Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Estrogens Down-regulate p27 in Breast Cancer Cells through Skp2 and through Nuclear Export Mediated by the ERK Pathway

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m302830200

ISSN

1083-351X

Autores

James S. Foster, Romaine I. Fernando, N. Ishida, Keiichi I. Nakayama, Jay Wimalasena,

Tópico(s)

DNA Repair Mechanisms

Resumo

The cyclin-dependent kinase (CDK) inhibitor p27 Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27 Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27 Kip1 in down-regulation of p27 Kip1 induced in MCF-7 cells by estrogens. 17β-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16 Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27 Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27 Kip1 expression. Under conditions of G1 blockade, p27 Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27 Kip1 (Ser10 → Ala; S10A) interfering with CRM1/p27 Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1–452) mutant prevented down-regulation of p27 Kip1 S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27 Kip1 in antiestrogen-treated cells and prevented accumulation of p27 Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27 Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27 Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27 Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway. The cyclin-dependent kinase (CDK) inhibitor p27 Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27 Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27 Kip1 in down-regulation of p27 Kip1 induced in MCF-7 cells by estrogens. 17β-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16 Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27 Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27 Kip1 expression. Under conditions of G1 blockade, p27 Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27 Kip1 (Ser10 → Ala; S10A) interfering with CRM1/p27 Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1–452) mutant prevented down-regulation of p27 Kip1 S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27 Kip1 in antiestrogen-treated cells and prevented accumulation of p27 Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27 Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27 Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27 Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway. Estrogenic steroids are essential for normal development and function of female reproductive tissues yet play a pivotal, causative role in the initiation and progression of breast cancer (1Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar, 2Colditz G.A. J. Natl. Cancer Inst. 1998; 90: 814-823Crossref PubMed Scopus (352) Google Scholar). Estrogens and antiestrogens exert growth regulatory actions in normal and malignant breast epithelial cells through regulation of events in the G1 phase of the cell cycle (for review, see Ref. 3Foster J.S. Henley D.C. Ahamed S. Wimalasena J. Trends Endocrinol. Metab. 2001; 12: 320-327Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). p27 Kip1 is a key element of the G1 phase regulatory apparatus. Removal of cyclin-dependent kinase (CDK) 1The abbreviations used are: CDK, cyclin-dependent kinase; BrdUrd, bromodeoxyuridine; E2, 17β-estradiol; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; Kip, kinase inhibitory protein; LMB, leptomycin B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pfu, plaque-forming units; SCF, Skp1/Cul-1/F box protein. inhibitory activity associated with p21 Cip1 and p27 Kip1 in G1 phase is essential to cyclin E-CDK2 activation and S phase entry (for review, see Refs. 4Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3227) Google Scholar and 5Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5220) Google Scholar). Along with other members of the kinase inhibitory protein (Kip) family (p21 Cip1 and p57Kip2) p27 Kip1 serves both as an inhibitor of CDK2 activity in G0 and early G1 and as an assembly factor for cyclin D-CDK4/6 complexes in early G1 (for review, see Ref. 5Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5220) Google Scholar). Functional expression of p27 Kip1 is required for morphogenesis and normal proliferative responses in the mammary epithelium (6Muraoka R.S. Lenferink A.E. Simpson J. Brantley D.M. Roebuck L.R. Yakes F.M. Arteaga C.L. J. Cell Biol. 2001; 153: 917-932Crossref PubMed Scopus (66) Google Scholar). Mammary glands of p27 Kip1 (–/–) mice are underdeveloped compared with the wild-type, whereas the mammary epithelium of p27 Kip1 (+/–) mice is hyperproliferative and susceptible to oncogene-induced tumorigenesis (6Muraoka R.S. Lenferink A.E. Simpson J. Brantley D.M. Roebuck L.R. Yakes F.M. Arteaga C.L. J. Cell Biol. 2001; 153: 917-932Crossref PubMed Scopus (66) Google Scholar, 7Muraoka R.S. Lenferink A.E. Law B. Hamilton E. Brantley D.M. Roebuck L.R. Arteaga C.L. Mol. Cell. Biol. 2002; 22: 2204-2219Crossref PubMed Scopus (105) Google Scholar). Breast cancer isolates frequently exhibit decreased p27 Kip1 expression in addition to elevated expression of cyclin E (8Nielsen N.H. Emdin S.O. Cajander J. Landberg G. Oncogene. 1997; 14: 295-304Crossref PubMed Scopus (143) Google Scholar, 9Nielsen N.H. Arnerlov C. Emdin S.O. Landberg G. Br. J. Cancer. 1996; 74: 874-880Crossref PubMed Scopus (170) Google Scholar, 10Catzavelos C. Bhattacharya N. Ung Y.C. Wilson J.A. Roncari L. Sandhu C. Shaw P. Yeger H. Morava-Protzner I. Kapusta L. Franssen E. Pritchard K.I. Slingerland J.M. Nat. Med. 1997; 3: 227-230Crossref PubMed Scopus (795) Google Scholar, 11Porter P.L. Malone K.E. Heagerty P.J. Alexander G.M. Gatti L.A. Firpo E.J. Daling J.R. Roberts J.M. Nat. Med. 1997; 3: 222-225Crossref PubMed Scopus (850) Google Scholar, 12Tan P. Cady B. Wanner M. Worland P. Cukor B. Magi-Galluzzi C. Lavin P. Draetta G. Pagano M. Loda M. Cancer Res. 1997; 57: 1259-1263PubMed Google Scholar). High levels of cyclin E-CDK2 activity in these tumors correlate with a high cyclin E/low p27 Kip1 phenotype (13Loden M. Nielsen N.H. Roos G. Emdin S.O. Landberg G. Oncogene. 1999; 18: 2557-2566Crossref PubMed Scopus (45) Google Scholar) as does poor disease outcome. Expression of p27 Kip1 is an independent prognostic indicator for breast carcinomas and for a range of other tumor types (for review, see Ref. 14Slingerland J. Pagano M. J. Cell. Physiol. 2000; 183: 10-17Crossref PubMed Scopus (640) Google Scholar). Estrogens elicit active cyclin E-CDK2, and G1/S transition in estrogen receptor-positive MCF-7 breast cancer cells corresponding with decreasing levels of p27 Kip1 protein (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) and a decrease in CDK inhibitory activity associated with both p27 Kip1 and p21 Cip1 (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 17Planas-Silva M.D. Weinberg R.A. Mol. Cell. Biol. 1997; 17: 4059-4069Crossref PubMed Scopus (238) Google Scholar). Levels of p21 Cip1 and p27 Kip1 increase in MCF-7 cells after treatment of proliferating cells with antiestrogen, resulting in decreased CDK2 activity and growth arrest (18Watts C.K. Brady A. Sarcevic B. deFazio A. Musgrove E.A. Sutherland R.L. Mol. Endocrinol. 1995; 9: 1804-1813Crossref PubMed Scopus (153) Google Scholar). Inhibition of expression of either p21 Cip1 or p27 Kip1 with antisense oligonucleotides maintains active CDK2 and prevents antiestrogen-mediated G1 blockade (19Cariou S. Donovan J.C. Flanagan W.M. Milic A. Bhattacharya N. Slingerland J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9042-9046Crossref PubMed Scopus (207) Google Scholar, 20Carroll J.S. Prall O.W. Musgrove E.A. Sutherland R.L. J. Biol. Chem. 2000; 275: 38221-38229Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). p27 Kip1 levels are regulated in distinct fashion at different points in the cell cycle. After mitogenic stimulation of quiescent cells p27 Kip1 levels fall beginning in mid-G1, primarily mediated through regulation of protein stability by ubiquitin/proteasome-mediated proteolysis (Ref. 21Pagano 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 (1742) Google Scholar; for review see Ref. 14Slingerland J. Pagano M. J. Cell. Physiol. 