EXPRESSION OF PROKARYOTIC HHAI DNA METHYLTRANSFERASE IS TRANSFORMING AND LETHAL TO NIH 3T3 CELLS

In neoplastic cells, levels of DNA methyltransferase activity are ofte n increased, and evidence is accruing to suggest an important role for this event in tumorigenesis, To evaluate this possibility further, an d to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bac terial HhaI methyltransferase in cultured murine fibroblasts. This enz yme is a pure de novo DNA methyltransferase that methylates the intern al C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to t he cells, However, surviving cell clones that express low levels of M, HhaI demonstrate increased tumorigenicity as assessed by soft agar cl oning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0 .4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 ce lls versus 0% for a mutant HhaI control) and tumorigenicity in nude mo use heterotransplants (75% for sense HhaI-transduced PA 317 cells vers us 18.5% for antisense controls), DNA isolated from the clonogenic sen se HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7 -38.9) increase in methylcytosine content at GCGC sites, These finding s suggest that eukaryotic cells tolerate a narrow window of increased ne novo DNA methylating capacity, above which cell death occurs and wi thin which cell transformation results, Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.