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• W2017322511 abstract "1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3. 1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) 1The abbreviations used are: 1,25-(OH)2D31,25-dihydroxyvitamin D3VDRvitamin D receptorCKIcyclin-dependent kinase inhibitorCdk2cyclin-dependent kinase 2UTRuntranslated regionPBSphosphate-buffered salineCAKCdk-activating kinase. exerts important effects on cellular proliferation and differentiation (1Pols H.A. Birkenhager J.C. Foekens J.A. van Leeuwen J.P. J. Steroid Biochem. Mol. Biol. 1990; 37: 873-876Crossref PubMed Scopus (102) Google Scholar, 2Bikle D.D. Endocr. Rev. 1992; 13: 765-784PubMed Google Scholar, 3Walters M.R. Endocr. Rev. 1992; 13: 719-764Crossref PubMed Google Scholar, 4Feldman D. Glorieux F.H. Pike J.W. Vitamin D. Academic Press, San Diego, CA1997: 1089-1105Google Scholar). 1,25-(OH)2D3 activates the vitamin D receptor (VDR), a ligand-dependent transcription factor that binds cis-acting DNA sequences known as vitamin D response elements (5Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar). Several established human prostate cancer cell lines, as well as primary cultures of benign and cancerous prostatic tissue, express functional VDRs and are growth inhibited by 1,25-(OH)2D3 (6Miller G.J. Stapleton G.E. Ferrara J.A. Lucia M.S. Pfister S. Hedlund T.E. Upadhya P. Cancer Res. 1992; 52: 515-520PubMed Google Scholar, 7Skowronski R.J. Peehl D.M. Feldman D. Endocrinology. 1993; 132: 1952-1960Crossref PubMed Google Scholar, 8Schwartz G.G. Oeler T.A. Uskokovic M.R. Bahnson R.R. Anticancer Res. 1994; 14: 1077-1081PubMed Google Scholar, 9Miller G.J. Stapleton G.E. Hedlund T.E. Moffatt K.A. Clin. Cancer Res. 1995; 1: 997-1003PubMed Google Scholar, 10Blutt S.E. Allegretto E.A. Pike J.W. Weigel N.L. Endocrinology. 1997; 138: 1491-1497Crossref PubMed Scopus (191) Google Scholar, 11Zhuang S.-H. Schwartz G.G. Cameron D. Burnstein K.L. Mol. Cell. Endocrinol. 1997; 126: 83-90Crossref PubMed Scopus (115) Google Scholar, 12Zhuang S.-H. Burnstein K.L. Endocrinology. 1998; 139: 1197-1207Crossref PubMed Scopus (203) Google Scholar). 1,25-dihydroxyvitamin D3 vitamin D receptor cyclin-dependent kinase inhibitor cyclin-dependent kinase 2 untranslated region phosphate-buffered saline Cdk-activating kinase. Our previous studies established that the initial growth inhibition of LNCaP and its androgen-independent derivative LNCaP-104R1 by 1,25-(OH)2D3 correlates with an increase in the levels of the cyclin-dependent kinase inhibitors (CKIs) p21WAF1,CIP1 and p27Kip1, a profound decrease in cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of pRb, and accumulation of cells in the G1 phase of the cell cycle (12Zhuang S.-H. Burnstein K.L. Endocrinology. 1998; 139: 1197-1207Crossref PubMed Scopus (203) Google Scholar, 13Yang E.S. Maiorino C.A. Roos B.A. Knight S.R. Burnstein K.L. Mol. Cell. Endocrinol. 2002; 186: 69-79Crossref PubMed Scopus (40) Google Scholar). 1,25-(OH)2D3 also decreased transcriptional activity of the E2F transcription factor family, which regulates the expression of genes necessary for S phase entry (12Zhuang S.-H. Burnstein K.L. Endocrinology. 1998; 139: 1197-1207Crossref PubMed Scopus (203) Google Scholar). The CKI p27 appears to play a more central role than p21 in growth inhibition mediated by 1,25-(OH)2D3 and its analogs (13Yang E.S. Maiorino C.A. Roos B.A. Knight S.R. Burnstein K.L. Mol. Cell. Endocrinol. 2002; 186: 69-79Crossref PubMed Scopus (40) Google Scholar, 14Campbell M.J. Elstner E. Holden S. Uskokovic M. Koeffler H.P. J. Mol. Endocrinol. 1997; 19: 15-27Crossref PubMed Scopus (181) Google Scholar). In several prostate cancer cell lines, 1,25-(OH)2D3 treatment results in persistent up-regulation of p27, whereas p21 is only induced transiently (12Zhuang S.-H. Burnstein K.L. Endocrinology. 