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W2123687793 abstract "Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mm butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at −82 and −69 relative to the transcription start site. We also found that a TATA element at −46 and two overlapping consensus Sp1 sites at −60 and −55 are essential for the basal promoter activity ofWAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion. Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mm butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at −82 and −69 relative to the transcription start site. We also found that a TATA element at −46 and two overlapping consensus Sp1 sites at −60 and −55 are essential for the basal promoter activity ofWAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion. Butyrate is one of the most abundant short chain fatty acids in the large intestine, generated by bacterial fermentation of dietary fibers (1Cummings J.H. Gut. 1981; 22: 763-779Crossref PubMed Scopus (772) Google Scholar). Butyrate shows potent effects on growth arrest and differentiation in vitro in various malignant tumor cell lines, such as breast cancer cells, hepatoma cells, and others (2Graham K.A. Buick R.N. J. Cell. Physiol. 1988; 136: 63-71Crossref PubMed Scopus (53) Google Scholar, 3Saito H. Morizane T. Watanabe T. Kagawa T. Miyaguchi S. Kumagai N. Tsuchiya M. Int. J. Cancer. 1991; 48: 291-296Crossref PubMed Scopus (54) Google Scholar, 4Leder A. Leder P. Cell. 1975; 5: 319-322Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 5Dong Q.G. Gong L.L. Wang H.J. Wang E.Z. Anti-Cancer Drugs. 1993; 4: 617-627Crossref PubMed Scopus (3) Google Scholar). In colorectal cancer cells, butyrate inhibits cell growth and induces differentiation marker proteins such as alkaline phosphatase and carcinoembryonic antigen (6Kim Y.S. Tsao D. Siddiqui B. Whitehead J.S. Arnstein P. Bennett J. Hicks J. Am. Cancer Soc. 1980; 45: 1185-1192Google Scholar, 7Tsao D. Morita A. Bella Jr., A. Luu P. Kim Y.S. Cancer Res. 1982; 42: 1052-1058PubMed Google Scholar, 8Morita A. Tsao D. Kim Y.S. Cancer Res. 1982; 42: 4540-4545PubMed Google Scholar, 9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar). Furthermore, butyrate arrests the cell cycle progression at the G1 phase (9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar) and decreases c-myc oncogene expression in human colon cancer cell lines (9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar, 10Herold K.M. Rothberg P.G. Oncogene. 1988; 3: 423-428PubMed Google Scholar). However, the precise mechanism of growth suppression by butyrate in colon cancer cells has not been clarified.WAF1/Cip1 protein potently inhibits the various G1cyclin-dependent kinases activities (11Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 12Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3157) Google Scholar, 13Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (707) Google Scholar) by suppressing the phosphorylation of retinoblastoma (RB) protein, thereby supposedly inhibiting the G1-S phase transition (11Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 14Dulić V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1414) Google Scholar). Besides its role as a kinase inhibitor, it has been reported recently that WAF1/Cip1 at low doses assembles kinase complexes and promotes a kinase activity (15LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1209) Google Scholar). Furthermore, the transcription of theWAF1/Cip1 gene is directly activated by wild-type p53 protein (16El-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 (7890) Google Scholar). Thus, WAF1/Cip1 could play a key role as a downstream mediator of the p53-induced cell growth arrest.Several studies have already shown the p53-independent induction ofWAF1/Cip1 by serum, transforming growth factor β, and other differentiation-inducers (17Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Li C.-Y. Suardet L. Little J.B. J. Biol. Chem. 1995; 270: 4971-4974Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In addition, butyrate has been reported to induce WAF1/Cip1 mRNA independently of p53 during differentiation of hematopoietic cells, hepatoma cells, and colon cancer cells in vitro (18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 21Hodin R.A. Meng S. Archer S. Tang R. Cell Growth Differ. 