Non-malignant and Tumor-derived Cells Differ in Their Requirement for p27Kip1 in Transforming Growth Factor-β-mediated G1 Arrest

Transforming growth factor β (TGF-β) induces G1 arrest in susceptible cells by multiple mechanisms that inhibit the G1 cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-β treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27Kip1 in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G1 arrest by TGF-β was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-β-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G1 phase. Maintenance of the G1 arrest was accompanied by increased association of the Cdk inhibitor p21WAF-1/Cip-1 and the retinoblastoma family member p130Rb2 in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G1 arrest by TGF-β in two tumor-derived lines. ASp27 transfection into two TGF-β-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G1 arrest despite the continued presence of TGF-β. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G1-to-S phase progression. Transforming growth factor β (TGF-β) induces G1 arrest in susceptible cells by multiple mechanisms that inhibit the G1 cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-β treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27Kip1 in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G1 arrest by TGF-β was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-β-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G1 phase. Maintenance of the G1 arrest was accompanied by increased association of the Cdk inhibitor p21WAF-1/Cip-1 and the retinoblastoma family member p130Rb2 in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G1 arrest by TGF-β in two tumor-derived lines. ASp27 transfection into two TGF-β-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G1 arrest despite the continued presence of TGF-β. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G1-to-S phase progression. transforming growth factor-β cyclin-dependent kinase kinase inhibitory protein human mammary epithelial cells mouse embryonic fibroblasts antisense p27 TGF-β1 mediates effects on diverse cellular processes such as proliferation, growth, and differentiation via cell surface receptors that in turn regulate the activity of SMAD transcription factors (reviewed in Ref. 1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3945) Google Scholar). In many normal cell types, including epithelial and melanocytic cells, TGF-β has a potent antiproliferative effect. In contrast to nontransformed cells, cancer-derived lines show reduced antiproliferative responses to TGF-β or have lost this response altogether (2Reiss M. Barcellos-Hoff M.H. Breast Cancer Res. Treat. 1997; 45: 81-95Crossref PubMed Scopus (141) Google Scholar). In most cases, the loss of TGF-β responsiveness occurs without inactivation of TGF-β receptors or the SMADs. Cell cycle deregulation is believed to contribute to the resistance of malignant cells to G1 arrest by TGF-β (reviewed in Ref.3Donovan J. Slingerland J. Breast Cancer Res. 2000; 2: 116-124Crossref PubMed Scopus (74) Google Scholar). TGF-β induces cell cycle arrest in the G1 phase via a number of pathways that lead ultimately to inhibition of the G1 cyclin-dependent kinases (Cdks). The Cdks are key mediators of progression through the cell cycle and are regulated by phosphorylation, cyclin binding, and by the binding of Cdk inhibitory proteins (reviewed in Ref. 4Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5071) Google Scholar). During G1-to-S phase progression, the D-type cyclins bind Cdk4 and Cdk6 and the E-type cyclins bind Cdk2, contributing to kinase activation and G1-to-S phase progression. The G1 phosphatase, Cdc25A, plays an essential role in Cdk activation by the removal of inhibitory phosphates from Cdk2 (5Hoffmann I. Draetta G. Karsenti E. EMBO (Eur. Mol. Biol. Organ.) J. 1994; 13: 4302-4310Crossref PubMed Scopus (420) Google Scholar) and possibly also from Cdks 4 and Cdk6 (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar). Cdc25A may be transcriptionally up-regulated by c-Myc (7Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Crossref PubMed Scopus (641) Google Scholar). Two families of Cdk inhibitory proteins oppose Cdk activation. p21WAF1/Cip-1, p27Kip1, and p57Kip2belong to the kinase inhibitory protein (KIP) family and contribute to the inhibition of cyclin E1-Cdk2 complexes in the G1 phase. p15INK4B, p16INK4A, p18INK4C, and p19INK4D belong to the inhibitors of Cdk4 family and act to inhibit Cdk4 and Cdk6 (reviewed in Ref. 4Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5071) Google Scholar). In several cell types, including human mammary epithelial cells (HMECs) and mink lung epithelial cells, TGF-β induces and stabilizes the p15 protein, which leads to its binding to and inhibition of Cdk4 and Cdk6 complexes (8Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (885) Google Scholar, 9Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1874) Google Scholar, 10Sandhu C. Garbe J. Daksis J. Pan C.H. Bhattacharya N. Yaswen P. Koh J. Slingerland J. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar). TGF-β also causes the accumulation of p27 in cyclin E1-Cdk2 complexes leading to Cdk2 inhibition (11Koff A. Ohtsuki M. Polyak K. Roberts J.M. Massague J. Science. 1993; 260: 536-539Crossref PubMed Scopus (509) Google Scholar, 12Slingerland J.M. Hengst L. Pan C.H. Alexander D. Stampfer M.R. Reed S.I. Mol. Cell. Biol. 1994; 14: 3683-3694Crossref PubMed Google Scholar). Changes in several essential cell cycle regulators cooperate to induce TGF-β arrest, including down-regulation of c-Myc (11Koff A. Ohtsuki M. Polyak K. Roberts J.M. Massague J. Science. 1993; 260: 536-539Crossref PubMed Scopus (509) Google Scholar, 13Pietenpol J.A. Stein R.W. Moran E. Yaciuk P. Schlegel R. Lyons R.M. Pittelkow M.R. Munger K. Howley P.M. Moses H.L. Cell. 1990; 61: 777-785Abstract Full Text PDF PubMed Scopus (491) Google Scholar), Cdc25A (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar), and cyclin D1 and, in some cell types, up-regulation of p21 (3Donovan J. Slingerland J. Breast Cancer Res. 2000; 2: 116-124Crossref PubMed Scopus (74) Google Scholar). Deregulation of various cell cycle targets including cyclin and Cdk overexpression, Cdk inhibitor inactivation, and Myc or Cdc25A overexpression are believed to contribute to TGF-β resistance in cancer (3Donovan J. Slingerland J. Breast Cancer Res. 2000; 2: 116-124Crossref PubMed Scopus (74) Google Scholar). Although mouse embryonic fibroblasts (MEFs) fromp27 −/− mice retain TGF-β sensitivity (14Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar), several studies have indicated an association between altered p27 regulation and the development of TGF-β resistance. Our previous work showed that the acquisition of TGF-β resistance in human mammary epithelial cells was associated with altered phosphorylation, altered Cdk inhibitory activity, and cytoplasmic mislocalization of the p27 protein (15Ciarallo S. Subramaniam V. Hung W. Lee J.H. Kotchetkov R. Sandhu C. Milic A. Slingerland J.M. Mol. Cell. Biol. 2002; 22: 2993-3002Crossref PubMed Scopus (55) Google Scholar). Although p27 gene mutations are rare in human tumors, increased proteasomal degradation of p27 is observed in a number of cancers, including breast, colon, and prostate, and the reduced p27 levels are associated with poor patient prognosis (reviewed in Refs. 16Slingerland J. Pagano M. J. Cell. Physiol. 2000; 183: 10-17Crossref PubMed Scopus (632) Google Scholar and 17Lee M.H. Yang H.Y. Cell. Mol. Life Sci. 2001; 58: 1907-1922Crossref PubMed Google Scholar). Relatively little is known about the compensatory mechanisms invoked by a nontransformed cell after a reduction in p27 protein levels, although a few reports support a role for compensation by other Cdk inhibitors to maintain normal cell cycle control. For example, in serum-starved p27 −/− mouse embryonic fibroblasts (MEFs), the accumulation of the retinoblastoma family member p130Rb2 in cyclin E-Cdk2 complexes compensated for p27 loss and enabled cells to undergo proliferative arrest in the G1 phase (18Coats S. White P. Fero M.L. Lacy S. Chung G. Randel E. Firpo E. Roberts J.M. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). The the requirement for G1 arrest by TGF-β in finite lifespan and HMECs and in cancer-derived lines. antisense p27 oligonucleotides to inhibit p27 show that HMECs, not the tumor cell maintain G1 arrest after p27 down-regulation via a compensatory accumulation of p21 and p130 in cyclin E-Cdk2 that p27 is to maintain TGF-β arrest in these malignant lines has a redundant in the finite lifespan that be compensated for by other Cdk inhibitory The and of normal finite lifespan human mammary epithelial cells from reduction has C. Garbe J. Daksis J. Pan C.H. Bhattacharya N. Yaswen P. Koh J. Slingerland J. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar, M. J. 9: Scopus Google Scholar). The human was from a growth phase and was by M. in with Bhattacharya N. M.R. Slingerland J.M. 1996; 13: Google Scholar). cells are human mammary epithelial cells from a patient with as L. J. Cancer Res. 1990; Google Scholar). cells by Cancer and in with as (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar). cells J. A. S. M. J. Cancer PubMed Scopus Google Scholar) in essential with and HMECs and cells with and cells with TGF-β from with for and with and with as T. R. D. Slingerland J. C. 1996; Google Scholar). Cell cycle was on a and Cell Cell and as C. Garbe J. Daksis J. Pan C.H. Bhattacharya N. Yaswen P. Koh J. Slingerland J. Stampfer M.R. Mol. Cell. Biol. 1997; 17: 2458-2467Crossref PubMed Google Scholar). protein was by for cyclin cyclin was from of protein with Cdk6 Cdk6 was from of protein and with the for of associated controls along The are of to p27 and p130 from to Cdk2, Cdc25A, and c-Myc from to cyclin D1 from to from S. Reed to cyclin and and p15 from E. and to from or cells with TGF-β for by antisense or p27 transfection for as S. Milic A. Bhattacharya N. Slingerland J.M. Sci. S. A. 2000; PubMed Scopus Google Scholar) in the presence of TGF-β by with TGF-β. and protein was after transfection and For and cells, the from or the transfection from to or the G1 arrest by TGF-β. was and with and as J.M. Hengst L. Pan C.H. Alexander D. Stampfer M.R. Reed S.I. Mol. Cell. Biol. 1994; 14: 3683-3694Crossref PubMed Google Scholar, V. E. Reed S.I. Science. PubMed Scopus Google Scholar). in the was an and in mouse was from kinase the TGF-β responsiveness of human mammary epithelial cells passage and cells are a finite lifespan mammary epithelial is a epithelial cell is a malignant cancer and is a malignant cell for in the or presence of TGF-β and cells sensitivity to G1 arrest with an reduction in the of cells in phase after of TGF-β treatment The and cell lines the or G1 arrest with TGF-β with reduction in the of cells in phase. The levels of the G1 and Cdk inhibitors by in and cells in the or after a to TGF-β D1 levels in and cells and in levels in the cancer-derived and cells with the and and cyclin D1 levels showed by TGF-β in in and In TGF-β cyclin D1 and cyclin levels by to The levels of p27 in the cancer-derived with and cells p27 levels and and TGF-β treatment not alter p27 protein levels in the p27 levels by in the in and in p21 levels in and with reduced levels in p21 levels in the and cells after of TGF-β The cells showed a in p21 levels of TGF-β treatment by a to levels as in the by cells showed a in p21 levels the p15 levels in the with the and are (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar, Bhattacharya N. M.R. Slingerland J.M. 1996; 13: Google Scholar). TGF-β treatment of led to a in p15 In cells, p15 not be in the that to p15 from the a of the showed that p15 levels increased by in TGF-β-treated cells that the p130Rb2 protein, p21WAF-1/Cip-1 and p27Kip1, has a Cdk inhibitory and also in and inhibit Cdk complexes (18Coats S. White P. Fero M.L. Lacy S. Chung G. Randel E. Firpo E. Roberts J.M. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar), p130 protein levels in and TGF-β-treated cells by p130 levels in with the cancer-derived lines TGF-β increased p130 levels in and also increased p130 in p130 levels not by TGF-β in the tumor-derived lines. was by The levels of and Cdk2 in cyclin complexes after of levels of cyclin from and TGF-β-treated and cells Cdk2 levels and not altered by TGF-β in in cell p21 levels in after TGF-β TGF-β treatment of and cells led to a in p21 binding to cyclin p27 increased in cyclin complexes in cell after TGF-β treatment cancer-derived lines showed a of cyclin p27 HMECs. p27 levels in and in p21 was also in and lines in p130 was in cyclin complexes in and TGF-β-treated cells Although the total p130 levels in and cells with the cancer-derived lines the levels of cyclin p130 in in the cancer lines. p130 binding to cyclin E1-Cdk2 was increased by TGF-β in and TGF-β treatment of led to a in cyclin The kinase activity of cyclin in was as Although of cyclin cyclin kinase in and that in the TGF-β treatment of the and led to a inhibition of cyclin kinase cells also showed reduction in kinase activity associated with the G1 kinase activity in the cells was reduced by TGF-β by kinase is in the levels of p15 and cyclin D1 in Cdk6 complexes from and TGF-β-treated cells Despite total cyclin D1 and Cdk6 in and cells, cyclin D1 was bound to Cdk6 in and in cyclin D1 levels in between that observed in and or TGF-β a reduction in the levels of cyclin D1 in cells, cyclin D1 association was not reduced by TGF-β in the and p15 was in The of p15 bound to Cdk6 increased after TGF-β arrest of the In p15 levels reduced and p15 levels not after TGF-β treatment of on of the p15 and cells p15 of a loss of the p15 gene (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Bhattacharya N. M.R. Slingerland J.M. 1996; 13: Google Scholar). The and cancer-derived lines in showed a number of in the regulation of cyclin cyclin and p15 with the The cyclin kinase increased despite the presence of increased cyclin E1-Cdk2 bound p27 in these complexes in and TGF-β-treated and lines. observed increased cyclin D1 bound to Cdk6 complexes and a to p15 in Cdk6 complexes after TGF-β treatment of the cancer-derived lines. to the levels of c-Myc has to with p27 many levels and to p15 K. J. 1998; PubMed Scopus Google Scholar, M. W. X. Mol. Cell. 2002; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, P. K. A. J. J. T. J. Massague J. M. Cell Biol. 2001; PubMed Scopus Google Scholar, J. Massague J. Mol. Cell. Biol. 1999; PubMed Google Scholar). c-Myc may also the Cdc25A gene (7Galaktionov K. Chen X. Beach D. 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Science. 1993; 260: 536-539Crossref PubMed Scopus (509) Google Scholar, 12Slingerland J.M. Hengst L. Pan C.H. Alexander D. Stampfer M.R. Reed S.I. Mol. Cell. Biol. 1994; 14: 3683-3694Crossref PubMed Google Scholar). in cell types, other in G1 regulators to contribute to TGF-β arrest (reviewed in Ref. J. Slingerland J. Breast Cancer Res. 2000; 2: 116-124Crossref PubMed Scopus (74) Google Scholar). the requirement for p27 in G1 arrest, the inhibition of p27 TGF-β arrest in and with TGF-β for by a transfection with or as TGF-β was after Cell cycle and protein after the transfection and p27 protein levels reduced by after ASp27 transfection and levels remained after transfection p27 levels in the and The transfection not alter protein levels of other including cyclin cyclin Cdk2, and Cdk6 not ASp27 and to the cell cycle such on arrested or showed that ASp27 transfection led to a in the of cells in G1 and an in the in phase in the tumor-derived and lines of these cells in phase after transfection with for the and of cyclin from and from p27 or and kinase of cyclin kinase accompanied cell cycle after inhibition of p27 in TGF-β-treated and cells for in In the cell cycle of the finite lifespan and the not altered by ASp27 showed that the cyclin kinase remained in G1 arrested and as in TGF-β-treated and controls for p27 is to maintain G1 arrest by TGF-β in these tumor-derived lines not in finite lifespan or mammary epithelial The of antisense and cells to the cell cycle was not by of the with led to cell cycle not early passage and passage in responsiveness to TGF-β M. Stampfer M.R. In Cell