DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells

Abstract

Transcriptional silencing by CpG island methylation is a prevalent mechanism of tumor-suppressor gene suppression in cancers. Genetic experiments have defined the importance of the DNA methyltransferase Dnmt1 for the maintenance of methylation in mouse cells and its role in neoplasia. In human bladder cancer cells, selective depletion of DNMT1 with antisense inhibitors has been shown to induce demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A. In contrast, targeted disruption of DNMT1 alleles in HCT116 human colon cancer cells produced clones that retained CpG island methylation and associated tumor-suppressor gene silencing, whereas HCT116 clones with inactivation of both DNMT1 and DNMT3B showed much lower levels of DNA methylation, suggesting that the two enzymes are highly cooperative. We used a combination of genetic (antisense and siRNA) and pharmacologic (5-aza-2'-deoxycytidine) inhibitors of DNA methyl transferases to study the contribution of the DNMT isotypes to cancer-cell methylation. Selective depletion of DNMT1 using either antisense or siRNA resulted in lower cellular maintenance methyltransferase activity, global and gene-specific demethylation and re-expression of tumor-suppressor genes in human cancer cells. Specific depletion of DNMT1 but not DNMT3A or DNMT3B markedly potentiated the ability of 5-aza-2'-deoxycytidine to reactivate silenced tumor-suppressor genes, indicating that inhibition of DNMT1 function is the principal means by which 5-aza-2'-deoxycytidine reactivates genes. These results indicate that DNMT1 is necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells.

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.…”

Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis

“…Following incorporation into DNA, 5-azadC promotes DNA hypomethylation by inhibiting the activity of the maintenance DNA methyltransferase DNMT1. 29 Recent evidence suggests that Antibody used in ChIP Figure 5 ChIP on crosslinked chromatin reveals ability of 5-azadC to modify histones associated with IkBa promoter in vivo. Crosslinked chromatin was prepared from control (denoted by a minus sign) or 48 h 5-AzdC treated (denoted by a plus sign) LX2 cells as described in Materials and methods.…”

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins

“…Numerous studies have implicated both DNMT1 and DNMT3B in the altered distribution of DNA methylation in cancer cells (Beaulieu et al, 2002;Robert et al, 2003). A human colon cancer cell line, HCT116 deficient in DNMT1 following targeted recombination, demonstrates demethylation of pericentromeric satellite sequences (Rhee et al, 2000), whereas HCT116 cells lacking both DNMT1 and DNMT3B contain demethylated satellite 2 and Alu repetitive sequences (Rhee et al, 2002) and demonstrate increased chromosomal instability (Karpf and Matsui, 2005).…”