Raloxifene Inhibits Estrogen-induced Up-regulation of Telomerase Activity in a Human Breast Cancer Cell Line
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m304363200
ISSN1083-351X
AutoresJun Kawagoe, Masahide Ohmichi, Toshifumi Takahashi, Chika Ohshima, Seiji Mabuchi, Kazuhiro Takahashi, Hideki Igarashi, Akiko Mori-Abe, Maki Saitoh, Botao Du, Tsuyoshi Ohta, Akiko Kimura, Satoru Kyo, Masakí Inoue, Hirohisa Kurachi,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoThe mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17β-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFκB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFκB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFκB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFκB. The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17β-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFκB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFκB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFκB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFκB. Chemoprevention, defined as the prevention of cancer by the administration of chemical compounds, is a new approach for the management of cancer. Breast cancer remains a significant health problem for women. The large chemoprevention clinical trial with the selective estrogen receptor modulator tamoxifen showed a 38% reduction in breast cancer incidence (1Cuzick J. Forbes J. Edwards R. Baum M. Cawthorn S. Coates A. Hamed A. Howell A. Powles T. IBISLancet. 2002; 360: 817-824Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 2Fisher B. Costantino J.P. Wickerham D.L. Redmond C.K. Kavanah M. Cronin W.M. Vogel V. Robidoux A. Dimitrov N. Atkins J. 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Because of its ideal tissue selectivity, raloxifene may have fewer side effects than tamoxifen. The MORE (Multiple Outcomes of Raloxifene Evaluation) trial was a randomized study designed to determine whether raloxifene would reduce the risk of fracture in postmenopausal women with osteoporosis (12Cauley J.A. Norton L. Lippman M.E. Eckert S. Krueger K.A. Purdie D.W. Farrerons J. Karasik A. Mellstrom D. Ng K.W. Stepan J.J. Powles T.J. Morrow M. Costa A. Silfen S.L. Walls E.L. Schmitt H. Muchmore D.B. Jordan V.C. Ste-Marie L.G. Breast Cancer Res. Treat. 2001; 65: 125-134Crossref PubMed Scopus (668) Google Scholar). The development of breast cancer was a secondary end point of the trial. At a median 48-month follow-up, raloxifene treatment resulted in a 72% reduction in breast cancer incidence without association with an increased risk of uterine endometrial cancer. However, the mechanism by which raloxifene acts to prevent breast cancer remains unclear. Telomerase is a cellular reverse transcriptase that catalyzes the synthesis and extension of telomeric DNA (13Greider C.W. Blackburn E.H. Cell. 1985; 43: 405-413Abstract Full Text PDF PubMed Scopus (2680) Google Scholar, 14Greider C.W. Blackburn E.H. Nature. 1989; 337: 331-337Crossref PubMed Scopus (1347) Google Scholar). This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, with the result that telomeres are progressively shortened with cell division in normal cells (15Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6650) Google Scholar, 16Shay J.W. Bacchetti S. Eur. J. Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2443) Google Scholar). Cells require a mechanism to maintain telomere stability to overcome replicative senescence, and telomerase activation may therefore be a rate-limiting or critical step in cellular immortalization and oncogenesis (17Harley C.B. Villeponteau B. Curr. Opin. Genet. Dev. 1995; 5: 249-255Crossref PubMed Scopus (303) Google Scholar). For example, telomerase activity is known to be involved in estrogen-induced carcinogenesis (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). The level of telomerase activity in cells can be regulated by modulating both the expression and phosphorylation of the catalytic subunit (hTERT). 