Inhibitory effects of 1alpha,25-dihydroxyvitamin D(3) on the G(1)-S phase-controlling machinery.

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) has been known for years, the molecular basis of the cell cycle blockade by 1alpha,25-dihydroxyvitamin D(3) remains largely unknown. Here we have investigated the mechanisms underlying the G(1) arrest induced upon 1alpha,25-dihydroxyvitamin D(3) treatment of the human breast cancer cell line MCF-7. Twenty-four-hour exposure of exponentially growing MCF-7 cells to 1alpha,25-dihydroxyvitamin D(3) impeded proliferation by preventing S phase entry, an effect that correlated with appearance of the growth-suppressing, hypophosphorylated form of the retinoblastoma protein (pRb), and modulation of cyclin-dependent kinase (cdk) activities of cdk-4, -6, and -2. Time course immunochemical and biochemical analyses of the cellular and molecular effects of 1alpha,25-dihydroxyvitamin D(3) treatment for up to 6 d revealed a dynamic chain of events, preventing activation of cyclin D1/cdk4, and loss of cyclin D3, which collectively lead to repression of the E2F transcription factors and thus negatively affected cyclin A protein expression. While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21(CIP1/WAF1), which blocked the remaining cyclin A(E)/cdk 2 complexes. Together with a rapid down-modulation of the c-Myc oncoprotein in response to 1alpha,25-dihydroxyvitamin D(3), these results demonstrate that 1alpha,25-dihydroxyvitamin D(3) inhibits cell proliferation by targeting several key regulators governing the G(1)/S transition.

[1]  R. Sheaff.  Regulation of mammalian cyclin-dependent kinase 2. , 1997, Methods in enzymology.

[2]  L. Dorssers,et al.  Inhibition of breast cancer cell growth by combined treatment with vitamin D3 analogues and tamoxifen. , 1994, Cancer research.

[3]  B. Mitchell,et al.  Transcriptional regulation of the c-myc protooncogene by 1,25-dihydroxyvitamin D3 in HL-60 promyelocytic leukemia cells. , 1987, The Journal of biological chemistry.

[4]  H. Okayama,et al.  Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4 , 1999, Oncogene.

[5]  E. Lees,et al.  The adenovirus E1A-associated kinase consists of cyclin E-p33cdk2 and cyclin A-p33cdk2 , 1993, Journal of virology.

[6]  C. O'keefe,et al.  Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function. , 1994, Genes & development.

[7]  G. Peters,et al.  Evidence for different modes of action of cyclin-dependent kinase inhibitors: p15 and p16 bind to kinases, p21 and p27 bind to cyclins. , 1995, Oncogene.

[8]  J. Nevins,et al.  A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  David O. Morgan,et al.  Principles of CDK regulation , 1995, Nature.

[10]  J. Harper,et al.  Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4. , 1996, Genes & development.

[11]  C. O'keefe,et al.  Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4. , 1996, Molecular biology of the cell.

[12]  M. Haussler,et al.  The Nuclear Vitamin D Receptor: Biological and Molecular Regulatory Properties Revealed , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[13]  J. Welsh,et al.  Apoptotic regression of MCF-7 xenografts in nude mice treated with the vitamin D3 analog, EB1089. , 1998, Endocrinology.

[14]  R. Simpson,et al.  c-myc Intron Element-binding Proteins Are Required for 1,25-Dihydroxyvitamin D3 Regulation of c-myc during HL-60 Cell Differentiation and the Involvement of HOXB4* , 1999, The Journal of Biological Chemistry.

[15]  L. Freedman,et al.  Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription , 1997, Molecular and cellular biology.

[16]  K. Helin,et al.  Regulation of cell proliferation by the E2F transcription factors. , 1998, Current opinion in genetics & development.

[17]  J. Bartek,et al.  Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity , 1995, Molecular and cellular biology.

[18]  A. Norman.  Receptors for 1α,25(OH)2D3: Past, Present, and Future , 1998 .

[19]  M. Pagano,et al.  Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumour cell lines. , 1994, Oncogene.

[20]  p21Waf1 inhibits the activity of cyclin dependent Kinase 2 by preventing its activating phosphorylation , 1998, Oncogene.

[21]  M. B. Kelly,et al.  Direct Activation of Protein Kinase C by 1α,25-Dihydroxyvitamin D3(*) , 1995, The Journal of Biological Chemistry.

