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Aberrant promoter methylation of a ras effector homologue gene (RASSF1) associated with loss of expression of transcript A (RASSF1A) is a common event in several types of cancer. Little is presently known about the molecular mechanisms mediating silencing of RASSF1A gene expression in cancer cells. We studied the expression of RASSFIA by reverse-transcribed polymerase chain reaction and methylation status of the RASSF1A promoter by bisulfite genomic sequencing in lung, breast, prostate, and HeLa cancer cell lines. RASSF1A is not expressed in MDA-MB231 breast cancer cells, A549 lung cancer cells, and LNCaP, DU 145, and PC3 prostate cancer cells while it is expressed in MDA-MB468 breast cancer cells, SkMes1 lung cancer cells and HeLa cells. The expression pattern correlated inversely with the methylation pattern of the RASSF1A promoter in the respective cell lines. The RASSF1A promoter was completely methylated in non- expressing MDA-MB231, A549, and LNCaP, DU 145, and PC3, partially methylated in MDA-MB468, while it was unmethylated in HeLa and SkMes1 which express RASSFIA. Treatment of LNCaP cells with a DNA methyltransferase inhibitor, 5-Aza-2’-deoxycytidine induced RASSF1A gene expression while treatment with a histone deacetylase inhibitor, trichostatin A failed to do so. We compared the levels of histones H3 and H4 acetylation and the interaction of histone deacetylases 1 and 2 (HDAC1, HDAC2) at the methylated and silenced RASSF1A promoter in MDA-MB231 and LNCaP cancer cells with an unmethylated and actively expressed RASSF1A promoter in HeLa cells using a chromatin immunoprecipitation assay combined with real time PCR. As compared to the HeLa cells, the RASSF1A promoter demonstrated reduced binding to acetylated histone H3 and to a lesser extent in acetylated histone H4 in MDA-MB231 and LNCaP cells. The RASSF1A promoter interacts with HDAC2 in both MDA-MB231 and LNCaP cells and with HDAC1 in LNCaP prostate cancer cells. As expected, the RASSF1A promoter in HeLa cells did not interact with either HDAC1 or HDAC2 proteins. These results are consistent with the recruitment of histone deacetylase-containing complexes by methylated DNA, resulting in a localized deacetylation. Since direct binding of specific transcriptional repressors to methylated DNA appears to be a major mechanism of transcriptional repression, we determined if methylcytosine binding proteins interact with RASSF1A promoter in MDA-MB231 and LNCaP cells. RASSF1A promoter interacts with MBD2 and MeCP2 in both cell lines and with MBD1 in LNCaP cells while HeLa cells did not interact with MBD1, MBD2, or MECP2 proteins.Our results suggest that the RASSF1A gene repression in cancer cells occurs through the interaction of methylcytosine binding proteins and histone deacetylase complexes with the methylated RASSF1A promoter. Further, there seems to be a cell-type specific difference in the interaction of transcriptional repressors involved in epigenetic silencing of RASSF1A gene.

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