1The abbreviations used are: hTERT, human telomerase reverse transcriptase subunit; E2, 17β-estradiol; ERE, estrogen-responsive element; ER, estrogen receptor; RT, reverse transcription; PI3K, phosphatidylinositol 3-kinase; NFκB, nuclear factor κB; IκB, inhibitor of NFκB; TPA, 12-O-tetradecanoylphorbol-13-acetate; CSS, charcoal-stripped serum; HA, hemagglutinin; NLS, nuclear localization signal; URR, upstream regulatory region. The hTERT promoter contains an imperfect palindromic estrogen-responsive element (ERE), and it was reported that estrogen activates telomerase via direct and indirect effects on hTERT in MCF-7 cells (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). However, the mechanism of the indirect effects remains unclear. It was reported that the hTERT promoter contains two putative NFκB-binding motifs (19Yin L. Hubbard A.K. Giardina C. J. Biol. Chem. 2000; 275: 36671-36675Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and that IGF-1 and IL-6 activate the PI3K/Akt/NFκB cascade in a human multiple myeloma cell line (20Akiyama M. Hideshima T. Hayashi T. Tai Y.T. Mitsiades C.S. Mitsiades N. Chauhan D. Richardson P. Munshi N.C. Anderson K.C. Cancer Res. 2002; 62: 3876-3882PubMed Google Scholar). Thus, it is possible that estrogen enhances the transcription of hTERT via the PI3K/Akt/NFκB cascade. It was reported that the region surrounding Ser-824 in hTERT conforms to a consensus sequence for phosphorylation by Akt and that Akt kinase enhances human telomerase activity through phosphorylation of hTERT (21Kang S.S. Kwon T. Kwon D.Y. Do S.I. J. Biol. Chem. 1999; 274: 13085-13090Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). In addition, it was reported that an Akt cascade mediates the estrogen-induced S phase entry and cyclin D1 promoter activity in MCF-7 cells (22Castoria G. Migliaccio A. Bilancio A. Di Domenico M. de Falco A. Lombardi M. Fiorentino R. Varricchio L. Barone M.V. Auricchio F. EMBO J. 2001; 20: 6050-6059Crossref PubMed Scopus (403) Google Scholar). Thus, it is possible that estrogen enhances human telomerase activity through an Akt cascade. Another possible mechanism for post-translational modulation of telomerase activity is via the interaction of hTERT with accessory proteins. Recently, it was reported that 14-3-3 proteins (23Yang J. Winkler K. Yoshida M. Kornbluth S. EMBO J. 1999; 18: 2174-2183Crossref PubMed Scopus (205) Google Scholar, 24Seimiya H. Sawada H. Muramatsu Y. Shimizu M. Ohko K. Yamane K. Tsuruo T. EMBO J. 2000; 19: 2652-2661Crossref PubMed Scopus (255) Google Scholar) and NFκB (25Akiyama M. Hideshima T. Hayashi T. Tai Y.-T. Mitsiades C.S. Mitsiades N. Chauhan D. Richardson P. Munshi N.C. Anderson K.C. Cancer Res. 2003; 63: 18-21PubMed Google Scholar) are post-translational modifiers of telomerase that function by controlling the intracellular localization of hTERT. These findings led us to examine whether estrogen induces up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFκB cascade but also by post-translational regulation via Akt-dependent phosphorylation of hTERT in MCF-7 cells. In addition, we attempted to clarify the mechanism by which raloxifene inhibits the induction of telomerase activity by estrogen. Materials—Raloxifene analog LY117018 was a kind gift from Eli Lilly Research Laboratories (Indianapolis, IN). 17β-Estradiol, TPA, and rabbit IgG were purchased from Sigma. ICI 182780 was obtained from TOCRIS (Ballwin, MO). LY294002 was purchased from Calbiochem. The anti-phospho-Akt, phospho-Akt substrate, and Akt antibodies were obtained from Cell Signaling (Beverly, MA). The anti-HA, IκB, phospho-IκB, NFκB p65, and hTERT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The IκBα phosphorylation inhibitor BAY-11-7082 was purchased from Alexis Biochemicals (San Diego, CA). The specific NFκB nuclear translocation inhibitor SN-50 was purchased from BIOMOL (Plymouth Meeting, PA). Hoechst 33258 was obtained from Molecular Probes (Eugene, OR). Constructs—pCR3 vector and pCR3-hTERT were kind gifts from Dr. Takashi Tsuruo (Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan) (24Seimiya H. Sawada H. Muramatsu Y. Shimizu M. Ohko K. Yamane K. Tsuruo T. EMBO J. 2000; 19: 2652-2661Crossref PubMed Scopus (255) Google Scholar). The pCR-FLAG-p50 and pCR-FLAG-p50ΔNLS constructs were kind gifts from Dr. Gourisankar Ghosh (Department of Chemistry and Biochemistry, University of California, San Diego, CA) (26Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (339) Google Scholar). Cell Culture—MCF-7 human breast cancer cells were obtained from the American Type Culture Collection. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate in the presence of 5% CO2 at 37 °C. Cell Proliferation Assay—The cells were plated at a density of 10 × 103 cells/well in 12-well plates and allowed to attach overnight. The cells were growth-arrested by phenol red-free Dulbecco's modified Eagle's medium with 10% charcoal-stripped serum (CSS) for 48 h and were then treated with vehicle, E2, raloxifene, or E2 + raloxifene by exchanging the culture medium containing these agent(s) with fresh medium every 48 h for 8 days. A Neubauer chamber was used to count the cell number, and a trypan blue exclusion test was carried out to determine the cell viability. All of the experiments were carried out in quadruplicate. The values shown are the means ± S.E. of three independent experiments performed in quadruplicate at three different passages of the cell lines. Stretch PCR Assay—For quantitative analysis of telomerase activity, stretch PCR assays were performed using the Telochaser system according to the manufacturer's protocol (Toyobo, Tokyo, Japan) as described previously (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). The PCR products were electrophoresed on a 7% polyacrylamide gel and visualized with SYBR green I nucleic acid gel stain (FMC BioProducts, Rockland, ME). To monitor the efficacy of PCR amplification, 10 ng of a internal control consisting of phage DNA (Toyobo) together with 50 pmol of specific primers (Toyobo) were added to the PCR mixture per reaction. Band intensity was measured using NIH Image software. RT-PCR Analysis—Total cellular RNA was isolated using Tri-Reagent (Molecular Research Center, Inc.). The expression of hTERT mRNA and glyceraldehyde-3-phosphate dehydrogenase mRNA was analyzed by semiquantitative RT-PCR amplification as described previously (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). Briefly, hTERT mRNAs were amplified using the primer pair 5′-CGGAAGAGTGTCTGGAGCAA-3′ and 5′-GGATGAAGCGGAGTCTGGA-3′. cDNA was synthesized from 1 μg of RNA using an RNA PCR kit version 2 (TaKaRa, Ohtsu, Japan) with random primers. Serially diluted cDNA reverse-transcribed from 1 μg of RNA was first amplified by RT-PCR to generate standard curves. The correlation between the band intensity and dose of cDNA template was linear under the conditions described below. Typically, 2-μl aliquots of the reverse-transcribed cDNA were amplified by 28 cycles of PCR in 50 μl of 1× buffer (10 mm Tris-HCl, pH 8.3, 2.5 mm MgCl2, and 50 mm KCl) containing 1 mm each dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq DNA polymerase (TaKaRa), and each specific primer at 0.2 μm. Each cycle consisted of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 45s. PCR products were resolved by electrophoresis in a 1% agarose gel. The efficiency of cDNA synthesis from each sample was estimated by PCR with glyceraldehyde-3-phosphate dehydrogenase-specific primers as described previously (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). Luciferase Assay—Plasmids pGL3–3328 and pGL3–2000 are hTERT promoter-luciferase reporters in which full-length or 5′-deleted promoters including a 77-bp 5′-untranslated region are cloned upstream of the luciferase gene in pGL3-Basic at MluI and BglII sites (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). pGL3-ERE-promoter was constructed by inserting head-in-tail tetramers of the ERE located at –2677 in the hTERT promoter, into the enhancer-less vector pGL3-promoter (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). pGL2-HPV31URR-luc vector is an HPV-31 enhancer and promoter-luciferase reporter containing the whole up-stream regulatory region (URR) of HPV-31 cloned upstream of the luciferase gene in pGL2-Basic (27Kyo S. Klumpp D.J. Inoue M. Kanaya T. Laimins L.A. J. Gen. Virol. 1997; 78: 401-411Crossref PubMed Scopus (69) Google Scholar). These reporter plasmids were transiently transfected into cells for 24 h using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. The cells were harvested and subjected to luciferase assays using a luciferase assay system (Promega) as described previously (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). A plasmid expressing the bacterial β-galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency. Western Blot Analysis—The cells were incubated in phenol red-free medium without serum for 16 h and then treated with various agents. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 μm sodium orthovanadate, 100 mm NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) (28Mabuchi S. Ohmichi M. Kimura A. Hisamoto K. Hayakawa J. Nishio Y. Adachi K. Takahashi K. Arimoto-Ishida E. Nakatsuji Y. Tasaka K. Murata Y. J. Biol. Chem. 2002; 277: 33490-33500Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The lysates were centrifuged at 12,000 × g at 4 °C for 15 min, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blocking was done in 10% bovine serum albumin in 1× Tris-buffered saline. Western blot analyses were performed with various specific primary antibodies. For detection of phosphorylated hTERT or association of hTERT with NFκB p65, cell lysates were prepared using HNTG buffer. The lysates were incubated with anti-hTERT antibody overnight and then immunoprecipitated for 2 h with protein A-Sepharose. Immune complexes were washed with ice-cold HNTG buffer, electrophoresed, and analyzed by immunoblotting with anti-phospho-Akt substrate antibody or anti-NFκB p65 antibody. Immunoreacted bands in the immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit or anti-mouse immunoglobulin by using the enhanced chemiluminescence Western blotting system. Fluorescence Microscopy—MCF-7 cells were grown on glass coverslips in six-well dishes. The cells were transfected with the pCR3-hTERT plasmid for 24 h and then incubated with various reagents. The cells were fixed with 10% formalin for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 3% bovine serum albumin for 1 h. Anti-HA antibody and Alexa Fluor secondary antibody were used at 2 μg/μl in blocking solution. The cells were counterstained with 10 mmol/liter Hoechst 33258 to visualize the nucleus. The samples were mounted on glass slides with Vectashield (Vector Laboratories), and the cells were examined using fluorescence microscopy. For quantification experiments, 100 cells were scored according to whether hTERT was higher in the nucleus (N), evenly distributed between nucleus and cytoplasm (N+C), or higher in the cytoplasm (C). The data represent the mean of three independent experiments. Statistics—Statistical analysis was performed by Student's t test, and p < 0.01 was considered significant. The data are expressed as the means ± S.E. Raloxifene Attenuates the E2-induced Up-regulation of Telomerase Activity and hTERT Expression—We first examined whether or not raloxifene regulates the proliferation of MCF-7 human breast cancer cells (Fig. 1A). E2 significantly induced cell growth at 10 nm. Although 10 nm raloxifene had no effect on the basal cell growth, it did significantly inhibit the E2-induced cell growth. To examine the effects of raloxifene on the estrogen-induced telomerase activity, MCF-7 human breast cancer cells were treated with 10 nm E2, 10 nm raloxifene, 10 nm E2 + 10 nm raloxifene, or 10 nm E2 + 1 μm ICI 182,780 (a highly selective ER antagonist) for 24 h (Fig. 1B). ICI 182,780 (positive control) attenuated the E2-induced telomerase activity (Fig. 1B, lane 5). Although raloxifene had no effect on basal telomerase activity (Fig. 1B, lane 3), it attenuated the E2-induced increase in telomerase activity (Fig. 1B, lane 4). Semiquantitative RT-PCR assays were performed to examine whether the attenuation of estrogen-induced telomerase activity by raloxifene was due to the attenuation by raloxifene of the estrogen-induced up-regulation of the expression of hTERT (Fig. 2A). As we previously reported (29Wang Z. Kyo S. Maida Y. Takakura M. Tanaka M. Yatabe N. Kanaya T. Nakamura M. Koike K. Hisamoto K. Ohmichi M. Inoue M. Oncogene. 2002; 22: 3517-3524Crossref Scopus (49) Google Scholar), treatment of MCF-7 cells with 1 μm tamoxifen for 24 h attenuated the up-regulation of hTERT mRNA induced by 10 nm E2 (Fig. 2A, lane 5). Treatment of MCF-7 cells with 10 nm raloxifene for 24 h also attenuated the up-regulation of hTERT mRNA induced by 10 nm E2 (Fig. 2A, lane 6).Fig. 2Raloxifene attenuates the E2-induced hTERT expression. A, MCF-7 cells were treated with 10 nm E2 (lane 2), 1 μm tamoxifen (lane 3), 10 nm raloxifene (lane 4), 10 nm E2 + 1 μm tamoxifen (lane 5), or 10 nm E2 + 10 nm raloxifene (lane 6). Twenty-four hours later, RNA was extracted, and RT-PCR assays were performed to detect hTERT mRNA. TAM, tamoxifen; RAL, raloxifene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. A representative example of an experiment that was repeated three times is shown. B, MCF-7 cells were transfected with luciferase reporter plasmids containing full-length (–3328) or 5′-deleted hTERT promoter (–2000) and treated with 10 nm E2 (lane 2), 10 nm raloxifene (lane 3), 10 nm E2 + 10 nm raloxifene (lane 4), 10 nm E2 + 1 μm ICI 182,780 (lane 5), or 10 nm E2 + 20 μm LY294002 (lane 6). Twenty-four hours later, the cells were collected, and luciferase assays were performed. The transcriptional activity of each reporter plasmid was normalized relative to β-galactosidase activity, and the activity in cells treated with vehicle was set at 1.0. The data are expressed as the mean fold activation ± S.E. of six transfections. C, MCF-7 cells were transfected with pGL3-ERE-promoter (pGL3-ERE-P) containing head-to-tail tetramers of ERE in the hTERT promoter or enhancer-less pGL3-promoter, and treated with 10 nm E2 (lane 2), 10 nm raloxifene (lane 3), 10 nm E2 + 10 nm raloxifene (lane 4), or 10 nm E2 + 1 μm ICI 182,780 (lane 5). Twenty-four hours later, the cell pellets were collected, and the luciferase assays were performed. The transcriptional activity was normalized relative to β-galactosidase activity, and the activity in cells treated with vehicle was set at 1.0. The data are expressed as the mean fold activation ± S.E. of six transfections. D, MCF-7 cells were transfected with 5′-deleted hTERT promoter (–2000) and treated with phenol red-free medium without serum (Serum free, lane 1), or with 10% CSS (lane 2), or 10% CSS + 10 nm raloxifene (lane 3). Twelve hours later, the cell pellets were collected, and the luciferase assays were performed. The transcriptional activity was normalized relative to β-galactosidase activity, and the activity in cells treated without serum was set at 1.0. The data are expressed as the mean fold activation ± S.E. of six transfections. E, MCF-7 cells were transfected with HPV31URR-luc and treated with 10 nm raloxifene (lane 2), 100 nm TPA (lane 3), or 100 nm TPA + 10 nm raloxifene (lane 4). Twenty-four hours later, the cell pellets were collected, and the luciferase assays were performed. The transcriptional activity was normalized relative to β-galactosidase activity, and the activity in cells treated with vehicle was set at 1.0. The data are expressed as the mean fold activation ± S.E. of six transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the effect of raloxifene on the estrogen-induced transcriptional activity of the hTERT promoter, luciferase assays of cells into which hTERT-promoter reporter plasmids were transfected were performed (Fig. 2B). We have previously reported that an imperfect palindromic ERE is located at –2677 in the hTERT promoter and is capable of direct association with ER (18Kyo S. Takakura M. Kanaya T. Zhuo W. Fujimoto K. Nishio Y. Orimo A. Inoue M. Cancer Res. 1999; 59: 5917-5921PubMed Google Scholar). Luciferase reporter plasmids containing the full-length hTERT 5′ regulatory region (pGL3–3328) or a deletion mutant lacking the imperfect palindromic ERE (pGL3–2000) were transfected into MCF-7 cells. ICI 182,780 attenuated the E2-induced transcriptional activation of pGL3–3328 (Fig. 2B, lane 5). Raloxifene also attenuated the E2-induced transcriptional activation of pGL3–3328 (Fig. 2B, lane 4). To examine whether the ERE at –2677 is involved in the E2-induced up-regulation of the hTERT promoter activity and the inhibitory effect of raloxifene, this putative ERE was cloned upstream of the SV40 promoter in a luciferase reporter plasmid (pGL3-ERE-promoter) and used for transfection. Although E2 had no effect on transcriptional activation of enhancer-less pGL3-promoter containing only SV40 promoter (data not shown), E2 treatment caused transcriptional activation of pGL3-ERE-promoter (Fig. 2C, lane 2), as we reported previously (28Mabuchi S. Ohmichi M. Kimura A. Hisamoto K. Hayakawa J. Nishio Y. Adachi K. Takahashi K. Arimoto-Ishida E. Nakatsuji Y. Tasaka K. Murata Y. J. Biol. Chem. 2002; 277: 33490-33500Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Treatment with raloxifene (Fig. 2C, lane 4) or ICI 182,780 (Fig. 2C, lane 5) attenuated the E2-induced transcriptional activation of the pGL3-ERE-promoter. E2 did activate the transcriptional activity of pGL3–2000 to some extent (Fig. 2B, lane 2), and raloxifene (Fig. 2B, lane 4) or ICI (Fig. 2B, lane 5) attenuated the E2-induced transcriptional activation of pGL3–2000. These results suggest that the ERE at –2677 is partially responsible for the E2-induced activation of the hTERT promoter and the inhibitory effect of raloxifene. We examined whether raloxifene has a general inhibitory effect on transcription. Raloxifene did not have an inhibitory effect on 10% CSS-induced hTERT promoter activity using a deletion mutant of the imperfect palindromic ERE (pGL3–2000) (Fig. 2D). In addition, we used HPV31URR-luc, which is a well characterized reporter for examining the AP-1 activity (27Kyo S. Klumpp D.J. Inoue M. Kanaya T. Laimins L.A. J. Gen. Virol. 1997; 78: 401-411Crossref PubMed Scopus (69) Google Scholar). Raloxifene did not have an inhibitory effect on TPA-induced HPV31 promoter activity (Fig. 2E). These results suggest that raloxifene does not have an inhibitory effect on the induction of AP-1 or SRE activity in promoters that lack an ERE. Raloxifene Inhibits E2-induced Akt Phosphorylation—Because it was previously reported that hTERT expression is induced via an Akt cascade (21Kang S.S. Kwon T. Kwon D.Y. Do S.I. J. Biol. Chem. 1999; 274: 13085-13090Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar), we first examined whether E2 induces Akt phosphorylation in MCF-7 cells. The cells were treated with E2 for various times and then used to p
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