Dev. Biol. PubMed Scopus Google Scholar), the ASp27 transfection with and passage of G1 arrest in the presence of TGF-β. The of the malignant tumor-derived lines to maintain after ASp27 was to have by in the TGF-β sensitivity of the cell The finite lifespan and malignant lines TGF-β as the and malignant cells and cells arrest by TGF-β after p27 mechanisms contributing to of TGF-β arrest in and despite the in p27 the levels of and p130 bound to cyclin in TGF-β-treated In cell types, ASp27 treatment reduced the levels of p27 in cyclin complexes for and in In the the reduction in cyclin p27 was associated with a in cyclin p21 and a in the of p130 in the cyclin complexes TGF-β-treated also showed increased cyclin p130 and p21 after loss of p27 and the G1 arrest In the cancer-derived and was increased p130 association with cyclin cyclin p21 in cells with the and cells and ASp27 led to cell cycle for of sensitivity to the growth inhibitory of TGF-β is in human tumor-derived cell lines and is to contribute to malignant tumor progression M. Rev. 1993; Google Scholar). Although increased p27 and TGF-β resistance have to early in previous work has not a between p27 deregulation and loss of G1 arrest by TGF-β during progression. the of the increased mice with the of in and cell cycle in mice that compensation by other cell cycle regulators may in the of p27 (11Koff A. Ohtsuki M. Polyak K. Roberts J.M. Massague J. Science. 1993; 260: 536-539Crossref PubMed Scopus (509) Google Scholar, K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar, M.L. M. M. P. Polyak K. Firpo E. L. V. R.M. K. Roberts J.M. Cell. 1996; 85: Full Text Full Text PDF PubMed Scopus Google Scholar, M. D. A. Cell. 1996; 85: Full Text Full Text PDF PubMed Scopus Google Scholar). mouse embryonic fibroblasts (MEFs) mice retain sensitivity to many growth inhibitory including TGF-β (14Nakayama K. Ishida N. Shirane M. Inomata A. Inoue T. Shishido N. Horii I. Loh D.Y. Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar). The that p27 is an essential of G1 arrest by TGF-β in two malignant the cancer cell and the inhibition of p27 led to cyclin E1-Cdk2 and cell cycle p27 was not essential for G1 arrest by TGF-β in two cell types, the finite lifespan and the In these cells, a compensatory in binding of and p130Rb2 to cyclin E1-Cdk2 complexes to contribute to of the G1 number of studies or cancer-derived cell lines support the that p27 loss or deregulation is associated with impaired TGF-β arrest of the kinase in human cells and cells led to the degradation of which was associated with TGF-β resistance T. C. M. J. 2000; PubMed Google Scholar). activation led to cytoplasmic mislocalization of p27 and to TGF-β resistance in epithelial cell lines X. T. 2000; PubMed Scopus Google Scholar). In the and in this that overexpression of protein kinase TGF-β in through protein kinase of leading to its cytoplasmic mislocalization J. J. T. Kotchetkov R. M. K. Lee J.H. S. E. Slingerland J. 2002; PubMed Scopus Google Scholar). overexpression in mink lung epithelial cells TGF-β and these cells failed to p27 in cyclin E1-Cdk2 complexes in response to TGF-β A. R. P. T. M. Nature. 1996; PubMed Scopus Google Scholar). In the observed in p27 regulation in the cancer-derived lines with two human mammary epithelial cell types, and cancer-derived a of p27 in cyclin In cyclin E1-Cdk2 complexes from cancer-derived lines kinase despite cyclin p27 and that the Cdk inhibitory activity of the may be impaired in and In the of a loss of p27 via antisense have a compensatory by p21 may be The p130Rb2 protein may an compensatory role in of after p27 loss in several cell types, including epithelial cells, as and −/− the accumulation of p130 in cyclin E-Cdk2 complexes compensated for the of p27 and to Cdk2 inhibition and G1 arrest after inhibition or (18Coats S. White P. Fero M.L. Lacy S. Chung G. Randel E. Firpo E. Roberts J.M. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, M. I. M.L. M. J. C. M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). studies support a role for p130 in the proliferative arrest by TGF-β. T. D. P. 1995; Google Scholar) showed an accumulation of p130 in complexes and of regulated during TGF-β arrest of human p130 in cyclin E1-Cdk2 complexes from and TGF-β-treated In cells TGF-β treatment a in the levels of cyclin despite p130 protein levels in the two malignant tumor-derived lines in the cells, the levels of p130 bound to cyclin in these cancer-derived lines in the mechanisms that regulate p130 binding to cyclin E1-Cdk2 complexes may between the and cancer-derived lines. p130 deregulation has and may have in several of human T. A. G. D. S. L. G. A. 1998; PubMed Scopus Google Scholar, N. K. T. S. T. G. 2001; PubMed Scopus Google Scholar, M. A. A. A. Cancer Res. 1999; Google Scholar). p130 regulation has in the of altered p27 regulation and may contribute to loss of responses to antiproliferative For example, the protein bind and p27 and and overexpression leads to TGF-β resistance A. R. P. T. M. Nature. 1996; PubMed Scopus Google Scholar). In p27 which p130 levels −/− to to after (18Coats S. White P. Fero M.L. Lacy S. Chung G. Randel E. Firpo E. Roberts J.M. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). deregulation of p130 and p27 lead to a loss of normal proliferative during tumor progression. studies may p130 deregulation for poor patient show reduced Our support the that deregulation of multiple G1 cell cycle regulators may be cells responsiveness to antiproliferative effects of TGF-β. Deregulation of several G1 regulators may ultimately be p27 essential for G1 arrest by TGF-β. p15 has to cooperate with p27 in G1 arrest by TGF-β (8Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (885) Google Scholar). p15 is not for G1 arrest by TGF-β (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar) and Bhattacharya N. M.R. Slingerland J.M. 1996; 13: Google Scholar) retain TGF-β responsiveness despite a of p15 p15 loss not cells on p27 for G1 Although and are ASp27 TGF-β arrest in the cells and not the cell cycle inhibitory pathways by TGF-β in that for loss not to be in of these may be the compensatory in p130 binding to The of p130 and p15 regulation in may alter the role of p27 from a redundant to essential of G1 arrest by TGF-β. c-Myc plays an role in the regulation of many cycle including cyclin and c-Myc overexpression causes TGF-β resistance K. J. 1998; PubMed Scopus Google Scholar). In the c-Myc levels may contribute to the reduced of p15 by TGF-β. c-Myc may also be to the impaired role of p130 J.M. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, C. Cell 1999; Google Scholar) and contribute to the increased of Cdc25A (7Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Crossref PubMed Scopus (641) Google Scholar) in and The increased Cdc25A levels in and may contribute to the increased cyclin E1-Cdk2 observed in these lines. Cdc25A down-regulation to G1 arrest by TGF-β (6Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar). Although TGF-β reduced Cdc25A levels in of the cell types, and Cdc25A levels after of TGF-β treatment and The levels of the Cdk2 Cdc25A in the TGF-β arrested cancer cells may susceptible to cyclin E1-Cdk2 activation after p27 P. S. G. M. Pagano M. M. J. 2000; PubMed Scopus Google Scholar) have increased in cancer Cdc25A and In was a between Cdk2 activity and Cdc25A in the Cdc25A and activity oppose the cyclin E1-Cdk2 inhibitory of In with Cdc25A overexpression, of p27 and may for continued responsiveness to such antiproliferative as TGF-β. In support the that a reduction in p27 levels may contribute to the loss of normal responsiveness to growth inhibitory during cancer progression. the reduction in p27 levels may be to cell cycle arrest responses other inhibitory mechanisms are Our antisense that normal mammary epithelial cells maintain antiproliferative responses in through activation of the inhibitory of p21 and p130 p27 levels are of these and other normal checkpoint controls during malignant progression may p27 essential for by TGF-β. M. M. and for the and cells, and W. M. and for the p27

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