1998; 139: 1197-1207Crossref PubMed Scopus (203) Google Scholar, 13Yang E.S. Maiorino C.A. Roos B.A. Knight S.R. Burnstein K.L. Mol. Cell. Endocrinol. 2002; 186: 69-79Crossref PubMed Scopus (40) Google Scholar, 14Campbell M.J. Elstner E. Holden S. Uskokovic M. Koeffler H.P. J. Mol. Endocrinol. 1997; 19: 15-27Crossref PubMed Scopus (181) Google Scholar). In addition, 1,25-(OH)2D3-mediated growth inhibition of the androgen-independent LNCaP-derivative LNCaP-104R1 cells occurs without induction of p21 (13Yang E.S. Maiorino C.A. Roos B.A. Knight S.R. Burnstein K.L. Mol. Cell. Endocrinol. 2002; 186: 69-79Crossref PubMed Scopus (40) Google Scholar). Loss of p27 expression correlates with prostate cancer recurrence, a more aggressive phenotype, and decreased patient prognosis and survival (15Guo Y. Sklar G.N. Borkowski A. Kyprianou N. Clin. Cancer Res. 1997; 3: 2269-2274PubMed Google Scholar, 16Cote R.J. Shi Y. Groshen S. Feng A.C. Cordon-Cardo C. Skinner D. Lieskovosky G. J. Nat. Cancer Inst. 1998; 90: 916-920Crossref PubMed Scopus (188) Google Scholar, 17Tsihlias J. Kapusta L.R. DeBoer G. Morava-Protzner I. Zbieranowski I. Bhattacharya N. Catzavelos G.C. Klotz L.H. Slingerland J.M. Cancer Res. 1998; 58: 542-548PubMed Google Scholar, 18Yang R.M. Naitoh J. Murphy M. Wang H.J. Phillipson J. deKernion J.B. Loda M. Reiter R.E. J. Urology. 1998; 159: 941-945Crossref PubMed Scopus (277) Google Scholar, 19Fernandez P.L. Arce Y. Farre X. Martinez A. Nadal A. Rey M.J. Peiro N. Campo E. Cardesa A. J. Pathol. 1999; 187: 563-566Crossref PubMed Scopus (47) Google Scholar, 20Cheng L. Lloyd R.V. Weaver A.L. Pisansky T.M. Cheville J.C. Ramnani D.M. Leibovich B.C. Blute M.L. Zincke H. Bostwick D.G. Clin. Cancer Res. 2000; 6: 1896-1899PubMed Google Scholar, 21Yang G. Ayala G. De Marzo A. Tian W. Frolov A. Wheeler T.M. Thompson T.C. Harper J.W. Clin. Cancer Res. 2002; 8: 3419-3426PubMed Google Scholar, 22Halvorsen O.J. Haukaas S.A. Akslen L.A. Clin. Cancer Res. 2003; 9: 1474-1479PubMed Google Scholar). Reduced p27 protein, however, is not the result of p27 gene mutations, which are quite rare in cancers (23Kawamata N. Morosetti R. Miller C.W. Park D. Spirin K.S. Nakamaki T. Takeuchi S. Hatta Y. Simpson J. Wilcyznski S. Cancer Res. 1995; 55: 2266-2269PubMed Google Scholar, 24Ponce-Castaneda M.V. Lee M.H. Latres E. Polyak K. Lacombe L. Montgomery K. Mathew S. Krauter K. Sheinfeld J. Massague J. Cancer Res. 1995; 55: 1211-1214PubMed Google Scholar, 25Ferrando A.A. Balbin M. Pendas A.M. Vizoso F. Velasco G. Lopez-Otin C. Hum. Genet. 1996; 97: 91-94Crossref PubMed Scopus (83) Google Scholar). Activation of oncogenic signaling pathways ultimately results in accelerated p27 proteolysis and decreased p27 levels (26Kawada M. Yamagoe S. Murakami Y. Suzuki K. Mizuno S. Uehara Y. Oncogene. 1997; 15: 629-637Crossref PubMed Scopus (176) Google Scholar, 27Davies M.A. Koul D. Dhesi H. Berman R. McDonnell T.J. McConkey D. Yung W.K.A. Steck P.A. Cancer Res. 1999; 59: 2551-2556PubMed Google Scholar, 28Donovan J.C.H. Milic A. Slingerland J.M. J. Biol. Chem. 2001; 276: 40888-40895Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 29Mamillapalli R. Gavrilova N. Mihaylova V.T. Tsvetkov L.M. Wu H. Zhang H. Sun H. Curr. Biol. 2001; 11: 263-267Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Understanding the mechanisms whereby p27 levels are regulated may yield new targets for potential anticancer agents. Because of the emerging role of p27 in controlling prostate cancer growth, we further investigated the mechanism of 1,25-(OH)2D3 regulation of this CKI in LNCaP cells. p27 is an important regulator of the G1 to S phase transition. p27 binds and inhibits cyclin E/Cdk2 and thereby negatively regulates S phase entry. To traverse G1, cellular p27 levels must decrease. A major mechanism to achieve this regulation involves ubiquitin-dependent p27 proteolysis (30Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1735) Google Scholar, 31Hengst L. Reed S.I. Science. 