Int. J. Cancer: 76, 571–578 (1998) r 1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer HUMAN CHORIONIC GONADOTROPIN (hCG) INHIBITS CISPLATIN-INDUCED APOPTOSIS IN OVARIAN CANCER CELLS: POSSIBLE ROLE OF UP-REGULATION OF INSULIN-LIKE GROWTH FACTOR-1 BY hCG Hideki KURODA, Masaki MANDAI, Ikuo KONISHI*, Yasuichiro YURA, Yuko TSURUTA, Atia A. HAMID, Kanako NANBU, Katsuko MATSUSHITA and Takahide MORI Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto, Japan Gonadotropins have been suggested to play a role in the development or progression of ovarian cancer, and we have previously reported the expression of luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor in 40% of epithelial ovarian carcinomas. To examine the biological effect of LH/hCG on ovarian cancer cells, apoptosis induced by cisplatin with or without hCG treatment was investigated in 2 ovarian cancer cell lines, OVCAR-3 and SK-OV-3. Stimulation of cell proliferation by hCG was also studied. In addition, to analyze further the mechanism of hCG signaling involved in apoptosis-inhibition, we examined the expression of LH/hCG receptors and the regulation by hCG for apoptosisinhibitory pathways, such as the bcl-2/bax system and the insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGFR) system. hCG did not increase cell proliferation in either cell line. However, hCG treatment suppressed cisplatin-induced apoptosis by 58% in the OVCAR-3 cells, as shown by immunofluorescent staining and quantitation of DNA fragmentation. LH/hCG receptor mRNA was expressed only in OVCAR-3, and no apoptosis-inhibitory effect of hCG was observed in the SK-OV-3 cells that did not express the receptor. In the OVCAR-3 cells, hCG significantly increased mRNA expression of IGF-1, but did not change mRNA levels of bcl-2/bax. Our findings suggest that LH/hCG influences the chemosensitivity of ovarian cancer cells through an apoptosis-inhibitory signal possibly via up-regulation of IGF-1 expression. Int. J. Cancer 76:571–578, 1998. r 1998 Wiley-Liss, Inc. Ovarian cancer is the leading cause of death from gynecological malignancies. Since the ovary is a target tissue of gonadotropins engaged in cyclic steroidogenesis, various hormonal conditions have been implicated in the biological behavior of ovarian cancer (Rao and Slotman, 1991). The possible relationship between infertility drugs and ovarian cancer risk has also been a subject for ongoing debate (Whittemore et al., 1992; Balasch and Barri, 1993; Cohen et al., 1993). There have been reports of ovarian cancer arising in infertile women during or after treatment with gonadotropins (Balasch and Barri, 1993; Kuroda et al., 1997b). Thus, the possible influence of gonadotropins on carcinogenesis or the progression of epithelial ovarian carcinomas is currently an important concern. Although whether ovarian cancer cells have specific binding sites for gonadotropins (Stouffer et al., 1984; Nakano et al., 1989) has been a subject of controversy, we have demonstrated that luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor is expressed at the mRNA and protein levels in 40% of epithelial ovarian carcinomas (Mandai et al., 1997). This finding prompted us to evaluate the functional aspects of LH/hCG signaling in ovarian cancer cells. Apoptosis is one of the fundamental forms of cell death, characterized by several morphological features such as fragmented nuclei, condensed chromatin and the protrusion of cytoplasm or formation of apoptotic body, which are not usually observed in another type of cell death, necrosis (Kerr et al., 1972). Apoptotic cell death generally is tightly regulated by normal physiological dynamics in various organs (Raff, 1992) or by differentiation processes (Ellis et al., 1991). LH/hCG has been reported to be involved in the inhibition of apoptosis in ovarian follicular cells (Tilly et al., 1995). In malignant neoplasms, it has been recognized that apoptotic cell death is induced by chemothera- peutic agents such as cisplatin, a key drug for the treatment of ovarian cancer (Gibb et al., 1997). The induction