Hypermethylation of the tumor necrosis factor receptor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclear factor kappaB sites in prostatic carcinoma.
DNA hypermethylation of CpG-rich promoter sequences is associated with tumor suppressor gene inactivation in many human cancers, notably in carcinoma of the prostate and the urinary bladder. Recently, the mouse homologue of the 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 apoptosis, which also is downregulated in different types of human carcinoma. We therefore investigated the methylation of the TNFRSF6 promoter in prostatic and bladder carcinomas and cell lines. In a restriction enzyme polymerase chain reaction assay, four of 32 prostatic carcinomas and three of 15 advanced bladder carcinomas showed evidence of hypermethylation at the rel/nuclear factor kappaB (NFkappaB) binding sites essential for promoter activity. The DU145 cell line derived from a metastasis of a prostate carcinoma also displayed hypermethylation in this assay, which was confirmed by bisulfite sequencing. Treatment of DU145 cells with the methylation inhibitor deoxyazacytidine slightly increased Fas protein expression, as detected by flow cytometry analysis. In vitro methylation of the TNFRSF6 promoter at the rel/NFkappaB sites completely abolished its activity. Thus, although the TNFRSF6 gene can be inactivated efficiently by DNA methylation, hypermethylation occurs neither frequently nor extensively in human carcinomas and appears to play a limited role in downregulation of Fas expression.
Santourlidis
S
S
Urologische Klinik, Heinrich-Heine-Universität, Düsseldorf, Germany.
Warskulat
U
U
Florl
A R
AR
Maas
S
S
Pulte
T
T
Fischer
J
J
Müller
W
W
Schulz
W A
WA
D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D000077209:Decitabine; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000235:genetics*; D020572:Oncogene Proteins v-rel / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013869:Thiolester Hydrolases / Q000235:genetics* / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D004273:DNA, Neoplasm; D016328:NF-kappa B; D020572:Oncogene Proteins v-rel; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor; D000077209:Decitabine; C402226:LYPLA1 protein, human; D013869:Thiolester Hydrolases; D001374:Azacitidine