1996; 7: 647-653PubMed Google Scholar). Butyrate can also dephosphorylate the retinoblastoma protein in mouse fibroblasts (22Buquet-Fagot C. Lallemand F. Charollais R.-H. Mester J. J. Cell. Physiol. 1996; 166: 631-636Crossref PubMed Scopus (28) Google Scholar). To investigate the mechanism of butyrate-induced growth arrest, we used a human colon cancer cell line WiDr harboring a point mutation in p53 at codon 273 (23Tamura T. Aoyama N. Saya H. Haga H. Futami S. Miyamoto M. Koh T. Ariyasu T. Tachi M. Kasuga M. Takahashi R. Oncogene. 1995; 11: 1939-1946PubMed Google Scholar) and examined the effect of butyrate on the expression of the WAF1/Cip1 gene.Our results demonstrate that WAF1/Cip1 mRNA is rapidly induced upon butyrate treatment, although WiDr lacks the wild-typep53 gene. We then found that butyrate markedly induces WAF1/Cip1 protein and causes G1-phase arrest. In addition, we observed that butyrate can strongly activate theWAF1/Cip1 promoter, and that the two p53-binding sites are not required for the transcriptional activation by butyrate. Using a series of mutant WAF1/Cip1 promoter constructs, we also found in p53-negative cell lines WiDr and human osteosarcoma cell line MG63 (24Chandar N. Billig B. McMaster J. Novak J. Br. J. Cancer. 1992; 65: 208-214Crossref PubMed Scopus (149) Google Scholar), that two Sp1 sites at −82 and −69 relative to the transcription start site are involved in the activation of theWAF1/Cip1 promoter by butyrate. Furthermore, the essential elements for the WAF1/Cip1 promoter activity have been shown to be two overlapping consensus Sp1 sites at −60 and −55 and TATA sequence at −46.DISCUSSIONMounting evidence indicates that mutations in p53 are among the most common genetic events in the development of human cancer (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). On the other hand, WAF1/Cip1 is well known to be induced by wild-type p53 (16El-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 (7890) Google Scholar). Hence, it might be plausible that little or no expression of WAF1/Cip1 is also a common event in cancer cells. Therefore, it would be of great value to identify the p53-independent pathway of WAF1/Cip1 induction, which could lead to an alternative pathway to suppress the oncogenic progression.In the present study, we have shown that treatment of either WiDr or MG63 cells with butyrate specifically induces WAF1/Cip1mRNA and protein, resulting in G1 arrest of the cell cycle progression in a p53-independent manner. A series of mutation analyses of the WAF1/Cip1 promoters have revealed that the main butyrate-responsive element is the Sp1 site between −82 and −77 relative to the transcription start site (the Sp1-3 site in this report), and the Sp1 site between −69 and −64 (Sp1-4 site) is also partially involved in this activation. In addition, we found that butyrate is capable of activating transcription from the luciferase reporter plasmid including only three Sp1 sites. These results strongly suggest that Sp1 is involved in the transcriptional activation of theWAF1/Cip1 promoter in response to butyrate; in fact, EMSA using MG63 cells revealed that Sp1 and Sp3 can specifically interact with this main butyrate-responsive element, the Sp1-3 site. However, the intensity and mobility pattern of the retarded bands were not changed by butyrate, which means that activation of theWAF1/Cip1 promoter by butyrate does not appear to be due to increasing the binding of Sp1 or Sp3. Additionally, butyrate could not affect the phosphorylation pattern of the Sp1 protein (data not shown), and CHX did not block the WAF1/Cip1 mRNA induction by butyrate in MG63 cells (Fig. 2 C, lanes 5 and 6). Hence, there is a possibility that Sp1-related or other unknown factors pre-exist and will be subject to modulation, such as phosphorylation, and involved in the activation of WAF1/Cip1 promoter in response to butyrate. On the other hand, very little is presently known of how the Sp1 modification affects transcription except for phosphorylation or glycosylation (37Jackson S.P. MacDonald J.J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar). Thus, further studies will be required to elucidate the mechanism of how butyrate modulates the potent transcriptional function of Sp1.Recently, several studies have reported the p53-independent induction of WAF1/Cip1 (17Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Li C.