STABILIZATION OF DNA METHYLTRANSFERASE LEVELS AND CPG ISLAND HYPERMETHYLATION PRECEDE SV40-INDUCED IMMORTALIZATION OF HUMAN FIBROBLASTS

De novo methylation of normally unmethylated CpG islands and increased expression of DNA (cytosine-5-)-methyltransferase (DNA MTase) are com mon characteristics of immortalized cell lines and human tumors. To ex amine the acquisition of these properties with respect to cellular imm ortalization, we studied CpG island methylation and DNA MTase expressi on in aging normal human fibroblasts and their SV40-infected preimmort al (precrisis) and immortal (postcrisis) derivatives. The levels of DN A MTase enzyme activity decreased by 50% as normal fibroblasts were cu ltured to senescence. By contrast, DNA MTase activity did not decrease in SV40-infected pre- or postcrisis cells but remained similar to tha t of young fibroblasts and 2-4-ford higher than that of senescent fibr oblasts. DNA MTase mRNA levels paralleled those of enzyme activity. Se veral loci were examined to determine the relationship between the dyn amics of DNA MTase expression and the appearance of de novo CpG island methylation. Ten CpG island loci examined were unmethylated in normal young fibroblasts. By contrast, four of these loci (the CALC1, MyoD, and IGF-2 genes on chromosome lip and the estrogen receptor gene on ch romosome 6q) were de novo methylated in fully immortalized, postcrisis cells. Two of these four were actually methylated in extended life sp an precrisis cells, and one, the estrogen receptor locus, exhibited de novo methylation with aging in normal fibroblasts. The data indicate that an ability to maintain DNA MTase levels is acquired with SV40-ind uced escape from senescence. Furthermore, aberrant CpG island methylat ion can be established prior to immortalization, either as a function of normal aging or in response to SV40-induced escape from senescence.