTT assay. Values are represented as the mean ± S:D. of three individual experiments, a, P<0.05 vs. control. 56 3.3 Role of p38 mitogen-activated protein kinase in ovarian cancer cells 3.3.1 p38 M A P K activation by GnRH-II in ovarian cancer cells The stimulation of p38 MAPK phosphorylation by GnRH-II was examined in a time-dependent manner at a concentration of 100 nM (Millar et al. 2001). Treatment with GnRH-II induced a significant activation of p38 MAPK at 2, 5, 10 and 15 min with the maximal increase (3.5-fold vs control) observed at 10 min (Fig. 1 6 ) . The total-p38 (non-phosphorylated form) level was not changed by GnRH-II treatment in this time period. To investigate the direct effect of GnRH-II on the activation of p38 MAPK, SB203580 (1 nM ), a specific inhibitor of p38 MAPK (Saklatvala et al. 1996), was added 20 min prior to GnRH-II treatment. As seen in Fig. 1 7 A , the pretreatment with SB203580 blocked the activation of p38 MAPK by GnRH-II, while p38 MAPK phosphorylation did not significantly differ in control samples treated without or with SB203580 in the absence of GnRH-II. Phosphorylation levels of p38 MAPK are expressed as a relative fold change in untreated and treated GnRH-II groups compared with their relative control groups (Fig. 1 7 B ) . The comparison of these shows a significant difference (PO.05) between GnRH-II and GnRH-II with SB203580. 3.3.2 Activation of AP-1 by GnRH-II requires p38 M A P K activity To investigate the relevance of GnRH-II-induced p38 MAPK activation with AP-1 transcriptional factor, the AP-l-TA-Luc vector containing an AP-1 response element was 5 7 transfected into OVCAR-3 cells. Treatment with GnRH-II (100 nM) resulted in activation of AP-1 transcription factor (1.5-fold vs control level) as demonstrated in Fig. 18. The pretreatment with SB203580 (1 uM) completely reversed the activation of AP-1 induced by GnRH-II treatment (Fig. 18). 3.3.3 Anti-proliferative effect of GnRH-II on ovarian cancer cells To determine the effect of GnRH-II on cell proliferation, OVCAR-3 cells were treated with increasing doses of GnRH-II (1 nM, 100 nM, 10 uM) for different time periods (2, 4 or 6 days). Treatment with GnRH-II resulted in a significant decrease of cell proliferation compared with control (Fig. 19). Cell proliferation was dose-dependently decreased at each concentration of GnRH-II. After 2 days of treatment with GnRH-II, cell proliferation was significantly decreased only at the 10 uM concentration (72.0 ± 13.4 %). After treatment with GnRH-II for 4 days, the inhibitory effect of proliferation was observed at all 3 concentrations of GnRH-II: 89.2 ± 1.4 % at 1 nM, 84.0 ± 1.4 % at 100 nM and 76.9 ± 9.0 % at 10 uM, respectively. The inhibitory effect on cell proliferation was also shown in the treatment groups of 1 nM (87.5 ± 4.7 %) and 100 nM GnRH-II (75.6 ± 5.0 %). The maximal antiproliferative effect was observed upon a 6-day treatment with 10 uM GnRH-II (66.4 ± 9.2 %). To investigate the relevance of the p38 signaling pathway in the proliferation of OVCAR-3 cells, SB203580 (1 uM) was added 20 min prior to GnRH-II (100 nM). This pretreatment with the p38 inhibitor completely reversed the antiproliferative effect of GnRH-II (Fig. 20). 58 3.3.4 Induction of apoptosis by GnRH-II OVCAR-3 cells were treated with GnRH-II for 2, 4 and 6 days and a DNA fragmentation assay was performed to quantify the induction of apoptosis. When the cells were treated with GnRH-II for 6 days, treatment with GnRH-II (100 nM) resulted in a significant increase in DNA fragmentation as quantified by ELISA (1.6-fold vs. control) ( F i g . 2 1 A ) . No significant difference was observed following 2 and 4 day treatments with GnRH-II. Further, pretreatment of OVCAR-3 cells with 1 pM SB203580 completely reversed the effect of GnRH-II-induced apoptosis ( F i g . 2 1 B ) . 5 9 T-p38 P-p38 C o n t r o l 2 5 10 15 20 30 60 90 ( m i n ) 5 C o n t r o l 2 5 10 15 20 30 60 90 Treatment with GnRH-II (min) F i g u r e 16. T i m e d e p e n d e n t e f f e c t o f G n R H - I I o n p 3 8 a c t i v a t i o n i n o v a r i a n c a n c e r c e l l l i n e . O V C A R - 3 c e l l s w e r e c u l t u r e d a n d t r e a t e d w i t h G n R H - I I ( 1 0 0 n M ) i n a t i m e d e p e n d e n t m a n n e r . T h e t o t a l ( T - p 3 8 ) a n d a c t i v a t e d p 3 8 ( P - p 3 8 ) l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y a n d t h e i n t e n s i t i e s o f t h e s i g n a l s w e r e q u a n t i t a t e d . P 3 8 l e v e l s a r e e x p r e s s e d a s r e l a t i v e f o l d c h a n g e t o b a s a l l e v e l s . T h e d a t a w e r e a n a l y z e d b y A N O V A f o l l o w e d b y T u k e y ' s m u l t i p l e c o m p a r i s o n t e s t . V a l u e s a r e r e p r e s e n t e d a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , ¥<0.05 vs c o n t r o l . 6 0 A B © 1 0 2 £ +-e x o 9 u i 1 •S-s 8 £ us w ft. 8 6 4 2 b i 1 - SB203580 + SB203580 GnRH-II treatment F i g u r e 1 7 . T h e e f f e c t o f S B 2 0 3 5 8 0 p r e t r e a t m e n t o n G n R H - I I - i n d u c e d p 3 8 M A P K a c t i v a t i o n . ( A ) O V C A R - 3 c e l l s w e r e p r e t r e a t e d w i t h 1 p M S B 2 0 3 5 8 0 f o r 2 0 m i n , f o l l o w e d b y s t i m u l a t i o n w i t h 1 0 0 n M G n R H - I I f o r 5 m i n . C o n t r o l c u l t u r e w a s t r e a t e d w i t h v e h i c l e . T h e t o t a l p 3 8 M A P K a n d a c t i v a t e d p 3 8 M A P K l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . p 3 8 M A P K l e v e l i s e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e t o b a s a l l e v e l . V a l u e s a r e r e p r e s e n t e d a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s ( n = 3 ) . ( B ) G n R H - I I -i n d u c e d p 3 8 M A P K p h o s p h o r y l a t i o n l e v e l i s e x p r e s s e d a s a r e l a t i v e f o l d c h a n g e , a , G n R H -II ( P O . 0 5 ) vs. c o n t r o l . ; b , G n R H - I I ( P O . 0 5 ) vs. G n R H - I I - t r e a t e d g r o u p w i t h o u t S B 2 0 3 5 8 0 . 6 1 p. < o 05 ha u 1.5 -3 0.5 Control GnRH-II SB203580 GnRH-II + SB203580 Treatment Figure 18. The effect of GnRH-II and SB203580 on AP-1 activation. Five hours after transfection with AP-l-TA-Luc vector, the medium was changed to 10 % FBS/DMEM, and 24 h after transfection, GnRH-II was added. Luc + 3-galactosidase assay were performed. Cells were treated with 1 pM SB203580 for 20 min, followed by stimulation with 100 nM GnRH-II for 5 min. Control culture was treated with vehicle. Values are represented as the mean + S.D. of three individual experiments, a, P<0.05 vs. control; b, P<0.05 vs. GnRH-II treatment. 6 2 120 • 2 days Concentration of GnRH-II F i g u r e 1 9 . The effect of GnRH-II on ovarian cancer cell proliferation. OVCAR-3 cells were cultured and treated with GnRH-II (1 nM, 100 nM, 10 uM). A [3H]thymidine incorporation assay was performed to quantify DNA synthesis. After 24 h culture to attach to the wells, the medium was changed and 20 ul of GnRH-II were added, resulting in final concentrations. GnRH-II was added every 12 h, and every 24 h the medium was changed. After 2, 4, and 6 days the cells were incubated with 1 (iCi[3H]thymidine for 6 h. Each experiment was repeated 3 times. Values are represented as the mean ± S.D. of three individual experiments, a, PO.05 vs. control. 6 3 C o n t r o l G n R H - I I D M S O SB203580 G n R H - I I + SB203580 Treatment F i g u r e 2 0 . The effect of p38 MAPK activation by GnRH-II on ovarian cancer cell proliferation. OVCAR-3 cells were pretreated with SB203580 prior to treatment with GnRH-II (100 nM). To quantify the DNA synthesis, a [3H]thymidine incorporation assay was performed as previously described. Treatment with GnRH-II for 6 days induced a significant decrease of growth in OVCAR-3 cells, but pretreatment with SB203580 reversed the effect of GnRH-II. Each experiment was repeated 3 times. Values are represented as the mean + S.D. of three individual experiments, a, P<0.05 vs. control; b, P<0.05 vs. GnRH-II treatment. 6 4 .2 •8 200 150 100 Control GnRH-II .a 8 2 0 0 1 5 0 1 0 0 5 0 O Time (day) C o n t r o l G n R H - I I D M S O S B 2 0 3 5 8 0 G n R H - I I + S B 2 0 3 5 8 0 Treatment with GnRH-II (100 nM) and SB203580 F i g u r e 2 1 . The effect of GnRH-II in the induction of apoptosis. To quantify the induction of apoptosis, OVCAR-3 cells were cultured and treated with GnRH-II (100 nM) for 2, 4, and 6 days (A). In addition, the cells were cultured for 6 days and pretreated with 1 pM SB203580 for 20 min prior to treatment with 100 nM GnRH-II ( B ) . The attached and detached cells were collected and DNA fragmentation was measured by cell death detection ELISA. Values are represented as the mean + S.D. of three individual experiments, a, PO.05 vs. control. 6 5 3.4 Investigation of the roles of other signaling components, and of the differential roles of MAPKs, in GnRH signaling 3.4.1 Roles of G a s and G a i in GnRH-induced M A P K activation and in mediating GnRH-induced inhibition of proliferation We recently demonstrated that GnRH-induced anti-proliferation is mediated via the PKC pathway (Kim et al. 2006), but the other components involved in this GnRH-induced anti-proliferative effect are still unclear. To shed light on the upstream signaling associated with GnRH-induced MAPK activation, we needed to investigate the involvement of the Gas and G ai proteins. Prior to GnRh-I and II treatment, we added two inhibitors: H-89, a specific inhibitor of PKA, or PTX (pertussis toxin), a specific inhibitor of G a , . Treatment with this specific activator of PKA, 8-bromo-cAMP, thus, resulted in an increase in ERK1/2 activity in OVCAR-3 cells. The pretreatment with H-89 reversed an activation of ERK1/2 by 8-bromo-cAMP, but did not block the activation by GnRH-I and II as shown in Fig. 22A. In addition, treatment with mastoparan, an activator of Gaj (Gil et al. 1991) resulted in an increase of ERK1/2 phosphorylation in OVCAR-3 cells (Fig. 23A). GnRH-induced activation of ERK 1/2 was not inhibited by PTX, but its activation induced by mastoparan was inhibited by PTX (Fig. 23A). Furthermore, GnRH-induced anti-proliferation was unaffected by H-89 or PTX, excluding the possibility of involvement of Gas or G ai protein in GnRH-induced anti-proliferation in ovarian cancer cells (Fig. 22B, 23B). 6 6 3.4.2 Role of M A P K activation in GnRH-induced apoptosis in ovarian cancer cells To explore GnRH-induced apoptotic events, the cells were incubated with Annexin V-FITC and 7-amino actinomycin D (7AAD) as described in the Materials and Methods. OVCAR-3 cells were treated with GnRH-I and II (100 nM) for 2 or 6 days. An increase in the number of apoptotic cells was only observed following 6 days (and not before 2 days) of continuous treatment with GnRH-I or II (Fig. 24B) which leads to the observation that apoptosis is time-dependent. The induction of apoptosis by GnRH appears to be time-dependent because the induction of apoptosis was not observed by 2-days of treatment with GnRH-I and II (Fig. 24A). To confirm the induction of apoptosis by GnRH, end-labeling of exposed 3' OH ends of DNA fragments was undertaken with TUNEL assay as described in the Materials and Methods (section 2.11). GnRH-I and II induced apoptosis following 6 days of treatment but not after 4 days in SKOV-3 cells (Fig. 25). These data confirm that GnRH-I and II induce apoptosis in ovarian cancer cells. To explore whether the activation of MAPK is involved in the induction of apoptosis, the cells were treated with the specific inhibitor of ERK1/2, PD98059, or the specific inhibitor of p38 MAPK, SB203580, as well as GnRH-I and II. GnRH-induced apoptosis was inhibited by pretreatment with SB203580, but not by PD98059. (Fig. 24B). 3.4.3 GnRH induces the kinetically distinct activation of MAPKs As shown before, because GnRH-I and II induced anti-proliferation in the early period 6 7 (1, 2, and 4 days), but resulted in the induction of apoptosis by 6 days (late period) as described in the present study, we explored whether the activation of ERK1/2 and p38 MAPK can vary in different time-dependent manners. To evaluate the effects of prolonged treatment with GnRH on MAPK activation in ovarian cancer cells, the phosphorylation of ERK 1/2 and p38 MAPK was examined following 1, 2, 4 or 6 days of treatment in OVCAR-3 cells. Following 1 or 2 days of treatment, GnRH-I and II stimulated the activation of ERK1/2, but not p38 MAPK ( F i g . 2 6 A , B ) . In contrast, treatment with GnRH-I and II for 4 or 6 days resulted in a small degree of stimulation of p38 MAPK phosphorylation, but ERK1/2 showed no activity ( F i g . 2 6 C , D ) . It is of interest to note that the high basal level of P-ERKl/2 were observed following 4 and 6 days of treatment with GnRH in OVCAR-3 cells, suggesting that ERK 1/2 is already activated in these cells, however, there is no good explanation for this observation. TGF-pl (1 ng/ml) was used as a control for MAPK activation, however, it seems to be different from GnRH in terms of its time-dependent effects on MAPK activation, whereas total ERK 1/2 or p38 (non-phosphorylated form) levels were not affected by GnRH treatment in these time periods. 3.4.4 Involvement of the EGF signaling pathway and protein tyrosine kinases in GnRH signaling To investigate whether the EGF signaling pathway is involved in GnRH-induced MAPK activation, SKOV-3 cells were treated with AG1478 in a dose-dependent manner prior to GnRH or EGF treatment. Interestingly, GnRH- or EGF-induced ERK1/2 activation was blocked at a higher concentration with AG 1478 (500 nM) treatment, whereas at a 68 lower concentration (1 nM) of AG1478, only GnRH-induced ERK1/2 activation was abolished (Fig. 27A). EGF-induced ERK1/2 activation was partially blocked at a intermediate concentration of 50 nM, indicating that AG1478 blocks the GnRH-induced ERK1/2 activation through a signaling pathway other than the EGF receptor signaling pathway. In addition, the phosphorylation of EGF receptor was examined using antibodies targeting phosphorylated-EGFR (P-EGFR) and total EGF receptor (T-EGFR) to investigate the effect of GnRH on EGF-induced EGF receptor phosphorylation. SKOV-3 cells were treated with EGF and GnRH-I and II (100 nM or 10 pM). EGF activated EGF receptor and co-treatment with GnRH-I and II did not abolish the EGF-induced EGF receptor activation (Fig. 28A). In addition, treatment with GnRH-I did not induced the activation of EGF receptor (Fig. 28B). To determine the involvement of protein tyrosine kinase pathway in GnRH-induced ERK1/2 activation, we performed a Western blot to examine GnRH-induced ERK1/2 activation in the presence or absence of genistein, a general inhibitor of protein tyrosine kinase activity (Reiss et al. 1997; Grosse et al. 2000; Benard et al. 2001). There was partial inhibition of GnRH-induced ERK1/2 activation following pretreatment with genistein for 30 min (Fig. 27B). To determine the effect of AG1478 on GnRH-induced anti-proliferation, SKOV-3 cells were treated with or without AG1478 (1 nM), followed by treatment with GnRH-I, -II (100 nM) for 24 h. Treatment with GnRH-I and II resulted in a significant decrease of cell proliferation compared to control, whereas co-treatment with AG1478 (1 nM) blocked GnRH-I and II-induced anti-proliferation (Fig. 29A). Genistein, was used to determine if tyrosine phosphorylation is involved in GnRH-6 9 induced anti-proliferation as previous reports have shown a role of PTK in GnRH-induced tyrosine phosphorylation (Johnson et al. 1995). Genistein (100 pM) partially reversed the anti-proliferative effect of GnRH-I and II in SKOV-3 cells ( F i g . 