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Androgens stimulate telomerase expression, activity and phosphorylation in ovarian adenocarcinoma cells
Molecular and Cellular Endocrinology 330 (2010) 10–16
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Androgens stimulate telomerase expression, activity and phosphorylation in ovarian adenocarcinoma cells Mitra Nourbakhsh a , Abolfazl Golestani a,∗ , Mahin Zahrai a , Mohammad Hossein Modarressi b , Zahra Malekpour a , Fatemeh Karami-Tehrani c a b c
Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, 1417613151 Tehran, Iran Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 17 July 2010 Accepted 22 July 2010 Keywords: Androgens Telomerase Ovarian cancer
a b s t r a c t Androgens have been implicated in increasing ovarian cancer risk. Most ovarian cancer cells have high telomerase activity which is effective in inducing ovarian carcinogenesis. The purpose of this study was to investigate the effects of testosterone and androstenedione on the viability of an ovarian adenocarcinoma cell line, the activity and expression of telomerase, and the phosphorylation status of its catalytic subunit in these cells. Results showed that androgens significantly increased the viability of ovarian cancer cells and that these hormones induced the expression, activity and phosphorylation of telomerase. This upregulation was blocked by phosphatidylinositol 3-kinase pathway inhibitors. These findings might have implications for understanding the role of androgens in ovarian carcinogenesis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A variety of disorders give rise to androgen excess in women, such as polycystic ovary syndrome, or idiopathic hyperandrogenism (Bulun and Adashi, 2008). It has been hypothesized that androgens increase the risk of ovarian carcinogenesis (Risch, 1998). Ovarian cancer is one of the most common cancers in women and the leading cause of death from gynecological malignancies (Roett and Evans, 2009). Epithelial ovarian cancer derives from malignant transformation of the epithelium of the ovarian surface (Cannistra, 2004), which is exposed to high concentrations of paracrine ovarian androgens (Edmondson et al., 2002). Androgen receptors are frequently found within normal and malignant ovarian epithelial cells (Cardillo et al., 1998). Various molecular changes occur during ovarian carcinogenesis including telomerase reactivation (Datar et al., 1999). Telomerase is a ribonucleoprotein polymerase that adds telomeric sequences onto chromosome ends (Harrington, 2003). This enzyme is present in early embryonic cells but is repressed upon cell differentiation (Hahn, 2003). Most human somatic cells do not express telomerase, except normal cells whose function requires ongoing proliferation, such as germ cells and stem cells (Cukusic et al., 2008). Since reestablishment of a mechanism to maintain telomere length is a necessary prerequisite to malignancy, the vast majority of human
∗ Corresponding author. Tel.: +98 21 88953004; fax: +98 21 64053385. E-mail address: [email protected] (A. Golestani). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.07.021
cancer cells maintain their telomeres by reactivating telomerase reverse transcriptase (hTERT) (Shin et al., 2006). hTERT is the catalytic subunit and the key determinant of telomerase activity as other components are usually expressed ubiquitously (Bertuch et al., 2003). The regulation of telomerase activity occurs at various levels, including transcriptional and posttranslational modifications (Cong et al., 2002). Transcriptional control of hTERT gene expression is the major and rate-limiting step in the activation of telomerase in most cells (Horikawa and Barrett, 2003). Accumulating evidence shows that telomerase activity can be regulated by hTERT phosphorylation (Cong et al., 2002). Various factors can regulate telomerase, and steroid hormones including androgens are suggested to be effective in regulating telomerase activity. By this mechanism, androgens may be responsible for tumorigenesis in hormone-dependent issues (Soda et al., 2000; Cong et al., 2002). In this study, we examined the effect of androstenedione and testosterone on the viability of the ovarian adenocarcinoma cell line OVCAR-3 and the expression and activity of telomerase in these cells. The effect of androgens on phosphorylation of hTERT was also investigated. Androgens can act in a non-genomic manner through activation of signaling pathways, including PI3K (phosphatidylinositol 3-OH kinase)/AKT (protein kinase B), as androgens can enhance the PI3K activity and increase phosphorylation of AKT (Baron et al., 2004). The PI3K/AKT pathway is frequently altered in ovarian cancer (Cho, 2009). The effect of AKT and PI3K inhibitors were also analyzed in androgen-induced upregulation and phosphorylation of telomerase.