of apoptosis by anticancer drugs plays an important role in therapeutic mechanisms. Therefore, if LH/hCG modulates the apoptotic pathway, it may influence the chemosensitivity of ovarian cancer cells. To address this hypothesis, we examined the effect of hCG on cisplatin-induced apoptosis by various methods including the quantitation of DNA fragmentation in 2 representative ovarian cancer cell lines, OVCAR-3 and SK-OV-3 (Gibb et al., 1997). The influence of hCG on the proliferation of these cell lines was also studied. To explore the mechanistic pathway of hCG signaling for apoptosis-inhibition, we examined the mRNA expression of LH/ hCG receptor in these cell lines. In addition, we investigated whether hCG regulates the gene expression of apoptosis-related systems such as bcl-2/bax and insulin-like growth factor-1 (IGF-1)/ IGF-1 receptor (IGFR). MATERIAL AND METHODS Cell cultures The ovarian cancer cell lines OVCAR-3 and SK-OV-3 were purchased from the ATCC (Rockville, MD). OVCAR-3 and SK-OV-3 cells were maintained in RPMI-1640 and McCoy’s 5a media, respectively, supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 10% fetal bovine serum, and 10 mg/ml insulin, at 37°C in 5% CO2. Equal numbers of each cell line (5 3 106 cells in 10 ml medium) were seeded in 10-cm culture dishes and allowed to attach for 48 hr. Treatment with cisplatin with or without hCG incubation For the induction of apoptosis with cisplatin with or without hCG, cells were precultured in medium with or without 0.1 µg/ml recombinant hCG (Roto Seiyaku, Osaka, Japan) for 24 hr and incubated in the medium containing 10 µM cisplatin for 2 hr. Then, each medium was replaced by cisplatin-free medium with or without 0.1 µg/ml hCG. After 24 hr of incubation in the cisplatinfree medium, apoptosis of the cells was assessed by various methods. Cell cultures not treated with drugs were used as controls. All the experiments were performed 3 times or more. In the preliminary experiments, the cisplatin concentration ranged from 5 to 100 µM. The cisplatin concentration of 10 µM corresponded to 50% lethal dose (LD50) for both the OVCAR-3 and SK-OV-3 cells and was appropriate for observation of apoptosis-inhibition by hCG. In the preliminary experiments, hCG concentration ranged from 0.01 to 10 µg/ml. Since the effect of hCG on apoptosis was observed at a concentration of 0.1 µg/ml or higher, the concentra- Grant sponsor: Grants-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan; Grant numbers: 09470358 and 097711274. *Correspondence to: Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Fax: (81)75761-3967. E-mail: firstname.lastname@example.org Received 23 October 1997; Revised 12 January 1998 572 KURODA ET AL. tion was then fixed at 0.1 µg/ml for all the subsequent experiments. In addition, the end point was set at 24 hr after cisplatin treatment; this period was shown to be appropriate for assessment of cisplatin-induced apoptosis (Gibb, et al., 1997). Count of viable cells after cisplatin treatment After the cisplatin treatment with or without hCG as described above, the cells were trypsinized and resuspended in 1 ml PBS (Nissui, Tokyo, Japan). The cells were stained by 0.02% erythrocin B, and the number of viable cells was counted in a hemocytometer chamber. Immunofluorescent staining for fragmented DNA (TUNEL) After the apoptosis-induction described above, floating and attached cells were collected, resuspended in 1 ml PBS and rinsed with PBS. The cells were then resuspended and centrifuged onto microscopic slides at 50g for 5 min using a cytospin centrifuge. The cells were then fixed with 4% paraformaldehyde at 4°C for 1 hr. After treatment with 0.5% Triton-X and RNase, immunofluorescent staining of fragmented DNA was carried out by the TdTmediated dUTP-biotin nick end labeling (TUNEL) method with a Mebstain Apoptosis Kit (Medical and Biological Laboratories, Nagoya, Japan), according to the manufacturer’s protocol. The number of cells positive for immunofluorescent staining was counted in 5 high-power fields (HPF)(320 objective, 310 eyepiece). DNA