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• W2083764314 abstract "Androgens are essential for the differentiation, growth, and maintenance of male-specific organs. The effects of androgens in cells are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Recently, transient transfection studies have shown that overexpression of cell cycle regulatory proteins affects the transcriptional activity of the AR. In this report, we characterize the transcriptional activity of endogenous AR through the cell cycle. We demonstrate that in G0, AR enhances transcription from an integrated steroid-responsive mouse mammary tumor virus promoter and also from an integrated androgen-specific probasin promoter. This activity is strongly reduced or abolished at the G1/S boundary. In S phase, the receptor regains activity, indicating that there is a transient regulatory event that inactivates the AR at the G1/S transition. This regulation is specific for the AR, since the related glucocorticoid receptor is transcriptionally active at the G1/S boundary. Not all of the effects of androgens are blocked, however, since androgens retain the ability to increase AR protein levels. The transcriptional inactivity of the AR at the G1/S junction coincides with a decrease in AR protein level, although activity can be partly rescued without an increase in receptor. Inhibition of histone deacetylases brings about this partial restoration of AR activity at the G1/S boundary, demonstrating the involvement of acetylation pathways in the cell cycle regulation of AR transcriptional activity. Finally, a model is proposed that explains the inactivity of the AR at the G1/S transition by integrating receptor levels, the action of cell cycle regulators, and the contribution of histone acetyltransferase-containing coactivators. Androgens are essential for the differentiation, growth, and maintenance of male-specific organs. The effects of androgens in cells are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Recently, transient transfection studies have shown that overexpression of cell cycle regulatory proteins affects the transcriptional activity of the AR. In this report, we characterize the transcriptional activity of endogenous AR through the cell cycle. We demonstrate that in G0, AR enhances transcription from an integrated steroid-responsive mouse mammary tumor virus promoter and also from an integrated androgen-specific probasin promoter. This activity is strongly reduced or abolished at the G1/S boundary. In S phase, the receptor regains activity, indicating that there is a transient regulatory event that inactivates the AR at the G1/S transition. This regulation is specific for the AR, since the related glucocorticoid receptor is transcriptionally active at the G1/S boundary. Not all of the effects of androgens are blocked, however, since androgens retain the ability to increase AR protein levels. The transcriptional inactivity of the AR at the G1/S junction coincides with a decrease in AR protein level, although activity can be partly rescued without an increase in receptor. Inhibition of histone deacetylases brings about this partial restoration of AR activity at the G1/S boundary, demonstrating the involvement of acetylation pathways in the cell cycle regulation of AR transcriptional activity. Finally, a model is proposed that explains the inactivity of the AR at the G1/S transition by integrating receptor levels, the action of cell cycle regulators, and the contribution of histone acetyltransferase-containing coactivators. androgen receptor mouse mammary tumor virus cyclin-dependent kinase retinoblastoma protein glucocorticoid receptor chloramphenicol acetyltransferase fluorescence activated cell sorting dihydrotestosterone dexamethasone no hormone trichostatin A p300/CBP-associated factor Androgens play a key role in the differentiation of male-specific tissues during mammalian development. In the adult, there is a continued requirement for androgens for the maintenance of some of these tissues (1Coffey D.S. Pienta K.J. Prog. Clin. Biol. Res. 1987; 239: 1-73PubMed Google Scholar). Androgen withdrawal leads, for instance, to increased apoptosis and regression of the prostate gland (2Koivisto P. Visakorpi T. Rantala I. Isola J. J. Pathol. 