M. Nourbakhsh et al. / Molecular and Cellular Endocrinology 330 (2010) 10–16 2. Materials and methods 2.1. Materials Human ovarian adenocarcinoma cell line OVCAR-3 was obtained from National Cell Bank, Pasteur Institute of Iran. Androstenedione, testosterone and 17-estradiol were purchased from Sigma (Germany). LY294002 was obtained from Upstate (USA) and AKT inhibitor I was obtained from Biosource (USA). The anti-hTERT antibody was purchased from ProSci (USA), anti--actin antibody from Affinity Bioreagents (USA), anti-phospho (Ser/Thr) Akt substrate antibody from cell signaling technology (USA) and the HRP-conjugated goat anti-rabbit IgG from Bethyl (USA). Protein A/G Plus-Agarose was purchased from Santa Cruz Biotechnology (USA). Western blotting materials were purchased from Roche Applied Science (Germany). Cell culture reagents were purchased from GIBCO Life Technologies (UK) and all the other reagents were obtained from Sigma or Merck (Germany). 2.2. Cell culture and treatment OVCAR-3 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 g/ml streptomycin sulfate and l-glutamine, at 37 ◦ C in a humidified 5% CO2 atmosphere. For hormone treatment experiments, cells were seeded at a density of 2 or 50 × 104 /well in 96- and 6-well plates, respectively, and allowed to attach overnight. For Western blotting and immunoprecipitation experiments, cells were treated in 25 and 75 cm2 flasks respectively. Prior to treatment, medium was removed and replaced with phenol-red free medium containing dextran-coated charcoal-stripped FBS. In dose-dependent experiments, cells were treated with serial concentrations (0.1–1000 nmol/l) of testosterone or androstenedione and harvested after 48 h. In time-dependent experiments, cells were treated with 10 nmol/l testosterone or 100 nmol/l androstenedione for 24, 48 and 72 h. To study the combined effect of hormones and inhibitors, cells were pretreated with 25 mol/l LY294002 (PI3K inhibitor) or Akt inhibitor I for 1 h followed by addition of androgens. 2.3. Cell viability assay After treatment with androgens, the cells were incubated for 3 h with 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) dye, which was converted by viable cells to blue formazan crystals. The crystals were solubilized by dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm. 2.4. Real-time reverse transcription Total cellular RNA was extracted from cells using RNeasy protect cell mini kit (Qiagen, Germany) and its quality was controlled. cDNA was synthesized from 1 g of total RNA using QuantiTect Rev. Transcription kit (Qiagen, Germany) according to manufacturer’s instructions. Real-time PCR was performed using SYBR premix Ex Taq (TaKaRa, Japan) and predesigned primers for hTERT and RPLP0 (ribosomal protein large P0) genes (QuantiTect Primer Assay, Qiagen, USA). Each cycle consisted of denaturation at 95 ◦ C for 10 s, and combined annealing and extension at 60 ◦ C for 30 s. A standard curve was generated from dilution series constructed from pooled cDNA. Relative changes in gene expression were quantified using CT method. Real-time PCR assays were carried out in triplicate and each experiment was performed at least three times. 2.5. PCR-ELISA telomerase assay Quantitative determination of telomerase activity was performed using the Telomeric Repeat Amplification Protocol (TRAP) in OVCAR-3 cells by TeloTAGGG telomerase PCR ELISA PLUS kit (Roche Applied Science), following manufacturer instructions. Briefly, 2 × 105 cells were lysed with 200 l lysis buffer and centrifuged at 16,000 × g for 20 min at 4 ◦ C. Two microlitres of cell extract was used for the TRAP reaction. Telomeric repeats were added to biotinylated synthetic primers by telomerase followed by amplification of the elongation product by PCR. PCR products were denatured and hybridized to a digoxigenin-labeled telomeric repeat-specific detection probe and then immobilized to a streptavidin-coated microplate. The immobilized PCR products were detected with an anti-digoxigenin antibody conjugated to peroxidase, and then it was visualized by peroxidase, which metabolizes tetramethylbenzidine (TMB) to form a colored reaction product. The absorbance of the samples was measured at 450 nm (with a reference wavelength of 690 nm) after addition of the stop reagent. Heat treated cell extract was used as the negative control, and its absorbance was subtracted from the absorbance of the samples. A template DNA with the same sequence as a telomerase product with 8 telomeric repeats was used as a positive control. A 216 bp internal standard was used as the internal amplification control and to detect DNA polymerase inhibitors. 2.6. Immunoprecipitation and immunoblot analysis Approximately 3 × 106 Cells from each sample were lysed on ice using a lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM EDTA, 0.5% Triton X-100, 10 mM
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sodium pyrophosphate, 100 M sodium orthovanadate, 100 mM sodium fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Kimura et al., 2004) and the lysates were centrifuged. The protein concentration of the lysate was estimated using the Bradford method (Bradford, 1976). To analyze the level of total hTERT in cell lysate, 30 g of protein from each sample lysate was loaded on to the SDS-PAGE and then immunoblotting was performed using anti-hTERT (1:1000) or -actin (1:1000) antibodies. For immunoprecipitation studies, antibody-conjugated beads were prepared by incubating Protein A/G Plus Agarose beads with 1:500 dilution of anti-hTERT polyclonal antibody while rotating for 2 h. Normal rabbit IgG was used as a control. The antibody-conjugated bead suspension was mixed with cellular extract from 107 cells, and incubated while rotating for 2 h at 4 ◦ C. The beads were then centrifuged and washed with cold lysis buffer. The immunoprecipitated samples were loaded onto an 8% SDS-polyacrylamide gel (SDS-PAGE) and then blotted on to polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using anti-phospho (Ser/Thr) Akt substrate antibody (1:1000) or anti-hTERT (1:1000). The immunoblots were visualized with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody using the enhanced chemiluminescence Western blotting substrate (Roche Applied Science). Densitometric analysis was performed with imageJ software (NIH). 2.7. Statistical analysis Statistical analyses were performed using SPSS version 16.0 software. Analysis of variance (ANOVA) was used to compare means in different groups. Assuming normal distribution, differences were statistically significant with p < 0.05. Data are expressed as the mean ± SE.
3. Results 3.1. Effect of androgens on the viability of OVCAR-3 cells To examine the effect of androgens on the viability of human ovarian cancer cells, OVCAR-3 cells were incubated with different concentrations of testosterone and androstenedione. Testosterone treatment for 48 h resulted in an increase in the viability of OVCAR-3 cells with an optimal concentration of 10 nM (p < 0.001). Androstenedione also increased cell viability with the maximum effect at a concentration of 100 nM (p < 0.001), which was higher than the effective concentration of testosterone (Fig. 1). 3.2. Androgen-induced hTERT mRNA and protein expression To investigate whether androgens were able to induce telomerase mRNA expression, OVCAR-3 cells were treated with various concentrations of androstenedione or testosterone. hTERT mRNA levels were analyzed with real-time PCR. Both the 10 nM concentration of testosterone and the 100 nM concentration of androstenedione significantly increased hTERT mRNA levels (Fig. 2a) (p < 0.05). In time-dependent experiments, the inducing effect of androgens was observed 48 h after treatment, and there was no significant increase before 24 h of treatment (Fig. 2b). The cells were also treated with 10 nM 17-estradiol as a positive control, which significantly increased the expression of hTERT 24 h after treatment. To confirm the results obtained from real-time RT-PCR experiments, hTERT protein expression was also analyzed by Western blotting. Although androstenedione and testosterone did not affect the expression of -actin, they induced the protein expression of telomerase (1.8- and 1.9-fold respectively) 48 h after treatment (p < 0.05) (Fig. 3). The effect of 17-estradiol on protein expression was also compared with that of androgens. 17-Estradiol significantly induced the protein expression of hTERT early after treatment, and this induction persisted at least for 48 h. 3.3. Androgen-induced telomerase activity The ability of androgens to enhance the telomerase activity in ovarian cancer cells was tested by treating the cells with various concentrations of androgens (Fig. 4a). As shown in Fig. 4a
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Fig. 1. Androstenedione and testosterone increase cellular viability of ovarian cancer cell line OVCAR-3. Cells were seeded in 96-well plates in complete medium. After 24 h, 0.1–1000 nmol/l androstenedione or testosterone was added, and the cells were incubated with hormone or vehicle alone (C) for 48 h. The MTT assay was used to assess cellular viability. The data shown is the mean ± SE from at least three separate experiments. * p < 0.01; ** p < 0.001.