extraction and gel electrophoresis for DNA fragmentation DNA fragmentation was analyzed by DNA ladder formation using an Arends et al. (1990) modified method. Briefly, after the apoptosis-induction described above, all cells were collected with a cell scraper. Extraction of cellular DNA was performed; cells were lysed in 0.5% Triton-X, 10 mM Tris-HCl (pH 7.5) and 20 mM EDTA for 2 hr on ice. After centrifugation at 14,000g for 30 min, the supernatants containing fragmented DNA were incubated with 200 mg/ml proteinase K for 1 hr at 50°C and extracted by the phenol/chloroform method. The DNA was then precipitated overnight at 220°C in 2 volumes of ethanol, 0.13 M NaCl and 50 mg glycogen. After treatment with 1 mg/ml boiled bovine pancreatic RNase A for 1 hr at 50°C, the DNA was loaded onto a 2% (w/v) horizontal agarose gel containing 0.3 mg/ml ethidium bromide and run in 13 TAE buffer. The gels were photographed under UV light. Since DNA was extracted from equal numbers of pre-treated cells, the relative amount of fragmented DNA in each dish was obtained by measuring the amount of low m.w. DNA. NIH Image 1.55 Plot Profile was used to measure the amount of low m.w. DNA (#900 bp). BrdU assay The effect of hCG on the proliferative activity of the 2 ovarian cancer cell lines was assessed by a bromodeoxyuridine (BrdU) incorporation assay using a 5-bromo-28-deoxy-uridine labeling and detection kit (Boehringer Mannheim, Indianapolis, IN). In 100 µl of each medium, 5 3 104 of OVCAR-3 and SKOV-3 cells were seeded in a 96-well microtiter plate. Cells were incubated at 37°C in 5% CO2 and allowed to attach for 24 hr. After incubation, the medium was carefully replaced by the medium with or without 0.1 µg/ml hCG for 24 hr. BrdU assay was then carried out according to the manufacturer’s protocol. RNA preparation and RT-nested PCR of LH/hCG receptor gene Total RNA was isolated by the method of Chomczynski and Sacchi (1987). Reverse transcription (RT)-nested polymerase chain reaction (PCR) was performed according to the method of Lin et al. (1994) with some modifications. Briefly, cDNA was prepared from 1 µg of total RNA by the random priming method using a first-strand cDNA synthesis kit (Pharmacia-LKB, Uppsala, Sweden). The nucleotide sequences of the primers used for PCR were as follows: the sense and antisense primers for first-round PCR were 58-GCATCTGTAACACAGGCATC-38 and 58-CATCTG- GTTCAGGAGCACAT-38, respectively: the sense and antisense primers for second-round PCR were 58-GCAGAAGATGCACAATGGAG-38 and 58-CTCTCAGCAAGCATGGAAGA-38, respectively. First-round PCR was carried out in a Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT) with a mixture consisting of cDNA derived from 50 ng of RNA, 8 pmol each of upstream and downstream primer, 200 mmol of dNTP and 0.1 unit of Taq DNA polymerase with reaction buffer (Takara Shuzo, Shiga, Japan) in a final volume of 10 µl. The PCR conditions consisted of denaturation for 5 min at 94°C, then 30 cycles of denaturation for 1 min at 94°C, annealing for 2 min at 55°C and extension for 1 min at 72°C, followed by a final extension reaction for 5 min at 72°C. One-tenth of the product of first-round PCR was used for second-round PCR, in which the PCR conditions were the same except that nested primers were used. Northern blotting for bcl-2 and bax mRNA expression OVCAR-3 cells were cultured in the presence or absence of 0.1 µg/ml hCG for 4, 8, 24, 48 and 72 hr. Ten micrograms of total RNA were separated by electropheresis in 1% agarose-formaldehyde gels and transferred onto a nylon membrane. The plasmids containing the coding region of bcl-2 and bax cDNA were kindly provided by Dr. Y. Tsujimoto (Osaka University) and Dr. C. Sakakura (Kyoto Prefectural University of Medicine), respectively. Probes for Northern blotting were prepared by PCR using the primers as follows. For bcl-2, the sense primer was 58-GGTGCCACCTGTGGTCCACCTG-38 and the antisense primer was 58CTTCACTTGTGGCCCAGATAGG-38. For bax, the sense and antisense primers were 58-AGCGGCGGTGATGGACGGGT-38 and 58-CTCAGCCCATCTTCTTCCAG-38, respectively. Complementary