2 9 B ) . 7 0 A T - E R K l / 2 P - E R K l / 2 H-89 GnRH-I GnRH-II H-89 H-89 c A M P H-89 +GnRH-I +GnRH-II +CAMP 120 100 80 £ 60 | 40 | 20 I a a b b M i l l C H-89 G n R H - I G n R H - I I H-89 H-89 + G n R H - I +GnRH-II F i g u r e 2 2 . E f f e c t o f 8 - b r o m o - c A M P a n d H - 8 9 o n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i - p r o l i f e r a t i o n . ( A ) O V C A R - 3 c e l l s w e r e p r e t r e a t e d w i t h o r w i t h o u t 1 u M H - 8 9 f o r 1 h , f o l l o w e d b y s t i m u l a t i o n w i t h G n R H - I o r I I ( 1 0 0 n M ) f o r 1 0 m i n . C e l l s w e r e a l s o t r e a t e d w i t h 8 - b r o m o - c A M P ( l O m M ) a s a p o s i t i v e c o n t r o l . T o t a l E R K 1 / 2 a n d a c t i v a t e d E R K 1 / 2 l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . ( B ) T h e c e l l s w e r e t r e a t e d w i t h G n R H - I o r I I ( 1 0 0 n M ) i n t h e p r e s e n c e o r a b s e n c e o f H - 8 9 (1 u M ) f o r 2 4 h , . A [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y w a s p e r f o r m e d t o q u a n t i f y S - p h a s e g r o w i n g c e l l s . T h e d a t a f r o m t h r e e i n d i v i d u a l e x p e r i m e n t s a r e p r e s e n t e d a s t h e m e a n ± S . D . . S t a t i s t i c a l a n a l y s i s w a s p e r f o r m e d b y o n e - w a y A N O V A f o l l o w e d b y T u k e y ' s m u l t i p l e c o m p a r i s o n t e s t , a , P < 0 . 0 5 vs. c o n t r o l ; b , P O . 0 5 vs. H - 8 9 a l o n e . 7 1 A C P T X G n R H - I G n R H - I I P T X P T X +GnRH - I +GnRH- I I F i g u r e 2 3 . E f f e c t o f m a s t o p a r a n a n d P T X o n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i -p r o l i f e r a t i o n . ( A ) O V C A R - 3 c e l l s w e r e t r e a t e d w i t h o r w i t h o u t 5 0 n g / m l P T X f o r 1 6 h , a n d t h e n s t i m u l a t e d w i t h G n R H - I o r I I o r 1 p M m a s t o p a r a n ( M P ) f o r 1 0 m i n . T - E R K 1 / 2 a n d P -E R K 1 / 2 l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . ( B ) T h e c e l l s w e r e t r e a t e d w i t h G n R H -I o r - I I ( 1 0 0 n M ) f o r 2 4 h i n t h e p r e s e n c e o r a b s e n c e o f P T X ( 5 0 n g / m l ) . A [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y w a s p e r f o r m e d t o q u a n t i f y S - p h a s e g r o w i n g c e l l s . D a t a a r e s h o w n a s t h e m e a n ± S . D . o f t h r e e i n d i v i d u a l e x p e r i m e n t s , a , P O . 0 5 vs. c o n t r o l ( 1 0 0 % ) ; b , P O . 0 5 vs. P T X a l o n e . 7 2 A B C SB PD Gn-I Gn-II SB SB PD PD + Gn-I +Gn-II +Gn-I +Gn-II Gn-I: GnRH-I Gn-II: GnRH-II F i g u r e 2 4 . E f f e c t o f M A P K i n h i b i t o r s o n G n R H - i n d u c e d a p o p t o s i s . T o e x p l o r e G n R H -i n d u c e d a p o p t o t i c e v e n t s , t h e c e l l s w e r e i n c u b a t e d w i t h A n n e x i n V - F I T C a n d 7 - a m i n o a c t i n o m y c i n D ( 7 A A D ) i n O V C A R - 3 c e l l s . ( A ) T h e n u m b e r o f a p o p t o t i c c e l l s w a s u n a f f e c t e d b y G n R H ( 1 0 0 n M ) t r e a t m e n t f o r 2 d a y s . ( B ) C e l l s w e r e t r e a t e d f o r 6 d a y s w i t h G n R H - I o r - I I i n t h e p r e s e n c e o r a b s e n c e o f P D 9 8 0 5 9 ( P D , 1 0 p M ) o r S B 2 0 3 5 8 0 ( S B , 1 p M ) . I n c r e a s i n g t h e d u r a t i o n o f e x p o s u r e t o G n R H i n c r e a s e d c e l l d e a t h . 7 3 4 days 6 days ( x 1 0 0 ) F i g u r e 2 5 . T i m e - d e p e n d e n t a p o p t o t i c e f f e c t s o f G n R H - I a n d G n R H - I I . T o c o n f i r m t h e i n d u c t i o n o f a p o p t o s i s b y G n R H , T U N E L a s s a y w a s p e r f o r m e d i n S K O V - 3 c e l l s . C e l l s w e r e t r e a t e d w i t h G n R H - I o r I I ( 1 0 0 n M ) f o r 4 o r 6 d a y s . T r e a t e d c e l l s w e r e f i x e d w i t h 4 % p a r a f o r m a l d e h y d e s o l u t i o n a n d i n c u b a t e d i n a 0 . 1 % T r i t o n p e r m e a b i l i z a t i o n s o l u t i o n o n i c e a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . C e l l w e r e t h e n r i n s e d t h r e e t i m e s i n P B S a n d i n c u b a t e d f o r a f u r t h e r 3 0 m i n w i t h 5 0 p i o f t h e C o n v e r t e r - P O D , f o l l o w e d b y 1 0 m i n w i t h D A B . G n R H - I a n d G n R H - I I i n d u c e d i n c r e a s e d t h e n u m b e r o f T U N E L - p o s i t i v e c e l l s ( c o l o r e d c e l l s ) f o l l o w i n g 6 d a y s c o n t i n u o u s t r e a t m e n t w i t h G n R H - I o r I I . 7 4 1 day T-p38 P-p38 T-ERK1/2 P-ERKl/2 II T G F II T G F 2 day T-p38 P-p38 T-ERK1/2 P-ERKl/2 11 TGF-J3 II TGF-P 4 day T-ERK1/2 P-ERKl/2 II T G F - 3 6 day T-p38 P-p38 T-ERK1/2 P-ERKl/2 II T G F - 3 II T G F - 3 F i g u r e 2 6 . E f f e c t o f G n R H - I o r II o n E R K 1 / 2 a n d p 3 8 a c t i v a t i o n . T o e v a l u a t e t h e e f f e c t s o f p r o l o n g e d t r e a t m e n t w i t h G n R H o n M A P K p h o s p h o r y l a t i o n i n o v a r i a n c a n c e r c e l l s , t h e p h o s p h o r y l a t i o n o f E R K 1 / 2 a n d p 3 8 M A P K w a s e x a m i n e d f o l l o w i n g 1, 2 , 4 o r 6 d a y s o f t r e a t m e n t i n O V C A R - 3 c e l l s . C e l l s w e r e t r e a t e d w i t h G n R H - I o r II ( 1 0 0 n M ) i n a t i m e -d e p e n d e n t m a n n e r . T o t a l a n d p h o s p h o r y l a t e d E R K 1 / 2 ( T - E R K 1 / 2 a n d P - E R K l / 2 ) a n d p 3 8 l e v e l s ( T - p 3 8 a n d P - p 3 8 ) w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . 7 5 A T-ERKI/2 m m m m m m a mmm\mmmmmm'»MM%Wt''^f^m^ " P-ERKl/2 **ttS*1 "'iii'Su*1 C D 500 50 1 100 100 500 50 1 500 50 1 (nM) AG1478 - - + + + - - + + + + + + GnRH-I _ _ _ _ _ + _ + * + * + * _ _ _ GnRH-II _ _ _ _ _ _ + _ _ _ + * + * + * D: DMSO * 100 nM T-ERKI/2, HimiifTr M r mmm S ^ ' M ^ L s ^ g g - , C D 500 50 1 0.1* 500 50 1 100** 100*** (nM) AG1478 - - + + + - + + + - -EGF — — — — — + + * + * + * — — *0.1: ng/ml ** GnRH-I B ***GnRH-II T-ERK1/2J P-ERKl/2 C DMSO Gen G-I G-II G-I Gen Gen Gen +G-II +G-I +G-II +G-I +G-II Gen: Genistein G-I: GnRH-I G-II: GnRH-II F i g u r e 2 7 . E f f e c t s o f A G 1 4 7 8 a n d G e n i s t e i n o n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n . ( A ) S K O V - 3 c e l l s w e r e t r e a t e d w i t h o r w i t h o u t A G 1 4 7 8 f o r 3 0 m i n i n a d o s e - d e p e n d e n t m a n n e r , f o l l o w e d b y s t i m u l a t i o n w i t h G n R H - I , - I I ( 1 0 0 n M ) o r E G F ( 0 . 1 n g / m l ) f o r 1 0 m i n . ( B ) T h e c e l l s w e r e p r e t r e a t e d w i t h G e n i s t e i n ( 1 0 0 n M ) f o r 3 0 m i n , f o l l o w e d b y s t i m u l a t i o n w i t h G n R H - I ( G - I ) , a n d G n R H - I I ( G - I I ) ( 1 0 0 n M ) f o r 1 0 m i n . T - E R K I / 2 a n d P - E R K l / 2 l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . 7 6 A F i g u r e 2 8 . E f f e c t s o f G n R H o n E G F r e c e p t o r a c t i v a t i o n . T o e v a l u a t e t h e e f f e c t s o f G n R H t r e a t m e n t w i t h E G F o n E G F r e c e p t o r p h o s p h o r y l a t i o n i n o v a r i a n c a n c e r c e l l s , t h e p h o s p h o r y l a t i o n o f E G F r e c e p t o r i n S K O V - 3 c e l l s . ( A ) T h e c e l l s w e r e t r e a t e d w i t h E G F a n d G n R H ( 1 0 0 n M o r 1 0 u M ) f o r 3 0 m i n . ( B ) T h e c e l l s w e r e t r e a t e d w i t h G n R H - I i n a t i m e - d e p e n d e n t m a n n e r . T - E G F R a n d P - E G F R l e v e l s w e r e a n a l y z e d b y i m m u n o b l o t a s s a y . 7 7 A s 120 is 100 80 H o 60 40 20 0 1 AG1478 GnRH-I GnRH-II AG1478 AG1478 + GnRH-I + GnRH-II 120 C Genistein G n R H - I GnRH-II Genistein Genistein + GnRH-I + GnRH-II F i g u r e 2 9 . E f f e c t s o f A G 1 4 7 8 a n d G e n i s t e i n o n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n . ( A ) S K O V - 3 c e l l s w e r e t r e a t e d w i t h o r w i t h o u t A G 1 4 7 8 ( 1 n M ) f o r 3 0 m i n , f o l l o w e d b y s t i m u l a t i o n w i t h G n R H - I , G n R H - I I ( 1 0 0 n M ) o r E G F ( 0 . 1 n g / m l ) f o r 2 4 h . ( B ) T h e c e l l s w e r e t r e a t e d w i t h G n R H - I o r G n R H - I I ( 1 0 0 n M ) f o r 2 4 h i n t h e p r e s e n c e o r a b s e n c e o f G e n i s t e i n ( 1 0 0 u M ) . A [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n a s s a y w a s p e r f o r m e d t o q u a n t i f y S -p h a s e g r o w i n g c e l l s , a , P O . 0 5 vs. c o n t r o l ( 1 0 0 % ) . 7 8 4. DISCUSSION 4.1 Roles of the GnRH-I receptor and PKC in GnRH signaling and anti-proliferative effects GnRH-I analogs have long been shown to have direct anti-proliferative effects on ovarian cancer cells (Balbi et al. 2004). It has been reported that GnRH-II has a stronger anti-proliferative effect than GnRH-I in ovarian cancer cell lines, suggesting that GnRH-II could be considered as a novel target for anti-proliferative therapeutic approaches (Grundker et al. 2002). However, the intracellular signaling generated by GnRH in order to induce the activation of MAPK and inhibit cancer cell growth is still poorly understood. In this context, the elucidation of the signaling pathways involved in the anti-proliferative effects of GnRH could provide new therapeutic insights for ovarian cancer prevention and/or treatment. Previous studies have shown that OVCAR-3 and SKOV-3 cells, ovarian adenocarcinoma cell lines, express the mRNA for GnRH-I receptors (Yin et al. 1998; Ngan et al. 1999) and respond to GnRH-I (Kim et al. 1999b; Kang et al. 2000b). The GnRH-I receptor is a member of the G protein-coupled receptor (GPCR) family. GPCRs are characterized by the presence of seven transmembrane (7TM) domains and transmit their signals through multiple G protein subunits (Goq, Gas, and Gai), often activating multiple signaling pathways. The mammalian GnRH-I receptor is unique within this family because it lacks the cytoplasmic carboxyl-terminal tail that is known to be responsible for internalization and desensitization following ligand stimulation (Millar et al. 2004). A search of the human genome database has revealed a putative GnRH-II 7 9 receptor gene located on chromosome 1 which shares 40% identity with the GnRH-I receptor gene (Neill et al. 2001). It is predicted that the GnRH-II receptor may contain a cytoplasmic tail and only five transmembrane domains (5TM lacking TM I and II) (Neill 2002; Millar 2003). Within the body, GnRH-II receptors binding GnRH-II were found more widely than GnRH-I receptors, including the brain (Millar et al. 1999; Kang et al. 2000b; Millar et al. 2001). However, a full length human GnRH-II receptor has yet to be cloned (Neill et al. 2001; Grundker et al. 2002; Leung et al. 2003; Millar 2003). Recent evidence revealed that GnRH-II may be a potent regulator of ovarian function (Siler-Khodr et al. 2003). In addition, it is of interest that treatment with GnRH-II analog in its lowest concentration (250 nM) induced a stimulation of human chorionic gonadotropin (hCG), whereas the release of hCG from human placental explants was inhibited at higher concentrations in a placental explant perfusion system (Siler-Khodr et al. 2003). It was in this same report that pulsatile release of GnRH-II from the early human placenta was shown. The different activities of GnRH-I and-II may be derived from the different culture systems used, the concentrations of GnRHs employed, and differential degradation of GnRHs in different tissues. Previous reports suggest that a specific receptor for GnRH-II (type II GnRH receptor) might exist in human ovarian cancer cells (Grundker et al. 2002; Millar 2003) and Neill et al. observed that a type II GnRH receptor gene is present in human and suggested several possible explanations including frame shift or mRNA editing or the elaborating of partial type II receptor (Neill et al. 2001). Grundker et al. further demonstrated that GnRH-II had a more potent anti-proliferative effect than GnRH-I in ovarian and endometrial tumors, and that GnRH-II receptors are present in OVCAR-3 cells, suggesting that the anti-proliferative effect of GnRH-II might be mediated through 8 0 GnRH-II receptors in these gynecological tumor cells (Grundker et al. 2002). Thus, whether this transcript encodes a functional receptor protein in any human tissue remains obscure as does the potential roles of the GnRH-II receptor in mediating the effects of GnRH-I and II. There is a discrepancy among the previous reports regarding the role of GnRH-I and II receptors. Enomoto et al. showed that a GnRH-II receptor is required to mediate the effect of GnRH-II (Enomoto et al. 2004), and Grundker et al. reported that the anti-proliferative effects induced by GnRH-II is not mediated through GnRH-I receptor (Grundker et al. 2004). Conversely, a recent study demonstrated that transient transfection with GnRH-II receptor inhibited the expression of GnRH-I receptor at the cell surface and impaired signaling via the GnRH-I receptor by reduction of GnRH-induced inositol phosphate accumulation. This indicated that the GnRH-I receptor may be a common receptor that mediates the effects of both GnRH-I and GnRH-II in ovarian cancer cell lines (Pawson et al. 2005). This discrepancy suggests that the role of GnRH I and II receptors may vary considerably in different cell types or conditions. In addition, a human GnRH-II receptor protein has not been identified because the human GnRH-II receptor transcript has a frame-shift resulting in a premature stop codon (Morgan et al. 2003). It has been noted that treatment with antide completely blocked the growth inhibitory effect of GnRH-II in neoplastic ovarian surface epithelial cells (Choi et al. 2001b). We investigated the role of the GnRH-I receptor and PKC signaling pathway in GnRH-induced MAPK activation and growth inhibition of ovarian cancer cells. The expression of GnRH-I receptor was monitored by immunoblot assay and the GnRH-I antagonist, antide, was used to block GnRH-I receptor-induced signaling. Antide is known to exert its 8 1 a n t a g o n i s t i c p r o p e r t i e s i n o v a r i a n c a n c e r c e l l s ( L i e t a l . 