RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells.
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.
Suzuki
Makoto
M
Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA.
Sunaga
Noriaki
N
Shames
David S
DS
Toyooka
Shinichi
S
Gazdar
Adi F
AF
Minna
John D
JD
D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human