androstenedione and testosterone significantly increased telomerase activity in a dose-dependent manner, with maximum effect at concentrations of 100 and 10 nM, respectively (p < 0.05). Although the increased telomerase activity by androgens began to increase 24 h after treatment, it did not become significant until 48 h. Androgens significantly increased the activity of telomerase 48 h after treatment, which continued for at least 72 h (Fig. 4b) (p < 0.05). We also examined the effect of pretreatment of cells with different concentrations of PI3K and Akt inhibitors on telomerase activity. The inhibitors significantly attenuated androgen-induced telomerase activity (Fig. 5a) (p < 0.05).
3.4. Phosphorylation of hTERT by androgens To investigate whether androgens were able to phosphorylate telomerase, hTERT was immunoprecipitated by anti-hTERT antibody and then subjected to Western blotting by anti-phospho (Ser/Thr) Akt substrate antibody or anti-hTERT antibody. Fortyeight-hour treatment of OVCAR-3 cells with testosterone and androstenedione significantly increased the phosphorylation of hTERT (2.1- and 1.7-fold, respectively) (p < 0.01, p < 0.05, respectively) (Fig. 5b). The anti-hTERT Western blot analysis shows equal precipitation of hTERT protein.
Fig. 2. Androgens induce hTERT mRNA expression. (a) OVCAR-3 cells were cultured in phenol-red free RPMI-1640 medium supplemented with dextran-coated charcoalstripped serum and then treated with various concentrations of androstenedione, testosterone or vehicle alone for 48 h. hTERT mRNA level was analyzed by real-time PCR and was normalized to RPLP0 (ribosomal protein, large, P0) mRNA level as the housekeeping gene. (b) The cells were treated with 100 nM androstenedione or 10 nM testosterone or 10 nM 17-estradiol as the positive control for 72 h, and every 24 h, the hTERT mRNA level was analyzed by real-time PCR and was normalized to RPLP0 RNA level. The data shown are mean ± SE from of least three separate experiments performed in triplicate. * p < 0.05; ** p < 0.01.
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Fig. 3. Androgens induce hTERT protein expression. OVCAR-3 cells were cultured in phenol-red free RPMI-1640 medium supplemented with dextran-coated charcoalstripped serum and then treated with 100 nM androstenedione, 10 nM testosterone or 10 nM 17-estradiol as the positive control. Total cellular lysates were subjected to 8% SDS-PAGE electrophoresis followed by immunoblotting using antibodies against hTERT (1:1000) or -actin (1:1000). Visualization was performed with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody using the enhanced chemiluminescence Western blotting substrate. Densitometric analysis was performed with imageJ software (NIH). The level for vehicle treated cells (control) was taken as 1.0. The data shown are mean ± SE from of at least three separate experiments performed in triplicate. * p < 0.05; ** p < 0.01.
Fig. 4. Androstenedione and testosterone increase the activity of telomerase in OVCAR-3 cells. (a) Cells were incubated with various concentrations of androstenedione or testosterone for 48 h. The telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. (b) OVCAR-3 cells were treated with androstenedione (100 nM) or testosterone (10 nM) for 72 h. The cells were harvested every 24 h, and their telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. The data are mean ± SE and are representative of three separate experiments. * p < 0.05.
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Fig. 5. (a) Androgen-induced telomerase activity is blocked by PI3K and Akt inhibitors. OVCAR-3 cells were pretreated with different concentrations of LY294002 (LY) or Akt inhibitor I (AktI) for 1 h and then incubated with androstenedione (A) (100 nM), testosterone (T) (10 nM) or vehicle alone (C) for 48 h. The telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. (b) Androgens phosphorylate hTERT, and the phosphorylation is blocked by PI3K and Akt inhibition. OVCAR-3 cells were incubated with androstenedione (100 nM) or testosterone (10 nM) with or without LY204002 or Akt inhibitor I. Cells were harvested and lysed after 48 h. The cell extracts were immunoprecipitated using anti-hTERT antibody (1:500) and Protein A/G Plus-Agarose. The immunoprecipitates were subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane and blotted with anti-phospho-Akt substrate (upper panel) or anti-hTERT (lower panel) antibodies. The data are mean ± SE and are representative of three separate experiments performed. * p < 0.05; ** p < 0.01.
To examine whether the androgen-induced telomerase phosphorylation is mediated via PI3K and AKT, OVCAR-3 cells were treated with LY294002 (PI3K inhibitor) and AKT inhibitor I prior to androgen treatment. The hTERT phosphorylation by androstenedione and testosterone was completely blocked by PI3K and Akt inhibitors (Fig. 5b). 4. Discussion In this study, we have shown that OVCAR-3 cells responded to testosterone and androstenedione treatment by increased viability, and both androgens were effective in stimulating OVCAR-3 cells, although the effective concentration of androstenedione was higher than testosterone. Androgens increase the risk of ovarian carcinogenesis. They can form small adenomas in the ovarian parenchyma and papillomas on the ovarian surface (Silva et al., 1997) and increase the expression of oncogenic GTPases (Sheach et al., 2009). Some epidemiologic studies support the involvement of androgens in ovarian cancer pathogenesis (Helzlouser et al., 1995). In the cancer and steroid hormone case-control study, case subjects were more likely than control subjects to report a history of polycystic ovary syndrome (Schildkraut et al., 1996). Long-term androgen administration in women can cause ovarian cancer (Hage et al., 2000; Dizon et al., 2006). The results of studies on the correlation between circulating androgens and ovarian cancer risk are controversial (Helzlouser et al., 1995; Rinaldi et al., 2007; Tworoger et al., 2008) because the levels of circulating androgen do not necessarily reflect exposure at the tissue level and paracrine androgens may be more important than endocrine sources. Epithelial cells, particularly those within inclusion cysts, appear to be appreciably exposed to paracrine ovarian androgens (Risch, 1998).