[22]  James M. Roberts,et al.  CDK inhibitors: positive and negative regulators of G1-phase progression. , 1999, Genes & development.

[23]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[24]  P. Yaswen,et al.  Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells , 1997, Molecular and cellular biology.

[25]  M. Uskoković,et al.  Signaling pathways for vitamin D-induced differentiation: implications for therapy of proliferative and neoplastic diseases. , 1993, Critical reviews in eukaryotic gene expression.

[26]  E. Sausville,et al.  Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. , 1996, Cancer research.

[27]  G. Peters,et al.  Induced Expression of p16INK4a Inhibits Both CDK4- and CDK2-Associated Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor Complexes , 1999, Molecular and Cellular Biology.

[28]  M. Meyerson,et al.  Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase , 1991, Nature.

[29]  C. M. Hansen,et al.  Seocalcitol (EB 1089): a vitamin D analogue of anti-cancer potential. Background, design, synthesis, pre-clinical and clinical evaluation. , 2000, Current pharmaceutical design.

[30]  A. Norman,et al.  Stimulation of Phosphorylation of Mitogen-Activated Protein Kinase by 1α,25-Dihydroxyvitamin D3 in Promyelocytic NB4 Leukemia Cells: A Structure-Function Study. , 1998, Endocrinology.

[31]  H. Koeffler,et al.  Effect of a novel vitamin D3 analog, EB1089, on G1 cell cycle regulatory proteins in HL-60 cells. , 2000, International journal of oncology.

[32]  D. Morgan,et al.  Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase , 1994, Molecular and cellular biology.

[33]  J. Bartek,et al.  The retinoblastoma protein pathway and the restriction point. , 1996, Current opinion in cell biology.

[34]  D. Dean,et al.  Active Transcriptional Repression by the Rb–E2F Complex Mediates G1 Arrest Triggered by p16INK4a, TGFβ, and Contact Inhibition , 1999, Cell.

[35]  G. Hannon,et al.  A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 , 1993, Nature.

[36]  L. Freedman,et al.  Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo , 1996, Molecular and cellular biology.

[37]  L. Binderup,et al.  EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro , 1998, British journal of pharmacology.

[38]  L. Freedman,et al.  Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. , 1996, Genes & development.

[39]  C. M. Hansen,et al.  EB 1089, a novel vitamin D analog with strong antiproliferative and differentiation-inducing effects on target cells. , 1997, Biochemical pharmacology.

[40]  J. Troppmair,et al.  Regulation of c-myc expression by Ras/Raf signalling , 1998, Oncogene.

[41]  J. Jones,et al.  Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. , 1996, Cancer research.

[42]  G. P. Studzinski,et al.  Cyclin-dependent kinase 6 is the principal target of p27/Kip1 regulation of the G1-phase traverse in 1,25-dihydroxyvitamin D3-treated HL60 cells. , 1997, Cancer research.

[43]  N. Mailand,et al.  Involvement of Myc Activity in a G1/S-Promoting Mechanism Parallel to the pRb/E2F Pathway , 2000, Molecular and Cellular Biology.

[44]  L. Tsai,et al.  The cdk2 kinase is required for the G1-to-S transition in mammalian cells. , 1993, Oncogene.

[45]  C. Carlberg.  The vitamin D(3) receptor in the context of the nuclear receptor superfamily : The central role of the retinoid X receptor. , 1996, Endocrine.

[46]  S. Inui,et al.  Synchronization of normal human keratinocyte in culture: its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle. , 1998, Journal of dermatological science.

[47]  James M. Roberts,et al.  Inhibitors of mammalian G1 cyclin-dependent kinases. , 1995, Genes & development.

[48]  M. Campbell,et al.  Inhibition of proliferation of prostate cancer cells by a 19-nor-hexafluoride vitamin D3 analogue involves the induction of p21waf1, p27kip1 and E-cadherin. , 1997, Journal of molecular endocrinology.

[49]  K. Burnstein,et al.  Antiproliferative Effect of 1α,25-Dihydroxyvitamin D3 in Human Prostate Cancer Cell Line LNCaP Involves Reduction of Cyclin-Dependent Kinase 2 Activity and Persistent G1 Accumulation. , 1998, Endocrinology.