1 9 9 4 ) , a n d p r e t r e a t m e n t w i t h a n t i d e r e d u c e d G n R H - I - i n d u c e d E R K 1 / 2 a c t i v a t i o n i n e n d o m e t r i a l e p i t h e l i a l c e l l s ( L u o e t a l . 2 0 0 4 ) . A l t h o u g h t h e r e i s a p r e v i o u s r e p o r t t h a t G n R H - I d o e s n o t a c t i v a t e E R K 1/2 p a t h w a y ( E m o n s e t a l . 1 9 9 8 ) , o t h e r s h a v e d o c u m e n t e d t h a t G n R H - I a c t i v a t e s E R K 1 / 2 i n v a r i o u s c e l l t y p e s i n c l u d i n g o v a r i a n c a n c e r c e l l s ( K i m u r a e t a l . 1 9 9 9 ; K r a u s e t a l . 2 0 0 1 ) . O u r o w n s t u d i e s h a v e s h o w n t h a t G n R H - I I a c t i v a t e s E R K 1 / 2 ( K i m e t a l . 2 0 0 4 b ) a n d t h a t t h i s p a t h w a y i s i n v o l v e d i n t h e e f f e c t o f G n R H - I I o n a n t i - p r o l i f e r a t i o n . U s i n g O V C A R - 3 a n d S K O V - 3 c e l l s , a n e o p l a s t i c O S E c e l l l i n e d e r i v e d f r o m o v a r i a n c a n c e r p a t i e n t s , w e f o u n d i n o u r p r e s e n t s t u d y t h a t p r e t r e a t m e n t w i t h a n t i d e b l o c k e d t h e G n R H - I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n . F u r t h e r m o r e , c o m b i n e d t r e a t m e n t s o f G n R H - I a n d G n R H - I I d i d n o t h a v e a n a d d i t i v e e f f e c t o n t h e a c t i v a t i o n o f E R K 1 / 2 ( d a t a n o t s h o w n ) . O u r p r e s e n t a n d p r e v i o u s s t u d i e s h a v e s h o w e d t h a t G n R H - I a n d - I I h a v e a n t i - p r o l i f e r a t i v e e f f e c t s o n o v a r i a n c a n c e r c e l l s ( K a n g e t a l . 2 0 0 0 a ; K i m e t a l . 2 0 0 4 a ) . C o - t r e a t m e n t w i t h a n t i d e p r e v e n t e d G n R H - I a n d I I - i n d u c e d a n t i - p r o l i f e r a t i o n . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e G n R H - I r e c e p t o r m e d i a t e s G n R H - I a n d I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i - p r o l i f e r a t i o n i n o v a r i a n c a n c e r . T o f u r t h e r e x a m i n e t h e r o l e o f t h e G n R H - I r e c e p t o r i n m e d i a t i n g t h e e f f e c t s o f G n R H -I a n d I I , w e e m p l o y e d t h e R N A i n t e r f e r e n c e t e c h n i q u e . S K O V - 3 a n d O V C A R - 3 c e l l s w e r e t r a n s f e c t e d w i t h s m a l l i n t e r f e r i n g ( s i ) R N A s t a r g e t e d a g a i n s t t h e G n R H - I r e c e p t o r g e n e . T h e s e q u e n c e o f t h e G n R H - I r e c e p t o r s i R N A u s e d i n t h i s s t u d y d o e s n o t h a v e a n y o v e r l a p p i n g s e q u e n c e h o m o l o g y w i t h t h e r e c e p t o r f o r G n R H - I I . 8 2 Immunoblot analysis was used to examine GnRH-I receptor gene expression in transfected cells and the proliferation of cells transfected with siRNAs was monitored by thymidine incorporation assay. The GnRH-I receptor was expressed in the cells treated with vehicle and scrambled siRNA, whereas its expression was significantly reduced in siRNA-transfected cells. This indicated that the transcription and translation of the GnRH-I receptor is reduced in these cells. Transfection with siRNAs targeted to the GnRH-I receptor blocked the anti-proliferative effect of GnRH-I and II. These data, together with the effects of antide, indicate that the anti-proliferative effect of GnRH-I and II on ovarian cancer cells is dependent on the GnRH-I receptor. The signal transduction pathway activated following the binding of GnRH-I to the GnRH-I receptor has been studied extensively (Millar et al. 2004). The receptor is coupled to the Gaq/i i protein that activates phospholipase CP, leading to the activation of PKC and various downstream signal transduction cascades, including the MAPK pathways (Kraus et al. 2001). Activation of the PKC pathway has been well documented in response to GnRH-I stimulation (Andrews and Conn 1986; Zheng et al. 1994), and GnRH-I induces the translocation of PKC and stimulates enzyme activity (Farshori et al. 2003). Activation of PKC appears to be an important second messenger mediating GnRH-I-induced ERK activation in pituitary cells (Harris et al. 1997b) and ovarian cancer cells (Chamson-Reig et al. 2003). In contrast, the GnRH-II signaling mechanism is poorly understood. Our results demonstrate that treatment of OVCAR-3 and SKOV-3 cells with GnRH-I or II for 10 min resulted in activation of ERK1/2. Furthermore, pretreatment with GF109203X, which inhibits PKC by competing with ATP (Barent et al. 1998), abolished the activation of ERK 1/2 induced by GnRH-I, II or TPA. Our results are consistent with the finding that 8 3 G n R H - I a c t i v a t e s t h e P K C p a t h w a y i n o v a r i a n t u m o r c e l l s ( C h a m s o n - R e i g e t a l . 2 0 0 3 ) a n d p i t u i t a r y c e l l s ( S u n d a r e s a n e t a l . 1 9 9 6 ) . A l t h o u g h t h e P K C i s o z y m e i n v o l v e d i n t h e a c t i v a t i o n o f E R K 1 / 2 b y G n R H - I a n d I I i n t h e s e c e l l s i s n o t y e t k n o w n , t h e s e r e s u l t s s u g g e s t t h a t b o t h G n R H i s o f o r m s a c t i v a t e E R K 1 /2 t h r o u g h a P K C - d e p e n d e n t p a t h w a y , p o s s i b l y c o u p l e d t o t h e G a q p r o t e i n . I n c o n t r a s t , K i m u r a e t a l . s h o w e d t h a t t h e a c t i v a t i o n o f E R K 1 / 2 b y G n R H - I a p p e a r s t o b e i n d e p e n d e n t o f P K C i n C a o v - 3 c e l l s ( K i m u r a e t a l . 1 9 9 9 ) . I n a d d i t i o n , a c t i v a t i o n o f t h e c -J u n N - t e r m i n a l k i n a s e ( J N K ) p a t h w a y w a s i n d e p e n d e n t o f P K C i n o v a r i a n a n d e n d o m e t r i a l c a n c e r c e l l s ( G r u n d k e r a n d E m o n s 2 0 0 3 ) . T h i s d e m o n s t r a t e s t h a t i n v o l v e m e n t o f t h e P K C p a t h w a y i s d e p e n d e n t o n t h e c e l l t y p e a n d t h e e x p r e s s i o n o f t h e r e q u i r e d s i g n a l i n g c o m p o n e n t s . T o i n v e s t i g a t e t h e r o l e o f P K C i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n , w e t r e a t e d o v a r i a n c a n c e r c e l l s w i t h G F 1 0 9 2 0 3 X i n a d o s e - d e p e n d e n t m a n n e r . I n h i b i t i o n o f P K C r e v e r s e d t h e a n t i - p r o l i f e r a t i v e e f f e c t s o f G n R H - I a n d - I I , f u r t h e r s u p p o r t i n g t h e r o l e o f a c t i v a t e d P K C i n t h e r e g u l a t i o n o f c e l l u l a r f u n c t i o n s i n r e s p o n s e t o b o t h f o r m s o f G n R H i n o v a r i a n c a n c e r c e l l s . H o w e v e r , i t h a s b e e n r e p o r t e d t h a t G F 1 0 9 2 0 3 X a l s o m o d u l a t e s G S K -3 P a c t i v i t y . It i n h i b i t e d t h e a c t i v a t i o n o f G S K - 3 P i n r a t e p i d i d y m a l a d i p o c y t e s ( H e r s k o w i t z 1 9 9 5 ) a n d h u m a n p r o s t a t e c a n c e r c e l l s ( L i a o a n d H u n g 2 0 0 4 ) , w h e r e a s i t i n d u c e d G S K - 3 o v e r a c t i v a t i o n i n b r a i n c e l l s ( X u e t a l . 2 0 0 5 ) . A l t h o u g h G F 1 0 9 2 0 3 X i s w e l l k n o w n a s a s p e c i f i c i n h i b i t o r o f P K C a , p a n d y ( A l e s s i 1 9 9 7 ) a n d t h e P K C - a c t i v a t i n g p h o r b o l e s t e r ( T P A ) - i n d u c e d a c t i v a t i o n o f E R K 1 / 2 w a s b l o c k e d b y G F 1 0 9 2 0 3 X i n t h i s s t u d y , t h e p o s s i b i l i t y t h a t G F 1 0 9 2 0 3 X e x e r t s e f f e c t s o n G S K - 3 P c a n n o t b e e x c l u d e d a n d i t i s n e c e s s a r y t o u s e o t h e r P K C i n h i b i t o r s ( f o r e x a m p l e , S t a u r o s p o r i n o r P h o r b o r 1 2 - m y r i s t a t e 8 4 1 3 - a c e t a t e ) t o c o n f i r m t h e r o l e o f P K C i n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i -p r o l i f e r a t i o n . I n s u m m a r y , w e d e m o n s t r a t e d t h a t a n t i d e a n d G F 1 0 9 2 0 3 X b l o c k e d t h e a c t i v a t i o n o f E R K 1 / 2 a n d a n t i - p r o l i f e r a t i o n i n d u c e d b y G n R H - I a n d I I . F u r t h e r m o r e , s i R N A t a r g e t i n g t h e G n R H - I r e c e p t o r a b o l i s h e d G n R H - I a n d I I - i n d u c e d a n t i - p r o l i f e r a t i o n . T a k e n t o g e t h e r , o u r r e s u l t s d e m o n s t r a t e t h a t t h e G n R H - I r e c e p t o r a n d P K C p l a y a n i m p o r t a n t r o l e i n m e d i a t i n g t h e c e l l u l a r r e s p o n s e s i n d u c e d b y G n R H - I I , a s w e l l a s G n R H - I , i n o v a r i a n c a n c e r c e l l s . 4.2 Role of ERK1/2 in GnRH-II signaling and the inhibition of ovarian cancer cell proliferaion G n R H - I h a s b e e n s h o w n t o h a v e a n i n h i b i t o r y e f f e c t o n c e l l g r o w t h i n h u m a n m a m m a r y , o v a r i a n , e n d o m e t r i a l a n d p r o s t a t e t u m o r s , a n d h a s b e e n i m p l i c a t e d a s a n a n t i - p r o l i f e r a t i v e r e g u l a t o r o f g y n e c o l o g i c a l c a n c e r s ( S a n t e n e t a l . 1 9 9 0 ; S a v i n o e t a l . 1 9 9 2 ; S c h a l l y 1 9 9 9 ; S c h a l l y e t a l . 2 0 0 1 ) . R e c e n t l y , i t h a s b e e n s h o w n t h a t a G n R H - I I a n a l o g i s s t a b l e a n d b i n d s t o h i g h a f f i n i t y b i n d i n g s i t e s i n t h e m a m m a l i a n o v a r y , s u g g e s t i n g t h a t G n R H - I I m a y b e a p o t e n t r e g u l a t o r o f o v a r i a n f u n c t i o n ( S i l e r - K h o d r e t a l . 2 0 0 3 ) . A l t h o u g h t h e b i o l o g i c a l f u n c t i o n o f G n R H - I I i s p o o r l y u n d e r s t o o d , a p p r o a c h e s t o d e f i n e i t s m e c h a n i s m o f a c t i o n m a y y i e l d a n i m p o r t a n t t h e r a p e u t i c c l u e i n t h e t r e a t m e n t o f o v a r i a n c a n c e r . O v e r 1 2 d i f f e r e n t t y p e s o f M A P K s h a v e b e e n i d e n t i f i e d i n m a m m a l i a n c e l l s . E R K 1 / 2 , J N K / S A P K 1 a n d p 3 8 / S A P K 2 a r e t h r e e o f t h e b e s t - c h a r a c t e r i z e d M A P K f a m i l y m e m b e r s t h a t e x e r t t h e i r e f f e c t s v i a t h e a c t i v a t i o n o f t r a n s c r i p t i o n f a c t o r s r e s u l t i n g i n c e l l u l a r 8 5 r e s p o n s e s s u c h a s c e l l p r o l i f e r a t i o n o r a p o p t o s i s ( C o b b a n d G o l d s m i t h 1 9 9 5 ; R o b i n s o n a n d C o b b 1 9 9 7 ; G a r r i n g t o n a n d J o h n s o n 1 9 9 9 ; L i n 2 0 0 3 ) . T h i s s t u d y d e m o n s t r a t e s t h a t t r e a t m e n t w i t h G n R H - I I a c t i v a t e d E R K 1 / 2 i n i m m o r t a l i z e d O S E a n d o v a r i a n c a n c e r c e l l l i n e s . I t i s o f i n t e r e s t t o n o t e t h a t G n R H - I I s e e m s t o a c t i v a t e E R K 1 / 2 i n a d i f f e r e n t t i m e m a n n e r i n t h e s e c e l l l i n e s , i n d i c a t i n g t h a t d i f f e r e n t s i g n a l i n g p a t h w a y s m a y b e i n v o l v e d i n t h e G n R H - I I - i n d u c e d E R K 1 /2 a c t i v a t i o n i n d i f f e r e n t o v a r i a n c e l l t y p e s . M o r e o v e r , P D 9 8 0 5 9 , a n i n h i b i t o r o f M E K , m a r k e d l y a t t e n u a t e d t h e a c t i v a t i o n o f E R K 1 / 2 b y G n R H - I I i n O V C A R - 3 c e l l s . T h e s e r e s u l t s a r e i n a g r e e m e n t w i t h p r e v i o u s s t u d i e s w h i c h d e m o n s t r a t e d t h a t G n R H - I a c t i v a t e s E R K 1 /2 i n n o r m a l a n d o v a r i a n c a n c e r c e l l s ( K i m u r a e t a l . 1 9 9 9 ; K a n g e t a l . 2 0 0 0 b ) . T h e p r e s e n t r e s u l t s i n d i c a t e t h a t t h e E R K 1 / 2 p a t h w a y m i g h t b e a n i m p o r t a n t s i g n a l i n g p a t h w a y m e d i a t i n g t h e e f f e c t s o f G n R H - I I i n o v a r i a n c a n c e r c e l l s . It h a s b e e n s h o w n t h a t t r e a t m e n t o f C a O V - 3 c e l l s w i t h G n R H r e s u l t s i n a n a c t i v a t i o n o f E R K a t 5 m i n w i t h m a x i m a l a c t i v a t i o n o c c u r r i n g a t 3 h a n d s u s t a i n e d u n t i l 2 4 h , w h e r e a s G n R H - I h a d n o e f f e c t o n t h e a c t i v a t i o n o f t h e J N K ( K i m u r a e t a l . 1 9 9 9 ) . I n a d d i t i o n , E R K 1 / 2 k i n a s e w a s a l s o a c t i v a t e d a n d a n i n c r e a s e i n p h o s p h o r y l a t i o n o f s o n o f s e v e n l e s s ( S o s ) , a n d S h e w a s o b s e r v e d f o l l o w i n g G n R H t r e a t m e n t . T r e a t m e n t w i t h a n M E K i n h i b i t o r , P D 9 8 0 5 9 , r e d u c e d t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H a n a l o g a n d t h e G n R H - i n d u c e d d e p h o s p h o r y l a t i o n o f t h e r e t i n o b l a s t o m a p r o t e i n , i n d i c a t i n g t h a t t h e a c t i v a t i o n o f E R K m a y p l a y a n i m p o r t a n t r o l e i n t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H ( K i m u r a e t a l . 1 9 9 9 ) . I n p a r a l l e l w i t h t h e p r e v i o u s s t u d y , E R K 1 / 2 m a y p l a y i n a c r i t i c a l r o l e i n G n R H - I I - i n d u c e d a n t i - p r o l i f e r a t i o n i n o v a r i a n c a n c e r c e l l s . F u r t h e r m o r e , G n R H - I a g o n i s t a c t i v a t e d J N K p a t h w a y i n e n d o m e t r i a l c a n c e r c e l l s ( G r u n d k e r a n d E m o n s 2 0 0 3 ) a n d a T 3 - l g o n a d o t r o p h c e l l l i n e ( L e v i e t a l . 