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RASSF1A (Ras association domain family 1A) gene, a tumor suppressor gene, is located at chromosome 3p21.3 and is epigenetically inactivated in many human cancers. The re-expression of RASSF1A can arrest tumor cells in G1/S, inhibiting their proliferation. Recent studies have identified RASSF1A promoter methylation as a major mechanism of its silencing. We report here that 5-fluoro-2’-deoxycytidine (FdCyd) treatment of EJ6 human bladder carcinoma cells, in which RASSF1A is not expressed, decreases the RASSF1A promoter methylation and increases the RASSF1A mRNA expression. Primers specific for methylated and unmethylated sequences in the region of the RASSF1A promoter CpG Island were used for methylation-specific PCR (MSP) analysis of bisulfite-modified DNA. An unmethylated signal was observed in EJ6 cells treated with 1, 10 or 100 μM FdCyd for 3 days and was stronger after 3 weeks of treatment with 10 or 100 μM FdCyd and 9 weeks of treatment with 1 μM FdCyd. A methylated signal was observed in both FdCyd-treated and untreated EJ6 cells, indicating both successful bisulfate conversion and a lack of complete demethylation in all cells. The bisulfite modified DNA samples from EJ6 cells that were treated with 100 μM FdCyd from 3 days to 12 weeks were used for quantitative DNA CpG methylation analysis of RASSF1A by methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). The average methylation of the four CpG sites examined was 0.96 in untreated EJ6 cells, 0.85 after treatment with 1 μM 5-azadeoxycytidine for 3 days. After the EJ6 cells were treated by FdCyd for 14 or 15 weeks at 1, 10 or 100 μM concentration, the average methylation was 0.80, 0.67 or 0.57 respectively. The expression of RASSF1A mRNA was examined by RT-PCR. A 245-bp amplification fragment of the RASSF1A message was undetected in untreated EJ6 cells and was detected after treatment of the cells with 100 μM FdCyd for 3 weeks. The signal was greater after longer exposures, up to 9 weeks. We compared the expression of RASSF1A mRNA (normalized to HPRT) in untreated and FdCyd-treated EJ6 cells to the expression in normal human fibroblasts (HF). In EJ6 cells treated with 100 μM FdCyd for 3 and 9 weeks, the relative RASSF1A mRNA expression was 2.0% and 39.9%, respectively, of the expression in HF. Thus, FdCyd, not only inhibits RASSF1A DNA methylation, but also increases RASSF1A mRNA expression. (Supported by CA81055 and CA33572.)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]