-Y. Suardet L. Little J.B. J. Biol. Chem. 1995; 270: 4971-4974Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The promoter analysis of the p53-independent pathways has also been reported (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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, 38Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (828) Google Scholar). Biggset al. (26Biggs 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) have reported that the region between −122 and −61 from the transcription start site, including Sp1-1 to Sp1-4 sites in our paper, is required for both the basal activity and the full activation of the WAF1/Cip1 promoter by phorbol esters and okadaic acid and suggested that Sp1 is involved in this activity by using gel mobility shift assays (26Biggs 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). Furthermore, Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) have identified the main transforming growth factor β-responsive element of the WAF1/Cip1 promoter, termed TβRE, as an element including Sp1 site between −82 and −77 (Sp1-3 site), by using a series of deleted or mutated constructs. It is of interest that TβRE in the WAF1/Cip1 promoter corresponds to the main butyrate-responsive element including the Sp1 site between −82 and −77 (Sp1-3 site). Sp1 protein is a ubiquitously expressed transcription factor that regulates a large number of constitutive and induced mammalian genes by interacting with specific GC-rich elements (GC boxes) (40Kadonaga J.T. Jones K.A. Tjian R. Trends Biochem. Sci. 1986; 11: 20-23Abstract Full Text PDF Scopus (875) Google Scholar, 41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar). It would thus be of great interest to clarify the mechanism by which butyrate and other WAF1/Cip1-inducing factors such as TGF-β act on the Sp1 transcription factor.In addition, we clearly showed that two overlapping Sp1 sites between −60 and −51 (Sp1-5-6 site) and TATA box are the most essential for the WAF1/Cip1 promoter activity. This discrepancy with the results of Biggs et al. (26Biggs 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) or Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) may be explained by the different cell lines or by small differences in the sequences of generated plasmids (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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).In summary, our results suggest that butyrate-induced growth arrest in WiDr cells is due to the p53-independent activation ofWAF1/Cip1 promoter mediated through specific Sp1 sites in the promoter region. Recently, we proposed a novel approach for chemotherapy or chemoprevention against cancer, which we termed “gene-regulating chemotherapy or chemoprevention” (42Sakai T. Jpn. J. Hyg. 1996; 50: 1036-1046Crossref PubMed Scopus (24) Google Scholar). Our strategy is to activate the potent function of growth-inhibitory genes, which are activating targets of p53. The WAF1/Cip1 gene is one of the good candidates, because WAF1/Cip1 appears to be rarely mutated in human common tumors (43Chedid M. Michieli P. Lengel C. Huppi K. Givol D. Oncogene. 1994; 9: 3021-3024PubMed Google Scholar, 44Li Y.-J. Laurent-Puig P. Salmon R.J. Thomas G. Hamelin R. Oncogene. 1995; 10: 599-601PubMed Google Scholar), whereas thep53 gene is frequently mutated (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). Therefore, in the future, clarification of the p53-independent activating pathway of theWAF1/Cip1 gene might contribute to the therapy or the prevention of cancer when p53 is mutated. Butyrate is one of the most abundant short chain fatty acids in the large intestine, generated by bacterial fermentation of dietary fibers (1Cummings J.H. Gut. 1981; 22: 763-779Crossref PubMed Scopus (772) Google Scholar). Butyrate shows potent effects on growth arrest and differentiation in vitro in various malignant tumor cell lines, such as breast cancer cells, hepatoma cells, and others (2Graham K.A. Buick R.N. J. Cell. Physiol. 1988; 136: 63-71Crossref PubMed Scopus (53) Google Scholar, 3Saito H. Morizane T. Watanabe T. Kagawa T. Miyaguchi S. Kumagai N. Tsuchiya M. Int. J. Cancer. 1991; 48: 291-296Crossref PubMed Scopus (54) Google Scholar, 4Leder A. Leder P. Cell. 1975; 5: 319-322Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 5Dong Q.G. Gong L.L. Wang H.J. Wang E.Z. Anti-Cancer Drugs. 1993; 4: 617-627Crossref PubMed Scopus (3) Google Scholar). In colorectal cancer cells, butyrate inhibits cell growth and induces differentiation marker proteins such as alkaline phosphatase and carcinoembryonic antigen (6Kim Y.S. Tsao D. Siddiqui B. Whitehead J.S. Arnstein P. Bennett J. Hicks J. Am. Cancer Soc. 1980; 45: 1185-1192Google Scholar, 7Tsao D. Morita A. Bella Jr., A. Luu P. Kim Y.S. Cancer Res. 1982; 42: 1052-1058PubMed Google Scholar, 8Morita A. Tsao D. Kim Y.S. Cancer Res. 1982; 42: 4540-4545PubMed Google Scholar, 9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar). Furthermore, butyrate arrests the cell cycle progression at the G1 phase (9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar) and decreases c-myc oncogene expression in human colon cancer cell lines (9Barnard J.A. Warwick G. Cell Growth Differ. 1993; 4: 495-501PubMed Google Scholar, 10Herold K.M. Rothberg P.G. Oncogene. 1988; 3: 423-428PubMed Google Scholar). However, the precise mechanism of growth suppression by butyrate in colon cancer cells has not been clarified. WAF1/Cip1 protein potently inhibits the various G1cyclin-dependent kinases activities (11Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 12Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3157) Google Scholar, 13Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (707) Google Scholar) by suppressing the phosphorylation of retinoblastoma (RB) protein, thereby supposedly inhibiting the G1-S phase transition (11Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 14Dulić V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1414) Google Scholar). Besides its role as a kinase inhibitor, it has been reported recently that WAF1/Cip1 at low doses assembles kinase complexes and promotes a kinase activity (15LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1209) Google Scholar). Furthermore, the transcription of theWAF1/Cip1 gene is directly activated by wild-type p53 protein (16El-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 (7890) Google Scholar). Thus, WAF1/Cip1 could play a key role as a downstream mediator of the p53-induced cell growth arrest. Several studies have already shown the p53-independent induction ofWAF1/Cip1 by serum, transforming growth factor β, and other differentiation-inducers (17Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Li C.-Y. Suardet L. Little J.B. J. Biol. Chem. 1995; 270: 4971-4974Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In addition, butyrate has been reported to induce WAF1/Cip1 mRNA independently of p53 during differentiation of hematopoietic cells, hepatoma cells, and colon cancer cells in vitro (18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 21Hodin R.A. Meng S. Archer S. Tang R. Cell Growth Differ. 1996; 7: 647-653PubMed Google Scholar). Butyrate can also dephosphorylate the retinoblastoma protein in mouse fibroblasts (22Buquet-Fagot C. Lallemand F. Charollais R.-H. Mester J. J. Cell. Physiol. 1996; 166: 631-636Crossref PubMed Scopus (28) Google Scholar). To investigate the mechanism of butyrate-induced growth arrest, we used a human colon cancer cell line WiDr harboring a point mutation in p53 at codon 273 (23Tamura T. Aoyama N. Saya H. Haga H. Futami S. Miyamoto M. Koh T. Ariyasu T. Tachi M. Kasuga M. Takahashi R. Oncogene. 1995; 11: 1939-1946PubMed Google Scholar) and examined the effect of butyrate on the expression of the WAF1/Cip1 gene. Our results demonstrate that WAF1/Cip1 mRNA is rapidly induced upon butyrate treatment, although WiDr lacks the wild-typep53 gene. We then found that butyrate markedly induces WAF1/Cip1 protein and causes G1-phase arrest. In addition, we observed that butyrate can strongly activate theWAF1/Cip1 promoter, and that the two p53-binding sites are not required for the transcriptional activation by butyrate. Using a series of mutant WAF1/Cip1 promoter constructs, we also found in p53-negative cell lines WiDr and human osteosarcoma cell line MG63 (24Chandar N. Billig B. McMaster J. Novak J. Br. J. Cancer. 1992; 65: 208-214Crossref PubMed Scopus (149) Google Scholar), that two Sp1 sites at −82 and −69 relative to the transcription start site are involved in the activation of theWAF1/Cip1 promoter by butyrate. Furthermore, the essential elements for the WAF1/Cip1 promoter activity have been shown to be two overlapping consensus Sp1 sites at −60 and −55 and TATA sequence at −46. DISCUSSIONMounting evidence indicates that mutations in p53 are among the most common genetic events in the development of human cancer (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). On the other hand, WAF1/Cip1 is well known to be induced by wild-type p53 (16El-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 (7890) Google Scholar). Hence, it might be plausible that little or no expression of WAF1/Cip1 is also a common event in cancer cells. Therefore, it would be of great value to identify the p53-independent pathway of WAF1/Cip1 induction, which could lead to an alternative pathway to suppress the oncogenic progression.In the present study, we have shown that treatment of either WiDr or MG63 cells with butyrate specifically induces WAF1/Cip1mRNA and protein, resulting in G1 arrest of the cell cycle progression in a p53-independent manner. A series of mutation analyses of the WAF1/Cip1 promoters have revealed that the main butyrate-responsive element is the Sp1 site between −82 and −77 relative to the transcription start site (the Sp1-3 site in this report), and the Sp1 site between −69 and −64 (Sp1-4 site) is also partially involved in this activation. In addition, we found that butyrate is capable of activating transcription from the luciferase reporter plasmid including only three Sp1 sites. These results strongly suggest that Sp1 is involved in the transcriptional activation of theWAF1/Cip1 promoter in response to butyrate; in fact, EMSA using MG63 cells revealed that Sp1 and Sp3 can specifically interact with this main butyrate-responsive element, the Sp1-3 site. However, the intensity and mobility pattern of the retarded bands were not changed by butyrate, which means that activation of theWAF1/Cip1 promoter by butyrate does not appear to be due to increasing the binding of Sp1 or Sp3. Additionally, butyrate could not affect the phosphorylation pattern of the Sp1 protein (data not shown), and CHX did not block the WAF1/Cip1 mRNA induction by butyrate in MG63 cells (Fig. 2 C, lanes 5 and 6). Hence, there is a possibility that Sp1-related or other unknown factors pre-exist and will be subject to modulation, such as phosphorylation, and involved in the activation of WAF1/Cip1 promoter in response to butyrate. On the other hand, very little is presently known of how the Sp1 modification affects transcription except for phosphorylation or glycosylation (37Jackson S.P. MacDonald J.J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar). Thus, further studies will be required to elucidate the mechanism of how butyrate modulates the potent transcriptional function of Sp1.Recently, several studies have reported the p53-independent induction of WAF1/Cip1 (17Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Li C.-Y. Suardet L. Little J.B. J. Biol. Chem. 1995; 270: 4971-4974Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The promoter analysis of the p53-independent pathways has also been reported (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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, 38Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (828) Google Scholar). Biggset al. (26Biggs 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) have reported that the region between −122 and −61 from the transcription start site, including Sp1-1 to Sp1-4 sites in our paper, is required for both the basal activity and the full activation of the WAF1/Cip1 promoter by phorbol esters and okadaic acid and suggested that Sp1 is involved in this activity by using gel mobility shift assays (26Biggs 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). Furthermore, Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) have identified the main transforming growth factor β-responsive element of the WAF1/Cip1 promoter, termed TβRE, as an element including Sp1 site between −82 and −77 (Sp1-3 site), by using a series of deleted or mutated constructs. It is of interest that TβRE in the WAF1/Cip1 promoter corresponds to the main butyrate-responsive element including the Sp1 site between −82 and −77 (Sp1-3 site). Sp1 protein is a ubiquitously expressed transcription factor that regulates a large number of constitutive and induced mammalian genes by interacting with specific GC-rich elements (GC boxes) (40Kadonaga J.T. Jones K.A. Tjian R. Trends Biochem. Sci. 1986; 11: 20-23Abstract Full Text PDF Scopus (875) Google Scholar, 41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar). It would thus be of great interest to clarify the mechanism by which butyrate and other WAF1/Cip1-inducing