5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of antimetabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However, because of 5-azacytidine's general toxicity, other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that, when present in DNA, it inhibited DNA methylation, led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here, the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed.
Purpose: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (À)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation. Experimental Design: Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor h (RARb), p16INK4a , and O 6 -methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2V -deoxy-5-aza-cytidine (5-aza-dCyd). Results: Genistein (2-20 Amol/L) reversed DNA hypermethylation and reactivated RARb, p16INK4a , and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARb by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 Amol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate-and methyl donor^dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARb, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth. Conclusions:These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.
Natural killer (NK)-cell malignancies are among the most aggressive lymphoid neoplasms with very poor prognosis. We performed array comparative genomic hybridization analysis on a number of NK cell lines and primary tumors to gain better understanding of the pathogenesis and tumor biology of these malignancies. We also obtained transcriptional profiles of genes residing in these regions and compared them with normal and activated NK cells. Only 30-50% of the genes residing in the gained or deleted regions showed corresponding increased or decreased expression. However, many of the upregulated genes in regions of gain are functionally important for the proliferation and growth of the neoplastic population. Genes downregulated in regions of loss included many transcription factors or repressors, tumor suppressors or negative regulators of the cell cycle. The minimal common region of deletion in 6q21 included three known genes (PRDM1, ATG5 and AIM1) showing generally low expression. Mutations resulting in truncated PRDM1 and changes in conserved amino-acid sequences of AIM1 were detected. Highly methylated CpG islands 5 0 of PRDM1 and AIM1 correlated with low expression of the transcripts. Reversal of methylation by Decitabine induced expression of PRDM1 and cell death. In conclusion, we have shown a general tumorpromoting effect of genetic alterations and have identified PRDM1 as the most likely target gene in del6q21. ATG5, an essential gene for autophagy and AIM1, a gene implicated in melanoma, may also participate in the functional abnormalities.
Transposable elements such as long terminal repeats (LTR) constitute about 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity, and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles, transposition events, and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.
As we have reported previously, both DNA and tRNA become hypomethylated in livers of rats fed a cancer promoting, methyl-deficient diet (MDD) for as short a period as one week. Within the same period, activities of tRNA and DNA methyltransferases (MTases) increase and levels of mRNAs for several genes believed to have roles in growth regulation are altered. These diet-induced changes in nucleic acid methylation and gene expression increased in extent when MDD was fed continuously for four weeks. We also observed hypomethylation of specific CCGG sites within several genes for which mRNA levels were increased. These included c-myc, c-fos and c-Ha-ras. To investigate the reversibility of such diet-induced alterations in methylation and gene expression, animals were fed MDD for four weeks, after which a diet supplemented with adequate sources of methyl groups (CSD) was fed for 1-3 weeks. One to two weeks after the restoration of an adequate diet, the overall extent of methylation of tRNA and DNA from livers of these rats did not differ from that of tRNA and DNA from livers of age matched animals continually maintained on CSD. At the same time, activities of MTases in the liver dropped to normal values. Levels of mRNAs for all genes studied returned to control levels within three weeks after ending MDD feeding, although at different rates. In contrast, MDD-induced hypomethylation of some HpaII sites in c-myc, c-fos and c-Ha-ras genes persisted after 3 weeks refeeding of an adequate diet. These results, which demonstrate that most of the effects of MDD on the parameters we have studied occur rapidly and are essentially reversible, are consistent with the role of MDDs as promoters of hepatocarcinogenesis. However, the finding that unmethylated sites persist in genes that play a role in growth regulation suggests a mechanism by which intermittent or long term exposure to MDDs could result in heritable phenotypic changes in some hepatocytes that lead to hyperplasia and tumorigenesis.
DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b -/-lymphomas, but not in Dnmt3b -/-pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b -/-lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b +/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.
This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4 mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25 -30 of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and er y thr oid differentiation.At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division.To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from F L cells induced to differentiate with dimethylsulfoxide and butyrate.In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or MezSO cannot be methylated in vitro using homologous enzymes.DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.
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