DNAs were hybridized sequentially to the membranes at 65°C in rapid hybridization buffer (Amersham, Aylesbury, UK), followed by final washes in 0.13 standard saline citrate (SSC) and SDS for 5 min at 65°C. The membrane was rehybridized with a human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) complementary DNA probe to normalize gene expressions. Densitometric analysis was performed using a BAS 2000 II Bioimage Analyzer (Fujix, Tokyo, Japan). Northern blotting and RT-PCR for IGF-1 and IGFR mRNA expression OVCAR-3 cells were cultured in the presence or absence of 0.1 µg/ml hCG for 4, 8, 24, 48 and 72 hr. For Northern blot hybridization of IGF-1 mRNA, 10 mg of total RNA were separated by electrophoresis in 1% agarose-formaldehyde gels and transferred onto a nylon membrane. The DNA probe for IGF-1 was prepared from human corpus luteum cDNA by RT-PCR as described above and was then sequenced. Complementary DNAs were hybridized sequentially to the membranes at 55°C in 53 saline sodium phosphate-EDTA, 1% SDS, 53 Denhardt’s solution and 0.5 mg/ml salmon sperm DNA, followed by final washes in 0.13 SSC and SDS for 5 min at 55°C. The filters were rehybridized with a G3PDH probe. Densitmetric analysis was performed using a BAS 2000 II Bioimage Analyzer. For RT-PCR, the total RNA and cDNA of each sample were prepared as described above. The nucleotide sequences of the primers for IGF-1 and IGFR were as follows (Ullrich et al., 1986; Sussenbach, 1989). For IGF-1, the sense and antisense primers were 58-TCTTGAAGGTGAAGATGCACACCA-38 and 58-AGCGAGCTGACTTGGCAGGCTTGA-38, respectively. For IGFR, the sense primer was 58-ACCCGGAGTACTTCAGCGCT-38, and the antisense primer was 58-CACAGAAGCTTCGTTGAGAA-38. The PCR conditions consisted of denaturation for 5 min at 94°C, 30 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 55°C and extension for 1 min at 72°C, followed by a final extension reaction for 5 min at 72°C. Statistical analysis Differences were assessed by Student’s t test and Wilcoxon test for signed rankings; p , 0.05 was considered significant. INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG 573 RESULTS Cell morphology after cisplatin treatment with or without hCG The OVCAR-3 cells grew as a cobble-stone-like monolayer with multilayering foci. Twenty-four hours after exposure to cisplatin, the OVCAR-3 cells exhibited formation of small, roughly spherical or ovoid cytoplasmic fragments that have been designated apoptotic bodies (Kerr et al., 1972)(Fig. 1a). In contrast, the OVCAR-3 cells treated with hCG before and after exposure to cisplatin showed significantly less apoptotic features (Fig. 1b). In the SK-OV-3 cells, incubation with cisplatin produced similar apoptotic features, but treatment with hCG did not change the morphology. Reduction of cell death due to cisplatin treatment by hCG The number of viable OVCAR-3 cells counted 24 hr after cisplatin treatment was 2.68 6 0.29 (M 6 SD) 3 106/dish in the cisplatin-treated cells without hCG, 3.43 6 0.21 3 106/dish in the cisplatin-treated cells with hCG and 4.20 6 0.59 3 106/dish in the cells not exposed to cisplatin (Fig. 2). Incubation with hCG before and after cisplatin treatment significantly decreased cell death (by 49%), compared with cisplatin treatment alone (p 5 0.0013). Inhibition of cisplatin-induced apoptosis by hCG: immunofluorescent staining of fragmented DNA (TUNEL) To distinguish necrosis and apoptosis, we initially performed the immunofluorescent staining of fragmented DNA (TUNEL). The OVCAR-3 cells exposed to cisplatin showed a significantly larger FIGURE 2 – Number of viable OVCAR-3 cells counted 24 hr after cisplatin treatment. The incubation with hCG before and after exposure to cisplatin reduced the cell death by 49%. number of cells stained for fragmented DNA compared with the non-exposed cells. The cells not exposed to cisplatin contained 5.2 6 3.6 apoptotic cells/HPF. The cells treated with cisplatin without hCG contained 23.4 6 4.0 cells/HPF with fragmented DNA (Fig. 3a). The cells incubated with hCG before