DE-NOVO METHYLATION OF CPG ISLAND SEQUENCES IN HUMAN FIBROBLASTS OVEREXPRESSING DNA (CYTOSINE-5)-METHYLTRANSFERASE

Recent studies showing a correlation between the levels of DNA (cytosi ne-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicit y have implicated this enzyme in the carcinogenic process. Moreover, h ypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progressio n. One proposed role for DNA MTase in tumorigenesis is therefore a dir ect role in the de novo methylation of these otherwise unmethylated Cp G islands. In this study, we sought to determine whether increased lev els of DNA MTase could directly affect CpG island methylation. A full- length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50 -fold the level of DNA MTase protein and enzyme activity of the parent al cell line or clones transfected with the control vector alone (Neo) . To determine the effects of DNA MTase overexpression on CpG island m ethylation, we examined 12 endogenous CpG island loci in the HMT clone s. HMT clones expressing greater than or equal to 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relati ve to at least 11 Neo clones at five CpG island loci. In the HMT clone s, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylat ion status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de n ovo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression throu gh CpG island methylation-mediated gene inactivation.