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 G 1 -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 of WAF1/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 (1). 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 (2-5). In colorectal cancer cells, butyrate inhibits cell growth and induces differentiation marker proteins such as alkaline phosphatase and carcinoembryonic antigen (6 -9). Furthermore, butyrate arrests the cell cycle progression at the G 1 phase (9) and decreases c-myc oncogene expression in human colon cancer cell lines (9, 10). However, the precise mechanism of growth suppression by butyrate in colon cancer cells has not been clarified. WAF1/Cip1 protein potently inhibits the various G 1 cyclindependent kinases activities (11-13) by suppressing the phosphorylation of retinoblastoma (RB) protein, thereby supposedly inhibiting the G 1 -S phase transition (11,14). 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 (15). Furthermore, the transcription of the WAF1/Cip1 gene is directly activated by wild-type p53 protein (16). 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 of WAF1/Cip1 by serum, transforming growth factor ␤, and other differentiation-inducers (17)(18)(19)(20). 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 (18,21). Butyrate can also dephosphorylate the retinoblastoma protein in mouse fibroblasts (22). To investigate the mechanism of butyrateinduced growth arrest, we used a human colon cancer cell line WiDr harboring a point mutation in p53 at codon 273 (23) and examined the effect of butyrate on t...

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Trichostatin A (TSA), a specific histone deacetylase inhibitor, induces histone hyperacetylation and modulates the expression of some genes. We examined the effects of TSA on MG63 cells. TSA induced growth arrest and expression of the p21/WAF1/Cip1 protein. A close correlation between the level of histone acetylation and induction of the p21/WAF1/Cip1 protein was detected. Using several mutant p21/WAF1/Cip1 promoter fragments, mutation of either of two Sp1 sites at -82 or -69 of the p21/WAF1/Cip1 promoter reduced the responsiveness to TSA. This finding indicates that TSA activates the p21/WAF1/Cip1 promoter through the Sp1 sites in a p53-independent manner.

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