FoxM1c Counteracts Oxidative Stress-induced Senescence and Stimulates Bmi-1 Expression
2008; Elsevier BV; Volume: 283; Issue: 24 Linguagem: Inglês
10.1074/jbc.m709604200
ISSN1083-351X
AutoresSamuel K.M. Li, David K. Smith, Wai Ying Leung, Alice M.S. Cheung, Eric W.‐F. Lam, Goberdhan P. Dimri, Kwok‐Ming Yao,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoThe Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether FoxM1 can protect cells against stress-induced premature senescence, we established NIH3T3 lines with doxycycline-inducible overexpression of FoxM1c. Treatment of these lines with sublethal doses (20 and 100 μm) of H2O2 induced senescence with senescence-associated β-galactosidase expression and elevated levels of p53 and p21. Induction of FoxM1c expression markedly suppressed senescence and expression of p53 and p21. Consistent with down-regulation of the p19Arf-p53 pathway, p19Arf levels decreased while expression of the Polycomb group protein Bmi-1 was induced. That Bmi-1 is a downstream target of FoxM1c was further supported by the dose-dependent induction of Bmi-1 by FoxM1c at both the protein and mRNA levels, and FoxM1 and Bmi-1 reached maximal levels in cells at the G2/M phase. Depletion of FoxM1 by RNA interference decreased Bmi-1 expression. Using Bmi-1 promoter reporters with wild-type and mutated c-Myc binding sites and short hairpin RNAs targeting c-Myc, we further demonstrated that FoxM1c activated Bmi-1 expression via c-Myc, which was recently reported to be regulated by FoxM1c. Our results reveal a functional link between FoxM1c, c-Myc, and Bmi-1, which are major regulators of tumorigenesis. This link has important implications for the regulation of cell proliferation and senescence by FoxM1 and Bmi-1. The Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether FoxM1 can protect cells against stress-induced premature senescence, we established NIH3T3 lines with doxycycline-inducible overexpression of FoxM1c. Treatment of these lines with sublethal doses (20 and 100 μm) of H2O2 induced senescence with senescence-associated β-galactosidase expression and elevated levels of p53 and p21. Induction of FoxM1c expression markedly suppressed senescence and expression of p53 and p21. Consistent with down-regulation of the p19Arf-p53 pathway, p19Arf levels decreased while expression of the Polycomb group protein Bmi-1 was induced. That Bmi-1 is a downstream target of FoxM1c was further supported by the dose-dependent induction of Bmi-1 by FoxM1c at both the protein and mRNA levels, and FoxM1 and Bmi-1 reached maximal levels in cells at the G2/M phase. Depletion of FoxM1 by RNA interference decreased Bmi-1 expression. Using Bmi-1 promoter reporters with wild-type and mutated c-Myc binding sites and short hairpin RNAs targeting c-Myc, we further demonstrated that FoxM1c activated Bmi-1 expression via c-Myc, which was recently reported to be regulated by FoxM1c. Our results reveal a functional link between FoxM1c, c-Myc, and Bmi-1, which are major regulators of tumorigenesis. This link has important implications for the regulation of cell proliferation and senescence by FoxM1 and Bmi-1. Forkhead box (Fox) M1, known previously as WIN, HFH-11, and Trident, is a transcription factor ubiquitously expressed in proliferating cells (1Yao K.M. Sha M. Lu Z. Wong G.G. J. Biol. Chem. 1997; 272: 19827-19836Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 2Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (309) Google Scholar, 3Korver W. 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Reverse transcription PCR (RT-PCR) 2The abbreviations used are: RT-PCR, reverse transcription PCR; MEF, mouse embryonic fibroblast; SA, senescence-associated; SIPS, stress-induced premature senescence; shRNA, short hairpin RNA; dox, doxycycline; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoside; tet, tetracycline. 