1 9 9 8 b ) b u t n o t i n o v a r i a n c a n c e r c e l l s 8 6 (Kimura et al. 1999). In this study, JNK/SAPK1 was not activated by GnRH-II in OVCAR-3 cells. This is in agreement with a previous report using GnRH-I agonist in CaOV-3 cells (Kimura et al. 1999). In addition, JNK/SAPK1 was not activated in SKOV-3 cells, an ovarian cancer cell line, suggesting that the JNK/SAPK1 pathway may not be involved in GnRH-I and -II signaling to induce cellular responses. Taken together, these results indicate that the effects of GnRH-I and GnRH-II on the activation of ERK1/2 and JNK/SAPK1 may be identical in ovarian cancer cells. Activated MAPKs translocate from the cytoplasm to the nucleus and activate transcription factors. ERK1/2 induces gene expression by the activation of transcription factors including the phosphorylation of ternary complex factors (TCFs) such as Elk-1 and SAP-1 (Janknecht et al. 1993; Gille et al. 1995a; Babu et al. 2000). Elk-1, an Ets family transcription factor, is a physiological substrate for ERK1/2 and mediates the c-fos and other co-regulated gene activity through the serum response element (SRE). Therefore, the ability of GnRH-II to activate a downstream pathway of ERK1/2 was examined using the immunoprecipitation method. In this study, the treatment with GnRH-II resulted in i substantial phosphorylation of Elk-1 fusion protein in vitro. However, PD98059, an inhibitor of MEK, did not abolish the effect of GnRH-II on the phosphorylation of Elk-1, suggesting that GnRH-II-induced Elk-phosphorylation is not mediated by the activation of ERK1/2 in ovarian cancer cells. To confirm its effect on the inhibition of tumor growth, [3H]thymidine incorporation and MTT assays were performed. After a 4-day treatment, GnRH-II (100 nM) inhibited the cell growth and cell viability. The importance of MEK-MAPK in cell proliferation and apoptosis is now widely recognized (Lewis et al. 1998). It has been reported that the inhibition of the ERK 1/2 signaling pathway with PD98059 may I 8 7 a f f e c t t h e g r o w t h o f p r o s t a t e a n d b r e a s t t u m o r s ( P r i c e e t a l . 1 9 9 9 ; R e d d y e t a l . 1 9 9 9 ) a n d P D 9 8 0 5 9 r e s t o r e d c e l l p r o l i f e r a t i o n i n h i b i t i o n ( T s u k a d a e t a l . 2 0 0 1 ) . I n a d d i t i o n , i t h a s b e e n o b s e r v e d t h a t P D 9 8 0 5 9 r e d u c e d t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I , s u g g e s t i n g t h a t t h e M E K - M A P K p a t h w a y h a s a c r i t i c a l r o l e i n t h e e f f e c t o f G n R H - I ( K i m u r a e t a l . 1 9 9 9 ) . T h e r e f o r e , w e i n v e s t i g a t e d t h e i n v o l v e m e n t o f t h e M E K - M A P K p a t h w a y i n t h e a n t i -p r o l i f e r a t i v e e f f e c t o f G n R H - I I o n o v a r i a n c a n c e r c e l l s . I n t h e p r e s e n t s t u d y , G n R H - I I -i n d u c e d g r o w t h i n h i b i t i o n i n o v a r i a n c a n c e r c e l l s w a s c o m p l e t e l y a b o l i s h e d b y P D 9 8 0 5 9 , s u g g e s t i n g t h a t E R K 1 / 2 m e d i a t e t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I I a n d t h a t t h e M E K -M A P K p a t h w a y m a y b e a n i n t e g r a l m e d i a t o r o f G n R H - i n d u c e d f u n c t i o n s s u c h a s c e l l g r o w t h a n d / o r a p o p t o s i s . I n t h e p r e v i o u s s t u d y , p 3 8 M A P K w a s i n v o l v e d i n t h e G n R H - I I -i n d u c e d i n h i b i t i o n o f c e l l g r o w t h t h r o u g h a c t i v a t o r p r o t e i n - 1 a c t i v a t i o n , w h i c h m a y b e r e l a t e d t o i n d u c t i o n o f a p o p t o s i s i n o v a r i a n c a n c e r c e l l s ( K i m e t a l . 2 0 0 4 a ) . It c a n b e a s s u m e d t h a t p 3 8 M A P K i s i n v o l v e d i n G n R H - I I - i n d u c e d a p o p t o t i c p a t h w a y a n d E R K 1 / 2 M A P K i s i n v o l v e d i n G n R H - I I - i n d u c e d c e l l g r o w t h i n h i b i t i o n i n o v a r i a n c a n c e r c e l l s . H o w e v e r , i t h a s b e e n r e p o r t e d t h a t t h e M A P K p a t h w a y s a r e r e g u l a t e d b y o t h e r s i g n a l i n g p a t h w a y s ( Z h a n g a n d L i u 2 0 0 2 ) a n d f u r t h e r s t u d y i s n e c e s s a r y t o i n v e s t i g a t e a p o s s i b l e i n v o l v e m e n t o f o t h e r p a t h w a y s i n t h i s c e l l u l a r r e s p o n s e . G n R H - I a n d I I r e c e p t o r s b e l o n g t o t h e G - p r o t e i n c o u p l e d r e c e p t o r f a m i l y , w h i c h i s k n o w n t o r e g u l a t e t h e M A P K c a s c a d e ( S u g d e n a n d C l e r k 1 9 9 7 ) . H o w e v e r , t h e i s s u e o f w h e t h e r t h e G n R H - I r e c e p t o r o r G n R H - I I r e c e p t o r m e d i a t e s t h e e f f e c t s o f G n R H - I I a n d w h e t h e r t h e G n R H - I I r e c e p t o r i s f u n c t i o n a l i n h u m a n s r e m a i n s u n s o l v e d . I n a d d i t i o n , G n R H - I I w a s m o r e s t a b l e t h a n G n R H - I i n m a m m a l i a n o v a r y a n d t h e e x i s t e n c e o f G n R H - I I 8 8 r e c e p t o r w a s p r o p o s e d ( S i l e r - K h o d r e t a l . 2 0 0 3 ) . T h e r e f o r e , t h e p o s s i b i l i t y t h a t t h e e f f e c t o f G n R H - I I i s m e d i a t e d b y f u n c t i o n a l G n R H - I I r e c e p t o r s r e m a i n s t o b e d e t e r m i n e d . I n c o n c l u s i o n , t h e p r e s e n t s t u d y d e m o n s t r a t e s t h a t t r e a t m e n t w i t h G n R H - I I i n d u c e s t h e p h o s p h o r y l a t i o n o f E R K 1 / 2 i n O V C A R - 3 c e l l s , w h i c h w a s r e d u c e d b y a M E K i n h i b i t o r , P D 9 8 0 5 9 . I n a n in vitro k i n a s e a s s a y , t r e a t m e n t w i t h G n R H - I I r e s u l t e d i n t h e p h o s p h o r y l a t i o n o f E l k - 1 a n d t h i s e f f e c t w a s n o t a l s o b l o c k e d b y P D 9 8 0 5 9 . T h i s s u g g e s t s t h a t E l k - 1 m a y n o t m e d i a t e c e l l u l a r r e s p o n s e s b y G n R H - I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n . F u r t h e r m o r e , t h e g r o w t h i n h i b i t o r y e f f e c t s o f G n R H - I I w e r e a t t e n u a t e d b y P D 9 8 0 5 9 . T h e s e r e s u l t s , t a k e n t o g e t h e r w i t h o u r p r e v i o u s s t u d y , s t r o n g l y s u g g e s t t h a t G n R H - I I - i n d u c e d M A P K a c t i v a t i o n i n c l u d i n g E R K 1 / 2 a n d p 3 8 , b u t n o t J N K / S A P K 1 m e d i a t e c e l l u l a r r e s p o n s e s s u c h a s g r o w t h i n h i b i t i o n a n d i n d u c t i o n o f a p o p t o s i s i n o v a r i a n c a n c e r c e l l s . 4.3 Role of p38 M A P K in GnRH-II signaling in ovarian cancer cells G n R H - I I m R N A i s e x p r e s s e d i n n o r m a l O S E , i m m o r t a l i z e d O S E ( I O S E ) c e l l s , p r i m a r y c u l t u r e s o f o v a r i a n t u m o r s a n d o v a r i a n c a n c e r c e l l l i n e s . I n a d d i t i o n , t r e a t m e n t s w i t h i n c r e a s i n g d o s e s ( 1 0 " 9 - 1 0 * 7 M ) o f G n R H - I I r e s u l t e d i n a g r o w t h - i n h i b i t i o n i n I O S E c e l l l i n e s , s u g g e s t i n g t h a t G n R H - I I , s i m i l a r t o G n R H - I , m a y h a v e a g r o w t h - r e g u l a t o r y e f f e c t i n n o r m a l a n d n e o p l a s t i c O S E c e l l s ( C h o i e t a l . 2 0 0 1 b ) . A l t h o u g h G n R H - I I m a y h a v e a l e s s e r e f f e c t t h a n G n R H - I o n t h e s e c r e t i o n o f h C G i n c y t o t r o p h o b l a s t i c c e l l s ( I s l a m i e t a l . 2 0 0 1 ) , t h e m R N A e x p r e s s i o n o f G n R H - I I w a s d e t e c t e d a t h i g h e r l e v e l s ( u p t o 3 0 t i m e s m o r e t h a n G n R H - I ) i n t h e k i d n e y , b o n e m a r r o w , a n d p r o s t a t e t h a n G n R H - I ( W h i t e e t a l . 1 9 9 8 ) . 8 9 Treatment with GnRH-II resulted in a significant anti-proliferative effect in a dose-dependent manner in OVCAR-3 cells. It appears that no significant difference in the anti-proliferative effect of GnRH-II was observed at various doses (1 nM to 10 uM). It is of note that the effect of GnRH-II on cell proliferation and apoptosis does not seem to be significantly different from that of GnRH-I. The discrepancy with a previous report (Grundker et al. 2002) that GnRH-II has a more potent anti-proliferative effect than GnRH-I might be a result of the use of different cell types and culture conditions in the two studies. It implies that treatment with GnRH may have different effect on other cell types including primary ovarian cancer cells. To investigate the signaling pathway involved in the anti-proliferative effect of GnRH-II, the activation of p38, one of the MAPKs, was measured by Western blot analysis using a phospho-specific p38 MAPK antibody following treatment with GnRH-II. p38 MAPK is activated by phosphorylation on threonine 180 and tyrosine 182 in the activation loop and modulates cell-cycle, transcriptional activity and programmed cell death in response to environmental stress, hormones, ligands that bind G-protein coupled receptors, and inflammatory cytokines (Johnson and Lapadat 2002). We observed that GnRH-II (100 nM) induced the activation of p38 MAPK (3.5-fold vs control) as early as 10 min, which is in agreement with a previous study performed in COS-7 cells (Millar et al. 2001). Furthermore, the activation of p38 MAPK was completely blocked by SB203580 (1 uM), a specific inhibitor of p38 MAPK (Saklatvala et al. 1996). Previously, it has been shown that GnRH-I induced the activation of p38 as well as ERK1/2 and JNK in pituitary cell lines (Reiss et al. 1997; Levi et al. 1998a; Roberson et al. 1999; Liu et al. 2002), and that this was reversed by chronic phorbol 9 0 e s t e r t r e a t m e n t . F u r t h e r r e s e a r c h i s r e q u i r e d t o e l u c i d a t e t h e r e l a t i v e e f f e c t o f G n R H - I v s . G n R H - I I o n a c t i v a t i o n o f t h e 3 8 M A P K p a t h w a y i n o v a r i a n c a n c e r c e l l s . I n t h i s s t u d y , t r e a t m e n t w i t h i n c r e a s i n g d o s e s o f G n R H - I I f o r 2 , 4 a n d 6 d a y s d e c r e a s e d t h e a m o u n t o f [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n D N A s y n t h e s i s i n O V C A R - 3 c e l l s . T h i s r e s u l t i s i n a g r e e m e n t w i t h t h e p r e v i o u s r e p o r t u s i n g c e l l c o u n t i n g m e t h o d w i t h t h e N e u b a u e r h e m o c y t o m e t e r ( G r u n d k e r e t a l . 2 0 0 2 ) . T o e l u c i d a t e t h e s i g n a l i n g p a t h w a y i n v o l v e d i n p 3 8 M A P K i n t h i s a n t i - p r o l i f e r a t i v e e f f e c t b y G n R H - I I , c e l l s w e r e p r e t r e a t e d w i t h S B 2 0 3 5 8 0 , f o l l o w e d b y t r e a t m e n t w i t h G n R H - I I ( 1 0 0 n M ) f o r 2 , 4 a n d 6 d a y s . I t i s o f i n t e r e s t t o n o t e t h a t a n a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I I w a s r e v e r s e d i n t h e p r e s e n c e o f S B 2 0 3 5 8 0 , i n d i c a t i n g t h a t t h e a c t i v a t i o n o f p 3 8 M A P K m a y p l a y a c r u c i a l r o l e i n t h e r e g u l a t i o n o f c e l l p r o l i f e r a t i o n i n o v a r i a n c a n c e r . It h a s b e e n r e p o r t e d t h a t t h e a c t i v a t i o n o f E R K i s a l s o i n v o l v e d i n t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I i n C a o v - 3 h u m a n o v a r i a n c a n c e r c e l l l i n e ( K i m u r a e t a l . 1 9 9 9 ) . A l t h o u g h o u r p r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t G n R H - I I a c t i v a t e d t h e E R K 1 / 2 b u t n o t J N K / S A P K 1 i n O V C A R - 3 c e l l s , w h e t h e r o r n o t o t h e r m e m b e r s o f t h e M A P K p a t h w a y s a r e i n v o l v e d i n t h e a n t i -p r o l i f e r a t i v e a c t i o n o f G n R H - I I r e m a i n s t o b e e l u c i d a t e d . T h e i m p o r t a n c e o f a p o p t o s i s i n t u m o r c e l l s h a s b e e n w i d e l y r e c o g n i z e d a s a c r i t i c a l p h e n o m e n o n t o r e g u l a t e c e l l p r o l i f e r a t i o n . It h a s b e e n r e p o r t e d t h a t G n R H - I I i n d u c e d a p o p t o s i s i n g o l d f i s h t e s t i s ( A n d r e u - V i e y r a a n d H a b i b i 2 0 0 1 ) . It i s n o t e w o r t h y t h a t t h e e f f e c t o f G n R H - I a n a l o g s i n t h e i n d u c t i o n o f a p o p t o s i s i s s t i l l c o n t r o v e r s i a l ( K l e i n m a n e t a l . 1 9 9 4 ; T h o m p s o n 1 9 9 5 ; M o t o m u r a 1 9 9 8 ; K i m e t a l . 1 9 9 9 a ; G r u n d k e r e t a l . 2 0 0 0 ) . R e c e n t s t u d i e s f r o m o u r l a b o r a t o r y f o u n d t h a t G n R H - I I h a d a n t i - p r o l i f e r a t i v e e f f e c t o n I O S E c e l l s ( C h o i e t a l . 2 0 0 1 b ) . I n t e r e s t i n g l y , t h e r e s u l t s p r e s e n t e d h e r e i n d e m o n s t r a t e 9 1 t h a t a s i g n i f i c a n t i n c r e a s e i n a p o p t o s i s ( 1 . 6 - f o l d ) w a s i n d u c e d f o l l o w i n g G n R H - I I t r e a t m e n t i n O V C A R - 3 c e l l s , w h i c h i s t h e f i r s t r e p o r t o f t h e i n d u c t i o n o f a p o p t o s i s b y G n R H - I I i n o v a r i a n c a n c e r c e l l s . T h e i n c r e a s e i n D N A f r a g m e n t a t i o n i n d u c e d b y G n R H -I I w a s c o m p l e t e l y i n h i b i t e d b y S B 2 0 3 5 8 0 p r e t r e a t m e n t , s u g g e s t i n g t h a t t h e p 3 8 M A P K s i g n a l i n g c a s c a d e p l a y s a r o l e i n t h e i n d u c t i o n o f a p o p t o s i s b y G n R H - I I i n t h e s e c e l l s . T o c l a r i f y t h e s i g n a l i n g c a s c a d e i n t h e i n d u c t i o n o f a p o p t o s i s , t h e r o l e o f A P - 1 i n G n R H - I I a c t i o n w a s f u r t h e r i n v e s t i g a t e d . A P - 1 i s t h o u g h t t o r e g u l a t e c e l l p r o l i f e r a t i o n , d i f f e r e n t i a t i o n a n d a p o p t o s i s i n d i f f e r e n t s y s t e m s ( S i k o r a e t a l . 1 9 9 3 ; S h a u l i a n a n d K a r i n 2 0 0 2 ) . I t i s k n o w n t h a t M A P K s c o n t r i b u t e t o t h e i n d u c t i o n o f A P - 1 a c t i v i t y w h i c h i s i n v o l v e d i n a p r o t e c t i v e f u n c t i o n a g a i n s t t h e e f f e c t s o f U V - i n d u c e d c e l l d a m a g e ( K a r i n 1 9 9 5 ; K a r i n a n d H u n t e r 1 9 9 5 ; R a i n g e a u d e t a l . 