Telomerase activity is regulated by different factors using various transcriptional and post-transcriptional mechanisms (Cong et al., 2002). As far as we know, the effect of androgens on telomerase had not been previously analyzed in ovarian cancer cells. We have shown here that the expression of telomerase was increased by testosterone and androstenedione. The results obtained from the hTERT protein level confirms the increased hTERT mRNA by androgens. Androstenedione was also effective in increasing hTERT expression, although its effective concentration was higher than testosterone. Androstenedione is a weak androgen (Bulun and Adashi, 2008), but epithelial ovarian cancer cells express the androgenic 17-hydroxysteroid dehydrogenase/17 ketosteroid reductase, which converts androstenedione to testosterone (Blomquist et al., 2002; Chura et al., 2009). The present study demonstrated that testosterone and androstenedione could increase the activity of telomerase in an epithelial ovarian cancer cell line, which was consistent with their action on hTERT expression. The effect of androgens on telomerase activity has been studied in prostate cells, and androgens have been shown to upregulate this enzyme in androgen sensitive prostate cancer cells (Soda et al., 2000; Bouchal et al., 2002; Kirschenbaum et al., 2006; Guo et al., 2003). Although Removal of testosterone by castration results in increased telomerase activity in the normal prostate (Meeker et al., 1996; Ravindranath et al., 2001), androgen ablation therapy has been shown to suppress hTERT in prostate carcinoma (Iczkowski et al., 2004). These hormones also increase the activity of telomerase in human primary hematopoietic cells (Calado et al., 2009). Some evidences suggest that androgens perform their action on telomerase indirectly and not by activating hTERT promoter construct (Guo et al., 2003). Thus hTERT induction may be due to the androgen dependent
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expression of growth factors which initiate signal transduction pathways (Verhoeven and Swinnen, 1999). In this study, telomerase increased activity and expression by androgens occurred at the 48-h time point, so it seems that activation of telomerase by androgens is indirect. Pretreatment with LY294002 which is a specific PI3K inhibitor or Akt inhibitor I, down regulated telomerase activity and reduced hTERT protein expression which suggests that androgens effect on telomerase might be mediated by PI3K and Akt. Of interest is the observation that androgens could phosphorylate telomerase catalytic subunit, so the up-regulated telomerase activity could be attributed to its phosphorylation as well as increased transcription by androgens. Phosphorylation seems to be an important mechanism to regulate telomerase activity. The activity of this enzyme in human breast cancer cells is markedly inhibited by treatment with protein phosphatase 2A (Li et al., 1997). In some studies it has been shown that induction of telomerase does not require net hTERT protein increase but requires its phosphorylation and translocation from cytoplasm to the nucleus (Liu et al., 2001). Protein kinase Akt can phosphorylate telomerase catalytic subunit in melanoma cells and increase its activity (Kang et al., 1999). Inhibition of PI3K and Akt can decrease telomerase activity and its phosphorylation in T-cells (Plunkett et al., 2007). To investigate whether hTERT phosphorylation by androgens is mediated by Akt kinase we used anti-Akt substrate antibody which recognizes the phospho-Ser/Thr preceded by Lys/Arg at positions -5 and -3. In addition, we observed that pretreatment of cells with LY294002 and Akt kinase inhibitor I inhibits hTERT phosphorylation. Thus the phosphorylation of telomerase by androgens might be secondary to the activation of PI3K/Akt signaling pathway. Estrogen induced telomerase activation is also mediated by PI3K/Akt (Kimura et al., 2004). Increased PI3K expression and activity is a common occurrence in ovarian cancer cells including OVCAR-3 cell line (Shayesteh et al., 1999; Wong et al., 2001). In addition, Akt protein kinase, a downstream effector of PI3K, is not only overexpressed but also constitutively phosphorylated in epithelial ovarian cancer cells (Yuan et al., 2000). In conclusion, these data provide evidence that androgens can increase ovarian cancer cell viability and up-regulate telomerase activity in these cells by both transcriptional and posttranslational mechanisms, and the PI3k/Akt pathway is involved in this upregulation. These data could explain the stimulatory effect of androgens on ovarian carcinogenesis. Further analyses are required to allow a more complete understanding of the molecular mechanism of androgen action and telomerase activation in ovarian cancer. Acknowledgment This research has been financially supported by Tehran University of Medical Sciences & Health Services Grant No. 86-1-30-5368. References Baron, S.r., Manin, M.l., Beaudoin, C., Leotoing, L., Communal, Y., Veyssiere, G., Morel, L., 2004. Androgen receptor mediates non-genomic activation of phosphatidylinositol 3-OH kinase in androgen-sensitive epithelial cells. J. Biol. Chem. 279, 14579–14586. Bertuch, A.A., Buckley, K., Lundblad, V., 2003. The way to the end matters—the role of telomerase in tumor progression. Cell Cycle 2, 36–38. Blomquist, C.H., Bonenfant, M., McGinley, D.M., Posalaky, Z., Lakatua, D.J., Tuli-Puri, S., Bealka, D.G., Tremblay, Y., 2002. Androgenic and estrogenic 17betahydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms. J. Steroid Biochem. Mol. Biol. 81, 343–351. Bouchal, J., Kolar, Z., Mad’arova, J., Hlobikova, A., Angerer, E.v., 2002. The effect of natural ligands of hormone receptors and their antagonists on telomerase activity in the androgen sensitive prostatic cancer cell line. Biochem. Pharmacol. 63, 1177–1181.
