title: Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma.
abstract: Allelic deletion or transcriptional silencing of RASSF1, a putative tumor suppressor at 3p21.3, has been found in a considerable proportion of lung, breast, and ovarian cancers. In this study, we analyzed the expression and mutation status of three RASSF1 isoforms (-A, -B, and -C) in 55 primary bladder carcinomas and 10 bladder and prostate cancer cell lines. The RASSF1A transcript was not found in 80% (4 of 5) and 100% (4 of 4) of bladder and prostate cell lines, respectively. Compared with normal bladder tissues, loss or significant reduction of RASSF1A was identified in 62% (34 of 55) of primary bladder carcinomas and 10 (83%) of 12 matched sets showed tumor-specific alteration of RASSF1A expression. Moreover, loss or abnormal down-regulation of RASSF1A correlated with advanced tumor stage. RASSF1B was undetectable in 60% (3 of 5) of bladder cell lines and in 31% (17 of 55) of primary tumors, but none of these tumors showed altered expression exclusively in RASSF1B. RASSF1C transcript was detected in all cell lines and primary tumors we examined. Expression of RASSF1A and RASSF1B was reactivated in all nonexpressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing analysis revealed that aberrant hypermethylation at the CpG island in the RASSF1A promoter is strongly associated with the loss of RASSF1A expression in cell lines and uncultured primary tumors. Methylation-specific PCR and BstUI digestion analyses also demonstrated that 97% (33 of 34) of RASSF1A-nonexpressing primary tumors are methylated. Although somatic mutations were not identified in RASSF1 transcripts expressed in unmethylated tumors, 24% (9 of 37) of methylated cell lines and primary tumors showed detectable reductions in genomic levels of RASSF1, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of bladder tumors. Together, our data suggest that RASSF1A inactivation may play a critical role in the malignant progression of human bladder carcinomas.
authors.affiliation: Department of Pathology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea.
authors.forename: M G
authors.initials: MG
authors.lastname: Lee
authors.forename: H Y
authors.initials: HY
authors.lastname: Kim
authors.forename: D S
authors.initials: DS
authors.lastname: Byun
authors.forename: S J
authors.initials: SJ
authors.lastname: Lee
authors.forename: C H
authors.initials: CH
authors.lastname: Lee
authors.forename: J I
authors.initials: JI
authors.lastname: Kim
authors.forename: S G
authors.initials: SG
authors.lastname: Chang
authors.forename: S G
authors.initials: SG
authors.lastname: Chi
mesh_terms: D000328:Adult; D001483:Base Sequence; D002893:Chromosomes, Human, Pair 3; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor / Q000235:genetics; D006801:Humans; D019656:Loss of Heterozygosity; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; C411762:RASSF1 protein, human; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins