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• W2039611809 abstract "An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1 during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21 WAF1/Cip1, suggesting that GKLF may be involved in the induction of p21 WAF1/Cip1. Indeed, GKLF activates p21 WAF1/Cip1 through a specific Sp1-likecis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisenseGklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated. An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1 during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21 WAF1/Cip1, suggesting that GKLF may be involved in the induction of p21 WAF1/Cip1. Indeed, GKLF activates p21 WAF1/Cip1 through a specific Sp1-likecis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisenseGklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated. cyclin-dependent kinase gut-enriched Krüppel-like factor Krüppel-like factor 4 DNA-binding domain chloramphenicol acetyltransferase nucleotide(s) kilobase(s) base pair(s) mouse embryo fibroblasts methyl methanesulfonate polymerase chain reaction human embryonic kidney electrophoretic mobility shift assay 1,3-bis]tris(hydroxymethyl)methylamino[propane cAMP-response element-binding protein-binding protein A principal function of the tumor suppressor p53 is to maintain genomic stability. It does so by eliciting cellular changes in response to various forms of stress such as DNA damage, hypoxia, and nucleotide deprivation (1.Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 2.Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, 3.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). The amount of p53 protein increases in response to these so-called genotoxic stresses. In addition, covalent modifications such as phosphorylation are involved in its activation (4.Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1177) Google Scholar, 5.Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Once activated, p53 exerts potent regulatory effects on diverse aspects of cellular events that cumulate in cell cycle arrest or apoptosis (3.Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). Many of these “downstream” events are dependent upon the ability of p53 to function as a transcription factor in activating the expression of “target” genes (2.Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, 6.El-Deiry W.S. Semin. Cancer Biol. 1998; 8: 345-357Crossref PubMed Scopus (727) Google Scholar). Notably, an important consequence of p53 activation is the transcriptional induction of the gene encoding the cyclin-dependent kinase (Cdk)1 inhibitor p21 (also called WAF1 or Cip1) (7.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar, 8.Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar). p21 WAF1/Cip1 inhibits the activity of several cyclin-Cdk complexes such as cyclin D1-Cdk4, cyclin E1-Cdk2, and cyclin A-Cdk2, which results in cell cycle arrest at the G1-S transition checkpoint (9.Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar, 10.Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar). The gut-enriched Krüppel-like factor (GKLF, KLF4) (11.Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) is a recently identified and developmentally regulated transcription factor, the expression of which is enriched in the epithelial cells of the gastrointestinal tract (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 13.Jenkins T.D. Opitz O.G. Okano J. Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14.Garrett-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 (275) Google Scholar), skin (14.Garrett-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 (275) Google Scholar, 15.Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (644) Google Scholar), and thymus (16.Panigada M. Porcellini M. Sutti F. Doneda L. Pozzoli O. Consalez G.G. Guttinger M. Grassi F. Mech. Dev. 1999; 81: 103-113Crossref PubMed Scopus (46) Google Scholar) and in vascular endothelial cells (17.Yet S.F. McA'Nulty M.M. Folta S.C. Yen H.W. Yoshizumi M. Hsieh C.M. Layne M.D. Chin M.T. Wang H. Perrella M.A. Jain M.K. Lee M.E. J. Biol. Chem. 1998; 273: 1026-1031Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Both the in vivo (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 13.Jenkins T.D. Opitz O.G. Okano J. Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14.Garrett-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 (275) Google Scholar, 15.Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (644) Google Scholar, 16.Panigada M. Porcellini M. Sutti F. Doneda L. Pozzoli O. Consalez G.G. Guttinger M. Grassi F. Mech. Dev. 1999; 81: 103-113Crossref PubMed Scopus (46) Google Scholar) and in vitro (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar) patterns of expression of Gklfare indicative of a growth arrest-associated nature. Upon stimulation of quiescent cultured cells by fresh serum, levels of GklfmRNA are decreased significantly during the G1-S transition phase of the cell cycle (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Conversely, constitutive expression of GKLF inhibits DNA synthesis (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). In vivo,Gklf transcripts are highly enriched in the population of terminally differentiated, post-mitotic epithelial cells of the intestinal tract and skin (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 13.Jenkins T.D. Opitz O.G. Okano J. Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14.Garrett-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 (275) Google Scholar, 15.Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (644) Google Scholar). Moreover, the intestinal expression of Gklf is down-regulated in two independent mouse models of intestinal tumorigenesis or hyperproliferation (18.Ton-That H. Kaestner K.H. Shields J.M. Mahatanankoon C.S. Yang V.W. FEBS Lett. 1997; 419: 239-243Crossref PubMed Scopus (97) Google Scholar, 19.Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (190) Google Scholar). Taken together, these studies suggest that GKLF is potentially a negative regulator of proliferation; however, the mechanism by which it accomplishes this task is not well defined. The established binding site for GKLF is rich in GC content (20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar) and overlaps with that for the transcription factor Sp1 (21.Zhang W. Shields J.M. Sogawa K. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 22.Mahatan C.S. Kaestner K.H. Geiman D.E. Yang V.W. Nucleic Acids Res. 1999; 27: 4562-4569Crossref PubMed Scopus (64) Google Scholar). By coincidence, the proximal promoter of the p21 WAF1/Cip1 gene contains a number of GC-rich elements (7.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar), some of which have been shown to bind Sp1 (23.Biggs J.R. Kudlow J.E. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 24.Datto M.B., Yu, Y. Wang X.F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 25.Li J.M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar, 26.Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar, 27.Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 28.Prowse D.M. Bolgan L. Molnar A. Dotto G.P. J. Biol. Chem. 1997; 272: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 29.Sowa Y. Orita T. Minamikawa S. Nakano K. Mizuno T. Nomura H. Sakai T. Biochem. Biophys. Res. Commun. 1997; 241: 142-150Crossref PubMed Scopus (287) Google Scholar). These Sp1-binding sites have been shown to be important in controlling expression of p21 WAF1/Cip1 in several physiologically diverse processes, including the gene's responsiveness to phorbol ester (23.Biggs J.R. Kudlow J.E. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), transforming growth factor-β (24.Datto M.B., Yu, Y. Wang X.F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 25.Li J.M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar, 26.Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar), and sodium butyrate (27.Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), and in keratinocyte differentiation (28.Prowse D.M. Bolgan L. Molnar A. Dotto G.P. J. Biol. Chem. 1997; 272: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). As both Gklf and p21 WAF1/Cip1 are growth arrest-associated genes, we sought to determine whether GKLF is involved in regulating p21 WAF1/Cip1 expression. We demonstrate that GKLF not only transactivates the p21 WAF1/Cip1 proximal promoter, but also mediates the activating effects of p53 in response to DNA damage on the same promoter. This study suggests that GKLF may be an important component of the p53 tumor suppressor network of regulatory proteins. The eukaryotic expression vector PMT3 and its derivatives containing various forms of GKLF were previously described (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar, 30.Shields J.M. Yang V.W. J. Biol. Chem. 