DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance
methyltransferase gene. Recent studies indicate that inhibiting DNMT1
protein expression may be a useful approach for understanding the role of DNA methylation in
tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1
protein expression in NCI-H1299
lung cancer and HCC1954
breast cancer cells. RNA interference-mediated knockdown of DNMT1
protein expression resulted in >80% reduction of promoter methylation in RASSF1A,
p16(ink4A), and CDH1 in NCI-H1299; and RASSF1A,
p16(ink4A), and HPP1 in HCC1954; and re-expression of
p16(ink4A), CDH1, RASSF1A, and SEMA3B in NCI-H1299; and
p16(ink4A), RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The
small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the
tumor cells still were programmed for these epigenetic changes. We saw no effects on soft
agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as
tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates
tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and
breast cancer.