2000; 183: 10-17Crossref PubMed Scopus (640) Google Scholar). p27 Kip1 protein expression is also regulated at the level of transcription (22Servant M.J. Coulombe P. Turgeon B. Meloche S. J. Cell Biol. 2000; 148: 543-556Crossref PubMed Scopus (124) Google Scholar), translation (23Hengst L. Reed S.I. Science. 1996; 271: 1861-1864Crossref PubMed Scopus (826) Google Scholar, 24Agrawal D. Hauser P. McPherson F. Dong F. Garcia A. Pledger W.J. Mol. Cell. Biol. 1996; 16: 4327-4336Crossref PubMed Scopus (162) Google Scholar), as well as through proteolytic processing independent of ubiquitination (25Shirane M. Harumiya Y. Ishida N. Hirai A. Miyamoto C. Hatakeyama S. Nakayama K. Kitagawa M. J. Biol. Chem. 1999; 274: 13886-13893Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) and independent of the proteasome (26Delmas C. Aragou N. Poussard S. Cottin P. Darbon J.M. Manenti S. J. Biol. Chem. 2003; 278: 12443-12451Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Patel Y.M. Lane M.D. J. Biol. Chem. 2000; 275: 17653-17660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Early in G1 inhibitory activity of p21 Cip1 and p27 Kip1 toward CDK2 is removed by sequestration of the proteins into newly formed cyclin D-CDK4/6 complexes (for review, see Refs. 4Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3227) Google Scholar and 5Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5220) Google Scholar). CDK2 activation in MCF-7 cells corresponds both with p21 Cip1 /p27 Kip1 sequestration in early G1 (16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 17Planas-Silva M.D. Weinberg R.A. Mol. Cell. Biol. 1997; 17: 4059-4069Crossref PubMed Scopus (238) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar, 29Ahamed S. Foster J.S. Bukovsky A. Diehl J.A. Wimalasena J. Mol. Carcinogen. 2002; 34: 45-58Crossref PubMed Scopus (10) Google Scholar) and with decreased p21 Cip1 /p27 Kip1 protein levels in mid- to late G1 (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 19Cariou S. Donovan J.C. Flanagan W.M. Milic A. Bhattacharya N. Slingerland J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9042-9046Crossref PubMed Scopus (207) Google Scholar), suggesting removal of CDK inhibitory activity in two phases after estrogen stimulation. Our recent studies indicate that the later phase of CDK inhibitor removal in MCF-7 cells requires the proteasome and proceeds in the absence of cyclin D-CDK4 complex formation or G1 transit (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). Further observations also indicated that down-regulation of p27 Kip1 expression in MCF-7 cells also corresponds with increased expression of Skp2 (29Ahamed S. Foster J.S. Bukovsky A. Diehl J.A. Wimalasena J. Mol. Carcinogen. 2002; 34: 45-58Crossref PubMed Scopus (10) Google Scholar), the F box component of the Skp1/Cul-1/F box protein (SCF) ubiquitin-protein ligase which binds p27 Kip1 phosphorylated on Thr187 allowing its ubiquitination and destruction in the proteasome (30Zhang H. Kobayashi R. Galaktionov K. Beach D. Cell. 1995; 82: 915-925Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 31Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1353) Google Scholar, 32Sutterluty H. Chatelain E. Marti A. Wirbelauer C. Senften M. Muller U. Krek W. Nat. Cell Biol. 1999; 1: 207-214Crossref PubMed Scopus (632) Google Scholar, 33Tsvetkov 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 (694) Google Scholar). Skp2 exhibits oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (34Signoretti S. Di Marcotullio L. Richardson A. Ramaswamy S. Isaac B. Rue M. Monti F. Loda M. Pagano M. J. Clin. Invest. 