1 9 9 5 ) . It i s i n t e r e s t i n g t h a t G n R H - I n o t o n l y s t i m u l a t e d t h e a c t i v a t i o n o f E l k - 1 t h r o u g h M A P K i n a T 3 c e l l s , b u t i t s a n a l o g a l s o a c t i v a t e d t h e A P - 1 t r a n s c r i p t i o n f a c t o r i n h u m a n e n d o m e t r i a l c a n c e r c e l l s ( R o b e r s o n e t a l . 1 9 9 5 ; G r u n d k e r e t a l . 2 0 0 1 a ) . O u r o b s e r v a t i o n o f a G n R H - I I e f f e c t o n A P - 1 a c t i v a t i o n i s c o n s i s t e n t w i t h t h e s e f i n d i n g s . T h e t r a n s f e c t i o n a s s a y u s i n g t h e A P - l - T A -L u c v e c t o r r e v e a l e d t h a t G n R H - I I i n d u c e d t h e a c t i v a t i o n o f A P - 1 ( 1 . 5 - f o l d vs c o n t r o l l e v e l ) . T h e p r e t r e a t m e n t w i t h S B 2 0 3 5 8 0 b l o c k e d t h e G n R H - I I - i n d u c e d e f f e c t o n t h e a c t i v a t i o n o f A P - 1 , s u g g e s t i n g t h a t G n R H - I I i n d u c e s a s e q u e n t i a l a c t i v a t i o n o f A P - 1 f o l l o w i n g a c t i v a t i o n o f p 3 8 M A P K . I n c o n c l u s i o n , w e h a v e i d e n t i f i e d t h e p 3 8 M A P K s i g n a l i n g p a t h w a y a s a n i n t e g r a l c o m p o n e n t i n t h e G n R H - I I s i g n a l i n g c a s c a d e , a n d d e m o n s t r a t e d t h a t t h e a n t i -p r o l i f e r a t i v e e f f e c t o f G n R H - I I i n o v a r i a n c a n c e r c e l l s m a y i n v o l v e , a t l e a s t i n p a r t , t h e a c t i v a t i o n o f p 3 8 M A P K . F u r t h e r , w e h a v e d e m o n s t r a t e d t h e i n d u c t i o n o f a p o p t o s i s b y 9 2 G n R H - I I i n o v a r i a n c a n c e r c e l l s , a s w e l l a s t h e G n R H - I I - i n d u c e d a c t i v a t i o n o f A P - 1 t r a n s c r i p t i o n f a c t o r v i a p 3 8 M A P K , i m p l i c a t i n g a p o t e n t i a l r o l e o f A P - 1 i n o v a r i a n c a n c e r c e l l g r o w t h . F u r t h e r s t u d y o f t h e r e l a t i o n s h i p b e t w e e n t h e G n R H - I I a n d s i g n a l i n g p a t h w a y s i n v o l v e d i n c e l l p r o l i f e r a t i o n a n d a p o p t o s i s w i l l p r o v i d e a b e t t e r i n s i g h t f o r t h e u n d e r s t a n d i n g o f o v a r i a n c a n c e r a n d t h e d e s i g n o f n o v e l t h e r a p e u t i c a p p r o a c h e s . 4.4 Investigation of GTP binding proteins and PTK in GnRH signaling P r e v i o u s s t u d i e s h a v e s h o w n t h a t t h e P T X - s e n s i t i v e G a i p r o t e i n m e d i a t e s t h e a n t i -p r o l i f e r a t i v e e f f e c t s o f G n R H - I ( G r u n d k e r e t a l . 2 0 0 1 b ) , a n d G n R H - i n d u c e d , E R K a c t i v a t i o n i n o t h e r o v a r i a n c a n c e r c e l l l i n e s ( K i m u r a e t a l . 1 9 9 9 ) . H o w e v e r , i t h a s a l s o b e e n n o t e d t h a t G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n i s G ( i / 0 ) i n d e p e n d e n t a n d P K C - d e p e n d e n t i n o t h e r o v a r i a n c a n c e r c e l l s ( C h a m s o n - R e i g e t a l . 2 0 0 3 ) , a n d t h a t t h e G n R H - I r e c e p t o r a c t i v a t e s p h o s p h o l i p a s e C v i a G a q i n a T 3 c e l l s ( H s i e h a n d M a r t i n 1 9 9 2 ) . I n a d d i t i o n , w e r e c e n t l y d e m o n s t r a t e d t h a t G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n i s m e d i a t e d b y t h e P K C p a t h w a y i n O V C A R - 3 a n d S K O V - 3 o v a r i a n c a n c e r c e l l s ( K i m e t a l . 2 0 0 6 ) . T o e x a m i n e t h e i n v o l v e m e n t o f o t h e r G p r o t e i n s i n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i - p r o l i f e r a t i o n i n t h e s e c e l l l i n e s , w e p e r f o r m e d a W e s t e r n b l o t a n d t h y m i d i n e i n c o r p o r a t i o n a s s a y s u s i n g H - 8 9 , a s p e c i f i c i n h i b i t o r o f P K A , o r P T X , a s p e c i f i c i n h i b i t o r o f G a j . P r e t r e a t m e n t w i t h H -8 9 r e v e r s e d t h e a c t i v a t i o n o f E R K 1 / 2 b y 8 - b r o m o - c A M P , b u t d i d n o t b l o c k G n R H - I - o r - I I -i n d u c e d a c t i v a t i o n i n O V C A R - 3 c e l l s . I n d e e d , E R K 1 / 2 a c t i v a t i o n i n d u c e d b y m a s t o p a r a n w a s i n h i b i t e d b y P T X , b u t G n R H - i n d u c e d a c t i v a t i o n o f E R K 1 /2 w a s n o t i n h i b i t e d b y P T X , s u g g e s t i n g t h a t G a i s u b u n i t i s n o t i n v o l v e d i n G n R H - i n d u c e d a c t i v a t i o n o f E R K 1 / 2 . 9 3 M o r e o v e r , G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n w a s n o t b l o c k e d b y H - 8 9 o r P T X , e x c l u d i n g t h e p o s s i b i l i t y t h a t G a s o r G a j a r e i n v o l v e d i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n i n t h i s c e l l l i n e . I n l i g h t o f o u r p r e v i o u s r e p o r t ( K i m e t a l . 2 0 0 6 ) , t h e p r e s e n t r e s u l t s i n d i c a t e t h a t G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i - p r o l i f e r a t i o n a r e m e d i a t e d b y t h e P K C p a t h w a y a n d d o n o t i n v o l v e G a s a n d G „ j s u b u n i t s T h i s d e m o n s t r a t e s t h a t G n R H s i g n a l i n g v a r i e s c o n s i d e r a b l y i n d i f f e r e n t c e l l t y p e s a n d m a k e s p o s s i b l e a d i v e r s i t y o f r e s p o n s e s d e p e n d i n g o n t h e p a t h w a y s t h a t a r e a c t i v a t e d d i s t i n c t s i t u a t i o n s . P r e v i o u s r e p o r t s h a v e s h o w n t h a t G n R H - I i n h i b i t s t h e e x p r e s s i o n o f E G F r e c e p t o r s a n d r e v e r s e s t h e e f f e c t s o f E G F v i a t h e a c t i v a t i o n o f t y r o s i n e p h o s p h a t a s e s ( L e e e t a l . 1 9 9 1 ; D o n d i e t a l . 1 9 9 6 ) . I n a d d i t i o n , E G F - i n d u c e d a u t o p h o s p h o r y l a t i o n o f E G F r e c e p t o r w a s a b o l i s h e d w i t h c o - t r e a t m e n t o f G n R H ( 1 0 p M ) ( G r u n d k e r e t a l . 2 0 0 1 b ) . H o w e v e r , t h e r e s u l t s i n t h i s s t u d y s h o w e d t h a t E G F - i n d u c e d E G F r e c e p t o r a c t i v a t i o n w a s n o t b l o c k e d w i t h a l o w c o n c e n t r a t i o n ( 1 0 0 n M ) o r a h i g h c o n c e n t r a t i o n ( 1 0 p M ) o f G n R H . T h i s d i s c r e p a n c y s u g g e s t s t h a t t h e e f f e c t o f G n R H o n E G F r e c e p t o r s m a y v a r y c o n s i d e r a b l y i n d i f f e r e n t c e l l t y p e s o r c o n d i t i o n s . I n t h i s s t u d y , w e i n v e s t i g a t e d w h e t h e r t h e E G F s i g n a l i n g p a t h w a y i s i n v o l v e d i n G n R H - i n d u c e d M A P K a c t i v a t i o n i n S K O V - 3 c e l l s . I n t e r e s t i n g l y , w e s h o w e d t h a t G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n w a s a b o l i s h e d w i t h a l o w e r c o n c e n t r a t i o n (1 n M ) o f A G 1 4 7 8 t h a t d i d n o t b l o c k E G F - i n d u c e d E R K 1 / 2 a c t i v a t i o n . H o w e v e r , E G F - i n d u c e d E R K 1 / 2 a c t i v a t i o n w a s b l o c k e d w i t h a h i g h e r c o n c e n t r a t i o n o f ( 5 0 0 n M ) A G 1 4 7 8 . G n R H - I i n d u c e s i t s a n t i - p r o l i f e r a t i v e e f f e c t b y i n t e r f e r i n g w i t h t h e E G F p a t h w a y w h i c h m e d i a t e s t h e m i t o g e n i c a c t i o n o f E G F i n p r o s t a t i c c a n c e r c e l l s a n d e n d o m e t r i a l c a n c e r c e l l s ( D o n d i e t a l . 1 9 9 6 ; G r u n d k e r e t a l . 2 0 0 1 a ) . B i n d i n g o f G n R H - I t o G n R H - I r e c e p t o r s i n h i b i t e d t h e e x p r e s s i o n o f c-fos a n d t h e a c t i v a t i o n o f M A P K b y E G F i n 9 4 g y n e c o l o g i c a l c a n c e r c e l l l i n e s ( E m o n s e t a l . 1 9 9 8 ; G r u n d k e r e t a l . 2 0 0 0 ) . T a k e n t o g e t h e r , t h e s e d a t a s u g g e s t t h a t t h e m e c h a n i s m o f G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d t h e s i g n a l i n g p a t h w a y s i n v o l v e d i n r e g u l a t i n g p r o l i f e r a t i o n v i a E R K 1 / 2 a c t i v a t i o n d i f f e r from t h a t o f E G F . A l t h o u g h A G 1 4 7 8 i s a w e l l k n o w n s p e c i f i c i n h i b i t o r o f t h e E G F r e c e p t o r , i t i s n o t c l e a r a s t o w h y a l o w e r c o n c e n t r a t i o n (1 n M ) o f A G 1 4 7 8 t h a t b l o c k s G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n d o e s n o t b l o c k E G F - i n d u c e d E R K 1 / 2 a c t i v a t i o n . It m a y i n d i c a t e t h a t A G 1 4 7 8 i n l o w c o n c e n t r a t i o n s b l o c k s a n o t h e r s i g n a l i n g p a t h w a y , w h i c h i s i n v o l v e d i n G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n i n s t e a d o f b l o c k i n g t h e E G F r e c e p t o r s i g n a l i n g . I t i s p o s s i b l e t h a t d i f f e r e n t E R K 1 /2 s u b s p e c i e s p a r t i c i p a t e i n d i f f e r e n t h o r m o n a l s t i m u l i a n d i n d u c e a v a r i e t y o f c e l l u l a r r e s p o n s e s i n o v a r i a n c a n c e r c e l l s . F u r t h e r s t u d i e s a r e r e q u i r e d t o c o n f i r m t h e s e o b s e r v a t i o n s a n d t o e x p l a i n t h e s i g n i f i c a n c e o f s u c h f i n d i n g s . E a r l i e r s t u d i e s h a v e s u g g e s t e d t h a t t y r o s i n e p h o s p h o r y l a t i o n m a y b e i n v o l v e d i n G n R H -i n d u c e d s i g n a l i n g ( J o h n s o n e t a l . 1 9 9 5 ; L e v i e t a l . 1 9 9 8 a ) . T o e x p l o r e w h e t h e r t y r o s i n e p h o s p h o r y l a t i o n i s i n v o l v e d i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n , g e n i s t e i n , a g e n e r a l i n h i b i t o r o f p r o t e i n t y r o s i n e k i n a s e s , w a s u s e d i n c o m b i n a t i o n w i t h G n R H . G e n i s t e i n ( 1 0 0 p M ) p a r t i a l l y r e v e r s e d t h e a n t i - p r o l i f e r a t i v e e f f e c t s i n d u c e d b y G n R H - I a n d - I I . I n a d d i t i o n , w h e n w e p e r f o r m e d a W e s t e r n b l o t t o e x a m i n e G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n i n t h e p r e s e n c e o r a b s e n c e o f g e n i s t e i n , t h e r e w a s p a r t i a l i n h i b i t i o n o f G n R H - i n d u c e d E R K 1 / 2 a c t i v a t i o n f o l l o w i n g p r e t r e a t m e n t w i t h g e n i s t e i n f o r 3 0 m i n . T h i s a g r e e s w i t h a p r e v i o u s r e p o r t s h o w e d t h a t g e n i s t e i n ( 2 0 0 p M ) r e d u c e d G n R H - I - i n d u c e d a c t i v a t i o n o f M A P K b y 5 0 % ( R e i s s e t a l . 1 9 9 7 ) . T h e s e r e s u l t s s u g g e s t t h a t p r o t e i n t y r o s i n e k i n a s e p l a y s a r o l e i n t h e s i g n a l i n g c a s c a d e c o n n e c t i n g t h e G n R H r e c e p t o r w i t h E R K 1 / 2 . 9 5 I n c o n c l u s i o n , t h i s s t u d y p r o v i d e s e v i d e n c e t h a t G a i a n d G a s s u b u n i t s m a y n o t b e i n v o l v e d i n t h e a n t i - p r o l i f e r a t i v e e f f e c t s o f G n R H - I a n d - I I . I n a d d i t i o n , t h e p r e s e n t d a t a s h o w t h a t P T K m a y p l a y a r o l e i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n . 4.5 Differential roles of ERKI/2 and p38 M A P K in GnRH signaling E x o g e n o u s l y a d m i n i s t e r e d G n R H a g o n i s t s a c t i n g o n t h e G n R H r e c e p t o r d e s e n s i t i z e a n d d o w n - r e g u l a t e G n R H r e c e p t o r s i n t h e p i t u i t a r y a n d d e c r e a s e L H a n d F S H p r o d u c t i o n , w h i c h i n d u c e s a d e c r e a s e i n t h e c i r c u l a t i n g s e x s t e r o i d l e v e l s ( N e i l l 2 0 0 2 ) . I n a d d i t i o n , e x o g e n o u s G n R H a g o n i s t s s e e m t o e x e r t a n t i - p r o l i f e r a t i v e e f f e c t s o n o v a r i a n c a n c e r c e l l s ( B a l b i e t a l . 2 0 0 4 ; K i m e t a l . 2 0 0 6 ) . G n R H h a s b e e n c o n s i d e r e d i m p o r t a n t f o r t h e d i r e c t s u p p r e s s i o n o f p r o l i f e r a t i o n a n d t h e i n d u c t i o n o f a p o p t o s i s i n t h e t r e a t m e n t o f g y n e c o l o g i c a l c a n c e r s ( K l e i n m a n e t a l . 1 9 9 4 ; M o t o m u r a 1 9 9 8 ; S c h a l l y e t a l . 2 0 0 1 ; K i m e t a l . 2 0 0 4 a ) . M o r e o v e r , t h e d e t e c t i o n o f G n R H a n d i t s r e c e p t o r i n b r e a s t , p l a c e n t a , o v a r y a n d p r o s t a t e t i s s u e s i m p l i e s t h a t G n R H m a y h a v e a d i r e c t e f f e c t o n p e r i p h e r a l t a r g e t s . A l t h o u g h n u m e r o u s s t u d i e s h a v e d e m o n s t r a t e d t h a t G n R H i n d u c e s a p o p t o s i s i n o v a r i a n c a n c e r c e l l s ( K i m e t a l . 1 9 9 9 a ; K a n g e t a l . 2 0 0 0 a ; K i m e t a l . 2 0 0 4 a ) , t h e e x i s t e n c e o f c o n f l i c t i n g r e s u l t s ( G r u n d k e r e t a l . 2 0 0 0 ) l e d u s t o i n v e s t i g a t e w h e t h e r G n R H i n d u c e s a p o p t o s i s i n S K O V - 3 a n d O V C A R - 3 c e l l s . T o e x p l o r e G n R H - i n d u c e d a p o p t o t i c e v e n t s , w e p e r f o r m e d f l o w c y t o m e t r y w i t h A n n e x i n V - F I T C a n d 7 - a m i n o a c t i n o m y c i n D ( 7 A A D ) i n O V C A R - 3 c e l l s . A l t h o u g h n o t o b s e r v e d a f t e r 2 d a y s o f t r e a t m e n t , G n R H - i n d u c e d a p o p t o s i s w a s o b s e r v e d f o l l o w i n g 6 d a y s o f c o n t i n u o u s t r e a t m e n t w i t h G n R H - I o r I I . G n R H - i n d u c e d a p o p t o s i s w a s c o n f i r m e d b y s h o w i n g t h a t G n R H - I a n d II i n d u c e d a p o p t o s i s w i t h i n t h e s a m e 9 6 t i m e f r a m e u s i n g T U N E L a s s a y i n S K O V - 3 c e l l s . T o e x p l o r e w h e t h e r t h e a c t i v a t i o n o f M A P K i s i n v o l v e d i n t h e i n d u c t i o n o f a p o p t o s i s , O V C A R - 3 c e l l s w e r e t r e a t e d w i t h P D 9 8 0 5 9 , a s p e c i f i c i n h i b i t o r o f M E K , o r S B 2 0 3 5 8 0 , a s p e c i f i c i n h i b i t o r o f p 3 8 M A P K . T h e s e s t u d i e s s h o w e d t h a t G n R H - i n d u c e d a p o p t o s i s w a s i n h i b i t e d b y p r e t r e a t m e n t w i t h S B 2 0 3 5 8 0 , b u t n o t b y P D 9 8 0 5 9 , s u g g e s t i n g t h a t p 3 8 M A P K p h o s p h o r y l a t i o n i s r e q u i r e d f o r G n R H - i n d u c e d a p o p t o s i s . O u r p r e v i o u s s t u d i e s h a v e s h o w n t h a t E R K 1 /2 a n d p 3 8 M A P K s a r e i m p o r t a n t p a t h w a y s m e d i a t i n g G n R H - I - i n d u c e d a n t i - p r o l i f e r a t i v e e f f e c t s ( K i m e t a l . 