[50]  F. Oesch,et al.  Differences in the mechanisms of growth control in contact-inhibited and serum-deprived human fibroblasts , 1997, Oncogene.

[51]  R. Bouillon,et al.  Action of 1,25(OH)2D3 on the cell cycle genes, cyclin D1, p21 and p27 in MCF-7 cells , 1998, Molecular and Cellular Endocrinology.

[52]  P. Farnham,et al.  Introduction to the E2F family: protein structure and gene regulation. , 1996, Current topics in microbiology and immunology.

[53]  J. Geysen,et al.  Proliferation and differentiation of cultured human keratinocytes is modulated by 1,25(OH)2D3 and synthetic vitamin D3 analogues in a cell density-, calcium- and serum-dependent manner. , 1997, Pharmacology & toxicology.

[54]  J. Massagué,et al.  Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-β in cells lacking the CDK inhibitor p15 , 1997, Nature.

[55]  E. A. Thompson,et al.  Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21Cip1. , 1997, Molecular endocrinology.

[56]  K. Umesono,et al.  Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling , 1992, Nature.

[57]  M. Tatsuka,et al.  Requirement for tyrosine phosphorylation of Cdk4 in Gl arrest induced by ultraviolet irradiation , 1995, Nature.

[58]  M. Garabedian,et al.  Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms , 1997, Molecular and cellular biology.

[59]  Gregory J. Hannon,et al.  pl5INK4B is a potentia| effector of TGF-β-induced cell cycle arrest , 1994, Nature.

[60]  P. Reitsma,et al.  Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite , 1983, Nature.

[61]  J. Nevins,et al.  Toward an understanding of the functional complexity of the E2F and retinoblastoma families. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[62]  M. Brattain,et al.  Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells , 1997, Oncogene.

[63]  James M. Roberts,et al.  Cloning of p27 Kip1 , a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals , 1994, Cell.

[64]  S. Mittnacht,et al.  Control of pRB phosphorylation. , 1998, Current opinion in genetics & development.

[65]  E. Lees,et al.  Active cdk6 complexes are predominantly nuclear and represent only a minority of the cdk6 in T cells , 1998, Oncogene.

[66]  S. Elledge,et al.  Inhibition of cyclin-dependent kinases by p21. , 1995, Molecular biology of the cell.

[67]  H. Matsushime,et al.  Inactivation of the Cyclin D-dependent Kinase in the Rat Fibroblast Cell Line, 3Y1, Induced by Contact Inhibition* , 1997, The Journal of Biological Chemistry.

[68]  D. Bikle,et al.  Antiestrogen potentiation of antiproliferative effects of vitamin D3 analogues in breast cancer cells. , 1996, Cancer research.

[69]  M. Kattan,et al.  A calcitriol analogue, EB1089, inhibits the growth of LNCaP tumors in nude mice. , 2000, Cancer research.

[70]  T. Kishi,et al.  Rapid control of transmembrane calcium influx by 1alpha,25-dihydroxyvitamin D3 and its analogues in rat osteoblast-like cells. , 1999, Biological & pharmaceutical bulletin.

[71]  M. Devonec,et al.  1,25-Dihydroxyvitamin D3 inhibitory effect on the growth of two human breast cancer cell lines (MCF-7, BT-20). , 1986, Journal of steroid biochemistry.

[72]  S. Shurtleff,et al.  D-type cyclin-dependent kinase activity in mammalian cells , 1994, Molecular and cellular biology.

[73]  M. Bissonnette,et al.  1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation* , 1999, The Journal of Biological Chemistry.

[74]  E. Harlow,et al.  The retinoblastoma protein is phosphorylated during specific phases of the cell cycle , 1989, Cell.

[75]  J. Bartek,et al.  Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression , 1996, Molecular and cellular biology.

[76]  M. Dyer,et al.  BCL-1/cyclin D1 oncoprotein oscillates and subverts the G1 phase control in B-cell neoplasms carrying the t(11;14) translocation. , 1994, Oncogene.

[77]  T. Nikaido,et al.  Vitamin D analogues up-regulate p21 and p27 during growth inhibition of pancreatic cancer cell lines. , 1997, British Journal of Cancer.

[78]  J. Welsh.  Induction of apoptosis in breast cancer cells in response to vitamin D and antiestrogens. , 1994, Biochemistry and cell biology = Biochimie et biologie cellulaire.