2The abbreviations used are: RT-PCR, reverse transcription PCR; MEF, mouse embryonic fibroblast; SA, senescence-associated; SIPS, stress-induced premature senescence; shRNA, short hairpin RNA; dox, doxycycline; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoside; tet, tetracycline. and RNase protection analyses indicated that FoxM1c is the predominant form expressed in various primary and secondary cell lines (e.g. human BJ1 and mouse NIH3T3 cells) and neonatal tissues rich in mitotically active cells. FoxM1b is the major isoform expressed in skin and testis (1Yao K.M. Sha M. Lu Z. Wong G.G. J. Biol. 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Huang S. Oncogene. 2007; 26: 6212-6219Crossref PubMed Scopus (157) Google Scholar). In HeLa cells, elevated levels of FoxM1c were found to accelerate transition through the G2/M phase of the cell cycle (6Leung T.W.C. Lin S.S.W. Tsang A.C.C. Tong C.S.W. Ching J.C.Y. Leung W.Y. Gimlich R. Wong G.G. Yao K.M. FEBS Lett. 2001; 507: 59-66Crossref PubMed Scopus (141) Google Scholar). The effect of overexpression of FoxM1c in mice has not been reported. FoxM1 levels display cell cycle phase-dependent changes; its expression is initiated just before entry into S phase and peaks at the G2/M phase of the cell cycle (3Korver W. Roose J. Clevers H. Nucleic Acids Res. 1997; 25: 1715-1719Crossref PubMed Scopus (207) Google Scholar, 6Leung T.W.C. Lin S.S.W. Tsang A.C.C. Tong C.S.W. Ching J.C.Y. Leung W.Y. Gimlich R. Wong G.G. Yao K.M. FEBS Lett. 2001; 507: 59-66Crossref PubMed Scopus (141) Google Scholar). Consistent with the predominant G2/M expression of FoxM1, loss-of-function analyses in FoxM1-/- mouse embryonic fibroblasts (MEFs) and in cancer cell lines by RNA interference have established a requirement for FoxM1 in mitosis (17Laoukili J. Kooistra M.R. Bras A. Kauw J. Kerkhoven R.M. Morrison A. Clevers H. Medema R.H. Nat. Cell Biol. 2005; 7: 126-136Crossref PubMed Scopus (624) Google Scholar, 18Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (495) Google Scholar, 19Wonsey D.R. Follettie M.T. Cancer Res. 2005; 65: 5181-5189Crossref PubMed Scopus (282) Google Scholar). FoxM1-depleted cells have difficulty executing mitosis and exhibit chromosomal instability and polyploidy. Further microarray and chromatin immunoprecipitation analyses revealed important G2/M-specific genes like cyclin B1, Cdc25B, CENPA, and Aurora B as direct targets of FoxM1 (17Laoukili J. Kooistra M.R. Bras A. 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Cancer Res. 2006; 66: 2153-2161Crossref PubMed Scopus (285) Google Scholar, 26Yoshida Y. Wang I.C. Yoder H.M. Davidson N.O. Costa R.H. Gastroenterology. 2007; 132: 1420-1431Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Besides exhibiting cell cycle arrest, FoxM1-/- MEFs show increased expression of p19Arf and senescence-associated (SA) β-galactosidase, which are typical markers of senescent cells (18Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (495) Google Scholar, 27Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5651) Google Scholar). The occurrence of senescence in FoxM1-deficient cells could be an indirect consequence of increased DNA damage (28Tan Y. Raychaudhuri P. Costa R.H. Mol. Cell. Biol. 2007; 27: 1007-1016Crossref PubMed Scopus (191) Google Scholar). Alternatively, FoxM1 may have a direct protective role against senescence that is lost in FoxM1-/- MEFs. To address whether FoxM1 can protect normal MEFs from stress-induced premature senescence (SIPS), we established doxycycline-inducible NIH3T3 lines to test the ability of elevated FoxM1c levels to protect against H2O2-induced premature senescence. Induction of FoxM1c expression dramatically suppressed SIPS and expression of p53 and p21Cip1. Consistent with a down-regulation of p53, expression of p19Arf, a well known positive regulator of p53 stability (29Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar, 30Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. 