2002; 110: 633-641Crossref PubMed Scopus (258) Google Scholar). Recent studies also indicate that p27 Kip1 ubiquitination can proceed in the absence of Skp2 (35Hara T. Kamura T. Nakayama K. Oshikawa K. Hatakeyama S. Nakayama K.I. J. Biol. Chem. 2001; 276: 48937-48943Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). In the studies described herein we examined p27 Kip1 down-regulation after estrogen stimulation of MCF-7 cells in detail with respect to the role of Skp2, as well as Skp2-independent mechanisms contributing to p27 Kip1 down-regulation. The results suggest that Skp2 participates in ubiquitination/degradation of p27 Kip1 in the nucleus of estrogen-treated MCF-7 cells and further demonstrate that Skp2 overexpression prevents antiestrogen-mediated growth arrest of MCF-7 cells by down-regulating p27 Kip1 expression and maintaining CDK2 activity. In contrast, estrogen-induced down-regulation of p27 Kip1 proceeds in the absence of any increase in Skp2 expression in MCF-7 cells subjected to G1 blockade with p16 Ink4a or roscovitine and appears to depend in this context upon export of p27 Kip1 to the cytoplasm. Our studies also indicate that this process requires phosphorylation of p27 Kip1 on Ser10 as well as participation of the Ras/Raf-1/extracellular signal-regulated kinase (ERK) signaling pathway at a stage beyond phosphorylation of p27 Kip1 on this residue. In addition, enforced activation of the Ras/Raf-1/ERK pathway elicited cytoplasmic relocalization of p27 Kip1 and prevented growth arrest by antiestrogen. Reagents and Antibodies—Cell culture media and antibiotics, 17β-estradiol (E2), histones, fetal bovine serum (FBS), glutathione-agarose beads, RNase A, propidium iodide, monoclonal (M2) and rabbit polyclonal anti-FLAG antibodies, and other chemicals were from Sigma Chemical Co. ICI 182,780 was supplied by Dr. Alan Wakeling at Zeneca Pharmaceuticals (Alderly Park, Cheshire, UK). Protein A/G beads, antibodies to CDK2 (M2), p27 Kip1 (C19), p21 Cip1 (C19), Skp2 (H435), Cul-1 (H213), ERK2 (C14), Cks-1 (FL79), and Skp1 (H6) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-actin, and anti-hemagglutinin epitope antibodies were from Roche Applied Science. Monoclonal antibody to p27 Kip1 was from BD Biosciences, and antibodies to green fluorescent protein (GFP) and E2F-1 were from Labvision/Neomarkers (Freemont, CA). Antibodies to active mitogen-activated protein kinases (MAPK) were from Promega (Madison, WI). [γ-32P]ATP, and Tran35S-label were from ICN (Irvine, CA). Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Fluorochrome-labeled secondary antibodies to mouse and rabbit IgG (Alexa Fluor 350 and 488) were from Molecular Probes (Eugene, OR). MG-132, Myc epitope antibodies (clone 9E10), PD98059, and roscovitine were from Calbiochem. Leptomycin B (LMB) was provided by M. Yoshida (36Kudo N. Wolff B. Sekimoto T. Schreiner E.P. Yoneda Y. Yanagida M. Horinouchi S. Yoshida M. Exp. Cell Res. 1998; 242: 540-547Crossref PubMed Scopus (715) Google Scholar). Cell Culture, Viral Vectors, and Transfections—MCF-7 cells were a gift from R. P. Shiu (37Dubik D. Shiu R.P. J. Biol. Chem. 1988; 263: 12705-12708Abstract Full Text PDF PubMed Google Scholar) and were maintained as described previously (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). MCF-7/tTA (Tet-off) cells were derived by transfection with pTet-off (Clontech). Cells were growth arrested by 2–3 days of culture in phenol red-free Dulbecco's modified Eagle's medium with 0.1% FBS and 20 nm ICI 182,780 as described previously (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar) (16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Chemical stock solutions were prepared in ethanol (17β-estradiol, ICI 182,780, LMB) or dimethyl sulfoxide (PD98059, roscovitine), and control cultures received equal amounts of solvents as vehicle controls where appropriate. Plasmid vectors for p16 Ink4a (pBI-p16) and cyclin E (pMTcycE) were provided by J. Lukas (38Santoni-Rugiu E. Falck J. Mailand N. Bartek J. Lukas J. Mol. Cell. Biol. 2000; 20: 3497-3509Crossref PubMed Scopus (150) Google Scholar), and vector for constitutively active ERK kinase (MEK, EE mutant) was provided by M. Cobb. Plasmids for Skp2 and FLAG-tagged p27 Kip1 (wild-type, and S10A/S10E mutants) have been described previously (39Nakayama K. Nagahama H. Minamishima Y.A. Matsumoto M. Nakamichi I. Kitagawa K. Shirane M. Tsunematsu R. Tsukiyama T. Ishida N. Kitagawa M. Nakayama K. Hatakeyama S. EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (640) Google Scholar, 40Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 41Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Plasmid vector for human Skp2 was provided by M. Pagano (31Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1353) Google Scholar). The dominant-negative ERK2 and RasN17 plasmids were provided by G. L. Johnson (42Knall C. Young S. Nick J.A. Buhl A.M. Worthen G.S. Johnson G.L. J. Biol. Chem. 1996; 271: 2832-2838Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), the Ras V12S35 plasmid was provided by C. M. Counter (43Hamad N.M. Elconin J.H. Karnoub A.E. Bai W. Rich J.N. Abraham R.T. Der C.J. Counter C.M. Genes Dev. 2002; 16: 2045-2057Crossref PubMed Scopus (352) Google Scholar), and the Cul-1(1–452) plasmid was provided by Z. Q. Pan (44Wu K. Fuchs S.Y. Chen A. Tan P. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. Biol. 2000; 20: 1382-1393Crossref PubMed Scopus (95) Google Scholar). The YFPp27 Kip1 expression plasmid was provided by J. Slingerland (45Connor 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 (162) Google Scholar). Transfections were carried out using LipofectAMINE PLUS transfection reagent (Invitrogen). Control and antisense Skp2 oligonucleotides (Sigma/Genosys) based upon a previous publication (31Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1353) Google Scholar) were prepared in partially phosphothiorated form to minimize destruction by cellular nucleases and were transfected into growth-arrested MCF-7 cells using Lipofectin transfection reagent (Invitrogen). The nucleotide sequences of the oligonucleotides were: antisense Skp2, 5′-CCTGGGGGATGTTCTCA-3′; control, 5′-CCGCTCATCGTATGACA-3′. Oligonucleotides were transfected for 3 h at a final concentration of 1 μm with 18 μl of liposome reagent. Replication-defective adenoviral vectors for expression of p16 Ink4a , p27 Kip1 , and control (β-galactosidase) adenovirus have been described previously (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar, 46Craig C. Kim M. Ohri E. Wersto R. Katayose D. Li Z. Choi Y.H. Mudahar B. Srivastava S. Seth P. Cowan K. Oncogene. 1998; 16: 265-272Crossref PubMed Scopus (121) Google Scholar, 47Craig C. Wersto R. Kim M. Ohri E. Li Z. Katayose D. Lee S.J. Trepel J. Cowan K. Seth P. Oncogene. 1997; 14: 2283-2289Crossref PubMed Scopus (195) Google Scholar) as has the adenoviral Skp2 vector (murine Skp2) (39Nakayama K. Nagahama H. Minamishima Y.A. Matsumoto M. Nakamichi I. Kitagawa K. Shirane M. Tsunematsu R. Tsukiyama T. Ishida N. Kitagawa M. Nakayama K. Hatakeyama S. EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (640) Google Scholar). Adenoviral vector for the constitutively active Raf-1caax mutant was provided by J. R. Nevins (48Leone G. DeGregori J. Sears R. Jakoi L. Nevins J.R. Nature. 1997; 387: 422-426Crossref PubMed Scopus (405) Google Scholar). Adenoviruses were propagated in human embryonic kidney 293 cells (ATCC), and viral lysates for use in experiments were titered by a standard plaque assay to determine plaque-forming units (pfu)/ml. Flow Cytometric Analysis—For flow cytometric analysis MCF-7 cells were harvested in saline-EDTA, fixed in cold 70% ethanol, and stored at –20 °C. Fixed cells were subsequently washed, treated with 100 μg/ml RNase A, and stained with 50 μg/ml propidium iodide. Analysis of DNA content was performed in a BD Biosciences FACScan with a minimum of 15,000 events collected for analysis using BD Biosciences Cell Quest software. For analysis of DNA synthesis bromodeoxyuridine (BrdUrd) was added to the medium as described in the text and BrdUrd incorporation measured by flow cytometry using fluorescein-labeled antibodies to BrdUrd (BD/Pharmingen) according to the manufacturer's protocol. Western Blot Analysis and Immune Complex Kinase Assays—Cells were lysed as described previously (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar) in ice-cold Nonidet P-40 lysis buffer (20 mm Tris, pH 7.5, 250 mm NaCl, 0.5% Nonidet P-40, 0.1 mm EDTA, 1 mm NaOV4, 10 mm NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride), with brief sonication and centrifugation to remove cellular debris. Nuclear/cytoplasmic