1996; 271: 1861-1864Crossref PubMed Scopus (823) Google Scholar). Two rate-limiting steps for this process include phosphorylation at Thr187 by Cdk2 and recognition of Thr187-phospho-p27 by the SCFskp2 ubiquitination system (17Tsihlias J. Kapusta L.R. DeBoer G. Morava-Protzner I. Zbieranowski I. Bhattacharya N. Catzavelos G.C. Klotz L.H. Slingerland J.M. Cancer Res. 1998; 58: 542-548PubMed Google Scholar, 32Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar, 33Vlach J. Hennecke S. Amati B. EMBO J. 1997; 16: 5334-5344Crossref PubMed Scopus (609) Google Scholar, 34Montagnoli A. Fiore F. Eytan E. Carrano A.C. Draetta G.F. Hershko A. Pagano M. Genes Dev. 1999; 13: 1181-1189Crossref PubMed Scopus (512) Google Scholar, 35Tsvetkov L.M. Yeh K.H. Lee S.J. Sun H. Zhang H. Curr. Biol. 1999; 9: 661-664Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 36Slingerland J.M. Pagano M. J. Cell. Physiol. 2000; 183: 10-17Crossref PubMed Scopus (636) Google Scholar). Recent studies have revealed a novel pathway for p27 degradation at the G0/early G1 phase of the cell cycle that is independent of Thr187 phosphorylation (37Hara T. Kamura T. Kakayama K. Oshikawa K. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 38Malek N.P. Sundberg H. McGrew S. Nakayama K. Kyriakidis T.R. Roberts J.M. Nature. 2001; 413: 323-327Crossref PubMed Scopus (228) Google Scholar, 39Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar). Although not well understood, this pathway is thought to be activated by mitogenic signaling during early G1 prior to Cdk2 activation. This initial phase of p27 degradation then facilitates Cdk2 activation, which results in further p27 degradation in late G1 and ultimately entry into S phase. Although this pathway still involves the ubiquitin-proteasome system, it is independent of Thr187 phosphorylation and may be Skp2-independent (37Hara T. Kamura T. Kakayama K. Oshikawa K. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 38Malek N.P. Sundberg H. McGrew S. Nakayama K. Kyriakidis T.R. Roberts J.M. Nature. 2001; 413: 323-327Crossref PubMed Scopus (228) Google Scholar, 39Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar). The exact mechanisms of this novel pathway for p27 degradation remain to be resolved. Evidence also exists for transcriptional (40Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (843) Google Scholar, 41Inoue T. Kamiyama J. Sakai T. J. Biol. Chem. 1999; 274: 32309-32317Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and translational (31Hengst L. Reed S.I. Science. 1996; 271: 1861-1864Crossref PubMed Scopus (823) Google Scholar, 42Millard S.S. Yan J.S. Nguyen H. Pagano M. Kiyokawa H. Koff A. J. Biol. Chem. 1997; 272: 7093-7098Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 43Millard S. Vidal A. Markus M. Koff A. Mol. Cell. Biol. 2000; 20: 5947-5959Crossref PubMed Scopus (121) Google Scholar, 44Miskimins W.K. Wang G. Hawkinson M. Miskimins R. Mol. Cell. Biol. 2001; 21: 4960-4967Crossref PubMed Scopus (84) Google Scholar, 45Gopfert U. Kullmann M. Hengst L. Hum. Mol. Genet. 2003; 12: 1-13Crossref PubMed Scopus (48) Google Scholar) regulation of p27. Synthesis of p27 mRNA is governed by the Forkhead transcription factor family (46Graff J.R. Konicek B.W. McNulty A.M. Wang Z. Houck K. Allen S. Paul J.D. Hbaiu A. Goode R.G. Sandusky G.E. Vessela R.L. Neubauer B.L. J. Biol. Chem. 2000; 275: 24500-24505Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 47Medema R.H. Kops G.J.P.L. Bos J.L. Burgering B.M.T. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar). These transcription factors can be negatively regulated by the phosphatidylinositol 3-kinase/Akt growth signaling pathway, which is dysregulated in many cancers, including prostate (27Davies M.A. Koul D. Dhesi H. Berman R. McDonnell T.J. McConkey D. Yung W.K.A. Steck P.A. Cancer Res. 1999; 59: 2551-2556PubMed Google Scholar). In addition, 1,25-(OH)2D3 can activate the p27 promoter by enhancing the binding of NF-Y and Sp1 transcription factors to the p27 promoter (41Inoue T. Kamiyama J. Sakai T. J. Biol. Chem. 1999; 274: 32309-32317Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The 5′-untranslated region (UTR) of the p27 mRNA regulates the translation of p27. In quiescent cells, p27 translation is enhanced despite an overall decrease in the synthesis of most other proteins (31Hengst L. Reed S.I. Science. 1996; 271: 1861-1864Crossref PubMed Scopus (823) Google Scholar, 42Millard S.S. Yan J.S. Nguyen H. Pagano M. Kiyokawa H. Koff A. J. Biol. Chem. 1997; 272: 7093-7098Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 43Millard S. Vidal A. Markus M. Koff A. Mol. Cell. Biol. 2000; 20: 5947-5959Crossref PubMed Scopus (121) Google Scholar, 44Miskimins W.K. Wang G. Hawkinson M. Miskimins R. Mol. Cell. Biol. 2001; 21: 4960-4967Crossref PubMed Scopus (84) Google Scholar, 45Gopfert U. Kullmann M. Hengst L. Hum. Mol. Genet. 2003; 12: 1-13Crossref PubMed Scopus (48) Google Scholar). The translation of most proteins involves recognition of the mRNA 5′ 7-methylguanosine cap by the eukaryotic initiation factor 4E. Eukaryotic initiation factor 4E activity is regulated by mitogenic stimulation (reviewed in Ref. 44Miskimins W.K. Wang G. Hawkinson M. Miskimins R. Mol. Cell. Biol. 2001; 21: 4960-4967Crossref PubMed Scopus (84) Google Scholar). In the absence of mitogenic signaling, this cap-dependent translation is decreased through down-regulation of eukaryotic initiation factor 4E activity. The 5′-UTR of p27, however, contains an internal ribosomal entry site, which allows cap-independent translation of p27. This is enhanced by binding of HuR, a member of the embryonic lethal, abnormal vision (ELAV) family of RNA-binding proteins and binding of heterogeneous nuclear ribonucleoproteins C1 and C2, factors implicated in mRNA stability and processing, to the 5′-UTR of p27 (42Millard S.S. Yan J.S. Nguyen H. Pagano M. Kiyokawa H. Koff A. J. Biol. Chem. 1997; 272: 7093-7098Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 43Millard S. Vidal A. Markus M. Koff A. Mol. Cell. Biol. 2000; 20: 5947-5959Crossref PubMed Scopus (121) Google Scholar, 44Miskimins W.K. Wang G. Hawkinson M. Miskimins R. Mol. Cell. Biol. 2001; 21: 4960-4967Crossref PubMed Scopus (84) Google Scholar, 45Gopfert U. Kullmann M. Hengst L. Hum. Mol. Genet. 2003; 12: 1-13Crossref PubMed Scopus (48) Google Scholar, 48Kullmann M. Gopfert U. Siewe B. Hengst L. Genes Dev. 2002; 16: 3087-3099Crossref PubMed Scopus (290) Google Scholar). To elucidate the mechanism of 1,25-(OH)2D3-mediated increase in p27 protein levels, we investigated 1,25-(OH)2D3 regulation of p27 transcription, translation, and degradation in LNCaP cells. The 1,25-(OH)2D3-mediated increase in p27 was post-translational, via an increase in p27 protein half-life. 1,25-(OH)2D3 treatment decreased Thr187 phosphorylation of p27, a critical phosphorylation event that targets p27 for Skp2-mediated proteolysis (36Slingerland J.M. Pagano M. J. Cell. Physiol. 2000; 183: 10-17Crossref PubMed Scopus (636) Google Scholar). In addition, 1,25-(OH)2D3 decreased the nuclear localization of Cdk2, the kinase that phosphorylates p27 at Thr187. Thus, p27 up-regulation by 1,25-(OH)2D3 results from increased p27 protein half-life via decreased Thr187 phosphorylation. 1,25-(OH)2D3-mediated decreases in nuclear Cdk2 translocation may directly reduce cyclin E- and A-dependent Cdk2 activity because of reduced nuclear Cdk2 pools available for cyclin binding. Cytoplasmic Cdk2 mislocalization would also contribute to reduced Thr187 phosphorylation of p27. Thus, a determinant of growth regulation by 1,25-(OH)2D3 may be the capacity of 1,25-(OH)2D3 to decrease the nuclear localization of Cdk2, thereby preventing activation of this kinase. Materials—Cell culture media (RPMI 1640 and Dulbecco's modified Eagle's medium-high glucose) were obtained from Invitrogen, and fetal bovine serum was from Hyclone (Logan, UT). 