Expression profiling of T24 cells revealed that 17 out of 313 human miRNAs were upregulated more than 3-fold by simultaneous treatment with the chromatin-modifying drugs 5-aza-2'-deoxycytidine and 4-phenylbutyric acid. One of these, miR-127, is embedded in a CpG island and is highly induced from its own promoter after treatment. miR-127 is usually expressed as part of a miRNA cluster in normal cells but not in cancer cells, suggesting that it is subject to epigenetic silencing. In addition, the proto-oncogene BCL6, a potential target of miR-127, was translationally downregulated after treatment. These results suggest that DNA demethylation and histone deacetylase inhibition can activate expression of miRNAs that may act as tumor suppressors.

Previous studies have shown that DNA methyltransferase (Dnmt) 1 is required for maintenance of bulk DNA methylation and is essential for mouse development. However, somatic disruption of DNMT1 in the human cancer cell line HCT116 was not lethal and caused only minor decreases in methylation. Here, we report the identification of a truncated DNMT1 protein, which was generated by the disruption of DNMT1 in HCT116 cells. The truncated protein, which had parts of the regulatory N-terminal domain deleted but preserved the catalytic C-terminal domain, was present at different levels in all DNMT1 single-knockout and DNMT1͞DNMT3b doubleknockout cell lines tested and retained hemimethylase activity. DNMT1 RNAi resulted in decreased cell viability in WT and knockout cells and further loss of DNA methylation in DNMT1 knockout cells. Furthermore, we observed a delay in methylation after replication and an increase in hemimethylation of specific CpG sites in cells expressing the truncated protein. Remethylation studies after drug-induced hypomethylation suggest a putative role of DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, which is lost in cells lacking full-length DNMT1. Our data suggest that DNMT1 might be essential for maintenance of DNA methylation, proliferation, and survival of cancer cells. DNA methylation ͉ epigeneticT he biological roles of the major mammalian DNA methyltransferase (Dnmt), DNMT1, have been enigmatic. Although gene-targeting studies in mice have clearly demonstrated an essential function of Dnmt1 in embryonic development, cell survival, and tumorigenesis (1), there have been controversial reports regarding the function of this enzyme in human cancer cells. In mice, Dnmt1 has been implicated in maintaining the majority of bulk DNA methylation, differentiation of ES cells, and imprinting (2, 3). Furthermore, deletion of Dnmt1 in mouse embryonic fibroblasts caused a decrease in genomic methylation, p53-dependent apoptosis, and deregulation of transcription (4). Heterozygosity for Dnmt1 in combination with administration of the DNMT inhibitor 5-aza-2Јdeoxycytidine (5-aza-CdR) greatly reduced the number of polyps in a mouse model for intestinal neoplasia (5).A series of RNAi experiments for DNMT1 have been described for various human cancer cell lines, which have shown apparently inconsistent results in regard to DNA methylation of tumor suppressor genes (6-10). The differences might be attributed to the techniques used but also to distinct sensitivities of individual cell lines (10). Furthermore, RNAi may not cause a complete depletion of the protein, so residual protein might still be available and capable of maintaining DNA methylation. Rhee et al. (11,12) generated a widely used series of HCT116 colon cancer cells with homozygous deletions for DNMT1 (DNMT1 Ϫ/Ϫ ) (11), DNMT3b (DNMT3b Ϫ/Ϫ ) (12), or both DNMT1 and DNMT3b (12) [double knockout (DKO)]. Surprisingly, somatic disruption of DNMT1 resulted in only a 20% decrease in overall genomic methylation with no discernible changes i...

DNA hypomethylation on pericentromeric satellite regions is an early and frequent event associated with heterochromatin instability during human hepatocarcinogenesis. A DNA methyltransferase, DNMT3b, is required for methylation on pericentromeric satellite regions during mouse development. To clarify the molecular mechanism underlying DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis, we examined mutations of the DNMT3b gene and mRNA expression levels of splice variants of DNMT3b in noncancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions, and in hepatocellular carcinomas (HCCs). Mutation of the DNMT3b gene was not found in HCCs. Overexpression of DNMT3b4, a splice variant of DNMT3b lacking conserved methyltransferase motifs IX and X, significantly correlated with DNA hypomethylation on pericentromeric satellite regions in precancerous conditions and HCCs (P ‫؍‬ 0.0001). In particular, the ratio of expression of DNMT3b4 to that of DNMT3b3, which is the major splice variant in normal liver tissues and retains conserved methyltransferase motifs I, IV, VI, IX, and X, showed significant correlation with DNA hypomethylation (P ‫؍‬ 0.009). Transfection of human epithelial 293 cells with DNMT3b4 cDNA induced DNA demethylation on satellite 2 in pericentromeric heterochromatin DNA. These results suggest that overexpression of DNMT3b4, which may lack DNA methyltransferase activity and compete with DNMT3b3 for targeting to pericentromeric satellite regions, results in DNA hypomethylation on these regions, even in precancerous stages, and plays a critical role in human hepatocarcinogenesis by inducing chromosomal instability. D NA methylation plays important roles in gene silencing, chromatin remodeling, and genome stability (1-4). Aberrant DNA methylation is one of the most consistent epigenetic changes in human cancers (1-4). Generally, the overall level of DNA methylation is lower in cancer cells than in normal cells (5, 6), although a number of tumor suppressor genes are silenced by DNA methylation on CpG islands around their promoter regions in cancer cells (1-4, 7, 8).We have carefully examined alterations of DNA methylation status on pericentromeric satellite regions and CpG islands of specific genes, and expression of DNA methyltransferases and methyl-CpG-binding proteins, in noncancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions (9, 10), and in hepatocellular carcinomas (HCCs) (11-17). Among these alterations, DNA hypomethylation on pericentromeric satellite regions was detected even in precancerous conditions and appears to be one of the earliest epigenetic changes during human hepatocarcinogenesis (17). Satellite regions are located in pericentromeric heterochromatin DNA, and DNA hypomethylation on these regions is known to result in centromeric decondensation, enhancing chromosome recombinations (18,19). In fact, frequent chromosome 1q copy gain wi...