1997; 183: 51-56Crossref PubMed Scopus (48) Google Scholar). This androgen dependence is retained in prostate cancer, where androgens are necessary for the onset and early development of the disease (3Culig Z. Hobisch A. Bartsch G. Klocker H. Urol. Res. 2000; 28: 211-219Crossref PubMed Scopus (105) Google Scholar). In newly diagnosed cases of prostate cancer, androgen ablation is the primary therapy used (4Trachtenberg J. Bruce A.W. Trachtenberg J. Adenocarcinoma of the prostate. Springer-Verlag New York Inc., New York1987: 173-184Crossref Google Scholar, 5Leewansangtong S. Soontrapa S. J. Med. Assoc. Thail. 1999; 82: 192-205PubMed Google Scholar), yet with time, androgen-independent tumors arise in individuals who undergo this therapy (6Scott W.W. Menon M. Walsh P.C. Cancer. 1980; 45: 1929-1936Crossref PubMed Scopus (119) Google Scholar, 7Akakura K. Bruchovsky N. Goldenberg S.L. Rennie P.S. Buckley A.R. Sullivan L.D. Cancer. 1993; 71: 2782-2790Crossref PubMed Scopus (372) Google Scholar). This has led to an intense investigation of the molecular mechanisms involved in androgen signaling. The actions of androgens are mediated by the androgen receptor (AR),1 a transcription factor that belongs to the nuclear hormone receptor superfamily. In the absence of androgens, the AR protein is primarily cytosolic and is found complexed with heat shock proteins that keep it inactive (8Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1539) Google Scholar). Upon binding to androgens, the receptor undergoes a conformational change that releases it from this inhibitory complex (9Ohara-Nemoto Y. Nemoto T. Sato N. Ota M. J Steroid Biochem. 1988; 31: 295-304Crossref PubMed Scopus (19) Google Scholar). AR then localizes to the nucleus, where it binds as a dimer to androgen response elements found on the promoters of target genes (10Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (233) Google Scholar). The ability of the AR to modulate gene transcription is enhanced by the recruitment of coactivators and possibly by the release of corepressors (11Chen J.D. Li H. Crit. Rev. Eukaryot. Gene Expr. 1998; 8: 169-190Crossref PubMed Scopus (102) Google Scholar, 12Collingwood T.N. Urnov F.D. Wolffe A.P. J. Mol. Endocrinol. 1999; 23: 255-275Crossref PubMed Scopus (268) Google Scholar). Coactivators can provide enhanced interactions with the basal transcriptional machinery through activation domains of their own. They also contribute intrinsic or associated histone acetyltransferase activities, thus allowing for chromatin remodeling (13Rosenfeld M.G. Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). We have previously shown that activation of AR brings about such nucleosome rearrangements on the mouse mammary tumor virus (MMTV) promoter and that this remodeling correlates with transcriptional activity (14List H.J. Lozano C., Lu, J. Danielsen M. Wellstein A. Riegel A.T. Exp. Cell Res. 1999; 250: 414-422Crossref PubMed Scopus (19) Google Scholar). We have also reported that the hyperacetylation of histones enhances the ability of the AR to remodel chromatin and modulate transcription (15List H.J. Smith C.L. Rodriguez O. Danielsen M. Riegel A.T. Exp. Cell Res. 1999; 252: 471-478Crossref PubMed Scopus (23) Google Scholar) and that anti-androgens inhibit chromatin remodeling, consequently blocking AR transcriptional activity (16List H.J. Smith C.L. Martinez E. Harris V.K. Danielsen M. Riegel A.T. Exp. Cell Res. 2000; 260: 160-165Crossref PubMed Scopus (11) Google Scholar). Thus, the functions of AR require the activity of histone acetylases. The AR itself seems to also be the target of acetylation, and its transcriptional activity may be enhanced in vivo by this modification (17Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). The rate of mammalian cell growth is largely determined by the length of the G1 phase of the cell cycle. Progression from G1 phase through the G1/S transition 2Throughout this paper, the terms “G1/S transition,” “G1/S boundary,” “G1/S junction,” and “G1/S” are used interchangeably. and into S phase is governed by the action of cyclins and cyclin-dependent kinases (CDKs) on the retinoblastoma protein (Rb) (18DeCaprio J.A. Ludlow J.W. Lynch D. Furukawa Y. Griffin J. Piwnica-Worms H. Huang C.M. Livingston D.M. Cell. 1989; 58: 1085-1095Abstract Full Text PDF PubMed Scopus (689) Google Scholar, 19Goodrich D.W. Wang N.P. Qian Y.W. Lee E.Y. Lee W.H. Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (614) Google Scholar). Cyclin D1-CDK4 complexes in middle to late G1 and then cyclin E-CDK2 complexes in G1/S and early S phase phosphorylate Rb, diminishing its ability to bind and repress the S-phase-promoting factor E2F (20Buchkovich K. Duffy L.A. Harlow E. Cell. 1989; 58: 1097-1105Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 21Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1095) Google Scholar, 22Hiebert S.W. Chellappan S.P. Horowitz J.M. Nevins J.R. Genes Dev. 1992; 6: 177-185Crossref PubMed Scopus (469) Google Scholar, 23DeCaprio J.A. Furukawa Y. Ajchenbaum F. Griffin J.D. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1795-1798Crossref PubMed Scopus (210) Google Scholar, 24Ekholm S.V. Reed S.I. Curr. Opin. Cell Biol. 2000; 12: 676-684Crossref PubMed Scopus (503) Google Scholar, 25Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). It is known that androgens influence growth, shortening the length of G1/G0 and accelerating entry into S phase, by affecting the expression and/or activity of cyclins and CDKs (3Culig Z. Hobisch A. Bartsch G. Klocker H. Urol. Res. 2000; 28: 211-219Crossref PubMed Scopus (105) Google Scholar, 26Gregory C.W. Johnson R.T., Jr. Presnell S.C. Mohler J.L. French F.S. J. Androl. 2001; 22: 537-548PubMed Google Scholar). Recently, it has been demonstrated that some cell cycle regulatory proteins can, in turn, influence AR transcriptional activity by acting as AR coregulators. These include the retinoblastoma protein, and cyclins D1 and E, molecules that show altered expression in many human cancers. Our laboratory and others have reported that expression of the retinoblastoma protein restores AR function in Rb-deficient cells (27Lu J. Danielsen M. J. Biol. Chem. 1998; 273: 31528-31533Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,28Yeh S. Miyamoto H. Nishimura K. Kang H. Ludlow J. Hsiao P. Wang C., Su, C. Chang C. Biochem. Biophys. Res. Commun. 1998; 248: 361-367Crossref PubMed Scopus (108) Google Scholar). Additionally, Knudsen et al. (29Knudson K.E. Carenee W.K. Arden K.C. Cancer Res. 1999; 59: 2297-2301PubMed Google Scholar) and Reutens et al. (30Reutens A.T., Fu, M. Wang C. Albanese C. McPhaul M.J. Sun Z. Balk S.P. Janne O.A. Palvimo J.J. Pestell R.G. Mol. Endocrinol. 2001; 15: 797-811Crossref PubMed Scopus (142) Google Scholar) have shown that overexpression of cyclin D1 (and to a lesser extent cyclin D3) inhibits AR function in a CDK-independent manner. Furthermore, Yamamoto et al. (31Yamamoto A. Hashimoto Y. Kohri K. Ogata E. Kato S. Ikeda K. Nakanishi M. J. Cell Biol. 2000; 150: 873-880Crossref PubMed Scopus (94) Google Scholar) determined that cyclin E overexpression, independently of its association with CDK2, results in the positive regulation of AR activity. Generally, these experiments used transient transfection techniques to introduce into cells expression vectors of both the AR and the cell cycle regulator under study and measured transcriptional effects on transient templates (27Lu J. Danielsen M. J. Biol. Chem. 1998; 273: 31528-31533Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Yeh S. Miyamoto H. Nishimura K. Kang H. Ludlow J. Hsiao P. Wang C., Su, C. Chang C. Biochem. Biophys. Res. Commun. 1998; 248: 361-367Crossref PubMed Scopus (108) Google Scholar, 29Knudson K.E. Carenee W.K. Arden K.C. Cancer Res. 1999; 59: 2297-2301PubMed Google Scholar, 30Reutens A.T., Fu, M. Wang C. Albanese C. McPhaul M.J. Sun Z. Balk S.P. Janne O.A. Palvimo J.J. Pestell R.G. Mol. Endocrinol. 2001; 15: 797-811Crossref PubMed Scopus (142) Google Scholar, 31Yamamoto A. Hashimoto Y. Kohri K. Ogata E. Kato S. Ikeda K. Nakanishi M. J. Cell Biol. 2000; 150: 873-880Crossref PubMed Scopus (94) Google Scholar). This approach results in overexpression of the cell cycle regulators throughout the cell cycle rather than the phase-specific expression found in normal cells. We have taken a more physiological approach by investigation of the regulation of the transcriptional activity of endogenous AR on integrated promoters during the cell cycle. In this report, we show that the transcriptional activity of endogenous AR varies through the cell cycle. We demonstrate that the AR is transcriptionally active in G0, loses over 90% of its activity during the G1/S transition, and then regains the ability to enhance transcription in S phase. We show that this transient negative regulation at the G1/S transition is specific for the AR, since the related glucocorticoid receptor (GR) maintains transcriptional activity at this boundary. The down-regulation of AR protein that we observe at G1/S may partially explain the lack of transcriptional activity. However, chemical inhibition of histone deacetylases rescues AR activity during G1/S without increasing the level of AR protein, suggesting that regulation of AR activity during the cell cycle also involves acetylation/deacetylation pathways. L929 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium supplemented with 3% calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. For the development of the L929-MMTVCAT stable cell line, L929 cells were transfected using Dosper liposomal reagent (Roche Molecular Biochemicals) with pMMTVCAT and pSV2neo (20:1 ratio), according to the manufacturer's protocol. To obtain the L929-ProbasinLuc cell line, L929 cells were transfected using LipofectAMINE 2000 reagent (Invitrogen) with p−286/+28PB-luciferase (32Rennie P.S. Bruchovsky N. Leco K.J. Sheppard P.C. McQueen S.A. Cheng H. Snoek R. Hamel A. Bock M.E. MacDonald B.S. Nickel B.E. Chang C. Liao S. Cattini P.A. Matusik R. Mol. Endocrinol. 1993; 7: 23-36Crossref PubMed Scopus (215) Google Scholar) and pSV2neo (20:1 ratio), according to the manufacturer's protocol. In both cases, the cells were split 48 h after transfection and selected in growth media supplemented with 400 mg/liter G418 sulfate (Cellgro). Single clones were picked with sterile pipette tips and expanded. Clones were screened for chloramphenicol acetyltransferase (CAT) or luciferase activity after a 24-h hormone induction. Single clones showing low basal reporter activity and at least 5-fold activation with DHT were used for further studies (clones L929-MMTVCAT #31 and L929-ProbasinLuc 2.9 were used in this study). L929-MMTVCAT and L929-ProbasinLuc cells were cultured in Dulbecco's modified Eagle's medium supplemented with 3% calf serum. All cell cycle arrests were carried out on ∼80% confluent cells (300,000 cells/well of a 24-well plate incubated overnight or the equivalent density on larger surface areas) according to the methods shown in Fig. 2A. After plating and overnight growth, cells were serum-starved (grown in 0.1% calf serum) for 48 h to induce entry into G0. (For control experiments, G0cells were exposed to 1 or 2 mm hydroxyurea for the second 24 h of starvation and during hormone induction as illustrated in Fig. 3B). During an additional 24 h of serum deprivation, cells were treated with steroids or left untreated. For G1/S arrests, cells were starved for 48 h as above. After this starvation, cells were exposed to 1 or 2 mmhydroxyurea in 10% serum for 24 h and for an additional 24 h in the presence or absence of hormone. Cells growing in serum were arrested along S phase by treatment with 1 or 2 mmhydroxyurea for 48 h. Cells were then induced with steroids for 24 h in the presence of hydroxyurea. In all cases, cells were washed with PBS after hormone treatment and harvested in 0.25m Tris-HCl, pH 7.8 (when only CAT or luciferase assays were performed) or trypsinized and collected (when additional fluorescence-activated cell sorting (FACS) or Western analysis was to be performed). Collected cells were aliquoted, spun down, and resuspended. For CAT/luciferase assays, cells were resuspended in 0.25m Tris-HCl, pH 7.8; aliquots for FACS were resuspended in citrate buffer (250 mm sucrose, 40 mm trisodium citrate-2H2O, 5% Me2SO, pH 7.6), and cells for Western analysis were lysed in modified radioimmune precipitation buffer (see below). All samples were stored frozen until analyzed. Citrate buffer samples were analyzed for DNA content at the Lombardi Cancer Center Flow Cytometry/Cell Sorting Shared Resource by propidium iodide staining in a FACSort (Becton Dickinson) (33Vindelov L.L. Virchows Arch. B Cell Pathol. 1977; 24: 227-242PubMed Google Scholar). Computer modeling of cell cycle phase distribution was performed at this facility using the software package ModFit (Verity).FIG. 3Transcriptional activity of AR and GR in the presence of hydroxyurea in G0 and G1/S cells. A, G0-arrested L929-MMTVCAT cells were exposed for 48 h to hydroxyurea during serum starvation. CAT activity in response to 1 nm DHT or 100 nm DEX was then measured (left panel). Transcriptional activity was measured in extracts from G1/S-arrested cells after a 24-h induction with 1 nm DHT or 100 nmDEX (right panel). The same amount of protein was used in all CAT assays. B, diagram of the cell synchronization protocol used in A. C, FACS analysis of cells used in A. Insets show DNA histograms for uninduced cells (NH) or for cells harvested before hormone induction at the 48-h time point shown in B(before induction). The percentage of cells arrested at the indicated stages of the cell cycle is shown in parenthesis. The results are representative of at least three independent experiments.D, cells were serum-starved for 72 h in the absence of hormone and for an additional 24 h in the presence of 1 nm DHT. AR activity was not affected by the longer starvation treatment.View Large Image Figure ViewerDownload (PPT) Cell extracts in 0.25m Tris-HCl buffer, pH 7.8, were frozen/thawed three times to lyse the cells. Protein concentrations were measured by the Bradford method (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). For CAT assays, equal amounts of protein from each extract (typically 1 or 2 μg) were combined with 1 μl of3H-labeled acetyl coenzyme A (1.33 Ci/mmol specific activity; DuPont), 19.0 μl of 2 mg/ml chloramphenicol, and 80 μl of 0.25 m Tris-HCl, pH 7.8. One ml of organic scintillation mixture Econoflour-2 (Packard Instrument Co.) was overlaid on the reaction mix, and vials were placed in a scintillation counter. As the reaction proceeds, the acetylated product is incorporated into the organic phase and is counted (35Zhang S.a.D., M. Lieberman B.A. Steroid Receptor Methods. 176. Humana Press, Totowa, NJ2001: 297-316Google Scholar). Samples were counted for three consecutive cycles in a β counter, and the results were expressed as the increase of the counts produced/min (cpm/min). For luciferase assays, equal amounts of protein from each extract were combined with 100 μl of luciferase assay substrate (Promega) and immediately counted in a luminometer. Cells were collected, spun, and washed in cold PBS. Cell pellets were dissolved in modified radioimmune precipitation buffer (20 mm Tris-HCl pH 7.8, 140 mm NaCl, 1 mm EDTA, 0.5% sodium deoxycholate, 0.5% Nonidet P-40) supplemented with 0.66 mg/ml Pefabloc (Invitrogen), 3.3 μg/ml leupeptin, and 1 mm dithiothreitol. Typically, 30 μg of total protein was loaded in each lane of a 4–20% gradient SDS-polyacrylamide gel and separated by electrophoresis. Protein was transferred to nitrocellulose membranes and confirmed by Ponceau Red staining. After blocking for at least 2 h in 5% milk, 0.2% polyvinyl pyrrolidone, membranes were blotted with the corresponding first antibody. For AR detection, 4 μg/ml PA1-111A, a rabbit polyclonal antibody that recognizes the N terminus of the AR (Affinity Bioreagents) was used. A 1:200 dilution of sc-1616, a goat polyclonal antibody, was used to probe for actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive bands were visualized using anti-rabbit or anti-goat horseradish peroxidase-conjugated secondary antibodies and ECL reagents (Amersham Biosciences). Immunoreactive bands were quantified using the software package ImageQuant. Several reports over the past few years show the involvement of G0, G1/S, and S phase cell cycle regulators in the control of androgen receptor activity. Generally, these experiments used transient transfection techniques to introduce into cells expression vectors of both the AR and the cell cycle regulator under study and measured transcriptional effects on transient templates (27Lu J. Danielsen M. J. Biol. Chem. 1998; 273: 31528-31533Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Yeh S. Miyamoto H. Nishimura K. Kang H. Ludlow J. Hsiao P. Wang C., Su, C. Chang C. Biochem. Biophys. Res. Commun. 1998; 248: 361-367Crossref PubMed Scopus (108) Google Scholar, 29Knudson K.E. Carenee W.K. Arden K.C. Cancer Res. 1999; 59: 2297-2301PubMed Google Scholar, 30Reutens A.T., Fu, M. Wang C. Albanese C. McPhaul M.J. Sun Z. Balk S.P. Janne O.A. Palvimo J.J. Pestell R.G. Mol. Endocrinol. 2001; 15: 797-811Crossref PubMed Scopus (142) Google Scholar, 31Yamamoto A. Hashimoto Y. Kohri K. Ogata E. Kato S. Ikeda K. Nakanishi M. J. Cell Biol. 2000; 150: 873-880Crossref PubMed Scopus (94) Google Scholar). Although such studies provide important information on the interaction of cell cycle regulators and the AR, they do not distinguish between effects seen due purely to overexpression and those that reflect interactions that occur during normal cell growth. Our approach was to investigate the regulation of the transcriptional activity of endogenous AR on integrated promoters during the cell cycle. To do this, we developed a cell line with an integrated AR-responsive CAT reporter gene. L929 cells that express endogenous AR were stably transfected with the androgen- and glucocorticoid-responsive reporter pMMTVCAT. The resulting clones were expanded and characterized. A cell line was established from a representative clone and is referred to here as L929-MMTVCAT. The presence of functional AR in these cells is shown in Fig. 1A (left panel), where treatment with androgens (1 nmdihydrotestosterone (DHT)) resulted in over 30-fold induction of CAT activity. This transcriptional activity was fully blocked by the anti-androgen cyproterone acetate, demonstrating the involvement of the AR in this response. Since L929 cells are known to also express endogenous GR, we measured transcriptional activity in response to glucocorticoids. CAT activity was induced over 20-fold in the presence of dexamethasone (DEX). This induction of MMTVCAT was due to the action of the GR, since the antiglucocorticoid ZK 98.299 fully blocked the response (Fig. 1A, right panel). To optimize the androgen response for cell cycle studies, a time course of AR activation was performed in serum-starved L929-MMTVCAT cells. Androgens clearly induced measurable CAT activity after 24 h (Fig. 1B). This time point was used in cell cycle experiments, since we observed that cells lose synchrony during prolonged arrest (data not shown). To measure the transcriptional activity of the AR in G0, G1/S, or S phase, cells were arrested prior to receptor activation, and cell cycle blocks were maintained during hormone treatment as described under “Experimental Procedures” and outlined in Fig. 2A. To ensure effective cell cycle arrests throughout the length of the experiments, we performed FACS analysis on arrested cells both prior to (data not shown) and after hormone induction as well as on uninduced controls (Fig. 2, B–D, right panels). Importantly, we observed that 24-h androgen treatment had no discernible effect on cell cycle distribution (compare NH histograms with DHT histograms, in Fig. 2, for example). This was expected, since the growth of L929 cells is affected negatively by glucocorticoids and positively by androgens only under chronic long term exposure (36Jung-Testas I. Baulieu E.E. Exp. Clin. Endocrinol. 1985; 86: 151-164Crossref PubMed Scopus (14) Google Scholar). As seen in Fig. 1A, we found that unsynchronized cells growing in the presence of 3% serum routinely showed 20–30-fold induction of CAT activity in response to 1 nm DHT. AR consistently had the highest activity in serum-starved G0cells, inducing CAT activity up to 100-fold in the presence of DHT (Fig. 2B, left panel). In contrast, the AR showed no detectable activity after treatment with 1 nm DHT, in cells arrested at the G1/S boundary (Fig. 2C). AR regained transcriptional activity when the cells were released from G1/S arrest (not shown) or were blocked along S phase by direct treatment with hydroxyurea without prior serum starvation (Fig. 2D). These data indicate that there is a transient regulatory event that prevents AR transcriptional