1,25-(OH)2D3 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Mouse anti-actin antibody (1378 996) was obtained from Roche Applied Science. Anti-human phospho-Thr187-p27 antibodies were obtained from Zymed Laboratories (San Francisco, CA). Anti-human p27, Cdk2, Skp2, protein A-agarose beads, and anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG antibodies with horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescence-conjugated secondary antibodies, mAb414 antibodies, and 4,6-diamidino-2-phenylindole were generously provided by Dr. Beatriz Fontoura (University of Miami School of Medicine, Miami, FL). Cell Culture—LNCaP-FGC cells (ATCC) were passaged and maintained in RPMI medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml l-glutamine. All of the cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. [3H]Thymidine Uptake Assays—LNCaP cells were treated with either ethanol vehicle or 10 nm 1,25(OH)2D3. Following the 48-h treatment period, the media were replaced with [3H]thymidine-containing medium, and the cells were incubated for 18 h. Acid soluble tritium was removed by a trichloroacetic acid wash; the cells were then lysed, and [3H]thymidine uptake was determined by scintillation counting. Reporter Plasmids and Luciferase Assay—The reporter plasmids p27PFLuc and control (41Inoue T. Kamiyama J. Sakai T. J. Biol. Chem. 1999; 274: 32309-32317Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) were the generous gifts of Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji). The reporter plasmids 5′-SvL-3′ and controls (43Millard S. Vidal A. Markus M. Koff A. Mol. Cell. Biol. 2000; 20: 5947-5959Crossref PubMed Scopus (121) Google Scholar) were provided by Dr. Andrew Koff (Memorial Sloan-Kettering Cancer Center, New York). For luciferase assays, the cells were transfected with 5.0 μg of reporter plasmid and 1.0 μg of cytomegalovirus-β-galactosidase vectors and treated with either ethanol vehicle or 10 nm 1,25-(OH)2D3 or serum-starved for the appropriate time periods. Following the treatment period, the cell lysates were collected, and luciferase activity was observed. β-Galactosidase activities were measured to normalize for transfection efficiency. Pulse-Chase Analysis—One day after plating, the cells were treated with either ethanol vehicle or 10 nm 1,25-(OH)2D3 for 24 h. Following the treatment period, the medium was replaced with Dulbecco's modified Eagle's medium (–met) and 10% dialyzed fetal bovine serum for 1 h. Next, the cells were pulsed for 1 h with 500 μCi of [35S]Met (PerkinElmer Life Sciences). Following the pulse, the cells were then chased for 0, 1, 3, and 6 h with Dulbecco's modified Eagle's medium (–met) medium containing 40 mm cold methionine and 10% dialyzed fetal bovine serum. After the chase times, the cells were lysed in sample buffer containing 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.5% Nonidet P-40, 10 μg/ml aprotonin, 10 μg/ml leupeptin, 50 mm NaF, 0.1 mm sodium orthovanadate. Extracts (500 μg protein) were precleared with 1 μg of normal rabbit IgG preadsorbed with protein A-agarose beads. Precleared extracts were then incubated with 1 μg of polyclonal p27 antibody and agitated overnight at 4 °C. The samples were then equilibrated with lysis buffer and 25 μl of packed volume of protein A-agarose beads for 4 h at 4 °C with agitation. Following three washes with lysis buffer, immune complexes were eluted by boiling in 20 μl of Laemmli gel loading buffer. The samples were then subjected to SDS-PAGE, transferred to nitrocellulose membrane filters, and exposed to autoradiography. Western Blot Analysis—The cells were treated with ethanol vehicle, 10 nm 1,25-(OH)2D3, or serum-starved for appropriate times, washed, and lysed in sample buffer. The protein concentrations were determined by the Bio-Rad Dc Protein Assay according to the manufacturer's instructions. 