Hypermethylation of the tumor necrosis factor receptor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclear factor kappa B sites in prostatic carcinoma

DNA hypermethylation of CpG-rich promoter sequences is associated with tumo r suppressor gene inactivation in many human cancers, notably in carcinoma of the prostate and the urinary bladder. Recently, the mouse homologue of t he tumor necrosis factor receptor superfamily 6 (TNFRSF6) gene was reported to be inactivated by DNA methylation in various cell types. The Fas (CD95, Apo-1) protein encoded by the TNFRSF6 gene is an important mediator of apo ptosis, which also is downregulated in different types of human carcinoma. We therefore investigated the methylation of the TNFRSF6 promoter in prosta tic and bladder carcinomas and cell lines. In a restriction enzyme polymera se chain reaction assay, four of 32 prostatic carcinomas and three of 15 ad vanced bladder carcinomas showed evidence of hypermethylation at the rel/nu clear factor kappaB (NF kappaB) binding sites essential for promoter activi ty. The DU145 cell line derived from a metastasis of a prostate carcinoma a lso displayed hypermethylation in this assay, which was confirmed by bisulf ite sequencing. Treatment of DU145 cells with the methylation inhibitor deo xyazacytidine slightly increased Fas protein expression, as detected by flo w cytometry analysis. In vitro methylation of the TNFRSF6 promoter at the r el/NF kappaB sites completely abolished its activity. Thus, although the TN FRSF6 gene can be inactivated efficiently by DNA methylation, hypermethylat ion occurs neither frequently nor extensively in human carcinomas and appea rs to play a limited role in downregulation of Fas expression. (C) 2001 Wil ey-Liss, Inc.