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Generation of Doxycycline-inducible FoxM1c-expressing NIH3T3 Cells—The NIH3T3 mouse fibroblastic cell line was purchased from The American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% bovine serum (Invitrogen). The pcDNA6/TR vector (Invitrogen), which expresses the tetracycline (tet) repressor under the regulation of the human cytomegalovirus promoter, was stably transfected into NIH3T3 cells using Lipofectamine™ 2000 (Invitrogen) to establish lines constitutively expressing the tet repressor. These lines were tested for tet repressor function by introducing the reporter construct pcDNA™4/TO-Luc by transient transfection. pcDNA™4/TO-Luc contains a luciferase gene under the control of a modified cytomegalovirus promoter carrying tet operator sequences, and its expression is therefore dox-inducible. One line (3T3/6TR) was selected based on strong inducibility of luciferase activity. The expression plasmid pcDNA™4/TO-FoxM1c was generated by replacing the Luc coding sequence with full-length FoxM1c coding sequence as reported previously (33Madureira P.A. Varshochi R. Constantinidou D. Francis R.E. Coombes C. Yao K.M. Lam E.W.F. J. Biol. Chem. 2006; 281: 25167-25176Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). pcDNA™4/TO-FoxM1c was then transfected into the 3T3/6TR line that expresses the tet repressor. Stable transfectants were selected and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% Tet-On™ approved fetal bovine serum (Clontech) and in the presence of 4 μg/ml blasticidin (Invitrogen) and 200 μg/ml zeocin (Invitrogen) as described by the manufacturer. Immunoblotting—Immunoblotting was performed as described previously (7Ma R.Y.M. Tong T.H.K. Cheung A.M.S. Tsang A.C.C. Leung W.Y. Yao K.M. J. Cell Sci. 2005; 118: 795-806Crossref PubMed Scopus (171) Google Scholar). Protein concentration was measured using the Bio-Rad protein assay kit according to the manufacturer's instruction. Protein samples were separated by SDS-PAGE in 7–12% Laemmli gels and electroblotted onto Hybond™-C Super nitrocellulose membranes (Amersham Biosciences). Blots were incubated with anti-FoxM1 antiserum (1:200, MPP2-C20 from Santa Cruz Biotechnology), anti-Cdc25B antiserum (1:200, H-85 from Santa Cruz), anti-cyclin B1 antiserum (1:200, GNS1 from Santa Cruz), anti-α-tubulin antiserum (1:500, DM1A from NeoMarker), anti-p53 (1:500, Pab1801 from Santa Cruz), anti-p21 (1:500, F5 from Santa Cruz), anti-p16 (1:500, F12 from Santa Cruz), anti-p19Arf antiserum (1:500, 54–57 from Calbiochem), anti-Bmi-1 antiserum (1:500, F6 from Upstate Biotechnology), or anti-c-Myc antiserum (1:500, C-33 from Santa Cruz). Senescence-associated β-Galactosidase Staining and Flow Cytometry—Phosphate-buffered saline-washed cells were fixed for 5 min (room temperature) in 2% formaldehyde with 0.2% glutaraldehyde. After washing with phosphate-buffered saline, cells were incubated at 37 °C for 12 h with fresh SA β-galactosidase stain solution (1 mg of X-gal/ml, 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, 150 mm NaCl, 2 mm MgCl2, 40 mm citric acid in phosphate-buffered saline, pH 6.0). DNA analysis by flow cytometry was performed as described previously (7Ma