Inhibition of the interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21(WAF1) promoter region

Expression of the cyclin-dependent kinase inhibitor p21(WAF1) can be up-reg ulated by activation of signal transducers and activators of transcription (STAT) proteins in response to IFN-gamma. In this study, we examined CpG me thylation at the p21(WAF1) promoter region in rhabdomyosarcomas (RMSs) usin g Southern blot analysis with the methylation-sensitive restriction enzyme HpaII, Sis-inducible element (SIE)-1, a STAT-responsive element located ups tream of the p21(WAF1) CpG island, was completely methylated at an internal CpG in 13 of 26 (50%) primary RMS tumors and 2 of 5 RMS cell lines. In con trast, all normal tissues examined showed a partial methylation pattern at SIE-I. Complete methylation within SIE-I strongly correlated with decreased p21(WAF1) mRNA expression in RMS. We further studied the effects of SIE-I hypermethylation on p21(WAF1) induction by STAT activation. CpG methylation within SIE-1 significantly inhibited binding, of activated STAT1 in electr ophoretic mobility shift assays and abrogated STAT-mediated transcription a ctivation in response to IFN-gamma in luciferase reporter gene assays. Acti vation of STAT1 in response to IFN-gamma resulted in increased p21(WAF1) ex pression and growth suppression in RMS cells containing unmethylated SIE-1 but failed to induce p21(WAF1) or growth inhibition in RD and A673 cells, b oth of which were completely methylated within SIE-I. However, demethylatio n at SIE-I. induced by a demethylating agent 5-aza-2'-deoxycytidine, reacti vated p21(WAF1) expression and restored the responsiveness to IFN-gamma in RD cells. Our results indicate a mechanism by which altered DNA methylation in the p21(WAF1) promoter region, by precluding STAT1 binding to SIE-I, di rectly inhibits the p21(WAF1) induction and cell growth regulation through the IFN-gamma/STAT signaling pathway in RMS cells.