Inhibition of the interferon-γ/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- regulated by activation of signal transducers and activators of transcription (STAT) proteins in response to IFN-γ. In this study, we examined CpG methylation at the p21(WAF)1 promoter region in rhabdomyosarcomas (RMSs) using Southern blot analysis with the methylation-sensitive restriction enzyme HpaII. Sis-inducible element (SIE)-1, a STAT-responsive element located upstream of the p21(WAF)1 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 contrast, all normal tissues examined showed a partial methylation pattern at SIE-1. Complete methylation within SIE-1 strongly correlated with decreased p21(WAF1) mRNA expression in RMS. We further studied the effects of SIE-1 hypermethylation on p21(WAF1) induction by STAT activation. CpG methylation within SIE-1 significantly inhibited binding of activated STAT1 in electrophoretic mobility shift assays and abrogated STAT-mediated transcription activation in response to IFN-γ in luciferase reporter gene assays. Activation of STAT1 in response to IFN-γ resulted in increased p21(WAF1) expression and growth suppression in RMS cells containing unmethylated SIE-1 but failed to induce p21(WAF1) or growth inhibition in RD and A673 cells, both of which were completely methylated within SIE-1. However, demethylation at SIE-1, induced by a demethylating agent 5-aza-2'- deoxycytidine, reactivated p21(WAF1) expression and restored the responsiveness to IFN-γ 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-1, directly inhibits the p21(WAF1) induction and cell growth regulation through the IFN-γ/STAT signaling pathway in RMS cells.