R.Y.M. Tong T.H.K. Cheung A.M.S. Tsang A.C.C. Leung W.Y. Yao K.M. J. Cell Sci. 2005; 118: 795-806Crossref PubMed Scopus (171) Google Scholar). RT-PCR—Total RNA was extracted from samples of ∼1 × 106 cells using TRIzol® reagent (Invitrogen). The quantity and quality of RNA samples were measured by absorbance at 260 and 280 nm. RNA samples with an A260:A280 ratio between 1.90 and 2.10 were stored at -80 °C until use. For synthesis of first-strand cDNAs, 2 μg of total RNA was incubated at 65 °C for 5 min and then chilled on ice immediately. The heat-denatured total RNA was used to perform the reverse transcription reaction using SuperScript™ III reverse transcriptase (Invitrogen) and oligo(dT)20 primer according to the manufacturer's instructions. For a typical semiquantitative RT-PCR reaction, 30 μl of master mix containing 1× reaction buffer IV, 0.2 mm of each dNTPs, 1.25 units of DyNAzyme™ II DNA polymerase (Finnzymes), and 1× PCR buffer for DyNAzyme™ II DNA polymerase was added into 20 μl of boiled RT sample containing 0.1 μg of total RNA. PCR was performed by initial denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. Mouse Bmi-1 and glyceraldehyde-3-phosphate dehydrogenase cDNAs were amplified using the following primers and cycle numbers: Bmi-1, 5′-agcagcaatgactgtgatgca-3′ and 5′-gctctccagcattcgtcagtc-3′, 26 cycles; glyceraldehyde-3-phosphate dehydrogenase, 5′-gaccatggagaaggccgggg-3′ and 5′-gacggacacattgggggtag-3′, 23 cycles. cDNA amounts and cycle numbers were optimized to ensure that amplification was within the linear range for quantitative analysis. Ten microliters of PCR-amplified products were electrophoresed in 1.5% (w/v) agarose gels with 0.25 μg/ml ethidium bromide. Experiments were repeated twice. RNA Interference—A plasmid-based approach by transient transfection using Lipofectamine™ 2000 was employed to deplete FoxM1 and c-Myc expression from different NIH3T3-derived cell lines. Each gene was silenced with two short hairpin RNA (shRNA)-expressing plasmids, and a control plasmid expressing an unrelated shRNA was used as negative control. For silencing FoxM1 expression, pTER A and pTER B were generated by cloning the two complementary hairpin sequences of 5′-accggttcatcctcatcag-3′ and 5′-aagacaggagagctatgct-3′, respectively, into the BglII and HindIII sites of the pTER vector (34van de Wetering M. Oving I. Muncan V. Pon Fong M.T. Brantjes H. van Leenen D. Holstege F.C. Brummelkamp T.R. Agami R. Clevers H. EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (461) Google Scholar). The pLKO.1 plasmids (c-Myc sh1, c-Myc sh2, Bmi-1 sh1, and Bmi-1 sh2 with oligo identification of TRCN0000042513, TRCN0000042514, TRCN0000012563, and TRCN0000012564, respectively) for silencing c-Myc and Bmi-1 expression were purchased from Open Biosystems. Transient Reporter Assays—Inducible NIH3T3 cells (5 × 104/ml) were seeded onto 24-well plates. After 24 h, cells were transiently transfected with the reporter constructs. For a typical transfection, 200 ng of Bmi-1 reporter (pGL3-BmiPrWT, pGL3-BmiPrMut, or pGL3-BmiPrΔMyc for the wild-type, E-box-mutated, or E-box-deleted constructs, respectively) and 4 ng of control reporter (pRL-tk) were mixed with 2 μl of Lipofectamine™ 2000. The next day, transfected cells were treated with dox (4 μg/ml) for 24 h. Thereafter, the Dual-Luciferase reporter assay system (Promega) was used to measure both firefly and Renilla luciferase activities, according to the manufacturer's instructions, using a microplate luminometer LB96V (EG&G Berthold). For co-transfection experiments, 100 ng of Bmi-1 reporter, 4 ng of control reporter, and various amounts of pcDNA3-FoxM1c (7Ma R.Y.M. Tong T.H.K. Cheung A.M.S. Tsang A.C.C. Leung