Requirement of Krüppel-like Factor 4 in Preventing Entry into Mitosis following DNA Damage
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m307631200
ISSN1083-351X
AutoresHong Seok Yoon, Vincent W. Yang,
Tópico(s)Epigenetics and DNA Methylation
ResumoPrevious studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after γ-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following γ-irradiation and does so by inhibiting cyclin B1 expression. Previous studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after γ-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following γ-irradiation and does so by inhibiting cyclin B1 expression. The eukaryotic cell cycle is divided into several phases: gap (G1), DNA replication (S), gap 2 (G2), mitosis (M), and a resting or quiescent phase (G0) (1Nurse P. Masui Y. Hartwell L. Nat. Med. 1998; 4: 1103-1106Crossref PubMed Scopus (122) Google Scholar). The events in the cell cycle are ordered into dependent pathways so that late events cannot begin until early events are completed. For example, mitosis is dependent on the completion of DNA replication. Control mechanisms that enforce the dependence of the cell cycle are called checkpoints (2Hartwell L.H. Weinert T.A. Science. 1989; 246: 629-634Crossref PubMed Scopus (2413) Google Scholar). Elimination of the checkpoints may result in cell death, infidelity in the distribution of chromosomes, or increasing susceptibility to environmental perturbations such as DNA-damaging agents. There are three major checkpoints in the cell cycle: G1/S, G2/M, and mitotic or spindle checkpoints (3Murray A. Curr. Opin. Cell Biol. 1994; 6: 872-876Crossref PubMed Scopus (164) Google Scholar, 4Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1760) Google Scholar, 5Russell P. Trends Biochem. Sci. 1998; 23: 399-402Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Controlling the checkpoints are three families of proteins: cyclins, cyclin-dependent kinases (Cdk), 1The abbreviations used are: Cdkcyclin-dependent kinaseChIPchromatin immunoprecipitationCkiCdk inhibitorPBSphosphate-buffered salineDPBSDulbecco's phosphate-buffered salineFBSfetal bovine serumGKLFgut-enriched Krüppel-like factorHDAChistone deacetylaseKLF4Krüppel-like factor 4siRNAsmall interfering RNATSAtrichostatin AGygray(s)ntnucleotide(s)GFPgreen fluorescent protein.1The abbreviations used are: Cdkcyclin-dependent kinaseChIPchromatin immunoprecipitationCkiCdk inhibitorPBSphosphate-buffered salineDPBSDulbecco's phosphate-buffered salineFBSfetal bovine serumGKLFgut-enriched Krüppel-like factorHDAChistone deacetylaseKLF4Krüppel-like factor 4siRNAsmall interfering RNATSAtrichostatin AGygray(s)ntnucleotide(s)GFPgreen fluorescent protein. and inhibitors of Cdk (Cki) (6Murray A.W. Kirschner M.W. Nature. 1989; 339: 275-280Crossref PubMed Scopus (863) Google Scholar, 7Sherr C.J. 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Cki are inhibitors of the cyclin-Cdk complexes and function by halting the cell cycle at checkpoints. cyclin-dependent kinase chromatin immunoprecipitation Cdk inhibitor phosphate-buffered saline Dulbecco's phosphate-buffered saline fetal bovine serum gut-enriched Krüppel-like factor histone deacetylase Krüppel-like factor 4 small interfering RNA trichostatin A gray(s) nucleotide(s) green fluorescent protein. cyclin-dependent kinase chromatin immunoprecipitation Cdk inhibitor phosphate-buffered saline Dulbecco's phosphate-buffered saline fetal bovine serum gut-enriched Krüppel-like factor histone deacetylase Krüppel-like factor 4 small interfering RNA trichostatin A gray(s) nucleotide(s) green fluorescent protein. In response to DNA damage such as that caused by ionizing radiation and chemotherapeutic drugs, cells are arrested at the transition from G1 to S phase and G2 to M phase (10Zhou B.B. Elledge S.J. 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The mechanisms by which p53 accomplishes this task are thought to be multiple and include its ability to transcriptionally suppress cdc2 and cyclin B1 (20Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (165) Google Scholar), which are required for entry into mitosis (21Nurse P. Nature. 