and after exposure to cisplatin contained 12.8 6 2.8 cells/HPF with fragmented DNA and showed a 58% decrease in the number of apoptotic cells (Fig. 3b), compared with the cells without hCG treatment (p 5 0.0004; Fig. 3c). FIGURE 1 – Morphological features of OVCAR-3 cells 24 hr after cisplatin treatment with or without incubation with hCG. (a) Cells treated with cisplatin alone. The formation of apoptotic bodies, i.e., small, roughly spherical or ovoid cytoplasmic fragments is evident (arrows). (b) Cells treated with hCG before and after exposure to cisplatin. Apoptotic features were significantly decreased. Scale bar 5 100 µm. Inhibition of cisplatin-induced apoptosis by hCG: quantitation of low m.w. DNA To confirm the suppressive effect of hCG on apoptosis, the amount of fragmented DNA in the apoptotic cells exposed to cisplatin with or without hCG was quantitated by electrophoresis for DNA fragmentation. The low m.w. DNA (#900 bp) in DNA ladders was measured with the NIH Image program. The amount of low m.w. DNA was significantly decreased (by 58%) in the hCG-treated OVCAR-3 cells compared with the non-treated cells (p 5 0.0117; Fig. 4a,b). In contrast, no reduction of low m.w. DNA by hCG treatment was observed in the SK-OV-3 cells (Fig. 4c). Effect of hCG on cell proliferation To address whether the reduction in cell death produced by hCG treatment was the result of an inhibition of apoptosis or an increase in cell number, the effect of hCG on the proliferative activity of the 2 cell lines was assayed by BrdU incorporation. The BrdU assay 574 KURODA ET AL. FIGURE 3 – Immunofluorescent staining for fragmented DNA (TUNEL) in OVCAR-3 cells. (a) Cells treated with cisplatin were extensively stained for fragmented DNA. (b) hCG incubation before and after exposure to cisplatin produced a decrease in the number of cells with fragmented DNA. (c) Significant difference in the number of cells stained for fragmented DNA with or without hCG treatment. Scale bar 5 100 µm. revealed no significant activation of cell proliferation in response to hCG stimulation in OVCAR-3 (p 5 0.1147) or SK-OV-3 cells (p 5 0.2934; Fig. 5a,b). LH/hCG receptor mRNA expression in ovarian cancer cells In the OVCAR-3 cells, RT-nested PCR amplified 2 differentsized fragments (Fig. 6; lane 3), both of which corresponded to the cDNA fragments of LH/hCG receptors (Lin et al., 1994). The larger fragment (342 bp) corresponded to the full-length cDNA, and the smaller fragment (156 bp) corresponded to the splice variant lacking exon 9 (Mandai et al., 1997). In contrast, neither of these two fragments was amplified in the SK-OV-3 cells (Fig. 6, lane 2). hCG up-regulates mRNA expression of IGF-1 but not that of bcl-2/bax In the OVCAR-3 cells, hCG treatment did not change the mRNA level of the bcl-2 or bax genes, as shown by Northern blotting (Fig. 7). In contrast, both Northern blotting and RT-PCR demonstrated that hCG treatment increased the mRNA expression of IGF-1 in OVCAR-3 cells; 48 hr of hCG incubation showed a 2-fold increase compared with control (Fig. 8a,b). mRNA expression of IGFR was found in OVCAR-3 cells, although hCG treatment did not change its expression level (Fig. 8b). DISCUSSION Our results demonstrate that cisplatin treatment induces apoptosis in the 2 representative ovarian cancer cell lines, OVCAR-3 and SK-OV-3. Both cell lines showed the same chemosensitivity to cisplatin; approximately half of the cells underwent apoptosis 24 hr after treatment with 10 µM cisplatin for 2 hr. These data are consistent with those of a previous report (Gibb et al., 1997). Strikingly, incubation with hCG before and after the exposure to cisplatin reduced cell death rate of OVCAR-3 cells, which express LH/hCG receptor mRNA. This was ascribed to the inhibition of apoptosis by hCG, as shown by immunofluorescent staining and the quantitation of DNA fragmentation; hCG treatment suppressed apoptosis by 58% compared with the untreated OVCAR-3 cells exposed to cisplatin. Although an inhibitory effect of hCG on the apoptotic pathway has previously been reported in