1997; 272: 18504-18507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 31.Geiman D.E. Ton-That H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar). They include full-length GKLF (PMT3-GKLF-(1–483)), truncated GKLF lacking the three zinc fingers (PMT3-GKLF-(1–401)), and truncated GKLF containing the zinc fingers only (PMT3-GKLF-(350–483)). pC53-SN3 and pC53-SX3, two cytomegalovirus-based expression constructs containing wild-type p53 and mutant p53 with a missense mutation at codon 143 in the DNA-binding domain (DBD) of p53, respectively, were kindly provided by B. Vogelstein and K. Kinzler (32.Baker S.J. Markowitz S. Fearon E.R. Willson J.K. Vogelstein B. Science. 1990; 249: 912-915Crossref PubMed Scopus (1610) Google Scholar). The reporter constructs linking various regions of the p21 WAF1/Cip1 promoter to chloramphenicol acetyltransferase (CAT) have previously been described (23.Biggs J.R. Kudlow J.E. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). They include the CAT reporter linked to either a 2320-nt 5′-flanking sequence of the p21 WAF1/Cip1 gene containing an upstream p53-binding site at nt −2301 (33.Gartel A.L. Tyner A.L. Exp. Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (580) Google Scholar) or the same 2320-nt 5′-flanking sequence with a small internal deletion of the sequence between nt −122 and −61 of the p21 WAF1/Cip1 promoter that removed the first four of the six Sp1 sites from the proximal promoter (33.Gartel A.L. Tyner A.L. Exp. Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (580) Google Scholar). Reporter constructs containing the proximal promoter region of the p21 WAF1/Cip1 gene with various 5′-end points as well as internal deletions or point mutations affecting the various Sp1 sites in the proximal promoters have all been described (23.Biggs J.R. Kudlow J.E. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The WWP-Luc and DM-Luc constructs are two luciferase reporters that contain 2.4 and 2.2 kb, respectively, of the 5′-flanking sequence of the p21 WAF1/Cip1 gene and were kindly provided by B. Vogelstein and K. Kinzler (7.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar). The DM-Luc construct lacks the upstream p53-binding site at nt −2301 in the p21 WAF1/Cip1 promoter (7.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar). The polyclonal rabbit anti-GKLF serum was described (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Anti-p53 serum was purchased from Santa Cruz Biotechnology (sc-6243), and the monoclonal antibody against p21 WAF1/Cip1 was purchased from Pharmingen (SXM30). The p53−/− and p53+/+ mouse embryo fibroblasts (MEFs) were generously provided by L. Donehower (34.Jones S.N. Sands A.T. Hancock A.R. Vogel H. Donehower L.A. Linke S.P. Wahl G.M. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14106-14111Crossref PubMed Scopus (78) Google Scholar). The 10(1)-p53 val135 cell line was provided by A. Levine (35.Sabbatini P. Lin J. Levine A.J. White E. Genes Dev. 1995; 9: 2184-2192Crossref PubMed Scopus (228) Google Scholar). This cell line, which was derived from the parental 10(1) cell line (36.Harvey D.M. Levine A.J. Genes Dev. 1991; 5: 2375-2385Crossref PubMed Scopus (417) Google Scholar), is an immortalized murine embryo fibroblast line that lacks endogenous p53 expression, but contains a stably transfected temperature-sensitive p53 protein, val135 (37.Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1645) Google Scholar). At the nonpermissive temperature of 38.5 °C, p53 val135 is transcriptionally inactive, whereas at the permissive temperature of 31.5 °C, it is transcriptionally competent (35.Sabbatini P. Lin J. Levine A.J. White E. Genes Dev. 1995; 9: 2184-2192Crossref PubMed Scopus (228) Google Scholar). The sense and antisense oligonucleotides to GKLF contain nucleotide sequences corresponding to amino acid codons 7–13 of GKLF in the sense and antisense orientations, respectively. At the center of this sequence (amino acid 10) is the second of two initiation methionine codons of GKLF, which was felt to be in a translationally more favorable context than the first (14.Garrett-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 (275) Google Scholar). The nucleotide sequence of the antisense oligonucleotide is 5′-GCT GAC AGC CAT GTC AGA CTC-3′, and that of the sense oligonucleotide is 5′-GAG TCT GAC ATG GCT GTC AGC-3′. Note that the underlined sequence represents the initiation