1990; 344: 503-508Crossref PubMed Scopus (2220) Google Scholar). A second mechanism is through the induction of 14-3-3σ expression (22Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. Mol. Cell. 1997; 1: 3-11Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 23Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (810) Google Scholar), which sequesters cyclin B1-Cdc2 complex in the cytoplasm (24Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1277) Google Scholar). Third, p21WAF1/CIP1 inhibits cyclin B1-Cdc2 and prevents entry into mitosis (25Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3161) Google Scholar, 26Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (856) Google Scholar). Last, 14-3-3σ and p21WAF1/CIP1 have also been shown to exert a cooperative effect in controlling the G2/M checkpoint (27Chan T.A. Hwang P.M. Hermeking H. Kinzler K.W. Vogelstein B. Genes Dev. 2000; 14: 1584-1588PubMed Google Scholar). Although the induction of p21WAF1/CIP1 expression has been shown to be a consequence of direct binding of p53 to its promoter, evidence implicates many other transcription factors in regulating p21WAF1/CIP1 transcription (28Gartel A.L. Tyner A.L. Exp. Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (576) Google Scholar). Among these is Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF), a member of the mammalian KLF family of transcription regulators (29Dang D.T. Pevsner J. Yang V.W. Int. J. Biochem. Cell Biol. 2000; 32: 1103-1121Crossref PubMed Scopus (361) Google Scholar, 30Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 31Kaczynski J. Cook T. Urrutia R. Genome Biol. 2003; 4: 206Crossref PubMed Scopus (732) Google Scholar). KLF4 was initially identified as an epithelially enriched gene with preferential expression in the terminally differentiated, postmitotic epithelial cells of the intestine and epidermis (32Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 33Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In cultured cells, expression of KLF4 is associated with conditions that lead to growth arrest such as serum deprivation or contact inhibition (32Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Consistent with these findings, constitutive expression of KLF4 inhibits DNA synthesis and reduced cell proliferation (32Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 34Geiman D.E. Ton-That H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar, 35Dang D.T. Chen X. Feng J. Torbenson M. Dang L.H. Yang V.W. Oncogene. 2003; 22: 3424-3430Crossref PubMed Scopus (142) Google Scholar). This is in part due to cell cycle arrest at the G1/S boundary as a result of the ability of KLF4 to transcriptionally activate expression of p21WAF1/CIP1 (36Chen X. Johns D.C. Geiman D.E. Marban E. Dang D.T. Hamlin G. Sun R. Yang V.W. J. Biol. Chem. 2001; 276: 30423-30428Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 37Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 38Chen X. Whitney E.M. Gao S.Y. Yang V.W. J. Mol. Biol. 2003; 326: 665-677Crossref PubMed Scopus (160) Google Scholar). In support of a checkpoint function for KLF4, we recently showed that its expression is activated in a p53-dependent fashion upon DNA damage by agents such as methyl methane sulfonate and γ-irradiation (37Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This induction is correlated with an increase in the levels of p21WAF1/CIP1 with consequent G1/S cell cycle arrest in cells with wild type p53 (39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Importantly, inhibition of KLF4 expression in such cells after γ-irradiation results in abrogation of the G1 arrest in a manner similar to the cell cycle profile seen in irradiated cells that are null for p53 (39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Conversely, conditional expression of KLF4 in irradiated cells null for p53 restored G1 arrest as if the they were wild type for p53 (39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). These findings indicate that KLF4 is a necessary and sufficient mediator of p53 for the G1/S cell cycle arrest resulting from DNA damage and does so by activating p21WAF1/CIP1 expression. Since p21WAF1/CIP1 has also been shown to be required for sustained G2 arrest following γ-irradiation (19Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2526) Google Scholar), we sought to determine in the present study whether KLF4 may also be involved in controlling the G2/M checkpoint after DNA damage. Cell Lines—The colon cancer cell lines wild type and null for p53, HCT116 p53+/+, and HCT116 p53-/-, respectively, were generous gifts from Dr. Bert Vogelstein (Johns Hopkins University) (19Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2526) Google Scholar). The cells were cultured in McCoy's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. EcR116 