Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma

Allelic deletion or transcriptional silencing of RASSF1, a putative tumor s uppressor at 3p21.3, has been found in a considerable proportion of lung, b reasts and ovarian cancers. In this study, we analyzed the expression and m utation status of three RASSF1 isoforms (-A, -B, and -C) in 55 primary blad der carcinomas and 10 bladder and prostate cancer cell lines. The RASSF1A t ranscript was not found in 80% (4 of 5) and 100% (4 of 4) of bladder and pr ostate cell lines, respectively. Compared with normal bladder tissues, loss or significant reduction of RASSF1A was identified in 62 % (34 of 55) of p rimary bladder carcinomas and 10 (83 %) of 12 matched sets, showed tumor-sp ecific alteration of RASSF1A expression. Moreover, loss or abnormal down-re gulation of RASSF1A correlated with advanced tumor stage. RASSF1B was undet ectable in 60 % (3 of 5) of bladder cell lines and in 31 % (17 of 55) of pr imary tumors, but none of these tumors showed altered expression exclusivel y in RASSF1B. RASSF1C transcript was detected in all cell lines and primary tumors we examined. Expression of R4SSF1A and RASSF1B was reactivated in a ll nonexpressor cell lines by treatment with the demethylating agent 5-aza- 2 ' -deoxycytidine. Bisulfite DNA sequencing analysis revealed that aberran t hypermethylation at the CpG island in the RASSF1A promoter is strongly as sociated with the loss of RASSF1A expression in cell lines and uncultured p rimary tumors. Methylation-specific PCR and BstUl digestion analyses also d emonstrated that 97% (33 of 34) of RASSFIA-nonexpressing primary tumors are methylated. Although somatic mutations were not identified in R4SSF1 trans cripts expressed in umnethylated tumors, 24% (9 of 37) of methylated cell l ines and primary tumors showed detectable reductions in genomic levels of R ASSFI. suggesting that RASSF1A inactivation might be caused by both epigene tic and genetic mechanisms in a subset of bladder tumors. Together, our dat a suggest that RASSF1A inactivation may play a critical role in the maligna nt progression of human bladder carcinomas.