2 0 0 4 a ; K i m e t a l . 2 0 0 4 b ) . I n t e r e s t i n g l y , G n R H - I a n d I I i n h i b i t e d p r o l i f e r a t i o n i n t h e e a r l y p e r i o d ( < 6 d a y s ) , b u t r e s u l t e d i n t h e i n d u c t i o n o f a p o p t o s i s a t a l a t e r t i m e - p o i n t ( > 6 d a y s ) . I n d e e d , o u r p r e v i o u s i r e s u l t s , a n d t h o s e o f t h e p r e s e n t s t u d y , s u g g e s t t h a t E R K 1 / 2 i s i n v o l v e d i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i v e e f f e c t s , w h e r e a s p 3 8 M A P K i s i n v o l v e d i n G n R H - i n d u c e d a p o p t o s i s . T h i s l e d u s t o e x a m i n e w h e t h e r t h e d i f f e r e n t i a l t i m e - d e p e n d e n t a c t i v a t i o n o f E R K 1 / 2 a n d p 3 8 M A P K c o u l d b e r e s p o n s i b l e f o r t h e s e d i f f e r i n g c e l l u l a r r e s p o n s e s . W e f i r s t f o c u s e d o n t h e t i m e - d e p e n d e n t p h o s p h o r y l a t i o n o f E R K 1 / 2 a n d p 3 8 M A P K f o l l o w i n g 1, 2 , 4 o r 6 d a y s o f t r e a t m e n t w i t h G n R H - I o r I I i n O V C A R - 3 c e l l s . T r e a t m e n t w i t h G n R H f o r 1 o r 2 d a y s i n d u c e d t h e a c t i v a t i o n o f E R K 1 / 2 , b u t n o t p 3 8 M A P K , w h i l e t r e a t m e n t f o r 4 d a y s r e s u l t e d i n a m a r k e d i n c r e a s e i n p 3 8 M A P K a c t i v i t y , b u t n o t E R K 1 / 2 a c t i v i t y . M o r e o v e r , i t i s o f i n t e r e s t t o n o t e t h a t t o t a l E R K 1 / 2 o r p 3 8 M A P K l e v e l s d o n o t s e e m t o b e a f f e c t e d b y G n R H t r e a t m e n t i n t h e s e t i m e p e r i o d s . T h e s e d a t a i n d i c a t e t h a t t h e t i m e - d e p e n d e n t a c t i v a t i o n o f E R K 1 / 2 o r p 3 8 M A P K m a y p l a y a r o l e i n d e t e r m i n i n g t h e c e l l u l a r r e s p o n s e i n o v a r i a n c a n c e r c e l l s . 9 7 In conclusion, GnRH-induced apoptosis in ovarian cancer cells is not mediated by the activation of ERK1/2, but likely results from the activation of p38 MAPK. These data suggest that different time-dependent effects of GnRH on ERK1/2 and p38 MAPK phosphorylation could result in early ERKl/2-dependent anti-proliferation and late p38 MAPK-dependent apoptosis. 9 8 V. Summary and Future Studies Summary Role of the GnRH-I receptor and protein kinase C pathway I n t h i s s t u d y , t h e r o l e o f t h e G n R H - I r e c e p t o r a n d P K C w a s i n v e s t i g a t e d i n G n R H - I a n d I I - i n d u c e d E R K 1 / 2 a c t i v a t i o n a n d a n t i - p r o l i f e r a t i o n i n o v a r i a n c a n c e r c e l l l i n e s . 1. A n t i d e a n d G F 1 0 9 2 0 3 X b l o c k e d t h e a c t i v a t i o n o f E R K 1 / 2 a n d a n t i - p r o l i f e r a t i o n i n d u c e d b y G n R H - I a n d I I . 2 . s i R N A t a r g e t i n g t h e G n R H - I r e c e p t o r a b o l i s h e d G n R H - I a n d I I - i n d u c e d a n t i -p r o l i f e r a t i o n . Role of ERKI/2 in GnRH-II involved in the inhibition of ovarian cancer cell proliferation I n t h i s s t u d y , t h e e f f e c t o f G n R H - I I o n t h e a c t i v a t i o n o f E R K I / 2 a n d J N K / S A P K 1 w a s e x a m i n e d i n o v a r i a n c a n c e r c e l l l i n e s . I n a d d i t i o n , t h e p o s s i b l e i n v o l v e m e n t o f t h e E R K I / 2 p a t h w a y i n m e d i a t i n g t h e a n t i - p r o l i f e r a t i v e e f f e c t s o f G n R H - I I w a s i n v e s t i g a t e d i n o v a r i a n c a n c e r c e l l l i n e s . 1. G n R H - I I i n d u c e s t h e p h o s p h o r y l a t i o n o f E R K I / 2 i n O V C A R - 3 c e l l s , w h i c h w a s r e d u c e d b y a M E K i n h i b i t o r , P D 9 8 0 5 9 . 2 . G n R H - I I r e s u l t e d i n t h e p h o s p h o r y l a t i o n o f E l k - 1 a n d t h i s e f f e c t w a s n o t b l o c k e d b y P D 9 8 0 5 9 . T h i s s u g g e s t s t h a t E l k - 1 m a y n o t m e d i a t e c e l l u l a r r e s p o n s e s b y G n R H - I I -i n d u c e d E R K 1 /2 a c t i v a t i o n . 3 . T h e g r o w t h i n h i b i t o r y e f f e c t s o f G n R H - I I w e r e a t t e n u a t e d b y P D 9 8 0 5 9 . 9 9 Role of p38 mitogen-activatedprotein kinase and apoptosis in ovarian cancer cells I n t h i s s t u d y , t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I I w a s e x a m i n e d i n t h e o v a r i a n a d e n o c a r c i n o m a O V C A R - 3 c e l l l i n e . I n a d d i t i o n t o c e l l g r o w t h , t h e p o s s i b l e e f f e c t o f G n R H - I I i n t h e a c t i v a t i o n o f p 3 8 M A P K a n d A P - 1 t r a n s c r i p t i o n a l f a c t o r w a s i n v e s t i g a t e d . 1. T h e p 3 8 M A P K s i g n a l i n g p a t h w a y w a s i d e n t i f i e d a s a n i n t e g r a l c o m p o n e n t i n t h e G n R H - I I s i g n a l i n g c a s c a d e . 2 . P 3 8 M A P K a c t i v a t i o n i s i n v o l v e d i n t h e a n t i - p r o l i f e r a t i v e e f f e c t o f G n R H - I I i n o v a r i a n c a n c e r c e l l s . 3 . G n R H - I I i n d u c e d a p o p t o s i s i n o v a r i a n c a n c e r c e l l s . 4 . G n R H - I I i n d u c e d t h e a c t i v a t i o n o f t h e A P - 1 t r a n s c r i p t i o n f a c t o r v i a p 3 8 M A P K . Role of G protein as and ai subunits, ERK1/2, p38 mitogen-activated protein kinase, and protein tyrosine kinase I n t h i s s t u d y , t h e r o l e o f G p r o t e i n s u b u n i t ( G a s a n d G a j ) a n d P T K w a s e x a m i n e d i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n i n o v a r i a n c a n c e r c e l l s . I n a d d i t i o n , t h e p o s s i b l e r o l e o f M A P K i n G n R H - i n d u c e d a p o p t o s i s w a s i n v e s t i g a t e d i n o v a r i a n c a n c e r c e l l s . 1. G a i a n d G a s s u b u n i t s a r e n o t i n v o l v e d i n t h e a n t i - p r o l i f e r a t i v e e f f e c t s o f G n R H - I a n d - I I . 2 . P T K m a y p l a y a r o l e i n G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n . 3 . G n R H - i n d u c e d a p o p t o s i s i n o v a r i a n c a n c e r c e l l s i s n o t m e d i a t e d b y t h e a c t i v a t i o n o f E R K 1 / 2 , b u t l i k e l y r e s u l t s f r o m t h e a c t i v a t i o n o f p 3 8 M A P K . T a k e n t o g e t h e r , t h e s e r e s u l t s d e m o n s t r a t e t h a t t h e G n R H - I r e c e p t o r a n d P K C , 100 E R K 1 / 2 , p 3 8 M A P K p l a y a n i m p o r t a n t r o l e i n m e d i a t i n g t h e c e l l u l a r r e s p o n s e s i n d u c e d b y G n R H - I I , a s w e l l a s G n R H - I , i n o v a r i a n c a n c e r c e l l s a s p r o p o s e d i n F i g . 3 0 a n d m a y b e a p o t e n t i a l t a r g e t i n t h e t r e a t m e n t o f o v a r i a n c a n c e r . 1 0 1 GnRH-I/-II Apoptosis ^ Cell Proliferation F i g u r e 3 0 . P r o p o s e d i n t r a c e l l u l a r s i g n a l i n g c a s c a d e o f G n R H - I a n d II i n o v a r i a n c a n c e r c e l l s . 1 0 2 Future study The intracellular pathway following MAPK activation by GnRH in ovarian cancer cells F u t u r e e x p e r i m e n t s s h o u l d f o c u s o n d e t e r m i n g t h e e x a c t m e c h a n i s m s f o r t h e d i f f e r e n t i a l r e g u l a t i o n b e t w e e n M A P K - i n d u c e d a n t i - p r o l i f e r a t i v e e f f e c t a n d c e l l g r o w t h e f f e c t . T h e r e l e v a n c e o f i n t r a c e l l u l a r f a c t o r s f o l l o w i n g M A P K a c t i v a t i o n b y v a r i o u s h o r m o n e s t i m u l i s h o u l d b e i n v e s t i g a t e d . The autocrine role of GnRH-I and II in ovarian cancer cells T h e e f f e c t o f t h e e x o g e n o u s G n R H t r e a t m e n t o n t h e p r o d u c t i o n o f e n d o g e n o u s G n R H n e e d s t o b e i n v e s t i g a t e d . The transcriptional gene regulation by GnRH-induced MAPK. F u t u r e s t u d i e s n e e d t o b e d o n e t o c l a r i f y t h e a c t i v a t i o n o f t r a n s c r i p t i o n a l f a c t o r s b y G n R H - i n d u c e d M A P K a c t i v a t i o n . The intracellular mechanisms involved in different ERK1/2 and p38 MAPK activation in a time dependent manner I n t h e p r e s e n t s t u d y , t h e a c t i v a t i o n o f E R K 1 / 2 a n d p 3 8 M A P K d i f f e r i n a t i m e d e p e n d e n t m a n n e r . T h e m e c h a n i s m o f t h e a c t i v a t i o n o f E R K 1 /2 a n d p 3 8 m a y b e a s s o c i a t e d w i t h d i f f e r e n t i n t r a c e l l u l a r p a t h w a y i n a d i f f e r e n t t i m e p e r i o d . F u t u r e e x p e r i m e n t s s h o u l d c l a r i f y t h e i n t r a c e l l u l a r m e c h a n i s m s i n v o l v e d i n t h e i r a c t i v a t i o n . 1 0 3 Involvement of other hormonal stimuli in activating MAPK by GnRH in ovarian cancer cells M A P K s a r e n o t o n l y a c t i v a t e d b y G n R H b u t o t h e r h o r m o n e s t i m u l i s u c h a s e s t r o g e n . T h e s e h o r m o n e s t i u m u l i n e e d t o b e c l a r i f i e d w h e t h e r t h e y i n c o r p o r a t e o r i n t e r v e n e t o i n d u c e c e l l u l a r r e s p o n s e s . Apoptotic pathway by GnRH-I and II in normal and ovarian cancer cells F u t u r e e x p e r i m e n t s a r e r e q u i r e d t o i n v e s t i g a t e t h e m e c h a n i s m o f G n R H -i n d u c e d p 3 8 M A P K a c t i v a t i o n t o c a u s e a p o p t o s i s . I n a d d i t i o n , t h e m o l e c u l a r m e c h a n i s m o f G n R H - i n d u c e d a p o p t o s i s i n n o r m a l a n d o v a r i a n c a n c e r c e l l s n e e d s t o b e s t u d i e d . The mechanism for regulating GnRH receptor expression F u t u r e e x p e r i m e n t s a r e r e q u i r e d t o i n v e s t i g a t e t h e l e v e l o f G n R H r e c e p t o r s i n v o l v e d i n t h e G n R H - i n d u c e d a n t i - p r o l i f e r a t i o n . The intracellular mechanisms of GnRH in primary ovarian cancer cells F u t u r e e x p e r i m e n t s a r e r e q u i r e d t o c o m p a r e t h e e f f e c t o f G n R H i n b e t w e e n o v a r i a n c a n c e r c e l l l i n e s a n d p r i m a r y o v a r i a n c a n c e r c e l l s . 1 0 4 The intracellular mechanisms involved in MAPK activation in normal ovarian surface epithelial cells T h e a c t i v a t i o n o f M A P K s m a y d i f f e r i n b e t w e e n n o r m a l o v a r i a n s u r f a c e e p i t h e l i a l c e l l s a n d o v a r i a n c a n c e r c e l l s . F u t u r e e x p e r i m e n t s s h o u l d c l a r i f y t h e i n t r a c e l l u l a r m e c h a n i s m s i n v o l v e d i n t h e i r a c t i v a t i o n . 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J B i o l C h e r r i 2 7 4 : 1 7 9 0 1 - 1 7 9 0 7 G r o s s e R , R o e l l e S , H e r r l i c h A , H o h n J a n d G u d e r m a n n T 2 0 0 0 E p i d e r m a l g r o w t h f a c t o r r e c e p t o r t y r o s i n e k i n a s e m e d i a t e s R a s a c t i v a t i o n b y g o n a d o t r o p i n - r e l e a s i n g h o r m o n e J B i o l C h e m 2 7 5 : 1 2 2 5 1 - 6 0 G r u n d k e r C a n d E m o n s G 2 0 0 3 R o l e o f g o n a d o t r o p i n - r e l e a s i n g h o r m o n e ( G n R H ) i n o v a r i a n c a n c e r R e p r o d u c t i v e B i o l o g y a n d E n d o c r i n o l o g y 1: 6 5 G r u n d k e r C , G u n t h e r t A R , M i l l a r R P a n d E m o n s G 2 0 0 2 E x p r e s s i o n o f g o n a d o t r o p i n -r e l e a s i n g h o r m o n e I I ( G n R H - I I ) r e c e p t o r i n h u m a n e n d o m e t r i a l a n d o v a r i a n c a n c e r c e l l s a n d e f f e c t s o f G n R H - I I o n t u m o r c e l l p r o l i f e r a t i o n J C l i n E n d o c r i n o l M e t a b 8 7 : 1 4 2 7 - 1 4 3 0 G r u n d k e r C , S c h l o t a w a L , V i e r e c k V , E i c k e N , H o r s t A , K a i r i e s B a n d E m o n s G 2 0 0 4 111 A n t i p r o l i f e r a t i v e e f f e c t s o f t h e G n R H a n t a g o n i s t c e t r o r e l i x a n d o f G n R H - I I o n h u m a n e n d o m e t r i a l a n d o v a r i a n c a n c e r c e l l s a r e n o t m e d i a t e d t h r o u g h t h e G n R H t y p e I r e c e p t o r E u r J E n d o c r i n o l 1 5 1 : 1 4 1 - 1 4 9 G r u n d k e r C , S c h l o t a w a L , V i e r e c k V a n d E m o n s G 2 0 0 1 a P r o t e i n k i n a s e C - i n d e p e n d e n t s t i m u l a t i o n o f a c t i v a t o r p r o t e i n - 1 a n d c - J u n N - t e r m i n a l k i n a s e a c t i v i t y i n h u m a n e n d o m e t r i a l c a n c e r c e l l s b y t h e L H R H a g o n i s t t r i p t o r e l i n E u r J E n d o c r i n o l 1 4 5 ( 5 ) : 6 5 1 - 8 : 6 5 1 - 6 5 8 G r u n d k e r C , S c h u l z K , G u n t h e r t A R a n d E m o n s G 2 0 0 0 L u t e i n i z i n g H o r m o n e - R e l e a s i n g H o r m o n e I n d u c e s N u c l e a r F a c t o r { k a p p a } B - A c t i v a t i o n a n d I n h i b i t s A p o p t o s i s i n O v a r i a n C a n c e r C e l l s J C l i n E n d o c r i n o l M e t a b 8 5 : 3 8 1 5 - 3 8 2 0 G r u n d k e r C , V o l k e r P a n d E m o n s G 2 0 0 1 b A n t i p r o l i f e r a t i v e S i g n a l i n g o f L u t e i n i z i n g H o r m o n e - R e l e a s i n g H o r m o n e i n H u m a n E n d o m e t r i a l a n d O v a r i a n C a n c e r C e l l s t h r o u g h G P r o t e i n { { a l p h a } } I - M e d i a t e d A c t i v a t i o n o f P h o s p h o t y r o s i n e P h o s p h a t a s e E n d o c r i n o l o g y 1 4 2 : 2 3 6 9 - 2 3 8 0 G u p t a S , C a m p b e l l D , D e r i j a r d B a n d D a v i s R J 1 9 9 5 T r a n s c r i p t i o n f a c t o r A T F 2 r e g u l a t i o n b y t h e J N K s i g n a l t r a n s d u c t i o n p a t h w a y S c i e n c e 2 6 7 : 3 8 9 - 3 9 3 H a r r i s D , R e i s s N a n d N a o r Z 1 9 9 7 a D i f f e r e n t i a l a c t i v a t i o n o f p r o t e i n k i n a s e C d e l t a a n d e p s i l o n g e n e e x p r e s s i o n b y g o n a d o t r o p i n - r e l e a s i n g h o r m o n e i n a l p h a T 3 - l c e l l s . A u t o r e g u l a t i o n b y p r o t e i n k i n a s e C J B i o l C h e m 2 7 2 : 1 3 5 3 4 - 1 3 5 4 0 H a r r i s D , R e i s s N a n d N a o r Z 1 9 9 7 b D i f f e r e n t i a l a c t i v a t i o n o f p r o t e i n k i n a s e C d e l t a a n d e p s i l o n g e n e e x p r e s s i o n b y g o n a d o t r o p i n - r e l e a s i n g h o r m o n e i n a l p h a T 3 - l c e l l s . A u t o r e g u l a t i o n b y p r o t e i n k i n a s e C J B i o l C h e m 2 7 2 : 1 3 5 3 4 - 4 0 H a r r i s o n G S , W i e r m a n M E , N e t t T M a n d G l o d e L M 2 0 0 4 G o n a d o t r o p i n - r e l e a s i n g h o r m o n e a n d i t s r e c e p t o r i n n o r m a l a n d m a l i g n a n t c e l l s E n d o c r R e l a t C a n c e r 1 1 : 7 2 5 - 7 4 8 1 1 2 H a z u m E a n d C o n n P M 1 9 8 8 M o l e c u l a r m e c h a n i s m o f g o n a d o t r o p i n r e l e a s i n g h o r m o n e ( G n R H ) a c t i o n . I . 