factors such as TGF-β act on the Sp1 transcription factor.In addition, we clearly showed that two overlapping Sp1 sites between −60 and −51 (Sp1-5-6 site) and TATA box are the most essential for the WAF1/Cip1 promoter activity. This discrepancy with the results of Biggs et al. (26Biggs 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) or Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) may be explained by the different cell lines or by small differences in the sequences of generated plasmids (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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).In summary, our results suggest that butyrate-induced growth arrest in WiDr cells is due to the p53-independent activation ofWAF1/Cip1 promoter mediated through specific Sp1 sites in the promoter region. Recently, we proposed a novel approach for chemotherapy or chemoprevention against cancer, which we termed “gene-regulating chemotherapy or chemoprevention” (42Sakai T. Jpn. J. Hyg. 1996; 50: 1036-1046Crossref PubMed Scopus (24) Google Scholar). Our strategy is to activate the potent function of growth-inhibitory genes, which are activating targets of p53. The WAF1/Cip1 gene is one of the good candidates, because WAF1/Cip1 appears to be rarely mutated in human common tumors (43Chedid M. Michieli P. Lengel C. Huppi K. Givol D. Oncogene. 1994; 9: 3021-3024PubMed Google Scholar, 44Li Y.-J. Laurent-Puig P. Salmon R.J. Thomas G. Hamelin R. Oncogene. 1995; 10: 599-601PubMed Google Scholar), whereas thep53 gene is frequently mutated (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). Therefore, in the future, clarification of the p53-independent activating pathway of theWAF1/Cip1 gene might contribute to the therapy or the prevention of cancer when p53 is mutated. Mounting evidence indicates that mutations in p53 are among the most common genetic events in the development of human cancer (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). On the other hand, WAF1/Cip1 is well known to be induced by wild-type p53 (16El-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 (7890) Google Scholar). Hence, it might be plausible that little or no expression of WAF1/Cip1 is also a common event in cancer cells. Therefore, it would be of great value to identify the p53-independent pathway of WAF1/Cip1 induction, which could lead to an alternative pathway to suppress the oncogenic progression. In the present study, we have shown that treatment of either WiDr or MG63 cells with butyrate specifically induces WAF1/Cip1mRNA and protein, resulting in G1 arrest of the cell cycle progression in a p53-independent manner. A series of mutation analyses of the WAF1/Cip1 promoters have revealed that the main butyrate-responsive element is the Sp1 site between −82 and −77 relative to the transcription start site (the Sp1-3 site in this report), and the Sp1 site between −69 and −64 (Sp1-4 site) is also partially involved in this activation. In addition, we found that butyrate is capable of activating transcription from the luciferase reporter plasmid including only three Sp1 sites. These results strongly suggest that Sp1 is involved in the transcriptional activation of theWAF1/Cip1 promoter in response to butyrate; in fact, EMSA using MG63 cells revealed that Sp1 and Sp3 can specifically interact with this main butyrate-responsive element, the Sp1-3 site. However, the intensity and mobility pattern of the retarded bands were not changed by butyrate, which means that activation of theWAF1/Cip1 promoter by butyrate does not appear to be due to increasing the binding of Sp1 or Sp3. Additionally, butyrate could not affect the phosphorylation pattern of the Sp1 protein (data not shown), and CHX did not block the WAF1/Cip1 mRNA induction by butyrate in MG63 cells (Fig. 2 C, lanes 5 and 6). Hence, there is a possibility that Sp1-related or other unknown factors pre-exist and will be subject to modulation, such as phosphorylation, and involved in the activation of WAF1/Cip1 promoter in response to butyrate. On the other hand, very little is presently known of how the Sp1 modification affects transcription except for phosphorylation or glycosylation (37Jackson S.P. MacDonald J.J. Lees-Miller S. Tjian R. Cell. 1990; 63: 155-165Abstract Full Text PDF PubMed Scopus (516) Google Scholar). Thus, further studies will be required to elucidate the mechanism of how butyrate modulates the potent transcriptional function of Sp1. Recently, several studies have reported the p53-independent induction of WAF1/Cip1 (17Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 18Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar, 19Jiang H. Lin J. Su Z.