Effect of carcinogen ethionine on enzymatic methylation of DNA sequences with various degrees of repetitiveness.

Abstract When P 815 mastocytoma cells are cultivated in McCoy 5 a medium in the presence of ethionine (0.0075–6 mM), neither inhibition of cell proliferation nor of DNA synthesis was observed. In order to study the effect of this carcinogen on enzymatic DNA methylation the cells were labelled simultaneously with l -(methyl- 3 H) -methionine and ( 14 C) deoxycytidine. The fractionation of isolated DNA was based on the reassociation kinetics of inverted repetitive sequences of type ABC ... CBA, ordinary repetitive sequences of type ABC ... ABC ... ABC, intermediary sequences and the unique sequences. The individual fractions were then hydrolyzed in 96% formic acid and the DNA bases were separated by paper chromatography. The relative rates of enzymatic DNA methylation were computed on the basis of ( 3 H) radioactivity in 5 -methylcytosine and ( 14 C) radioactivity in cytosine. They showed that the inverted repetitive sequences are approximately 50% higher methylated than the ordinary repetitive sequences, and about 300% higher than the intermediary and unique ones. In cells grown in presence of ethionine, this pattern changes dramatically. The enzymatic methylation of inverted repetitive sequences decreases to the level of methylation of ordinary repetitive ones, at a concentration as low as 0.01 mM . Although the other sequence classes are lower methylated in cells grown in presence of ethionine than in controls, the effect of this compound here is less profound. Because of a possible regulatory role of the inverted repetitive sequences in mammalian genome their hypomethylation may be related to ethionine induced re-expression of certain genes.