methionine codon at amino acid 10 (12). For the serum deprivation experiments, the content of fetal calf serum in the cell medium was reduced from 10 to 0.5% to induce a growth-arrested state (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). To cause DNA damage, methyl methanesulfonate (MMS) was added to cells at a concentration of 100 μg/ml, which has previously been shown to result in cell cycle arrest (38.Fornace A.J. Alamo I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). After various treatment periods, total RNA was isolated from cells using Triazol (Life Technologies, Inc.). Twenty μg of RNA from each sample were studied by Northern blot analyses using conditions previously described (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Blots were probed with a full-length cDNA encoding GKLF (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar), p21 WAF1/Cip1 (7.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar), or glyceraldehyde-3-phosphate dehydrogenase (CLONTECH). The conditions for Western blot analysis were also previously described, using a 1:1000 dilution of an affinity-purified polyclonal anti-GKLF serum (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). All transfections were performed by lipofection as described (20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar, 21.Zhang W. Shields J.M. Sogawa K. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 30.Shields J.M. Yang V.W. J. Biol. Chem. 1997; 272: 18504-18507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,31.Geiman D.E. Ton-That H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar). Unless otherwise specified, all reactions contained 5 μg each of the reporter and effector constructs/10-cm dish. Luciferase and CAT assays were performed as described (20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar, 21.Zhang W. Shields J.M. Sogawa K. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). RNA was extracted from human embryonic kidney (HEK) 293 cells and human colonic carcinoma HT29 cells (39.von Kleist S. Chany E. Burtin P. King M. Fogh J. J. Natl. Cancer Inst. 1975; 55: 555-560Crossref PubMed Scopus (121) Google Scholar). The content of Gklf transcript from each cell line was determined using reverse transcription-PCR. The content of the β-actin transcript was similarly determined as a control. One μg of RNA was reverse-transcribed in an 80-μl volume containing 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 10 mm dithiothreitol, 3 mm MgCl2, 0.5 mm dGTP, 0.5 mm dATP, 0.5 mmdTTP, 0.5 mm dCTP, 80 units of RNase inhibitor, 100 pmol of random primer, pd(N)6, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) at 42 °C for 1 h. The cDNA was then amplified in a 50-μl reaction that contained 10 mm Tris-HCl, pH 8.3, 50 mm KCl, 10 mm MgCl2, 0.1% gelatin, 2.5 units of REDTaq DNA polymerase (Sigma), 0.2 mm dGTP, 0.2 mm dATP, 0.2 mm dTTP, 0.2 mmdCTP, and 40 pm each of the forward and reverse primers (see below) at the following settings: 94 °C for 45 s, 45 °C for 1 min, and 72 °C for 1.5 min for a total of 40 cycles. The PCR products were then visualized on a 1.5% agarose gel stained with ethidium bromide. The primers used in the PCR were synthesized according to the published cDNA sequences encoding human GKLF and β-actin (GenBankTM Data Bank accession numbers AF105036 and X00351, respectively). The forward primer sequence for GKLF is 5′-AGGTCGGACCACCTCGCCTTACACATG-3′, and the reverse primer sequence is 5′-AAGGTAAAGAGAATACAAGGTGATCTTTTATGC-3′. The length of the expected PCR product was 345 bp. The forward and reverse primer sequences for β-actin are 5′-TACGCCAACACAGTGCTGTCTGG-3′ and 5′-TACTCCTGCTTGCTGATCCACAT-3′, respectively, with the expected PCR product measuring 206 bp. EMSAs were performed as described (20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar). Preparation of nuclear extracts from COS-1 cells transfected with PMT3 expression constructs containing full-length GKLF, truncated GKLF containing only the zinc fingers or lacking the zinc fingers, or PMT3 vector alone was as described previously (20.Shields J.M. Yang V.W. Nucleic Acids Res. 1998; 26: 796-802Crossref PubMed Scopus (153) Google Scholar,21.Zhang W. Shields J.M. Sogawa K. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Purified p53 containing the DBD was kindly provided by N. Pavletich (40.Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (449) Google Scholar). This domain contains the core portion of p53 between amino acids 102 and 292, which binds with high affinity to a p53 recognition site (40.Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (449) Google Scholar, 41.Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2158) Google Scholar). The purification of recombinant p53 DBD expressed from the pET3d bacterial expression vector (Novagen) in transformed Escherichia coli BL21(D3) cells was as described previously (40.Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (449) Google Scholar). The protein was supplied at a concentration of 14 mg/ml in a solution of 50 mm BisTris propane HCl, pH 6.8, 200 mm sodium phosphate, and 5 mmdithiothreitol and had a >98% purity of the core domain. The wild-type p21 oligonucleotide used in EMSA contains the sequence between nt −129 and −99 of the p21 WAF1/Cip1 promoter, which includes both Sp1-1 and Sp1-2 sites (27.Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The mutant p21 oligonucleotide contains a 3-bp substitution in the Sp1-1 site. The sequences in the sense orientation for the two oligonucleotides are shown below. The oligonucleotide probe containing the binding site for p53 was derived from the p53-response sequence in the promoter of the humanGADD45 gene (42.Hollander M.C. Alamo I. Jackman J. Wang M.G. McBride O.W. Fornace A.J. J. Biol. Chem. 1993; 268: 24385-24393Abstract Full Text PDF PubMed Google Scholar) and has the sequence 5′-TACAGAACATGTCTAAGCATGCTGGGG-3′ in the sense orientation. When indicated, unlabeled competitor oligonucleotides were added in 10-, 20-, or 50-fold molar excess of the probe to the reaction. [35S]Methionine-labeled p53 was synthesized by the TNT Coupled Reticulocyte Lysate system (Promega) using a full-length cDNA encoding p53 cloned in pBluescript (provided by B. Vogelstein). Ten μl of the translation product were mixed with 50 μg of nuclear extracts prepared from transfected COS-1 cells in a final volume of 100 μl containing 20 mm HEPES, pH 7.5, 40 mm KCl, 3 mm MgCl2, 1 mm dithiothreitol, and 5% glycerol at 4 °C for 2 h. At the completion of the incubation, 15 μg of affinity-purified anti-GKLF serum or preimmune serum were added to the reaction, which was gently rotated overnight at 4 °C. Fifty μl of packed protein A-Sepharose beads (Amersham Pharmacia Biotech) were then added to each reaction, and the incubation was continued for 1 h at 4 °C. The beads were subsequently collected by centrifugation, washed three times with the incubation buffer, and resuspended in sample buffer before electrophoresis. Previously, we showed that the levels of theGklf transcript were low in actively proliferating cells, but were increased in cells that had been deprived of serum (12.Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). Results of the Northern blot analysis in Fig.1 A recapitulate this event. Fig. 1 A also shows that upon serum deprivation, the levels of the p21 WAF1/Cip1 transcript rose concomitantly with those ofGklf. To determine whether Gklf is induced during growth arrest under a different condition, we treated NIH 3T3 cells with MMS, which causes DNA damage and subsequently cell cycle arrest (38.Fornace A.J. Alamo I. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). As shown in Fig. 1 B, the levels of GklfmRNA were increased 2 h after the addition of MMS, as were those of p21 WAF1/Cip1 mRNA. When normalized to the expression of the control glyceraldehyde-3-phosphate dehydrogenase gene, which was not affected by the treatment, the degree of induction of p21 WAF1/Cip1 was higher than that of Gklf between 2 and 8 h of MMS treatment (Fig. 1 B, bar graph). This contrasts with the changes in mRNA levels of the two genes during the initial 30 min of treatment, in which the rise inGklf preceded that in p21 WAF1/Cip1 (Fig.1 C). These results suggest that both Gklf and p21 WAF1/Cip1 respond similarly to signals elicited during growth arrest due to DNA damage. However, the induction ofGklf begins slightly earlier than that of p21 WAF1/Cip1 during the initial phase of DNA damage. To determine whether the inductive responses ofGklf and p21 WAF1/Cip1 to MMS trea" @default.
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• W2039611809 title "The Gut-enriched Krüppel-like Factor (Krüppel-like Factor 4) Mediates the Transactivating Effect of p53 on the p21 Promoter" @default.
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