granulosa cells of normal ovarian follicles (Tilly et al., 1995), this effect has never been reported in malignant tumor cells. In the present study, the apoptosis-suppressive effect of hCG was not observed in another ovarian cancer cell line, SK-OV-3, which does not express the LH/hCG receptor. These findings suggest that the inhibition of apoptosis by hCG in ovarian cancer cells depends on the expression of LH/hCG receptor. INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG 575 FIGURE 4 – DNA ladder formation after treatment with cisplatin in ovarian cancer cell lines, OVCAR-3 and SK-OV-3 (lane 1, cells in control medium without cisplatin; lane 2, cells treated with cisplatin alone; lane 3, cells treated with hCG before and after exposure to cisplatin.). In the OVCAR-3 cells exposed to cisplatin, compared with no hCG treatment (a, lane 2), the amount of low m.w. DNA was significantly decreased by hCG incubation (a, lane 3). Quantitation of the of low m.w. DNA (#900 bp) with the NIH Image program showed a 58% decrease with hCG treatment in OVCAR-3 cells (b). In the SK-OV-3 cells, there was no difference in DNA fragmentation between the cells treated with cisplatin alone (c, lane 2) and those treated with cisplatin plus hCG (c, lane 3). To clarify whether the effect of hCG is an attenuation of apoptosis and not an increase in cell number, the stimulation of mitogenic activity by hCG was analyzed by BrdU assay. hCG treatment for 24 hr showed no stimulatory effect on the proliferation of the LH/hCG receptor-positive OVCAR-3 or that of the receptor-negative SK-OV-3. There have been several reports on the effects of gonadotropins on the growth of ovarian cancer cells (Simon et al., 1983; Ohtani et al., 1992; Wimalasena et al., 1992; Kataoka et al., 1994), and all these authors supported the concept of the mitogenic action of follicle-stimulating hormone (FSH). How- ever, whether LH/hCG has an influence on cell proliferation has been a subject of controversy; a stimulatory effect was seen in several ovarian cancer cell lines (Simon et al., 1983; Wimalasena et al., 1992) but not in others (Ohtani et al., 1992; Kataoka et al., 1994). LH/hCG receptor expression was not examined in those cell lines. Therefore, the discrepancy in proliferative effect may be due to a difference of LH/hCG responsiveness among the cancer cell lines or to the effect of FSH contained in non-recombinant hCG, which was substituted for recombinant hCG in our present experiment. 576 KURODA ET AL. FIGURE 5 – Evaluation of proliferation of OVCAR-3 (a) and SK-OV-3 (b) cells in response to hCG assessed by BrdU assay. Neither of the 2 cell lines revealed significant differences in growth after hCG treatment. FIGURE 7 – Northern blot analysis of the mRNA expression of bcl-2 and bax in OVCAR-3 cells. hCG treatment did not change the gene expressions of bcl-2 (a) or bax (b). 1, 0; 2, 4; 3, 8; 4, 24; 5, 48; and 6, 72 hr of hCG treatment. FIGURE 6 – RT-nested PCR detection of LH/hCG receptor mRNA. In the OVCAR-3 cells, 2 different-sized fragments of cDNA were amplified; the larger one (342 bp) corresponded to the full-length LH/hCG receptor mRNA, and the smaller fragment (156 bp) corresponded to a splice variant lacking exon 9 (lane 3). No fragments were detected in the SK-OV-3 cells (lane 2). Lane 1, m.w. marker; lane 4, ovarian corpus luteums as positive control. To explore the mechanistic pathway of apoptosis-suppression by hCG, we examined the bcl-2/bax system and the IGF-1/IGFR system in the ovarian cancer cell line OVCAR-3. The results demonstrated that hCG treatment up-regulated mRNA expression of IGF-1 but did not change bcl-2/bax expression. Apoptosis is regulated by the expression of several gene cascades such as those of Fas, myc, p53, bcl-2, bax and bcl-xL, followed by an activation of proteases such as interleukin-1 beta-converting enzyme (ICE). Both cell lines have been shown to contain p53 genetic alterations: a transition in codon 248 of exon 7 with associated allelic loss in OVCAR-3 and a probable genetic rearrangement leading to absence of