W.Y. Yao K.M. J. Cell Sci. 2005; 118: 795-806Crossref PubMed Scopus (171) Google Scholar) were mixed with 2 μl of Lipofectamine™ 2000, and pcDNA3 was added to normalize the amount of vector across all transfections. All experiments were conducted in triplicate using independent cultures, and the results shown are the mean ± S.E. Establishment of Doxycycline-inducible FoxM1c-expressing NIH3T3 Cell Lines—FoxM1 function is essential for the proper execution of mitosis in MEFs (17Laoukili J. Kooistra M.R. Bras A. Kauw J. Kerkhoven R.M. Morrison A. Clevers H. Medema R.H. Nat. Cell Biol. 2005; 7: 126-136Crossref PubMed Scopus (624) Google Scholar, 18Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (495) Google Scholar). Early passage FoxM1-/- MEFs exhibited premature senescence characterized by increased expression of SA β-galactosidase and p19Arf. This senescent phenotype suggests that FoxM1 expression is required to protect MEFs from senescence. To investigate the ability of FoxM1 in counteracting SIPS in MEFs, we established two NIH3T3 lines (A and B) that exhibited doxycycline (a tetracycline derivative)-inducible FoxM1c expression (Fig. 1). The c instead of the b isoform was investigated because FoxM1c is the predominant isoform expressed in mitotically active cells (7Ma R.Y.M. Tong T.H.K. Cheung A.M.S. Tsang A.C.C. Leung W.Y. Yao K.M. J. Cell Sci. 2005; 118: 795-806Crossref PubMed Scopus (171) Google Scholar). For both lines, treatment with dox from 2 to 8 μg/ml for 24 h gradually induced FoxM1 expression. FoxM1 induction was dose-dependent; stimulation at 8 μg/ml was 8.6- and 7.0-fold over the uninduced levels in lines A and B, respectively (Fig. 1). The uninduced levels in both lines were higher than the basal levels in the parental lines (NIH3T3 with no introduced plasmid and NIH3T3/6TR stably transfected with the tet repressor construct), which required longer exposure time to visualize (data not shown). The functional effect of FoxM1c overexpression was reflected by parallel increases in levels of Cdc25B and cyclin B1 (Fig. 1), both of which are direct transcriptional targets of FoxM1c (7Ma R.Y.M. Tong T.H.K. Cheung A.M.S. Tsang A.C.C. Leung W.Y. Yao K.M. J. Cell Sci. 2005; 118: 795-806Crossref PubMed Scopus (171) Google Scholar, 17Laoukili J. Kooistra M.R. Bras A. Kauw J. Kerkhoven R.M. Morrison A. Clevers H. Medema R.H. Nat. Cell Biol. 2005; 7: 126-136Crossref PubMed Scopus (624) Google Scholar, 18Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (495) Google Scholar).FIGURE 4FoxM1c regulates Bmi-1 expression.A, line A cells induced with dox at the indicated concentrations (0–8 mg/ml) for 24 h were harvested for immunoblot analysis of FoxM1, Bmi-1, and tubulin expression. B, levels of Bmi-1 and glyceraldehyde-3-phosphate dehydrogenase transcripts (Gadph) were determined by semiquantitative RT-PCR analysis. Untreated and dox-treated NIH3T3/6TR cells were analyzed in parallel as controls. C, knock down of FoxM1 expression using shRNAs decreased Bmi-1 expression. pTER plasmids encoding hairpins A and B that target against FoxM1 were separately transfected into NIH3T3 cells and the cell lysates harvested for Western blot analysis of FoxM1, p53, p21Cip, p19Arf, Bmi-1, and tubulin expression. pTERGL3, which targets against firefly luciferase, was included as negative control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2Induced FoxM1c expression counteracts premature senescence induced by H2O2 treatment. Control (NIH3T3/6TR; A, B, G, and H), line A (C–F), and line B (I–L) cells were seeded onto coverslips and untreated (A, B, G, and H) or treated with H2O2 at 20 μm (C, D, I, and J) or 100 μm (E, F, K, and L) for 48 