Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16ink4A and p21WAF/Cip1 by distinct mechanisms

A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5' CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzyme (DNA MeTase) are also prevalent features of human neoplasia, However, direct evidence that elevated DNA MeTase levels alter gene expression and influence oncogenesis has been difficult to obtain, in part due to the lack of specific DNA MeTase inhibitors. Here we show that specific reduction of cellular DNA MeTase levels in human cancer cells with potent antisense inhibitors: 1) causes demethylation of the p16(ink4A) gene promoter; 2) causes re-expression of the p16(ink4A) protein; 3) leads to accumulation of the hypophosphorylated form of the retinoblastoma protein (pRb); and 4) inhibits cell proliferation. Stepwise reduction of cellular DNA MeTase protein levels also induced a corresponding rapid increase in the cell cycle regulator p21(WAF/Cip1) protein demonstrating a regulatory link between DNA MeTase and the growth regulator p21(WAF/Cip1) that is independent of methylation of DNA. These results suggest that the elevated levels of DNA MeTase seen in cancer cells can inhibit tumor suppressors by distinct mechanisms involving either transcriptional inactivation through DNA methylation or by a methylation independent regulation.