50 μg of cell extract proteins were subjected to standard SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were processed for Western blotting using standard procedures. Briefly, the filters were incubated overnight at 4 °C in blocking solution (5% dry milk, 0.1% Tween in 1× wash buffer (20 mm Tris, 50 mm NaCl, 2.5 mm EDTA)) followed by incubation with the primary antibody for 1 h. For phospho-specific antibodies, the filters were blocked overnight in 5% bovine serum albumin, 0.1% Tween in PBS. Actin antibody (Roche Applied Science) was used at 0.5 μg/ml. p27 and Cdk2 antibodies were used at 1.0 μg/ml. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody, and the proteins were visualized using the ECL system (Amersham Biosciences) following the supplier's instructions. In Vitro Cdk2 Kinase Assay—LNCaP cells plated at 40–50% confluency were treated with either ethanol vehicle or 10 nm 1,25-(OH)2D3. Following the treatment period, the cells were washed in PBS and solubilized in TNE buffer (50 mm Tris, pH 7.5, 140 mm NaCl, 5 mm EDTA) containing 1% Nonidet P-40, 1:100 dilution of the protease inhibitor mixture (Sigma), 50 mm NaF, and 0.1 mm sodium orthovanadate. The protein concentrations of the lysates were determined as described above. 200 μg of proteins were then incubated with 3 μg of rabbit anti-human Cdk2 or rabbit anti-human cyclin E antibodies for 1 h at 4 °C. Following this incubation, the samples were adsorbed with 40 μl of anti-rabbit IgG-agarose beads (Sigma) for 1 h at 4 °C. After washing once with TNE buffer and three times with kinase buffer (50 mm Tris, pH 7.4, 10 mm MgCl2), immune complexes were incubated in 30 μl of kinase buffer containing 1 μg of histone H1, 25 μm ATP, and 10 μCi of [γ-32P]ATP for 30 min at 30 °C. The reactions were stopped by the addition of 4× Laemmli buffer. After 5 min of boiling, the samples were then subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Phosphorylated histone H1 was visualized by autoradiography. After the autoradiography, the membrane was subjected to Western blotting for Cdk2 and cyclin E. Subcellular Fractionation—The cells were treated with ethanol vehicle, 10 nm 1,25-(OH)2D3 or serum-starved for appropriate times. Following the treatment periods, the cells were washed in PBS and lysed in transport buffer (20 mm HEPES, pH 7.4, 110 mm potassium acetate, 2 mm MgCl2) containing 15 μg/ml digitonin (Calbiochem, Cambridge, MA) and 10 μg/ml aprotonin, 10 μg/ml leupeptin, 50 mm NaF, and 0.1 mm sodium orthovanadate. The lysates were then centrifuged for 5 min at 800 × g at 4 °C, and the supernatant was collected (cytosolic fraction). The nuclear fraction was obtained by sonication of the pellet in transport buffer. The protein concentrations of both fractions were determined as described above. 25 μg of cytosolic and 50 μg nuclear proteins were then subjected to SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were processed for Western blotting of Cdk2, p27, nucleolin (for fractionation purity), and actin (loading control). Immunofluorescence—One day after plating on coverslips, the cells were treated with ethanol vehicle, 10 nm 1,25-(OH)2D3, or serum-starved for appropriate times. Following the treatment period, the cells were fixed in 2% formaldehyde in PBS for 10 min. Next, the cells were permeablized in PBS containing 1% bovine serum albumin, 0.1% Triton X-100 for 5 min. The cells were then incubated for 1 h with rabbit anti-Cdk2 (1:50 dilution in PBS) and mouse anti-nucleoporin antibodies (mAB414), washed in PBS, and incubated for 1 h with Cy3-conjugated