DE-NOVO METHYLATION OF SELECTIVE CPG DINUCLEOTIDE CLUSTERS IN TRANSFORMED-CELLS MEDIATED BY AN ACTIVATED N-RAS

We have explored a possible role of an activated N-ras oncogene in abe rrant methylation of CpG clusters in DNAs of transformed cells. Using three lines of hamster cells transformed by Rous sarcoma virus (RSV) a nd the method of detection of CpG islands as clustered sites for methy lation-sensitive restriction enzymes we have demonstrated that in each cell line the transcribed RSV proviruses are integrated in the vicini ty of sequence containing the cluster of unmethylated CpG dinucleotide s. Two out of three examined CpG clusters had hypermethylation pattern s in N-ras-neo- but not in neo-transfected variants of the cell lines. De novo methylation of CpG dinucleotides correlated with transcriptio nal inactivation of adjacent RSV proviruses that was related neither t o the lack of transcriptional factors binding RSV long terminal repeat (LTR) nor to the transcriptional incompetence of the LTR, as measured by reporter gene assays with the LTR cloned from DNA of these cells. These data suggest that activation of N-ras signal transduction pathwa y in transformed cells may be relevant to long-term inactivation of se lective genes by hypermethylation of their CpG islands.