p53-/- cells were established by stably transfecting pVgRXR (36Chen X. Johns D.C. Geiman D.E. Marban E. Dang D.T. Hamlin G. Sun R. Yang V.W. J. Biol. Chem. 2001; 276: 30423-30428Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), which contains VgEcR and RXR that form a receptor for the insect hormone, ecdysone, into the parental HCT116 cell line and selected with 100 μg/ml Zeocin (Invitrogen). The level of RXR expression was determined by Western blot analysis. γ-Irradiation—γ-Irradiation of cultured cells was performed using a 137Cs γ-irradiator at 0.8 Gy/min for 15 min, for a total of 12 Gy. Cells were harvested at 0, 24, 48, 72, and 96 h after γ-irradiation for subsequent assays. Medium was changed at the time of collection for the remaining plates. FACS Analysis—Cell cycle analysis was performed as previously described (39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Cells were rinsed in Dulbecco's phosphate-buffered saline (DPBS; Mediatech, Inc.), treated with trypsin, and resuspended in McCoy's medium containing 10% FBS. Cells were then collected by centrifugation, washed with DPBS, collected again by centrifugation, resuspended in 70% ethanol, and fixed at -20 °C overnight. Cells were pelleted once again by centrifugation and resuspended in a solution containing 50 μg/ml propidium iodide, 50 μg/ml RNase A, 0.1% Triton X-100, and 0.1 mm EDTA at room temperature for 30 min. Flow cytometry was performed on a FACSCalibur cytometer (Becton Dickinson). Measurement of Mitotic Indices—At each time point, cells were fixed in 3% formaldehyde for 15 min. Cold 100% methanol was then added, and cells were incubated at room temperature for 20 min. Cells were then rinsed three times with DPBS. A Hoechst 33258 solution (10 μg/ml) was added to each dish to a final concentration of 0.2 μg/ml, which was incubated at room temperature for 15 min. After the incubation, cells were rinsed five times with DPBS, and nuclei were visualized by fluorescence microscopy (Nikon). A minimum of 400 cells were examined per experiment. Mitotic figures were scored for cells with condensed chromosomes. Mitotic trapping experiments were performed by adding nocodazole to the culture media to a final concentration of 0.2 μg/ml. Media containing nocodazole were replaced every 24 h. Mitotic figures were examined following nuclear staining. Adenovirus Infection—The recombinant adenoviruses containing GFP and KLF4 (AdEGI-KLF4) or GFP alone (AdEGI) were described previously (36Chen X. Johns D.C. Geiman D.E. Marban E. Dang D.T. Hamlin G. Sun R. Yang V.W. J. Biol. Chem. 2001; 276: 30423-30428Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 40Johns D.C. Marx R. Mains R.E. O'Rourke B. Marban E. J. Neurosci. 1999; 19: 1691-1697Crossref PubMed Google Scholar). EcR116 p53-/- cells were grown to 40% confluence in 10-cm dishes and replenished with fresh media containing 2% FBS followed by the addition of 108 plaque-forming units of recombinant virus per dish. Infected cells were incubated at 37 °C for 6 h, at which time cells were γ-irradiated, and the media were changed. Cells were treated with 5 μm ponasterone A (Invitrogen) for 0, 24, 48, 72, and 96 h and then collected for further analysis. Medium was changed at the time of collection for the remaining plates. Preparation of Small Interfering RNA (siRNA) and Transfection— The KLF4-specific siRNA was described previously (39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). A nonspecific, double-stranded siRNA with identical length was also generated based on the sequence of an unrelated protein and used as a control. HCT116 p53+/+ cells were grown to 40% confluence in 10-cm dishes, γ-irradiated for a total of 12 Gy, and transfected with annealed siRNA using DMRIE-C reagent (Invitrogen) for 6 h as recommended by the manufacturer. McCoy's media containing 20% FBS and 2% penicillin/streptomycin were added to each dish to a final concentration of 10% FBS and 1% penicillin/streptomycin. Cells were harvested at 0, 24, 48, 72, and 96 h for further assays. Medium was changed at the time of collection for the remaining plates. Western Blot Analysis—Cell protein extraction and Western blot analyses were performed using standard procedures. Protein samples were mixed with loading buffer (100 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm dithiothreitol, 0.01% bromphenol blue, and 10% glycerol), heated at 100 °C for 5 min, and loaded onto a SDS-polyacrylamide gel in electrophoresis