cell fractionation was carried out by lysis in hypotonic buffer with 0.02% digitonin as described previously (41Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Western blots were performed as described in detail previously (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). Equal protein loading was verified for all lysate blots by Ponceau S staining and/or by reprobing for actin expression. In experiments measuring expression of ectopic FLAG-p27 Kip1 by Western blotting transfection was verified by coexpression of GFP (pEGFPN1, Clontech) and analysis by fluorescent microscopy/Western blotting with anti-GFP antibodies. Histone kinase assays were performed with anti-CDK2 immune precipitates as described (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar) using equal amounts of lysate proteins. Metabolic Labeling and Pulse-Chase Analysis of p27Kip1 Stability— MCF-7 cells in 6-well plates were growth arrested and, to facilitate detection of labeled p27 Kip1 , were infected with Adp27 at 5 pfu/cell with or without Adp16, AdSkp2, or control adenovirus as required. 12 h after infection cells were treated as described, and at appropriate times cultures were labeled for 1 h with [35S]Met/Cys as described previously (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). For the chase, monolayers were washed twice with fresh, complete medium and incubated for the indicated times before lysis in ice-cold Nonidet P-40 lysis buffer as described above. Immunoprecipitations were performed with 1 μg of p27 Kip1 (C19) antibodies and protein A/G-agarose beads and analyzed as described (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). Subcellular Localization of p27Kip1 by Microscopy—MCF-7 cells grown on glass coverslips were transfected with the YFPp27 Kip1 plasmid vector (45Connor 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 (162) Google Scholar) or FLAG-p27 Kip1 vectors (40Ishida N. Kitagawa M. Hatakeyama S. Nakayama K. J. Biol. Chem. 2000; 275: 25146-25154Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 41Ishida N. Hara T. Kamura T. Yoshida M. Nakayama K. Nakayama K.I. J. Biol. Chem. 2002; 277: 14355-14358Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) alone or in combination with appropriate vectors as indicated. After treatment the cells were fixed with 3% paraformaldehyde, mounted on slides, and cells with nuclear versus nuclear/cytoplasmic YFPp27 Kip1 were enumerated visually on an Olympus IMT-2 fluorescent microscope. For immunofluorescent localization of FLAG-p27 Kip1 or epitope-tagged cotransfectants, fixed cells were permeabilized with 0.25% Triton X-100 and stained with the appropriate antibodies and fluorochrome-labeled secondary antibodies. For quantification, a minimum of 400 cells were counted for each determination, and results were derived from at least three independent experiments. Statistical analysis was performed with Graph-Pad Prism 3.0 software. Skp2 Expression in Response to E2 Is Prevented by G1 Blockade, but p27Kip1 Down-regulation Is Maintained—Estrogen stimulation of G0/G1-arrested MCF-7 cells leads to down-regulation of p27 Kip1 protein expression corresponding with cyclin E-CDK2 activation and preceding entry into S phase (15Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (212) Google Scholar, 16Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). As we reported previously, p27 Kip1 down-regulation in MCF-7 cells is prevented by chemical inhibitors of the proteasome but is not affected in MCF-7 cells transduced with an adenoviral vector for p16 Ink4a despite effective inhibition of cyclin D-CDK complex formation, CDK2 activation, and cell cycle transit (28Foster J.S. Henley D.C. Bukovsky A. Seth P. Wimalasena J. Mol. Cell. Biol. 2001; 21: 794-810Crossref PubMed Scopus (156) Google Scholar). Degradation of p27 Kip1 via the ubiquitin-proteasome pathway is facilitated by interaction with the F box protein Skp2, which in conjunction with CKS1 binds p27 Kip1 phosphorylated on Thr187 by CDK2 and mediates interactions with the SCF ubiquitin-protein ligase (30Zhang H.

Referência(s)