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A clone of the human transforming Ha-ras oncogene (pT24-C3) was methylated "in vitro" at its HpaII and HhaI sites and co-transfected with a selectable marker into NIH/3T3 cells. In the resulting colonies the normal or transformed morphology correlated respectively with a methylated or unmethylated status of the Ha-ras 1 promoter. A transfected, morphologically normal NIH/3T3 colony treated with 5'-azacytidine acquired a transformed phenotype growing in agar and in nude mice and showed demethylation of the promoter region of Ha-ras 1 gene. The cells of the reactivated colony produced human Ha-ras 1 mRNA and p21, and their DNA displayed transforming activity for NIH/3T3 recipient cells that was due to the original human Ha-ras 1 oncogene. We conclude that the methylation status of the promoter region of the human Ha-ras 1 modulates the expression and consequently the transforming activity of the oncogene.
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