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r e l e a s i n g h o r m o n e a n d g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r s a c t i v a t e p h o s p h o l i p a s e C b y c o u p l i n g t o t h e g u a n o s i n e t r i p h o s p h a t e -b i n d i n g p r o t e i n s G q a n d G i l M o l e c u l a r E n d o c r i n o l o g y ( B a l t i m o r e , M d . ) 6 : 1 6 7 3 - 1 6 8 1 113 H u a n g S C , T a n g M J , H s u K F , C h e n g Y M a n d C h o u C Y 2 0 0 2 a F a s a n d i t s l i g a n d , c a s p a s e s , a n d b c l - 2 e x p r e s s i o n i n g o n a d o t r o p i n - r e l e a s i n g h o r m o n e a g o n i s t - t r e a t e d u t e r i n e l e i o m y o m a J C l i n E n d o c r i n o l M e t a b 8 7 : 4 5 8 0 - 4 5 8 6 H u a n g Y T , H w a n g J J , L e e L T , L i e b o w C , L e e P P , K e F C , L o T B , S c h a l l y A V a n d L e e M T 2 0 0 2 b I n h i b i t o r y e f f e c t s o f a l u t e i n i z i n g h o r m o n e - r e l e a s i n g h o r m o n e a g o n i s t o n b a s a l a n d e p i d e r m a l g r o w t h f a c t o r - i n d u c e d c e l l p r o l i f e r a t i o n a n d m e t a s t a s i s - a s s o c i a t e d p r o p e r t i e s i n h u m a n e p i d e r m o i d c a r c i n o m a A 4 3 1 c e l l s I n t J C a n c e r 9 9 : 5 0 5 - 5 1 3 H u b b a r d K B a n d H e p l e r J R 2 0 0 6 C e l l s i g n a l l i n g d i v e r s i t y o f t h e G q [ a l p h a ] f a m i l y o f h e t e r o t r i m e r i c G p r o t e i n s C e l l u l a r S i g n a l l i n g 1 8 : 1 3 5 - 1 5 0 I m a i A , T a k a g i A , H o r i b e S , T a k a g i H a n d T a m a y a T 1 9 9 8 a E v i d e n c e f o r T i g h t C o u p l i n g o f G o n a d o t r o p i n - R e l e a s i n g H o r m o n e R e c e p t o r t o S t i m u l a t e d F a s L i g a n d E x p r e s s i o n i n R e p r o d u c t i v e T r a c t T u m o r s : P o s s i b l e M e c h a n i s m f o r H o r m o n a l C o n t r o l o f A p o p t o t i c C e l l D e a t h J C l i n E n d o c r i n o l M e t a b 8 3 : 4 2 7 - 4 3 1 I m a i A , T a k a g i A , H o r i b e S , T a k a g i H a n d T a m a y a T 1 9 9 8 b F a s a n d F a s l i g a n d s y s t e m m a y m e d i a t e a n t i p r o l i f e r a t i v e a c t i v i t y o f g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r i n e n d o m e t r i a l c a n c e r c e l l s I n t J O n c o l 1 3 : 9 7 - 1 0 0 I m a i A , T a k a g i H , H o r i b e S , F u s e y a T a n d T a m a y a T 1 9 9 6 C o u p l i n g o f g o n a d o t r o p i n -r e l e a s i n g h o r m o n e r e c e p t o r t o G i p r o t e i n i n h u m a n r e p r o d u c t i v e t r a c t t u m o r s J C l i n E n d o c r i n o l M e t a b 8 1 : 3 2 4 9 - 3 2 5 3 I n n e r G , B u r g e r C , M u l l e r R , O r t m a n n O , P e t e r U , K a k a r S S , N e i l l J D , S c h u l z K D a n d E m o n s G 1 9 9 5 E x p r e s s i o n o f t h e m e s s e n g e r R N A s f o r l u t e i n i z i n g h o r m o n e - r e l e a s i n g h o r m o n e ( L H R H ) a n d i t s r e c e p t o r i n h u m a n o v a r i a n e p i t h e l i a l c a r c i n o m a C a n c e r R e s 5 5 : 8 1 7 - 8 2 2 I s l a m i D , C h a r d o n n e n s D , C a m p a n a A a n d B i s c h o f P 2 0 0 1 C o m p a r i s o n o f t h e e f f e c t s o f G n R H - I a n d G n R H - I I o n H C G s y n t h e s i s a n d s e c r e t i o n b y first t r i m e s t e r t r o p h o b l a s t M o i 1 1 4 H u m R e p r o d 7 : 3 - 9 I w a s h i t a M , E v a n s M I a n d C a t t K J 1 9 8 6 C h a r a c t e r i z a t i o n o f a g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r s i t e i n t e r m p l a c e n t a a n d c h o r i o n i c v i l l i J C l i n E n d o c r i n o l M e t a b 6 2 : 1 2 7 -1 3 3 J a n k n e c h t R , E r n s t W H , P i n g o u d V a n d N o r d h e i m A 1 9 9 3 A c t i v a t i o n o f t e r n a r y c o m p l e x f a c t o r E l k - 1 b y M A P k i n a s e s E m b o J 1 2 : 5 0 9 7 - 5 1 0 4 J i a n g Y , M a W , W a n Y , K o z a s a T , H a t t o r i S a n d H u a n g X Y 1 9 9 8 T h e G p r o t e i n G a l p h a l 2 s t i m u l a t e s B r u t o n ' s t y r o s i n e k i n a s e a n d a r a s G A P t h r o u g h a c o n s e r v e d P H / B M d o m a i n N a t u r e 3 9 5 : 8 0 8 - 8 1 3 . J o h n s o n G L a n d L a p a d a t R 2 0 0 2 M i t o g e n - A c t i v a t e d P r o t e i n K i n a s e P a t h w a y s M e d i a t e d b y E R K , J N K , a n d p 3 8 P r o t e i n K i n a s e s S c i e n c e 2 9 8 : 1 9 1 1 - 1 9 1 2 J o h n s o n M S , W o l b e r s W B , N o b l e J , F e n n e l l M a n d M i t c h e l l R 1 9 9 5 E f f e c t o f t y r o s i n e k i n a s e i n h i b i t o r s o n l u t e i n i z i n g h o r m o n e - r e l e a s i n g h o r m o n e ( L H R H ) - i n d u c e d g o n a d o t r o p i n r e l e a s e f r o m t h e a n t e r i o r p i t u i t a r y M o i C e l l E n d o c r i n o l 1 0 9 : 6 9 - 7 5 J o r i s s e n R N , W a l k e r F , P o u l i o t N , G a r r e t t T P , W a r d C W a n d B u r g e s s A W 2 0 0 3 E p i d e r m a l g r o w t h f a c t o r r e c e p t o r : m e c h a n i s m s o f a c t i v a t i o n a n d s i g n a l l i n g E x p C e l l R e s 2 8 4 : 3 1 - 5 3 K a k a r S S , M u s g r o v e L C , D e v o r D C , S e l l e r s J C a n d N e i l l J D 1 9 9 2 C l o n i n g , s e q u e n c i n g , a n d e x p r e s s i o n o f h u m a n g o n a d o t r o p i n r e l e a s i n g h o r m o n e ( G n R H ) r e c e p t o r B i o c h e m B i o p h y s R e s C o m m u n 1 8 9 : 2 8 9 - 2 9 5 K a n g S K , C h e n g K W , N a t h w a n i P S , C h o i K C a n d L e u n g P C 2 0 0 0 a A u t o c r i n e r o l e o f g o n a d o t r o p i n - r e l e a s i n g h o r m o n e a n d i t s r e c e p t o r i n o v a r i a n c a n c e r c e l l g r o w t h E n d o c r i n e 1 3 : 2 9 7 - 3 0 4 1 1 5 K a n g S K , C h o i K - C , T a i C - J , A u e r s p e r g N a n d L e u n g P C K 2 0 0 1 a E s t r a d i o l R e g u l a t e s G o n a d o t r o p i n - R e l e a s i n g H o r m o n e ( G n R H ) a n d i t s R e c e p t o r G e n e E x p r e s s i o n a n d A n t a g o n i z e s t h e G r o w t h I n h i b i t o r y E f f e c t s o f G n R H i n H u m a n O v a r i a n S u r f a c e E p i t h e l i a l a n d O v a r i a n C a n c e r C e l l s E n d o c r i n o l o g y 1 4 2 : 5 8 0 - 5 8 8 K a n g S K , T a i C - J , C h e n g K W a n d L e u n g P C K 2 0 0 0 b G o n a d o t r o p i n - r e l e a s i n g h o r m o n e a c t i v a t e s m i t o g e n - a c t i v a t e d p r o t e i n k i n a s e i n h u m a n o v a r i a n a n d p l a c e n t a l c e l l s M o l e c u l a r a n d C e l l u l a r E n d o c r i n o l o g y 1 7 0 : 1 4 3 - 1 5 1 K a n g S K , T a i C - J , N a t h w a n i P S , C h o i K - C a n d L e u n g P C K 2 0 0 1 b S t i m u l a t i o n o f M i t o g e n -A c t i v a t e d P r o t e i n K i n a s e b y G o n a d o t r o p i n - R e l e a s i n g H o r m o n e i n H u m a n G r a n u l o s a -L u t e a l C e l l s E n d o c r i n o l o g y 1 4 2 : 6 7 1 - 6 7 9 K a r i n M 1 9 9 5 T h e R e g u l a t i o n o f A P - 1 A c t i v i t y b y M i t o g e n - a c t i v a t e d P r o t e i n K i n a s e s J . 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C h e m . 2 7 0 : 1 6 4 8 3 - 1 6 4 8 6 K a r i n M a n d H u n t e r T 1 9 9 5 T r a n s c r i p t i o n a l c o n t r o l b y p r o t e i n p h o s p h o r y l a t i o n : s i g n a l t r a n s m i s s i o n f r o m t h e c e l l s u r f a c e t o t h e n u c l e u s C u r r B i o l 5 : 7 4 7 - 7 5 7 K e n n e d y N a n d D a v i s R 2 0 0 3 R o l e o f J N K i n t u m o r d e v e l o p m e n t C e l l C y c l e 2 : 1 9 9 - 2 0 1 K i m J H , P a r k D C , K i m J W , C h o i Y K , L e w Y O , K i m D H , J u n g J K , L i m Y A a n d N a m k o o n g S E 1 9 9 9 a A n t i t u m o r E f f e c t o f G n R H A g o n i s t i n E p i t h e l i a l O v a r i a n C a n c e r G y n e c o l o g i c O n c o l o g y 7 4 : 1 7 0 - 1 8 0 K i m J H , P a r k D C , K i m J W , C h o i Y K , L e w Y O , K i m D H , J u n g J K , L i m Y A a n d N a m k o o n g S E 1 9 9 9 b A n t i t u m o r e f f e c t o f G n R H a g o n i s t i n e p i t h e l i a l o v a r i a n c a n c e r G y n e c o l O n c o l 7 4 : 1 7 0 - 8 0 K i m K - Y , C h o i K - C , P a r k S - H , C h o u C - S , A u e r s p e r g N a n d L e u n g P C K 2 0 0 4 a T y p e II G o n a d o t r o p i n - R e l e a s i n g H o r m o n e S t i m u l a t e s p 3 8 M i t o g e n - A c t i v a t e d P r o t e i n K i n a s e a n d A p o p t o s i s i n O v a r i a n C a n c e r C e l l s J C l i n E n d o c r i n o l M e t a b 8 9 : 3 0 2 0 - 3 0 2 6 116 K i m K Y , C h o i K C , A u e r s p e r g N a n d L e u n g P C 2 0 0 6 M e c h a n i s m o f g o n a d o t r o p i n - r e l e a s i n g h o r m o n e ( G n R H ) - I a n d - I I - i n d u c e d c e l l g r o w t h i n h i b i t i o n i n o v a r i a n c a n c e r c e l l s : r o l e o f t h e G n R H - I r e c e p t o r a n d p r o t e i n k i n a s e C p a t h w a y E n d o c r R e l a t C a n c e r 1 3 : 2 1 1 - 2 0 K i m K Y , C h o i K C , P a r k S H , A u e r s p e r g N a n d L e u n g P C 2 0 0 4 b E x t r a c e l l u l a r s i g n a l -r e g u l a t e d p r o t e i n k i n a s e , b u t n o t c - J u n N - t e r m i n a l k i n a s e , i s a c t i v a t e d b y t y p e I I g o n a d o t r o p i n - r e l e a s i n g h o r m o n e i n v o l v e d i n t h e i n h i b i t i o n o f o v a r i a n c a n c e r c e l l p r o l i f e r a t i o n J C l i n E n d o c r i n o l M e t a b K i m u r a A , O h m i c h i M , K u r a c h i H , D c e g a m i H , H a y a k a w a J , T a s a k a K , K a n d a Y , N i s h i o Y , J i k i h a r a H , M a t s u u r a N a n d M u r a t a Y 1 9 9 9 R o l e o f M i t o g e n - a c t i v a t e d P r o t e i n K i n a s e / E x t r a c e l l u l a r S i g n a l - r e g u l a t e d K i n a s e C a s c a d e i n G o n a d o t r o p i n - r e l e a s i n g H o r m o n e -i n d u c e d G r o w t h I n h i b i t i o n o f a H u m a n O v a r i a n C a n c e r C e l l L i n e C a n c e r R e s 5 9 : 5 1 3 3 -5 1 4 2 K l a p p e r L N , K i r s c h b a u m M H , S e l a M a n d Y a r d e n Y 2 0 0 0 B i o c h e m i c a l a n d c l i n i c a l i m p l i c a t i o n s o f t h e E r b B / H E R s i g n a l i n g n e t w o r k o f g r o w t h f a c t o r r e c e p t o r s A d v C a n c e r R e s 7 7 : 2 5 - 7 9 K l e i n m a n D , D o u v d e v a n i A , S c h a l l y A , L e v y J a n d S h a r o n i Y 1 9 9 4 D i r e c t g r o w t h i n h i b i t i o n o f h u m a n e n d o m e t r i a l c a n c e r c e l l s b y t h e g o n a d o t r o p i n - r e l e a s i n g h o r m o n e a n t a g o n i s t S B - 7 5 : r o l e o f a p o p t o s i s A m J O b s t e t G y n e c o l 1 7 0 : 9 6 - 1 0 2 K r a u s S , N a o r Z a n d S e g e r R 2 0 0 1 I n t r a c e l l u l a r S i g n a l i n g P a t h w a y s M e d i a t e d b y t h e G o n a d o t r o p i n - R e l e a s i n g H o r m o n e ( G n R H ) R e c e p t o r A r c h M e d R e s 3 2 : 4 9 9 - 5 0 9 K r a u s S , N a o r Z a n d S e g e r R 2 0 0 6 G o n a d o t r o p i n - r e l e a s i n g h o r m o n e i n a p o p t o s i s o f p r o s t a t e c a n c e r c e l l s C a n c e r L e t t 2 3 4 : 1 0 9 - 1 2 3 K u h n B a n d G u d e r m a n n T 1 9 9 9 T h e L u t e i n i z i n g H o r m o n e R e c e p t o r A c t i v a t e s P h o s p h o l i p a s e C v i a P r e f e r e n t i a l C o u p l i n g t o G < s u b > i < / s u b > < s u b > 2 < / s u b > B i o c h e m i s t r y 3 8 : 1 2 4 9 0 - 1 2 4 9 8 117 L e e M , L i e b o w C , K a m e r A a n d S c h a l l y A 1 9 9 1 E f f e c t s o f E p i d e r m a l G r o w t h F a c t o r a n d A n a l o g u e s o f L u t e i n i z i n g H o r m o n e - R e l e a s i n g H o r m o n e a n d S o m a t o s t a t i n o n P h o s p h o r y l a t i o n a n d D e p h o s p h o r y l a t i o n o f T y r o s i n e R e s i d u e s o f S p e c i f i c P r o t e i n S u b s t r a t e s i n V a r i o u s T u m o r s P N A S 8 8 : 1 6 5 6 - 1 6 6 0 L e e S A , D r i t s c h i l o A a n d J u n g M 2 0 0 1 R o l e o f A T M i n o x i d a t i v e s t r e s s - m e d i a t e d c - J u n p h o s p h o r y l a t i o n i n r e s p o n s e t o i o n i z i n g r a d i a t i o n a n d C d C 1 2 J B i o l C h e m 2 7 6 : 1 1 7 8 3 - 9 0 L e u n g P C K , C h e n g C K a n d Z h u X - M 2 0 0 3 M u l t i - f a c t o r i a l r o l e o f G n R H - I a n d G n R H - I I i n t h e h u m a n o v a r y M o l e c u l a r a n d C e l l u l a r E n d o c r i n o l o g y 2 0 2 : 1 4 5 - 1 5 3 L e v i N L , H a n o c h T , B e n a r d O , R o z e n b l a t M , H a r r i s D , R e i s s N , N a o r Z a n d S e g e r R 1 9 9 8 a S t i m u l a t i o n o f J u n N - T e r m i n a l K i n a s e ( J N K ) b y G o n a d o t r o p i n - R e l e a s i n g H o r m o n e i n P i t u i t a r y { a l p h a } T 3 - 1 C e l l L i n e I s M e d i a t e d b y P r o t e i n K i n a s e C , c - S r c , a n d C D C 4 2 M o i E n d o c r i n o l 1 2 : 8 1 5 - 8 2 4 L e v i N L , H a n o c h T , B e n a r d O , R o z e n b l a t M , H a r r i s D , R e i s s N , N a o r Z a n d S e g e r R 1 9 9 8 b S t i m u l a t i o n o f J u n N - t e r m i n a l k i n a s e ( J N K ) b y g o n a d o t r o p i n - r e l e a s i n g h o r m o n e i n p i t u i t a r y a l p h a T 3 - 1 c e l l l i n e i s m e d i a t e d b y p r o t e i n k i n a s e C , c - S r c , a n d C D C 4 2 M o i E n d o c r i n o l 1 2 : 8 1 5 - 2 4 L e w i s T , S h a p i r o P a n d A h n N 1 9 9 8 S i g n a l t r a n s d u c t i o n t h r o u g h M A P k i n a s e c a s