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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Androgens stimulate telomerase expression, activity and phosphorylation in ovarian adenocarcinoma cells Mitra Nourbakhsh a , Abolfazl Golestani a,∗ , Mahin Zahrai a , Mohammad Hossein Modarressi b , Zahra Malekpour a , Fatemeh Karami-Tehrani c a b c
Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, 1417613151 Tehran, Iran Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 17 July 2010 Accepted 22 July 2010 Keywords: Androgens Telomerase Ovarian cancer
a b s t r a c t Androgens have been implicated in increasing ovarian cancer risk. Most ovarian cancer cells have high telomerase activity which is effective in inducing ovarian carcinogenesis. The purpose of this study was to investigate the effects of testosterone and androstenedione on the viability of an ovarian adenocarcinoma cell line, the activity and expression of telomerase, and the phosphorylation status of its catalytic subunit in these cells. Results showed that androgens significantly increased the viability of ovarian cancer cells and that these hormones induced the expression, activity and phosphorylation of telomerase. This upregulation was blocked by phosphatidylinositol 3-kinase pathway inhibitors. These findings might have implications for understanding the role of androgens in ovarian carcinogenesis. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A variety of disorders give rise to androgen excess in women, such as polycystic ovary syndrome, or idiopathic hyperandrogenism (Bulun and Adashi, 2008). It has been hypothesized that androgens increase the risk of ovarian carcinogenesis (Risch, 1998). Ovarian cancer is one of the most common cancers in women and the leading cause of death from gynecological malignancies (Roett and Evans, 2009). Epithelial ovarian cancer derives from malignant transformation of the epithelium of the ovarian surface (Cannistra, 2004), which is exposed to high concentrations of paracrine ovarian androgens (Edmondson et al., 2002). Androgen receptors are frequently found within normal and malignant ovarian epithelial cells (Cardillo et al., 1998). Various molecular changes occur during ovarian carcinogenesis including telomerase reactivation (Datar et al., 1999). Telomerase is a ribonucleoprotein polymerase that adds telomeric sequences onto chromosome ends (Harrington, 2003). This enzyme is present in early embryonic cells but is repressed upon cell differentiation (Hahn, 2003). Most human somatic cells do not express telomerase, except normal cells whose function requires ongoing proliferation, such as germ cells and stem cells (Cukusic et al., 2008). Since reestablishment of a mechanism to maintain telomere length is a necessary prerequisite to malignancy, the vast majority of human
∗ Corresponding author. Tel.: +98 21 88953004; fax: +98 21 64053385. E-mail address: [email protected] (A. Golestani). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.07.021
cancer cells maintain their telomeres by reactivating telomerase reverse transcriptase (hTERT) (Shin et al., 2006). hTERT is the catalytic subunit and the key determinant of telomerase activity as other components are usually expressed ubiquitously (Bertuch et al., 2003). The regulation of telomerase activity occurs at various levels, including transcriptional and posttranslational modifications (Cong et al., 2002). Transcriptional control of hTERT gene expression is the major and rate-limiting step in the activation of telomerase in most cells (Horikawa and Barrett, 2003). Accumulating evidence shows that telomerase activity can be regulated by hTERT phosphorylation (Cong et al., 2002). Various factors can regulate telomerase, and steroid hormones including androgens are suggested to be effective in regulating telomerase activity. By this mechanism, androgens may be responsible for tumorigenesis in hormone-dependent issues (Soda et al., 2000; Cong et al., 2002). In this study, we examined the effect of androstenedione and testosterone on the viability of the ovarian adenocarcinoma cell line OVCAR-3 and the expression and activity of telomerase in these cells. The effect of androgens on phosphorylation of hTERT was also investigated. Androgens can act in a non-genomic manner through activation of signaling pathways, including PI3K (phosphatidylinositol 3-OH kinase)/AKT (protein kinase B), as androgens can enhance the PI3K activity and increase phosphorylation of AKT (Baron et al., 2004). The PI3K/AKT pathway is frequently altered in ovarian cancer (Cho, 2009). The effect of AKT and PI3K inhibitors were also analyzed in androgen-induced upregulation and phosphorylation of telomerase.
M. Nourbakhsh et al. / Molecular and Cellular Endocrinology 330 (2010) 10–16 2. Materials and methods 2.1. Materials Human ovarian adenocarcinoma cell line OVCAR-3 was obtained from National Cell Bank, Pasteur Institute of Iran. Androstenedione, testosterone and 17-estradiol were purchased from Sigma (Germany). LY294002 was obtained from Upstate (USA) and AKT inhibitor I was obtained from Biosource (USA). The anti-hTERT antibody was purchased from ProSci (USA), anti--actin antibody from Affinity Bioreagents (USA), anti-phospho (Ser/Thr) Akt substrate antibody from cell signaling technology (USA) and the HRP-conjugated goat anti-rabbit IgG from Bethyl (USA). Protein A/G Plus-Agarose was purchased from Santa Cruz Biotechnology (USA). Western blotting materials were purchased from Roche Applied Science (Germany). Cell culture reagents were purchased from GIBCO Life Technologies (UK) and all the other reagents were obtained from Sigma or Merck (Germany). 2.2. Cell culture and treatment OVCAR-3 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 g/ml streptomycin sulfate and l-glutamine, at 37 ◦ C in a humidified 5% CO2 atmosphere. For hormone treatment experiments, cells were seeded at a density of 2 or 50 × 104 /well in 96- and 6-well plates, respectively, and allowed to attach overnight. For Western blotting and immunoprecipitation experiments, cells were treated in 25 and 75 cm2 flasks respectively. Prior to treatment, medium was removed and replaced with phenol-red free medium containing dextran-coated charcoal-stripped FBS. In dose-dependent experiments, cells were treated with serial concentrations (0.1–1000 nmol/l) of testosterone or androstenedione and harvested after 48 h. In time-dependent experiments, cells were treated with 10 nmol/l testosterone or 100 nmol/l androstenedione for 24, 48 and 72 h. To study the combined effect of hormones and inhibitors, cells were pretreated with 25 mol/l LY294002 (PI3K inhibitor) or Akt inhibitor I for 1 h followed by addition of androgens. 