-z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 20Li C.-Y. Suardet L. Little J.B. J. Biol. Chem. 1995; 270: 4971-4974Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The promoter analysis of the p53-independent pathways has also been reported (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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, 38Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (828) Google Scholar). Biggset al. (26Biggs 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) have reported that the region between −122 and −61 from the transcription start site, including Sp1-1 to Sp1-4 sites in our paper, is required for both the basal activity and the full activation of the WAF1/Cip1 promoter by phorbol esters and okadaic acid and suggested that Sp1 is involved in this activity by using gel mobility shift assays (26Biggs 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). Furthermore, Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) have identified the main transforming growth factor β-responsive element of the WAF1/Cip1 promoter, termed TβRE, as an element including Sp1 site between −82 and −77 (Sp1-3 site), by using a series of deleted or mutated constructs. It is of interest that TβRE in the WAF1/Cip1 promoter corresponds to the main butyrate-responsive element including the Sp1 site between −82 and −77 (Sp1-3 site). Sp1 protein is a ubiquitously expressed transcription factor that regulates a large number of constitutive and induced mammalian genes by interacting with specific GC-rich elements (GC boxes) (40Kadonaga J.T. Jones K.A. Tjian R. Trends Biochem. Sci. 1986; 11: 20-23Abstract Full Text PDF Scopus (875) Google Scholar, 41Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar). It would thus be of great interest to clarify the mechanism by which butyrate and other WAF1/Cip1-inducing factors such as TGF-β act on the Sp1 transcription factor. In addition, we clearly showed that two overlapping Sp1 sites between −60 and −51 (Sp1-5-6 site) and TATA box are the most essential for the WAF1/Cip1 promoter activity. This discrepancy with the results of Biggs et al. (26Biggs 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) or Datto et al. (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) may be explained by the different cell lines or by small differences in the sequences of generated plasmids (25Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 26Biggs 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). In summary, our results suggest that butyrate-induced growth arrest in WiDr cells is due to the p53-independent activation ofWAF1/Cip1 promoter mediated through specific Sp1 sites in the promoter region. Recently, we proposed a novel approach for chemotherapy or chemoprevention against cancer, which we termed “gene-regulating chemotherapy or chemoprevention” (42Sakai T. Jpn. J. Hyg. 1996; 50: 1036-1046Crossref PubMed Scopus (24) Google Scholar). Our strategy is to activate the potent function of growth-inhibitory genes, which are activating targets of p53. The WAF1/Cip1 gene is one of the good candidates, because WAF1/Cip1 appears to be rarely mutated in human common tumors (43Chedid M. Michieli P. Lengel C. Huppi K. Givol D. Oncogene. 1994; 9: 3021-3024PubMed Google Scholar, 44Li Y.-J. Laurent-Puig P. Salmon R.J. Thomas G. Hamelin R. Oncogene. 1995; 10: 599-601PubMed Google Scholar), whereas thep53 gene is frequently mutated (35Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 36Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1892) Google Scholar). Therefore, in the future, clarification of the p53-independent activating pathway of theWAF1/Cip1 gene might contribute to the therapy or the prevention of cancer when p53 is mutated. We thank Dr. K. Kawai for his continuous encouragement. We also thank Drs. R. Takahashi and E. Hara for useful advice on the Western blot technique." @default.
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W2123687793 title "Butyrate Activates the WAF1/Cip1 Gene Promoter through Sp1 Sites in a p53-negative Human Colon Cancer Cell Line" @default.
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