h. Afterward, cells were incubated in H2O2-free medium for 24 h in the absence (A, C, E, G, I, and K) or presence (B, D, F, H, J, and L) of dox (4 μg/ml) before their fixation for assay of senescent cells by SA β-galactosidase staining. The blue senescent cells became larger, flatter, and more vacuolated in appearance. The histograms show the percentage of senescent cells and indicate a clear suppression of stress-induced premature senescence with dox-induced FoxM1c overexpression. The mean ± S.E. of three independent experiments are shown. M and N, DNA analyses of line A cells before and after dox induction. Untreated and line A cells treated with dox (4 mg/ml for 24 h) were harvested and stained with propidium iodide for flow cytometric analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1Characterization of inducible NIH3T3 cell lines. Western blot analysis was performed to detect FoxM1, Cdc25B, cyclin B1, and tubulin expression in two dox-inducible FoxM1c-expressing cell lines (A and B). Both lines were treated for 24 h with dox at the indicated concentrations (0–8 μg/ml). Untransfected NIH3T3 cells and NIH3T3 cells transfected with the tet expression plasmid pcDNA6/TR (both treated with dox at 4 μg/ml for 24 h) were used as negative controls. Background levels of FoxM1 expression were detectable in the control cells after longer exposure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FoxM1 Overexpression Protects Cells against Oxidative Stress-induced Senescence—We subjected both FoxM1-inducible NIH3T3 cell lines to low doses of H2O2 to cause SIPS (35Chen Q.M. Bartholomew J.C. Campisi J. Acosta M. Reagen J.D. Ames B.N. Biochem. J. 1998; 332: 43-50Crossref PubMed Scopus (352) Google Scholar, 36Duan J. Duan J. Zhang Z. Tong T. Int. J. Biochem. Cell Biol. 2005; 37: 1407-1420Crossref PubMed Scopus (146) Google Scholar). Treatment with H2O2 at 20 and 100 μm for 48 h followed by 24 h of incubation in H2O2-free medium induced SA β-galactosidase expression in ∼40–60% of cells (Fig. 2, histograms). The blue senescent cells became larger, flatter, and more vacuolated in appearance (compare Fig. 2, C, E, I, and K against A and G). To address whether FoxM1c induction could slow down the senescence process, both lines were incubated for 24 h in the presence of dox (4 μg/ml) (after 48 h of H2O2 treatment) before their harvest for X-gal staining. Dox-induced FoxM1c expression in both lines clearly decreased the percentage of senescent cells (Fig. 2), and the majority of cells retained the spindle shape characteristic of fibroblasts (Fig. 2, D, F, J, and L). DNA analysis of non-treated and dox-treated cells of line A using flow cytometry did not reveal a decrease in percentage of cells at the G0/G1 phase upon FoxM1c induction, suggesting that the protective function was due to a direct effect on cellular senescence rather than an indirect effect of a perturbation in cell cycle progression (Fig. 2, M and N). FoxM1c Suppresses the p19Arf-p53 Pathway and Induces Bmi-1 Expression—To study the molecular mechanism underlying the counteractive effect of FoxM1c on SIPS, immunoblotting was performed to measure the levels of p53, p21Cip1, p19Arf, and Bmi-1, which are all components within the p19Arf-p53 pathway believed to be dominant in MEFs (37Campisi J. Trends Cell Biol. 2001; 11: S27-S31Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 38Itahana K. Campisi J. Dimri G.P. Biogerontology. 2004; 5: 1-10Crossref PubMed Scopus (266) Google Scholar, 39Ben-Porath I. Weinberg R.A. Int. J. Biochem. Cell Biol. 2005; 37: 961-976Crossref PubMed Scopus (792) Google Scholar). H2O2 treatment at 20 and 100 μm was found
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