Induced overexpression of P21(WAFI) causes de novo expression of MUC2 gene in a colon carcinoma cell line by inhibition of promoter methylation

Abstract

We found recently a correlation between expression ofp21(WAF1) and the intestinal mucin MUC2 in colonic carcinoma tissues and cell lines (IntJ.Cancer 82:868, 1999). Further, stable transfection of a colonic carcinoma cell line with a p21 plasmid increased expression of MUC2 (DDW 1999, Abstact 1703). Since MUC2 gene is regulated by methylation of its promoter region and p21 has been described as a putative inhibitor of DNA methylation in vitro(Science277:1996,1997), we investigated the hypothesis that p21 protein modulates the methylation status of the promoter region of MUC2 gene in vivo. Methods: The MUC2colon carcinoma cell line DLD1 was used for controled overexpression of p21 with the tet-off system. Expression of p21 was either analysed in Western blot and MUC2 expression by RT·PCR and Northern blot or both proteins were detected by immunohistochemistry. Methylation status of MUC2 promoter was determined in bisulfite-treated genomic DNA by SNuPE method. Results: Induction ofp21 in DLD1 cells leads to de novo expression of MUC2gene on mRNA and protein level only in cells strongly expressing p21 protein. Concomitantly, of nine completely methylated CpG sites within the MUC2 promoter region two become completely demethylated after overexpression of p21 protein. Conclusion: These results indicate that p21 overexpression induces de novo expression of MUC2 in DLDI cells by inhibiting MUC2 promoter methylation. This modulatory effect of p21 on methylation shown here for the first time in vivo may be operative in regulation of other genes in carcinogenesis or differentiation.