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methylCpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs). The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarci- Aberrant DNA cytosine methylation is one of the most consistent epigenetic changes in human cancers. 1 Generally, the overall DNA methylation level is lower in cancer cells than in normal cells. 2 However, some loci tend to show increased DNA methylation in cancer cells, 3-6 whereas others are often hypomethylated in human cancers. 7 DNA methylation may play roles in carcinogenesis by virtue of 3 mechanisms: 1) DNA cytosine methylation facilitates gene mutation, as 5-methylcytosine is deaminated to thymine 8 ; 2) aberrant DNA methylation may be associated with allelic loss 3-7,9-11 ; and 3) DNA methylation occurs frequently in CpG islands near regulatory regions of genes and affects the transcription of specific genes. 1,12,13 Recently, 2 types of DNA methylation were described; methylation of CpG islands in a cancer-specific manner (type C methylation) differs from age-dependent methylation (type A methylation). 14 CpG islands of the p16 and hMLH1 genes and the methylated in tumor (MINT) 1, 2, 12, 25, and 31 clones show type C methylation, 14 although the genes whose expression were regulated by MINT clones were not identified. A high level of type C methylation in human cancer has been referred to as the CpG island methylator phenotype (CIMP). 14,15 With respect to hepatocarcinogenesis, we reported previously that aberrant DNA methylation on chromosome 16, a hot spot for loss of heterozygosity (LOH) in hepatocellular carcinomas (HCCs), is observed frequently in chroni...

Alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. DNA methyltransferase (DNMT) 1 is a major enzyme involved in establishing genomic methylation patterns. Most of the studies concerning DNMT1 expression in human cancers have been performed only at the mRNA level. To directly examine DNMT1 protein expression levels during human hepatocarcinogenesis, 16 histologically normal liver tissues, 51 noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 53 hepatocellular carcinomas (HCCs) were subjected to immunohistochemic examination. If more than 20% of the cells exhibited nuclear DNMT1 staining, the tissue sample was considered to be DNMT1-positive. DNMT1 immunoreactivity was observed in 23 (43%) of the HCCs, but in none (0%) of the histologically normal liver or noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis. The incidence of increased DNMT1 protein expression in HCCs correlated significantly with poor tumor differentiation (p ‫؍‬ 0.0006) and portal vein involvement (p ‫؍‬ 0.0002). Moreover, the recurrence-free (p ‫؍‬ 0.0001) and overall (p < 0.0001) survival rates of patients with HCCs exhibiting increased DNMT1 protein expression were significantly lower than those of patients with HCCs that did not exhibit increased expression. Increased DNMT1 protein expression may play a critical role in the malignant progression of HCCs and be a biologic predictor of both HCC recurrence and a poor prognosis in HCC patients. © 2003 Wiley-Liss, Inc. Key words: DNA methylation, chronic hepatitis, liver cirrhosis, hepatocarcinogenesisAberrant DNA methylation is one of the most consistent epigenetic changes in human cancers. [1][2][3][4] Generally, the overall DNA methylation level is lower in cancers than in normal tissues. 5 However, some loci tend to exhibit increased DNA methylation in cancers, 6 -8 whereas others are often hypomethylated. 9 DNA methylation may play a role in carcinogenesis by virtue of 3 mechanisms: (i) DNA cytosine methylation facilitates gene mutation, as 5-methylcytosine is deaminated to thymine; 10 (ii) aberrant DNA methylation may be associated with allelic loss 9,11-13 and (iii) DNA methylation occurs frequently in CpG islands near regulatory regions of genes and affects the transcription of specific genes. [1][2][3][4] To date, 3 enzymes that possess DNA methyltransferase (DNMT) activity-DNMT1, 14 DNMT3a and DNMT3b, 15 -have been confirmed. Of these, DNMT1 is the major and best known. As DNMT1 has a preference for hemimethylated, rather than unmethylated, substrates in vitro 16 and targets replication foci by binding to proliferating cell nuclear antigen (PCNA), 17 it was recognized as the "maintenance" DNMT that copies methylation patterns after DNA replication. However, some researchers have proposed that DNMT1 possesses both maintenance and de novo DNA methylation activity in vivo, regardless of its preference for substrates in vitro. 4,18 Moreover, recent studies...