Inhibition of the interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region.
Expression of the cyclin-dependent kinase inhibitor p21WAF1 can be up-regulated by activation of signal transducers and activators of transcription (STAT) proteins in response to IFN-gamma. In this study, we examined CpG methylation at the p21WAF1 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 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 contrast, all normal tissues examined showed a partial methylation pattern at SIE-1. Complete methylation within SIE-1 strongly correlated with decreased p21WAF1 mRNA expression in RMS. We further studied the effects of SIE-1 hypermethylation on p21WAF1 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-gamma in luciferase reporter gene assays. Activation of STAT1 in response to IFN-gamma resulted in increased p21WAF1 expression and growth suppression in RMS cells containing unmethylated SIE-1 but failed to induce p21WAF1 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 p21WAF1 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-1, directly inhibits the p21WAF1 induction and cell growth regulation through the IFN-gamma/STAT signaling pathway in RMS cells.
Chen
B
B
Department of Pathology, University of Arkansas for Medical Sciences and Arkansas Children's Hospital, Little Rock 72202, USA.
He
L
L
Savell
V H
VH
Jenkins
J J
JJ
Parham
D M
DM
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015139:Blotting, Southern; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors; D008156:Luciferases; D019042:Muscle Neoplasms / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000235:genetics; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000378:metabolism; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000964:Antimetabolites, Antineoplastic; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D000077209:Decitabine; D007371:Interferon-gamma; D008156:Luciferases; D001374:Azacitidine