c a d e s A d v C a n c e r R e s 7 4 : 4 9 - 1 3 9 L i S , V u a g n a t B , G r u a z N , E s h k o l A , S i z o n e n k o P a n d A u b e r t M 1 9 9 4 B i n d i n g k i n e t i c s o f t h e l o n g - a c t i n g g o n a d o t r o p i n - r e l e a s i n g h o r m o n e ( G n R H ) a n t a g o n i s t a n t i d e t o r a t p i t u i t a r y G n R H r e c e p t o r s E n d o c r i n o l o g y 1 3 5 : 4 5 - 5 2 L i a o Y a n d H u n g M C 2 0 0 4 A n e w r o l e o f p r o t e i n p h o s p h a t a s e 2 a i n a d e n o v i r a l E l A p r o t e i n - m e d i a t e d s e n s i t i z a t i o n t o a n t i c a n c e r d r u g - i n d u c e d a p o p t o s i s i n h u m a n b r e a s t c a n c e r c e l l s C a n c e r R e s 6 4 : 5 9 3 8 - 5 9 4 2 1 1 8 L i n A 2 0 0 3 A c t i v a t i o n o f t h e J N K s i g n a l i n g p a t h w a y : b r e a k i n g t h e b r a k e o n a p o p t o s i s B i o e s s a y s 2 5 : 1 7 - 2 4 L i n X , V o y n o - Y a s e n e t s k a y a T A , H o o l e y R , L i n C Y , O r l o w s k i J a n d B a r b e r D L 1 9 9 9 G a l p h a l 2 d i f f e r e n t i a l l y r e g u l a t e s N a + - H + e x c h a n g e r i s o f o r m s . J B i o l C h e m 2 7 1 : : 2 2 6 0 4 -2 2 6 1 0 L i u F , A u s t i n D A , M e l l o n P L , O l e f s k y J M a n d W e b s t e r N J G 2 0 0 2 G n R H A c t i v a t e s E R K I / 2 L e a d i n g t o t h e I n d u c t i o n o f c - f o s a n d L H { b e t a } P r o t e i n E x p r e s s i o n i n L { b e t a } T 2 C e l l s M o i E n d o c r i n o l 1 6 : 4 1 9 - 4 3 4 L u o X , D i n g L a n d C h e g i n i N 2 0 0 4 G o n a d o t r o p i n - r e l e a s i n g h o r m o n e a n d T G F - b e t a a c t i v a t e M A P k i n a s e a n d d i f f e r e n t i a l l y r e g u l a t e f i b r o n e c t i n e x p r e s s i o n i n e n d o m e t r i a l e p i t h e l i a l a n d s t r o m a l c e l l s A m J P h y s i o l E n d o c r i n o l M e t a b 2 8 7 : E 9 9 1 - E 1 0 0 1 M a i h l e N J , B a r o n A T , B a r r e t t e B A , B o a r d m a n C H , C h r i s t e n s e n T A , C o r a E M , F a u p e l -B a d g e r J M , G r e e n w o o d T , J u n e j a S C , L a f k y J M , L e e H , R e i t e r J L a n d P o d r a t z K C 2 0 0 2 E G F / E r b B r e c e p t o r f a m i l y i n o v a r i a n c a n c e r C a n c e r T r e a t R e s 1 0 7 : 2 4 7 - 2 5 8 M a n s o u r S , M a t t e n W , H e r m a n n A , C a n d i a J , R o n g S , F u k a s a w a K , V a n d e W o u d e G a n d A h n N 1 9 9 4 T r a n s f o r m a t i o n o f m a m m a l i a n c e l l s b y c o n s t i t u t i v e l y a c t i v e M A P k i n a s e k i n a s e S c i e n c e 2 6 5 : 9 6 6 - 9 7 0 M a t s u o H , B a b a Y , N a i r R M , A r i m u r a A a n d S c h a l l y A V 1 9 7 1 S t r u c t u r e o f t h e p o r c i n e L H -a n d F S H - r e l e a s i n g h o r m o n e . 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C a r b o n e A a n d L a V e c c h i a C 2 0 0 3 F a m i l y h i s t o r y o f c a n c e r a n d r i s k o f o v a r i a n c a n c e r E u r J C a n c e r 3 9 : 5 0 5 - 5 1 0 N e i l l J D 2 0 0 2 M i n i r e v i e w : G n R H a n d G n R H R e c e p t o r G e n e s i n t h e H u m a n G e n o m e E n d o c r i n o l o g y 1 4 3 : 7 3 7 - 7 4 3 N e i l l J D , D u c k L W , S e l l e r s J C a n d M u s g r o v e L C 2 0 0 1 A G o n a d o t r o p i n - R e l e a s i n g H o r m o n e ( G n R H ) R e c e p t o r S p e c i f i c f o r G n R H II i n P r i m a t e s B i o c h e m B i o p h y s R e s C o m m u n 2 8 2 : 1 0 1 2 - 1 0 1 8 N g a n E S , C h e n g P K , L e u n g P C a n d C h o w B K 1 9 9 9 S t e r o i d o g e n i c f a c t o r - 1 i n t e r a c t s w i t h a g o n a d o t r o p e - s p e c i f i c e l e m e n t w i t h i n t h e f i r s t e x o n o f t h e h u m a n g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r g e n e t o m e d i a t e g o n a d o t r o p e - s p e c i f i c e x p r e s s i o n E n d o c r i n o l o g y 1 4 0 : 2 4 5 2 - 2 4 6 2 P a h w a G S , V o l l m e r G , K n u p p e n R a n d E m o n s G 1 9 8 9 P h o t o a f f i n i t y l a b e l l i n g o f g o n a d o t r o p i n r e l e a s i n g h o r m o n e b i n d i n g s i t e s i n h u m a n e p i t h e l i a l o v a r i a n c a r c i n o m a t a 1 2 1 B i o c h e m B i o p h y s R e s C o m m u n 1 6 1 : 1 0 8 6 - 1 0 9 2 P a l D , M i l l e r B T a n d P a r k e n i n g T A 1 9 9 2 T o p o g r a p h i c a l m a p p i n g o f G n R H r e c e p t o r s o n d i s p e r s e d m o u s e p i t u i t a r y c e l l s b y b a c k s c a t t e r e d e l e c t r o n i m a g i n g A n a t R e c 2 3 3 : 8 9 - 9 6 P a w s o n A J , M a u d s l e y S , M o r g a n K , D a v i d s o n L , N a o r Z a n d M i l l a r R P 2 0 0 5 I n h i b i t i o n o f h u m a n t y p e i g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r ( G n R H R ) f u n c t i o n b y e x p r e s s i o n o f a h u m a n t y p e I I G n R H R g e n e f r a g m e n t E n d o c r i n o l o g y 1 4 6 : 2 6 3 9 - 2 9 4 9 P r i c e D T , R o c c a G D , G u o C , B a l l o M S , S c h w i n n D A a n d L u t t r e l l L M 1 9 9 9 A c t i v a t i o n o f e x t r a c e l l u l a r s i g n a l - r e g u l a t e d k i n a s e i n h u m a n p r o s t a t e c a n c e r J U r o l . 1 6 2 : 1 5 3 7 - 1 5 4 2 P u r d i e D M , B a i n C J , S i s k i n d V , W e b b P M a n d G r e e n A C 2 0 0 3 O v u l a t i o n a n d r i s k o f e p i t h e l i a l o v a r i a n c a n c e r I n t J C a n c e r 1 0 4 : 2 2 8 - 2 3 2 Q a y u m A , G u l l i c k W , C l a y t o n R C , S i k o r a K a n d W a x m a n J 1 9 9 0 T h e e f f e c t s o f g o n a d o t r o p h i n r e l e a s i n g h o r m o n e a n a l o g u e s i n p r o s t a t e c a n c e r a r e m e d i a t e d t h r o u g h s p e c i f i c t u m o u r r e c e p t o r s B r J C a n c e r 6 2 : 9 6 - 9 9 R a i n g e a u d J , G u p t a S , D i c k e n s M a n d H a n J 1 9 9 5 P r o - i n f l a m m a t o r y C y t o k i n e s a n d E n v i r o n m e n t a l S t r e s s C a u s e p 3 8 M i t o g e n - a c t i v a t e d P r o t e i n K i n a s e A c t i v a t i o n b y D u a l P h o s p h o r y l a t i o n o n T y r o s i n e a n d T h r e o n i n e J . 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F e r t i l S t e r i l 6 2 : 4 3 3 - 4 4 8 S i k o r a E , G r a s s i l l i E , B e l l e s i a E , T r o i a n o L a n d F r a n c e s c h i C 1 9 9 3 S t u d i e s o f t h e R e l a t i o n s h i p B e t w e e n C e l l P r o l i f e r a t i o n a n d C e l l D e a t h . I I I . A P - 1 D N A - B i n d i n g A c t i v i t y d u r i n g C o n c a n a v a l i n A - I n d u c e d P r o l i f e r a t i o n o r D e x a m e t h a s o n e - I n d u c e d A p o p t o s i s o f R a t T h y m o c y t e s B i o c h e m i c a l a n d B i o p h y s i c a l R e s e a r c h C o m m u n i c a t i o n s 1 9 2 : 3 8 6 - 3 9 1 1 2 5 S i l e r - K h o d r T M , G r a y s o n M a n d E d d y C A 2 0 0 3 A c t i o n o f G o n a d o t r o p i n - R e l e a s i n g H o r m o n e I I o n t h e B a b o o n O v a r y B i o l R e p r o d 6 8 : 1 1 5 0 - 1 1 5 6 S t o j i l k o v i c S S , T o m i c M , K u k u l j a n M a n d C a t t K J 1 9 9 4 C o n t r o l o f c a l c i u m s p i k i n g f r e q u e n c y i n p i t u i t a r y g o n a d o t r o p h s b y a s i n g l e - p o o l c y t o p l a s m i c o s c i l l a t o r M o i P h a r m a c o l 4 5 : 1 0 1 3 - 1 0 2 1 S u g d e n P H a n d C l e r k A 1 9 9 7 R e g u l a t i o n o f t h e E R K S u b g r o u p o f M A P K i n a s e C a s c a d e s T h r o u g h G P r o t e i n - C o u p l e d R e c e p t o r s C e l l u l a r S i g n a l l i n g 9 : 3 3 7 - 3 5 1 S u n Y M , F l a n a g a n C A , I l l i n g N , O t t T R , S e l l a r R , F r o m m e B J , H a p g o o d J , S h a r p P , S e a l f o n S C a n d M i l l a r R P 2 0 0 1 A c h i c k e n g o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r t h a t c o n f e r s a g o n i s t a c t i v i t y t o m a m m a l i a n a n t a g o n i s t s . I d e n t i f i c a t i o n o f D - L y s ( 6 ) i n t h e l i g a n d a n d e x t r a c e l l u l a r l o o p t w o o f t h e r e c e p t o r a s d e t e r m i n a n t s . 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F l o w c y t o m e t r i c d e t e c t i o n o f p h o s p h a t i d y l s e r i n e e x p r e s s i o n o n e a r l y a p o p t o t i c c e l l s u s i n g f l u o r e s c e i n l a b e l l e d A n n e x i n V J I m m u n o l M e t h o d s 1 8 4 : 3 9 - 5 1 W a n g L , B o g e r d J , C h o i H S , S e o n g J Y , S o h J M , C h u n S Y , B l o m e n r o h r M, T r o s k i e B E , M i l l a r R P , Y u W H , M c C a n n S M a n d K w o n H B 2 0 0 1 T h r e e d i s t i n c t t y p e s o f G n R H r e c e p t o r c h a r a c t e r i z e d i n t h e b u l l f r o g P r o c N a t l A c a d S c i U S A 9 8 : 3 6 1 - 3 6 6 W a n g Q F , T i l l y K I , T i l l y J L , P r e f f e r F , S c h n e y e r A L , C r o w l e y W F J a n d S l u s s P M 1 9 9 6 A c t i v i n i n h i b i t s b a s a l a n d a n d r o g e n - s t i m u l a t e d p r o l i f e r a t i o n a n d i n d u c e s a p o p t o s i s i n t h e h u m a n p r o s t a t i c c a n c e r c e l l l i n e , L N C a P E n d o c r i n o l o g y 1 3 7 : 5 4 7 6 - 5 4 8 3 W a n g Y , M a t s u o H , K u r a c h i O a n d M a r u p T 2 0 0 2 D o w n - r e g u l a t i o n o f p r o l i f e r a t i o n a n d u p -r e g u l a t i o n o f a p o p t o s i s b y g o n a d o t r o p i n - r e l e a s i n g h o r m o n e a g o n i s t i n c u l t u r e d u t e r i n e l e i o m y o m a c e l l s E u r J E n d o c r i n o l 1 4 6 : 4 4 7 - 4 5 6 W a s y l y k B , H a g m a n J a n d G u t i e r r e z - H a r t m a n n A 1 9 9 8 E t s t r a n s c r i p t i o n f a c t o r s : n u c l e a r e f f e c t o r s o f t h e R a s - M A P - k i n a s e s i g n a l i n g p a t h w a y T r e n d s B i o c h e m S c i 2 3 : 2 1 3 - 2 1 6 W e i l C , C r i m L W , W i l s o n C E a n d C a u t y C 1 9 9 2 E v i d e n c e o f G n R H r e c e p t o r s i n c u l t u r e d 1 2 7 p i t u i t a r y c e l l s o f t h e w i n t e r f l o u n d e r ( P s e u d o p l e u r o n e c t e s a m e r i c a n u s W . ) G e n C o m p E n d o c r i n o l 8 5 : 1 5 6 - 1 6 4 W e l l s A 1 9 9 9 E G F r e c e p t o r I n t J B i o c h e m C e l l B i o l 3 1 : 6 3 7 - 6 4 3 W h i t e R B , E i s e n J A , K a s t e n T L a n d F e r n a l d R D 1 9 9 8 S e c o n d g e n e f o r g o n a d o t r o p i n -r e l e a s i n g h o r m o n e i n h u m a n s P N A S 9 5 : 3 0 5 - 3 0 9 W h i t m a r s h A J , S h o r e P , S h a r r o c k s A D a n d D a v i s R J 1 9 9 5 I n t e g r a t i o n o f M A P k i n a s e s i g n a l t r a n s d u c t i o n p a t h w a y s a t t h e s e r u m r e s p o n s e e l e m e n t S c i e n c e 2 6 9 : 4 0 3 - 4 0 7 W o r m a l d P J , E i d n e K A a n d M i l l a r R P 1 9 8 5 G o n a d o t r o p i n - r e l e a s i n g h o r m o n e r e c e p t o r s i n h u m a n p i t u i t a r y : l i g a n d s t r u c t u r a l r e q u i r e m e n t s , m o l e c u l a r s i z e , a n d c a t i o n i c e f f e c t s J C l i n E n d o c r i n o l M e t a b 6 1 : 1 1 9 0 - 1 1 9 4 X i a Z , D i c k e n s M , R a i n g e a u d J , D a v i s R J a n d G r e e n b e r g M E 1 9 9 5 O p p o s i n g e f f e c t s o f E R K a n d J N K - p 3 8 M A P k i n a s e s o n a p o p t o s i s S c i e n c e 2 7 0 : 1 3 2 6 - 1 3 3 1 X u G G , D e n g Y Q , L i u S J , L i H L a n d W a n g J Z 2 0 0 5 P r o l o n g e d A l z h e i m e r - l i k e t a u h y p e r p h o s p h o r y l a t i o n i n d u c e d b y s i m u l t a n e o u s i n h i b i t i o n o f p h o s p h o i n o s i t o l - 3 k i n a s e a n d p r o t e i n k i n a s e C i n N 2 a c e l l s A c t a B i o c h i m B i o p h y s S i n ( S h a n g h a i ) 3 7 : 3 4 9 - 5 4 Y a r d e n Y a n d S l i w k o w s k i M X 2 0 0 1 U n t a n g l i n g t h e E r b B s i g n a l l i n g n e t w o r k N a t R e v M o i C e l l B i o l 2 : 1 2 7 - 3 7 Y e n A , R o b e r s o n M , V a r v a y a n i s S a n d L e e A 1 9 9 8 R e t i n o i c a c i d i n d u c e d m i t o g e n - a c t i v a t e d p r o t e i n ( M A P ) / e x t r a c e l l u l a r s i g n a l - r e g u l a t e d k i n a s e ( E R K ) k i n a s e - d e p e n d e n t M A P k i n a s e a c t i v a t i o n n e e d e d t o e l i c i t H L - 6 0 c e l l d i f f e r e n t i a t i o n a n d g r o w t h a r r e s t C a n c e r R e s 5 8 : 3 1 6 3 - 3 1 7 2 Y e u n g C M , A n B S , C h e n g C K , C h o w B K a n d L e u n g P C 2 0 0 5 E x p r e s s i o n a n d 1 2 8 t r a n s c r i p t i o n a l r e g u l a t i o n o f t h e G n R H r e c e p t o r g e n e i n h u m a n n e u r o n a l c e l l s M o i H u m R e p r o d 1 1 : 8 3 7 - 8 4 2 Y i n H , C h e n g K W , H w a H L , P e n g C , A u e r s p e r g N a n d L e u n g P C 1 9 9 8 E x p r e s s i o n o f t h e m e s s e n g e r R N A f o r g o n a d o t r o p i n - r e l e a s i n g h o r m o n e a n d i t s r e c e p t o r i n h u m a n c a n c e r c e l l l i n e s L i f e S c i 6 2 : 2 0 1 5 - 2 3 Z h a n g W a n d L i u H T 2 0 0 2 M A P K s i g n a l p a t h w a y s i n t h e r e g u l a t i o n o f c e l l p r o l i f e r a t i o n i n m a m m a l i a n c e l l s C e l l R e s 1 2 : 9 - 1 8 Z h e n g L , S t o j i l k o v i c S S , H u n y a d y L , K r s m a n o v i c L Z a n d C a t t K J 1 9 9 4 S e q u e n t i a l a c t i v a t i o n o f p h o s p h o l i p a s e - C a n d - D i n a g o n i s t - s t i m u l a t e d g o n a d o t r o p h s E n d o c r i n o l o g y 1 3 4 : 1 4 4 6 - 1 4 5 4 Z h u X a n d B i r n b a u m e r L 1 9 9 6 G p r o t e i n s u b u n i t s a n d t h e s t i m u l a t i o n o f p h o s p h o l i p a s e C b y G s - a n d G i - c o u p l e d r e c e p t o r s : L a c k o f r e c e p t o r s e l e c t i v i t y o f G a l p h a 1 6 a n d e v i d e n c e f o r a s y n e r g i c i n t e r a c t i o n b e t w e e n G b e t a g a m m a a n d t h e a l p h a ^ u b u n i t o f a r e c e p t o r -a c t i v a t e d G p r o t e i n 1 0 . 1 0 7 3 / p n a s . 9 3 . 7 . 2 8 2 7 P N A S 9 3 : 2 8 2 7 - 2 8 3 1 Z i d a n J , Z o h a r S , M i j i r i t z k y I , K r a i S a n d B i l e n c a B 2 0 0 2 T r e a t i n g r e l a p s e d e p i t h e l i a l o v a r i a n c a n c e r w i t h l u t e i n i z i n g h o r m o n e - r e l e a s i n g a g o n i s t ( g o s e r e l i n ) a f t e r f a i l u r e o f c h e m o t h e r a p y I s r M e d A s s o c J 4 : 5 9 7 - 5 9 9 1 2 9 

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