RNA Interference-Mediated Knockdown of DNA Methyltransferase 1 Leads to Promoter Demethylation and Gene Re-Expression in Human Lung and Breast Cancer Cells

Abstract

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 downregulate 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, p16ink4A , and CDH1 in NCI-H1299; and RASSF1A, p16ink4A , 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.

UHRF1‐mediated tumor suppressor gene inactivation in nonsmall cell lung cancer

“…DDNMT3B and RASSF1. The importance of both DNMT1 and DNMT3B expression in the maintenance of aberrant CpG island methylation of TSGs in mammalian cell models 20,[53][54][55] has been demonstrated. Our data together with previous reports [20][21][22] suggest that DNMT1 and DNMT3B can regulate DNA methylation in a promoter-specific manner.…”

Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells

“…Then, cells were treated or not with E2 or ICI for 16 h for qPCR and for 1 h for ChIP experiments. siRNA directed against DNMT1 were chosen as in (Suzuki et al, 2004) (Eurogentec).…”

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

“…Altered expressions of DNMTs have been linked to the cancer-associated hypermethylation of TSGs (Gius et al, 2004;Suzuki et al, 2004;Pruitt et al, 2005;Oka et al, 2006), as well as the hypomethylation of melanoma antigen gene MAGE-A1 (Loriot et al, 2006). We sought to determine which DNMT was involved in CSE-induced changes in the methylation pattern of SNCG.…”

“…DNMT1 is the most abundant form of DNMTs in mammalian cells. DNMT1 functions primarily in maintenance methylation during DNA replication using hemimethylated DNA as substrates (Lyko et al, 1999;Robertson et al, 1999;Rountee et al, 2000;Robert et al, 2003;Suzuki et al, 2004). The reactions of de novo methylation using unmethylated DNA are thought to be primarily catalysed by DNMT3A and DNMT3B in mammals during early development (Okano et al, 1999;Bachman et al, 2001;Brueckner and Lyko, 2004;Hermann et al, 2004;Dodge et al, 2005).…”