detectable p53 in SKOV-3 cells (Brown et al., 1993). This suggests that inhibition of cisplatin-induced apoptosis by hCG is independent of p53 dysfunction. The suppression of ICE activation is regarded as a possible mechanism for apoptosis-inhibition by IGF-1/IGFR (Jung et al., 1996). hCG has also been shown to be INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG FIGURE 8 – Northern blot hybridization and RT-PCR analysis for mRNA expression of IGF-1 and IGFR in OVCAR-3 cells. hCG treatment significantly increased mRNA expression of IGF-1, as shown by Northern blotting (a) and RT-PCR (b). Expression of IGFR was not changed by hCG treatment (b). 1, 0; 2, 4; 3, 8; 4, 24; 5, 48; and 6, 72 hr of hCG treatment. involved in the apoptosis-suppression of ovarian follicular cells through the up-regulation of IGF-1/IGFR and/or down-regulation of bax (Tilly et al., 1995). IGF-1 has recently been shown to alter the drug sensitivity of human breast cancer cells by inhibition of apoptosis without changing the bcl-2 and bax mRNA levels (Dunn et al., 1997). Since OVCAR-3 cells express IGFR, it is likely that hCG signaling is involved in the inhibition of apoptosis, possibly via the autocrine or paracrine activation of the IGF-1/IGFR system. Serum levels of gonadotropin are elevated in ovarian cancer patients after menopause or after oophorectomy, and our data 577 suggest that high LH levels may be involved in the chemoresistance of ovarian cancer cells. Gonadotropin-releasing hormone (GnRH) analogues are drugs used for the treatment of leiomyoma or endometriosis to down-regulate the levels of serum gonadotropins. Several studies suggested that GnRH analogues are effective in some patients with ovarian cancer (Lind et al., 1992). The GnRH analogue has been shown to bind directly to the GnRH receptor on the ovarian cancer cells and inhibit mitogenic activity (Emons et al., 1993). However, another possible mechanism of the anti-tumor effect of GnRH analogue on ovarian cancer is the enhancement of susceptibility to anti-cancer drugs. If this is indeed the case, GnRH analogues could be applied for patients with LH/hCG receptorpositive ovarian cancer. A randomized trial did not show a significant benefit of the drug use on the survival of ovarian cancer patients (Emons et al., 1996). In our hospital, patients with advanced ovarian cancer are currently enrolled in a clinical trial of cisplatin-based chemotherapy with or without GnRH analogue, along with the analysis of LH/hCG receptor status of the tumor. Epidemiological data indicate that the incidence of ovarian cancer increases around the peri-menopausal period, when the serum levels of gonadotropins are elevated. Several ovarian carcinomas have been reported to develop in infertile women during gonadotropin treatment (Kuroda et al., 1997b). Since ovarian carcinomas are believed to arise from the surface epithelial cells of the ovary, a possible effect of gonadotropins on these cells should be considered. Expression of FSH receptor has been demonstrated in ovarian surface epithelial cells (Zheng et al., 1996). If LH/hCG stimulation also inhibits apoptosis in normal surface epithelium of the ovary, constant exposure to LH/hCG may lead to ovarian carcinogenesis; escape from physiological apoptosis is considered to promote the carcinogenic process (Bedi et al., 1995). We have detected mRNA expression of LH/hCG receptors and specific binding sites for hCG in normal ovarian surface epithelial cells in vitro (Kuroda et al., 1997a), and the influence of hCG on the apoptosis of these cells is now under investigation. ACKNOWLEDGEMENTS This work was supported by Grants-in-Aid for Scientific Research to I.K. (09470358) and to M.M. (09771274) from the Ministry of Education, Science and Culture, Japan. REFERENCES ARENDS, M.J., MORRIS, R.G. and WYLLIE, A.H., Apoptosis: the role of the endonuclease. Amer. J. Pathol., 136, 593–608 (1990). BALASCH, J. and BARRI, P.N., Follicular stimulation and ovarian cancer? Hum. Reprod., 8, 990–996 (1993). 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