buffer containing 25 mm Tris-HCl, pH 8.3, 250 mm glycine, and 0.1% SDS. Protein was then transferred to polyvinylidene difluoride membranes using the Trans-Blot semidry system (Bio-Rad). The membranes were immunoblotted with primary antibodies against KLF4 (32Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar), p53, p21WAF1/CIP1, Cdc2, cyclin B1, or β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Following incubation with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; 1:10,000 dilution; Santa Cruz Biotechnology), proteins were visualized with the SuperSignal West Pico chemiluminescent substrate kit (Pierce). Cdc2 Kinase Assay—Cdc2 kinase activities were determined as previously described (20Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (165) Google Scholar). Lysates were prepared by lysing pelleted cells in a lysis buffer containing 50 mm HEPES, pH 7.0, 250 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 1 mm phenylmethanesulfonyl fluoride, 2 μg/ml aprotinin, 25 μg/ml leupeptin, 5 μg/ml pepstatin A, and 1 mm dithiothreitol. Lysates were incubated with a Cdc2 monoclonal antibody. Immune complexes were isolated with protein G-conjugated affinity gel (Sigma). The gel pellets were collected with centrifugation, washed three times with lysis buffer, and incubated for 30 min at 37 °C in 20 mm HEPES, pH 7.9, 5 mm MgCl2, 1 μg of histone H1 (Hoffman LaRoch), 1 mm EDTA, 100 μm ATP, and 10 μCi of [γ-32P]ATP in a total volume of 20 μl. Phosphorylated histone H1 was resolved by SDS-PAGE (12.5% acrylamide) and visualized with autoradiography. Transfection Assay—Co-transfection experiments were performed with the -287 cyclin B1 promoter-luciferase reporter, pGL-cyclin B1 (-287) (42Katula K.S. Wright K.L. Paul H. Surman D.R. Nuckolls F.J. Smith J.W. Ting J.P. Yates J. Cogswell J.P. Cell Growth Differ. 1997; 8: 811-820PubMed Google Scholar), the expression construct containing KLF4, PMT3-KLF4 (37Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), and an internal control Renilla luciferase. Site-directed mutagenesis was used to introduce a 3-nt mutation into the putative KLF4-binding site in the cyclin B1 promoter (nt -137 to -142; 5′-GGGGCG-3′ to 5′-GGTTAG-3′) using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Two mutagenic primers containing the desired mutation were synthesized (5′-GCCTCACTGTGGCCCCTTACCTCTCGAACGCCT-3′ and 5′-AGGCGTTCGAGAGGGGATTGGCCACAGTGAGGC-3′) and annealed to pGL-cyclin B1 (-287). Mutagenesis was performed using the protocol provided by the manufacturer. The mutated sequence was confirmed by DNA sequencing. Luciferase activities were determined 1 day following transfection using the manufacturer's recommendation (Promega). All firefly luciferase activities were standardized to the Renilla luciferase internal control. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed based on a published protocol (43Chun A.C. Jin D.Y. J. Biol. Chem. 2003; 278: 37439-37450Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). HCT116 p53+/+ cells were irradiated or not for the amount of 12 Gy and maintained in culture for an additional 48 h. Cells were washed twice with PBS, followed by the addition of 1% formaldehyde in PBS, and incubated for 15 min at room temperature. At the end of the incubation, the cross-linking was terminated by the addition of glycine to a final concentration of 0.125 m. Cells were then washed twice with PBS and lysed with 1 ml of radioimmune precipitation assay buffer that contained 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors. Cell lysates were then sonicated to yield chromatin fragments of ∼600 bp, as assessed by agarose gel electrophoresis. Insoluble materials were removed by centrifugation, at 13,000 × g for 10 min, and the supernatant was transferred to a new tube. The supernatant was diluted 10-fold with dilution buffer (20 mm Tris-HCl, pH 8.1, 150 mm NaCl, 2 mm EDTA, and 1% Triton X-100), and the diluted extracts were precleared by incubation with 45 μl of protein G-Sepharose beads (Sigma) and sheared salmon sperm DNA. After centrifugation for 5 min at 7,000 × g, the supernatant was transferred to a fresh tube. Immunoprecipitation was performed by rocking overnight at 4 °C the precleared cell lysates with the appropriate antibodies. Immune complexes were then precipitated with protein G-Sepharose beads and sheared salmon sperm DNA. The beads were collected by centrifugation and washed twice sequentially with radioimmune precipitation assay wash buffer I (20 mm Tris-HCl, pH 8.1, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, and 2 mm EDTA), wash buffer II (20 mm Tris-HCl, pH 8.1, 500 mm NaCl, 0.1% SDS, 1% Triton X-100, and 2 mm EDTA), wash buffer III (10 mm Tris-HCl, pH 8.1, 0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, and 1 mm EDTA), and wash buffer IV (10 mm Tris-HCl, pH 8.1, and 1 mm EDTA). The immunoprecipitates were eluted by adding 200 μl of elution buffer (1% SDS, 0.1 m NaHCO3) to the washed beads followed by incubation at 65 °C overnight. After centrifugation for 5 min at 7,000 x g, DNA was extracted from the supernatant using a Qiagen PCR purification kit. A 3-μl DNA sample was then subjected to amplification using primers encompassing nt -287 and +23 of the cyclin B1 promoter (5′-TCTTGCCCGGCTAACCTTTCCAGG-3′ and 5′-TTCCGCCGCAGCACGCCGAGAAGA-3′). Trichostatin A (TSA) Treatment—HCT116 p53+/+ cells were irradiated or not at day 0 and fed with media containing 2 μm TSA or vehicle alone for 2 days. Cell extracts were then harvested for Western blot analysis of cyclin B1 and β-actin content. p53 Is Required for Preventing Mitotic Entry in HCT116 Cells following γ-Irradiation—The tumor suppressor p53 exerts critical checkpoint functions. To demonstrate a dependence of sustained G2 arrest on p53 following DNA damage, we irradiated HCT116 p53+/+ and HCT116 p53-/- with a 12-Gy γ-ray and examined daily the cell cycle profiles up to 4 days following irradiation. Consistent with previous findings (19Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2526) Google Scholar, 39Yoon H.S. Chen X. Yang V.W. J. Biol. Chem. 2003; 278: 2101-2105Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), parental cells (p53+/+) arrested in either G1 with 2n DNA content or G2 with 4n DNA content from day 1 to 4 after irradiation, as expected for cells with intact checkpoint functions (results not shown). Cells with deleted p53 alleles (p53-/-) were arrested mostly in G2 for the same duration. However, although a substantial number of cells were arrested in G2 for both genotypes, morphological examination shows a steady increase in the number of HCT116 p53-/- cells that entered mitosis beginning at day 2 following irradiation (Fig. 1B). In contrast, irradiated HCT116 p53+/+ cells had a persistent decrease in the percentage of cells in mitosis from days 1 to 4 when compared with that before irradiation (Fig. 1B). Control unirradiated cells displayed a steady and equal level of mitotic indices for both genotypes (Fig. 1A). Both HCT116 p53+/+ and p53-/- cells had an intact mitotic apparatus as evidenced by their ability to be become trapped in mitosis temporarily by the microtubule-disrupting agent, nocodazole (19Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2526) Google Scholar) (Fig. 1C). However, only HCT116 p53-/- cells were able to enter mitosis following γ-irradiation in the presence of nocodazole (Fig. 1D). These results indicate that p53 is required for sustaining G2 arrest and preventing mitotic entry in HCT116 cells following DNA damage. Levels of G2/M Markers in HCT116 p53+/+ Cells Are Decreased after γ-Irradiation—To determine the mechanism by which p53 sustains G2 arrest and prevents mitosis in irradiated HCT116 cells, we performed Western blot analysis on several markers of the G2/M checkpoint. In particular, Cdc2 and cyclin B1 were selected, since they have been shown to be required for entry into mitosis. As seen in Fig. 2, there was a persistent increase in the levels of p53 in irradiated HCT116 p53+/+ cells from day 1 to 4 following irradiation when compared with unirradiated cells. The levels of KLF4 and p21WAF1/CIP1 were concomitantly increased in irradiated cells. In contrast, there was a significant and sustained decrease in the levels of cyclin B1 in irradiated cells. The levels of Cdc2 were also decreased following γ-irradiation but to a lesser extent than that of cyclin B1. These results suggest that the reduced levels of Cdc2 and cyclin B1 contributed to the sustained G2 arrest observed in irradiated HCT116 p53+/+ cells. Inhibition of KLF4 Expression in HCT116 p53+/+ Cells Results in Abrogation of G2/M Arrest following γ-Irradiation— Previous studies show that p21WAF1/CIP1, the expression of which is p53-dependent, is required for the sustained G2 arrest following γ-irradiation (19Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2526) Google Scholar). Since p21WAF1/CIP1 is a target of KLF4 (36Ch
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