goat anti-rabbit (1:1000) and fluorescein isothiocyanate-conjugated donkey anti-mouse antibodies. Following three additional washes, the coverslips were incubated with 4,6-diamidino-2-phenylindole for 5 min and mounted on glass slides in anti-fade medium. The images were then collected using confocal microscopy (Zeiss). Quantification and Statistical Analysis—Western blot data were quantified using densitometry. The data were analyzed via either a two-tailed unpaired t test or a one-way analysis of variance followed by a Bonferroni post test using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). For confocal image analysis, fluorescence intensities were determined using ImageJ (rsb.info.nih.gov/ij/). For the 24-h time point, 85 control cells and 81 treated cells were quantified. For the 48-h time point, 108 control cells and 89 treated cells were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*, p < 0.05; **, p < 0.001). 1,25-(OH)2 Vitamin D3 Does Not Regulate the Transcription or Translation of p27—We previously reported that 1,25-(OH)2D3 inhibits the growth of the LNCaP prostate cancer cell line (11Zhuang S.-H. Schwartz G.G. Cameron D. Burnstein K.L. Mol. Cell. Endocrinol. 1997; 126: 83-90Crossref PubMed Scopus (115) Google Scholar, 12Zhuang S.-H. Burnstein K.L. Endocrinology. 1998; 139: 1197-1207Crossref PubMed Scopus (203) Google Scholar). [3H[Thymidine incorporation in 1,25-(OH)2D3-treated LNCaP cells was decreased ∼85% of control (Fig. 1A). This decrease in proliferation by 1,25-(OH)2D3 is associated with an increased percentage of LNCaP cells in G1 and a concomitant decrease in S and G2/M (Fig. 1B). Consistent with inhibition of G1 to S progression, 1,25-(OH)2D3 increases the levels of the CKI p27Kip1, enhances the association of this CKI with Cdk2, and decreases Cdk2 activ" @default.
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• W2017322511 title "Vitamin D Inhibits G1 to S Progression in LNCaP Prostate Cancer Cells through p27Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm" @default.
• W2017322511 cites W1965049479 @default.
• W2017322511 cites W1972422162 @default.
• W2017322511 cites W1978052913 @default.
• W2017322511 cites W1980123375 @default.
• W2017322511 cites W1986527803 @default.
• W2017322511 cites W1994282776 @default.
• W2017322511 cites W1998625763 @default.
• W2017322511 cites W2005744028 @default.
• W2017322511 cites W2013850508 @default.
• W2017322511 cites W2027451946 @default.
• W2017322511 cites W2031646117 @default.
• W2017322511 cites W2039169535 @default.
• W2017322511 cites W2043092024 @default.
• W2017322511 cites W2044312503 @default.
• W2017322511 cites W2063955417 @default.
• W2017322511 cites W2065760788 @default.
• W2017322511 cites W2067223570 @default.
• W2017322511 cites W2071429904 @default.
• W2017322511 cites W2073996456 @default.
• W2017322511 cites W2079494240 @default.
• W2017322511 cites W2088749263 @default.
• W2017322511 cites W2089448004 @default.
• W2017322511 cites W2091403769 @default.
• W2017322511 cites W2099990598 @default.
• W2017322511 cites W2101305429 @default.
• W2017322511 cites W2102323032 @default.
• W2017322511 cites W2110062940 @default.
• W2017322511 cites W2115704169 @default.
• W2017322511 cites W2118040139 @default.
• W2017322511 cites W2119634498 @default.
• W2017322511 cites W2129129118 @default.
• W2017322511 cites W2129391217 @default.
• W2017322511 cites W2132568558 @default.
• W2017322511 cites W2133528029 @default.
• W2017322511 cites W2147868370 @default.
• W2017322511 cites W2151559596 @default.
• W2017322511 cites W2152398347 @default.
• W2017322511 cites W2158253569 @default.
• W2017322511 cites W2162741492 @default.
• W2017322511 cites W2163931326 @default.
• W2017322511 cites W2166525118 @default.
• W2017322511 cites W4301651270 @default.
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