2.3. Cell viability assay After treatment with androgens, the cells were incubated for 3 h with 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) dye, which was converted by viable cells to blue formazan crystals. The crystals were solubilized by dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm. 2.4. Real-time reverse transcription Total cellular RNA was extracted from cells using RNeasy protect cell mini kit (Qiagen, Germany) and its quality was controlled. cDNA was synthesized from 1 g of total RNA using QuantiTect Rev. Transcription kit (Qiagen, Germany) according to manufacturer’s instructions. Real-time PCR was performed using SYBR premix Ex Taq (TaKaRa, Japan) and predesigned primers for hTERT and RPLP0 (ribosomal protein large P0) genes (QuantiTect Primer Assay, Qiagen, USA). Each cycle consisted of denaturation at 95 ◦ C for 10 s, and combined annealing and extension at 60 ◦ C for 30 s. A standard curve was generated from dilution series constructed from pooled cDNA. Relative changes in gene expression were quantified using CT method. Real-time PCR assays were carried out in triplicate and each experiment was performed at least three times. 2.5. PCR-ELISA telomerase assay Quantitative determination of telomerase activity was performed using the Telomeric Repeat Amplification Protocol (TRAP) in OVCAR-3 cells by TeloTAGGG telomerase PCR ELISA PLUS kit (Roche Applied Science), following manufacturer instructions. Briefly, 2 × 105 cells were lysed with 200 l lysis buffer and centrifuged at 16,000 × g for 20 min at 4 ◦ C. Two microlitres of cell extract was used for the TRAP reaction. Telomeric repeats were added to biotinylated synthetic primers by telomerase followed by amplification of the elongation product by PCR. PCR products were denatured and hybridized to a digoxigenin-labeled telomeric repeat-specific detection probe and then immobilized to a streptavidin-coated microplate. The immobilized PCR products were detected with an anti-digoxigenin antibody conjugated to peroxidase, and then it was visualized by peroxidase, which metabolizes tetramethylbenzidine (TMB) to form a colored reaction product. The absorbance of the samples was measured at 450 nm (with a reference wavelength of 690 nm) after addition of the stop reagent. Heat treated cell extract was used as the negative control, and its absorbance was subtracted from the absorbance of the samples. A template DNA with the same sequence as a telomerase product with 8 telomeric repeats was used as a positive control. A 216 bp internal standard was used as the internal amplification control and to detect DNA polymerase inhibitors. 2.6. Immunoprecipitation and immunoblot analysis Approximately 3 × 106 Cells from each sample were lysed on ice using a lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM EDTA, 0.5% Triton X-100, 10 mM
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sodium pyrophosphate, 100 M sodium orthovanadate, 100 mM sodium fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Kimura et al., 2004) and the lysates were centrifuged. The protein concentration of the lysate was estimated using the Bradford method (Bradford, 1976). To analyze the level of total hTERT in cell lysate, 30 g of protein from each sample lysate was loaded on to the SDS-PAGE and then immunoblotting was performed using anti-hTERT (1:1000) or -actin (1:1000) antibodies. For immunoprecipitation studies, antibody-conjugated beads were prepared by incubating Protein A/G Plus Agarose beads with 1:500 dilution of anti-hTERT polyclonal antibody while rotating for 2 h. Normal rabbit IgG was used as a control. The antibody-conjugated bead suspension was mixed with cellular extract from 107 cells, and incubated while rotating for 2 h at 4 ◦ C. The beads were then centrifuged and washed with cold lysis buffer. The immunoprecipitated samples were loaded onto an 8% SDS-polyacrylamide gel (SDS-PAGE) and then blotted on to polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using anti-phospho (Ser/Thr) Akt substrate antibody (1:1000) or anti-hTERT (1:1000). The immunoblots were visualized with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody using the enhanced chemiluminescence Western blotting substrate (Roche Applied Science). Densitometric analysis was performed with imageJ software (NIH). 2.7. Statistical analysis Statistical analyses were performed using SPSS version 16.0 software. Analysis of variance (ANOVA) was used to compare means in different groups. Assuming normal distribution, differences were statistically significant with p < 0.05. Data are expressed as the mean ± SE.
3. Results 3.1. Effect of androgens on the viability of OVCAR-3 cells To examine the effect of androgens on the viability of human ovarian cancer cells, OVCAR-3 cells were incubated with different concentrations of testosterone and androstenedione. Testosterone treatment for 48 h resulted in an increase in the viability of OVCAR-3 cells with an optimal concentration of 10 nM (p < 0.001). Androstenedione also increased cell viability with the maximum effect at a concentration of 100 nM (p < 0.001), which was higher than the effective concentration of testosterone (Fig. 1). 3.2. Androgen-induced hTERT mRNA and protein expression To investigate whether androgens were able to induce telomerase mRNA expression, OVCAR-3 cells were treated with various concentrations of androstenedione or testosterone. hTERT mRNA levels were analyzed with real-time PCR. Both the 10 nM concentration of testosterone and the 100 nM concentration of androstenedione significantly increased hTERT mRNA levels (Fig. 2a) (p < 0.05). In time-dependent experiments, the inducing effect of androgens was observed 48 h after treatment, and there was no significant increase before 24 h of treatment (Fig. 2b). The cells were also treated with 10 nM 17-estradiol as a positive control, which significantly increased the expression of hTERT 24 h after treatment. To confirm the results obtained from real-time RT-PCR experiments, hTERT protein expression was also analyzed by Western blotting. Although androstenedione and testosterone did not affect the expression of -actin, they induced the protein expression of telomerase (1.8- and 1.9-fold respectively) 48 h after treatment (p < 0.05) (Fig. 3). The effect of 17-estradiol on protein expression was also compared with that of androgens. 17-Estradiol significantly induced the protein expression of hTERT early after treatment, and this induction persisted at least for 48 h. 3.3. Androgen-induced telomerase activity The ability of androgens to enhance the telomerase activity in ovarian cancer cells was tested by treating the cells with various concentrations of androgens (Fig. 4a). As shown in Fig. 4a
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Fig. 1. Androstenedione and testosterone increase cellular viability of ovarian cancer cell line OVCAR-3. Cells were seeded in 96-well plates in complete medium. After 24 h, 0.1–1000 nmol/l androstenedione or testosterone was added, and the cells were incubated with hormone or vehicle alone (C) for 48 h. The MTT assay was used to assess cellular viability. The data shown is the mean ± SE from at least three separate experiments. * p < 0.01; ** p < 0.001.