activity at the G1/S boundary. The anti-androgen cyproterone acetate inhibited DHT-induced activity in G0cells and did not show any agonistic activity in cells synchronized at the G1/S boundary (data not shown). As seen in Table I, in three independent experiments, the transcriptional activity of the AR at the G1/S junction was decreased 92–100% compared with its activity in G0. This shows that at the G1/S transition AR function is strongly and consistently inhibited.Table IAR transcriptional activityExperimentG0 activityG1/S activityDecreasecpm/mincpm/min%145Undetectable∼1002751.6983342.792 Open table in a new tab To ensure that the inactivity of the AR in cells arrested at the G1/S transition was not the result of nonspecific actions of the arresting drug, we tested the effects of hydroxyurea on AR activity during G0. L929-MMTVCAT cells were prearrested in G0 by serum starvation for 24 h. During the next 24 h, the cells were exposed to 2 mm hydroxyurea with continued serum starvation. In the final 24 h of treatment, cells were induced with androgens during continued exposure to hydroxyurea and serum starvation (Fig.3B). AR transcriptional activity in G0 cells was unaffected by the presence of hydroxyurea, giving androgen inductions within the range usually obtained with cells in this phase of the cell cycle (Fig.3A, left panel). These data demonstrate that the loss of AR function observed at G1/S is not a nonspecific or toxic effect of the drug per se. Indeed, a similar lack of AR inhibition by hydroxyurea is seen in cells arrested along S phase with this drug (Fig. 2D). To test whether the prolonged treatment of G1/S cells (96 h compared with 72 h for G0 and S phase cells; see Fig.2A) could account for the inactivity of the AR, cells were serum-starved for 72 h and then exposed to hormone during an additional 24 h of starvation. As can be seen in Fig.3D, the transcriptional activity of the AR was unaffected by the 96-h starvation treatment. Indeed, we have prolonged starvation for an additional 24 h as well as performed 72-h hydroxyurea treatments in serum with no effects on AR activity (not shown). To test whether there was a general shut down of transcription or translation at the G1/S boundary or whether this regulation was specific to the androgen pathway, we arrested cells at the G1/S boundary and then treated them with either glucocorticoids or androgens. Treatment of cells synchronized at the G1/S boundary with 100 nm DEX or 1 nm DHT for 24 h did not alter their distribution along the cell cycle (Fig.3C). GR was transcriptionally active in cells arrested at the G1/S transition, inducing CAT activity over 20-fold, yet no AR activity was detected in androgen-treated cells in the same experiment (Fig. 3A, right panel). These data show that there is a preferential negative regulation of the AR over the GR at the G1/S transition. They also demonstrate that there is not an inherent deficiency in the transcription or the translation of the CAT message or protein, respectively, in G1/S boundary-arrested cells, since glucocorticoid treatment results in CAT activity. The MMTV long terminal repeat is a promiscuous promoter that not only responds to androgens and glucocorticoids but also to mineralocorticoids and progestins (37Ham J. Thomson A. Needham M. Webb P. Parker M. Nucleic Acids Res. 1988; 16: 5263-5276Crossref PubMed Scopus (269) Google Scholar, 38Beato M. Chalepakis G. Schauer M. Slater E.P. J. Steroid Biochem. 1989; 32: 737-747Crossref PubMed Scopus (252) Google Scholar). The results presented above demonstrate that the strong inhibition of transcriptional activity seen on the MMTV promoter at the G1/S boundary is specific for the AR. To evaluate whether a similar temporal regulation of AR is observed on promoters that respond only to the AR, we obtained" @default.
• W2083764314 created "2016-06-24" @default.
• W2083764314 creator A5002464705 @default.
• W2083764314 creator A5016242084 @default.
• W2083764314 date "2002-08-01" @default.
• W2083764314 modified "2023-10-17" @default.
• W2083764314 title "Loss of Androgen Receptor Transcriptional Activity at the G1/S Transition" @default.
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