Methylation induced by the RAS oncogene. Rattus norvegicus

Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but up-regulated after treatment with the demethylating agent 5-Aza-CdR and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, Mmp2, Ppicap, syndecan 4, Timp2, Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and in a methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed ten days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable. Keywords: expression analysis Overall design: Gene expression was analyzed in immortal (208F) and HRAS oncogene-transformed (FE8) rat fibroblasts after demethylation by 5-aza-2'-deoxycytidine. Genes potentially methylated in RAS-transformed cells were identified by bisulphite sequencing