androstenedione and testosterone significantly increased telomerase activity in a dose-dependent manner, with maximum effect at concentrations of 100 and 10 nM, respectively (p < 0.05). Although the increased telomerase activity by androgens began to increase 24 h after treatment, it did not become significant until 48 h. Androgens significantly increased the activity of telomerase 48 h after treatment, which continued for at least 72 h (Fig. 4b) (p < 0.05). We also examined the effect of pretreatment of cells with different concentrations of PI3K and Akt inhibitors on telomerase activity. The inhibitors significantly attenuated androgen-induced telomerase activity (Fig. 5a) (p < 0.05).
3.4. Phosphorylation of hTERT by androgens To investigate whether androgens were able to phosphorylate telomerase, hTERT was immunoprecipitated by anti-hTERT antibody and then subjected to Western blotting by anti-phospho (Ser/Thr) Akt substrate antibody or anti-hTERT antibody. Fortyeight-hour treatment of OVCAR-3 cells with testosterone and androstenedione significantly increased the phosphorylation of hTERT (2.1- and 1.7-fold, respectively) (p < 0.01, p < 0.05, respectively) (Fig. 5b). The anti-hTERT Western blot analysis shows equal precipitation of hTERT protein.
Fig. 2. Androgens induce hTERT mRNA expression. (a) OVCAR-3 cells were cultured in phenol-red free RPMI-1640 medium supplemented with dextran-coated charcoalstripped serum and then treated with various concentrations of androstenedione, testosterone or vehicle alone for 48 h. hTERT mRNA level was analyzed by real-time PCR and was normalized to RPLP0 (ribosomal protein, large, P0) mRNA level as the housekeeping gene. (b) The cells were treated with 100 nM androstenedione or 10 nM testosterone or 10 nM 17-estradiol as the positive control for 72 h, and every 24 h, the hTERT mRNA level was analyzed by real-time PCR and was normalized to RPLP0 RNA level. The data shown are mean ± SE from of least three separate experiments performed in triplicate. * p < 0.05; ** p < 0.01.
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Fig. 3. Androgens induce hTERT protein expression. OVCAR-3 cells were cultured in phenol-red free RPMI-1640 medium supplemented with dextran-coated charcoalstripped serum and then treated with 100 nM androstenedione, 10 nM testosterone or 10 nM 17-estradiol as the positive control. Total cellular lysates were subjected to 8% SDS-PAGE electrophoresis followed by immunoblotting using antibodies against hTERT (1:1000) or -actin (1:1000). Visualization was performed with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody using the enhanced chemiluminescence Western blotting substrate. Densitometric analysis was performed with imageJ software (NIH). The level for vehicle treated cells (control) was taken as 1.0. The data shown are mean ± SE from of at least three separate experiments performed in triplicate. * p < 0.05; ** p < 0.01.
Fig. 4. Androstenedione and testosterone increase the activity of telomerase in OVCAR-3 cells. (a) Cells were incubated with various concentrations of androstenedione or testosterone for 48 h. The telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. (b) OVCAR-3 cells were treated with androstenedione (100 nM) or testosterone (10 nM) for 72 h. The cells were harvested every 24 h, and their telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. The data are mean ± SE and are representative of three separate experiments. * p < 0.05.
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Fig. 5. (a) Androgen-induced telomerase activity is blocked by PI3K and Akt inhibitors. OVCAR-3 cells were pretreated with different concentrations of LY294002 (LY) or Akt inhibitor I (AktI) for 1 h and then incubated with androstenedione (A) (100 nM), testosterone (T) (10 nM) or vehicle alone (C) for 48 h. The telomerase activity was measured using TeloTAGGG telomerase PCR ELISA PLUS kit. (b) Androgens phosphorylate hTERT, and the phosphorylation is blocked by PI3K and Akt inhibition. OVCAR-3 cells were incubated with androstenedione (100 nM) or testosterone (10 nM) with or without LY204002 or Akt inhibitor I. Cells were harvested and lysed after 48 h. The cell extracts were immunoprecipitated using anti-hTERT antibody (1:500) and Protein A/G Plus-Agarose. The immunoprecipitates were subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane and blotted with anti-phospho-Akt substrate (upper panel) or anti-hTERT (lower panel) antibodies. The data are mean ± SE and are representative of three separate experiments performed. * p < 0.05; ** p < 0.01.
To examine whether the androgen-induced telomerase phosphorylation is mediated via PI3K and AKT, OVCAR-3 cells were treated with LY294002 (PI3K inhibitor) and AKT inhibitor I prior to androgen treatment. The hTERT phosphorylation by androstenedione and testosterone was completely blocked by PI3K and Akt inhibitors (Fig. 5b). 4. Discussion In this study, we have shown that OVCAR-3 cells responded to testosterone and androstenedione treatment by increased viability, and both androgens were effective in stimulating OVCAR-3 cells, although the effective concentration of androstenedione was higher than testosterone. Androgens increase the risk of ovarian carcinogenesis. They can form small adenomas in the ovarian parenchyma and papillomas on the ovarian surface (Silva et al., 1997) and increase the expression of oncogenic GTPases (Sheach et al., 2009). Some epidemiologic studies support the involvement of androgens in ovarian cancer pathogenesis (Helzlouser et al., 1995). In the cancer and steroid hormone case-control study, case subjects were more likely than control subjects to report a history of polycystic ovary syndrome (Schildkraut et al., 1996). Long-term androgen administration in women can cause ovarian cancer (Hage et al., 2000; Dizon et al., 2006). The results of studies on the correlation between circulating androgens and ovarian cancer risk are controversial (Helzlouser et al., 1995; Rinaldi et al., 2007; Tworoger et al., 2008) because the levels of circulating androgen do not necessarily reflect exposure at the tissue level and paracrine androgens may be more important than endocrine sources. Epithelial cells, particularly those within inclusion cysts, appear to be appreciably exposed to paracrine ovarian androgens (Risch, 1998).
Telomerase activity is regulated by different factors using various transcriptional and post-transcriptional mechanisms (Cong et al., 2002). As far as we know, the effect of androgens on telomerase had not been previously analyzed in ovarian cancer cells. We have shown here that the expression of telomerase was increased by testosterone and androstenedione. The results obtained from the hTERT protein level confirms the increased hTERT mRNA by androgens. Androstenedione was also effective in increasing hTERT expression, although its effective concentration was higher than testosterone. Androstenedione is a weak androgen (Bulun and Adashi, 2008), but epithelial ovarian cancer cells express the androgenic 17-hydroxysteroid dehydrogenase/17 ketosteroid reductase, which converts androstenedione to testosterone (Blomquist et al., 2002; Chura et al., 2009). The present study demonstrated that testosterone and androstenedione could increase the activity of telomerase in an epithelial ovarian cancer cell line, which was consistent with their action on hTERT expression. The effect of androgens on telomerase activity has been studied in prostate cells, and androgens have been shown to upregulate this enzyme in androgen sensitive prostate cancer cells (Soda et al., 2000; Bouchal et al., 2002; Kirschenbaum et al., 2006; Guo et al., 2003). Although Removal of testosterone by castration results in increased telomerase activity in the normal prostate (Meeker et al., 1996; Ravindranath et al., 2001), androgen ablation therapy has been shown to suppress hTERT in prostate carcinoma (Iczkowski et al., 2004). These hormones also increase the activity of telomerase in human primary hematopoietic cells (Calado et al., 2009). Some evidences suggest that androgens perform their action on telomerase indirectly and not by activating hTERT promoter construct (Guo et al., 2003). Thus hTERT induction may be due to the androgen dependent
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expression of growth factors which initiate signal transduction pathways (Verhoeven and Swinnen, 1999). In this study, telomerase increased activity and expression by androgens occurred at the 48-h time point, so it seems that activation of telomerase by androgens is indirect. Pretreatment with LY294002 which is a specific PI3K inhibitor or Akt inhibitor I, down regulated telomerase activity and reduced hTERT protein expression which suggests that androgens effect on telomerase might be mediated by PI3K and Akt. Of interest is the observation that androgens could phosphorylate telomerase catalytic subunit, so the up-regulated telomerase activity could be attributed to its phosphorylation as well as increased transcription by androgens. Phosphorylation seems to be an important mechanism to regulate telomerase activity. The activity of this enzyme in human breast cancer cells is markedly inhibited by treatment with protein phosphatase 2A (Li et al., 1997). In some studies it has been shown that induction of telomerase does not require net hTERT protein increase but requires its phosphorylation and translocation from cytoplasm to the nucleus (Liu et al., 2001). Protein kinase Akt can phosphorylate telomerase catalytic subunit in melanoma cells and increase its activity (Kang et al., 1999). Inhibition of PI3K and Akt can decrease telomerase activity and its phosphorylation in T-cells (Plunkett et al., 2007). To investigate whether hTERT phosphorylation by androgens is mediated by Akt kinase we used anti-Akt substrate antibody which recognizes the phospho-Ser/Thr preceded by Lys/Arg at positions -5 and -3. In addition, we observed that pretreatment of cells with LY294002 and Akt kinase inhibitor I inhibits hTERT phosphorylation. Thus the phosphorylation of telomerase by androgens might be secondary to the activation of PI3K/Akt signaling pathway. Estrogen induced telomerase activation is also mediated by PI3K/Akt (Kimura et al., 2004). Increased PI3K expression and activity is a common occurrence in ovarian cancer cells including OVCAR-3 cell line (Shayesteh et al., 1999; Wong et al., 2001). In addition, Akt protein kinase, a downstream effector of PI3K, is not only overexpressed but also constitutively phosphorylated in epithelial ovarian cancer cells (Yuan et al., 2000). In conclusion, these data provide evidence that androgens can increase ovarian cancer cell viability and up-regulate telomerase activity in these cells by both transcriptional and posttranslational mechanisms, and the PI3k/Akt pathway is involved in this upregulation. These data could explain the stimulatory effect of androgens on ovarian carcinogenesis. Further analyses are required to allow a more complete understanding of the molecular mechanism of androgen action and telomerase activation in ovarian cancer. Acknowledgment This research has been financially supported by Tehran University of Medical Sciences & Health Services Grant No. 86-1-30-5368. References Baron, S.r., Manin, M.l., Beaudoin, C., Leotoing, L., Communal, Y., Veyssiere, G., Morel, L., 2004. Androgen receptor mediates non-genomic activation of phosphatidylinositol 3-OH kinase in androgen-sensitive epithelial cells. J. Biol. Chem. 279, 14579–14586. Bertuch, A.A., Buckley, K., Lundblad, V., 2003. The way to the end matters—the role of telomerase in tumor progression. Cell Cycle 2, 36–38. Blomquist, C.H., Bonenfant, M., McGinley, D.M., Posalaky, Z., Lakatua, D.J., Tuli-Puri, S., Bealka, D.G., Tremblay, Y., 2002. Androgenic and estrogenic 17betahydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms. J. Steroid Biochem. Mol. Biol. 81, 343–351. Bouchal, J., Kolar, Z., Mad’arova, J., Hlobikova, A., Angerer, E.v., 2002. The effect of natural ligands of hormone receptors and their antagonists on telomerase activity in the androgen sensitive prostatic cancer cell line. Biochem. Pharmacol. 63, 1177–1181.
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