Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA in mammalian cells is enzymatically methylated at the 5-position of cytosine via S-adenosylmethionine and DNA methyltransferase. Several chemical carcinogens have been shown to inhibit this reaction, altering DNA methylation. We have been studying the mechanism by which carcinogens alter the methylation of DNA in order to better understand the cellular regulation of DNA methylase activity and to understand the role, if any, of DNA methylation in the carcinogenic process. We have utilized an in vitro assay for DNA methylase isolated from purified rat-liver nuclei. Ethionine, a liver carcinogen, given to rats 17 hr after partial hepatectomy inhibited the incorporation of [methyl-3H]-methionine into 5-methylcytosine residues of DNA. DNA isolated from these ethionine-treated rats was able to accept methyl groups from S-adenosylmethionine 8 times more than control DNA. It was further demonstrated that S-adenosylethionine competitively inhibited the DNA methylase resulting in hypomethylated DNA. N-Methyl-N-nitro-N-nitrosoguanidine reacted with the DNA methylase at the sulfhydryl sites inactivating the enzyme. Methylnitrosourea did not react directly with the methylase enzyme, but when reacted with DNA, the DNA methylase activity was inhibited by the carcinogen alkylated DNA. Sodium selenite also inhibited the enzyme non-competitively with a Ki of 6.7 microM. 5-Azacytidine prevented the 2 to 3 fold increase in DNA methylase seen 2 days following partial hepatectomy. All of these data with various carcinogens, altering DNA methylation by different mechanisms, support the hypothesis that DNA methylation plays a role in the initiation of carcinogenesis.
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PMID:Studies on DNA methyltransferase and alteration of the enzyme activity by chemical carcinogens. 243 29

Azacitidine is a pyrimidine ring analog of cytidine that is incorporated into RNA causing alteration in RNA synthesis and processing and resulting in inhibition of protein synthesis. Azacitidine as the deoxynucleotide is also incorporated into DNA inhibiting its synthesis and blocking cytosine methylation by noncompetitive inhibition of DNA methyltransferase. The resulting hypomethylation of DNA is thought to induce gene activation and expression and cell differentiation. This may be an underlying factor in azacitidine's antileukemic activity and also contributes to its carcinogenic and tumor-promoting properties in experimental models.
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PMID:Biochemistry of azacitidine: a review. 244 43

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.
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PMID:Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells. 615 82

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.
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PMID:Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes. 617 80

One of the most readily quantitated indices of myeloid maturation in HL-60 cells is their ability to respond to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity. HL-60 cells exposed to the antileukemic drug, 5-azacytidine (3 to 5 microM) for 24 hr and subsequently cultured in its absence for 2 to 3 days develop an enhanced ability to respond to 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity detectable as an increase both in hexose monophosphate shunt activity and in the proportion of the population producing superoxide anion. 5-Azacytidine treatment also causes marked inhibition of DNA methyltransferase, and thus DNA synthesized by HL-60 cells during the 24-hr period of analogue treatment is essentially devoid of methylated cytosine residues. This suggests, as does our previous finding that a general inhibitor of transmethylation reactions, L-ethionine, can induce differentiation of HL-60 cells, that changes in gene expression triggered by these compounds may be linked to their ability to alter patterns of DNA methylation. Since at least 50% of HL-60 cells capable of forming colonies in soft agar after a 24-hr exposure to 5-azacytidine yield progeny that mature (i.e., produce superoxide anion in response to 12-O-tetradecanoylphorbol-13-acetate) 2 weeks after 5-azacytidine treatment, the results also indicate that the changes induced in HL-60 cells by limited exposure to 5-azacytidine are heritable and can influence gene expression many generations after treatment has been terminated.
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PMID:Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (HL-60). 618 56

We have previously shown that treatment of normal and neoplastic cells with the antileukemic drug, 5-azacytidine, led to the rapid synthesis of a low molecular weight RNA containing 5-azacytosine. This fraudulent RNA inhibited tRNA (cytosine-5)-methyltransferase early after drug administration. The absence of tRNA (cytosine-5)-methyltransferase activity resulted in the synthesis of tRNA specifically deficient in 5-methylcytosine. Here, we show that treatment of L1210 cells, grown intraperitoneally in mice, with 5-azacytidine led to a rapid and prolonged inactivation of DNA (cytosine-5)-methyltransferase activity and to the synthesis of undermethylated DNA. DNA isolated from the treated tissue was found to inactivate the DNA methylase (decreased Vmax) in in vitro DNA (cytosine-5)-methyltransferase assays. Kinetic analysis showed noncompetitive inhibition of the substrate by the inhibitor. The persistence of DNA undermethylation after treatment with 5-azadeoxycytidine or 5-azacytidine in animals has not been measured directly; therefore, we have investigated this phenomenon in the intact animal. Prolonged treatment with 5-azacytidine was required to maintain a a fraction of undermethylated sites in DNA of L1210 cells in vivo for up to 4 months or longer after drug withdrawal. Such treatment led to instability of DNA methylation levels in L1210 cells in vivo. At least a partial restoration of DNA 5-methylcytosine levels was observed after acute and chronic 5-azacytidine treatment, respectively. 5-Azacytidine was also found to induce DNA hypomethylation in regenerating, but not in normal adult mouse liver cells. Our results show that: 1) it was extremely difficult to decrease the DNA methylation level to less than 50% of control; and 2) it was also difficult to maintain stable DNA methylation levels in vivo after exposure to the drug.
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PMID:Long term instability and molecular mechanism of 5-azacytidine-induced DNA hypomethylation in normal and neoplastic tissues in vivo. 620 75

5-Azacytidine (5-aza-CR) and 5-aza-2'-deoxycytidine (5-aza-CdR), analogs of cytidine modified in position 5, were originally developed as antitumor agents, and have been useful in the treatment of both childhood and adult leukemias. These agents are cytotoxic per se, but also induce differentiation in several experimental systems, most notably the induction of muscle, adipocytes, and chondrocytes in cultures of drug-treated mouse embryo fibroblasts. The mechanisms underlying this drug-induced differentiation have been difficult to unravel, a fact which limits the rational design of differentiation therapy as a modulator of cancer using these agents. Induction of new developmental pathways in cultured cells involves stable, heritable changes, presumably of an epigenetic nature. Our early studies demonstrated that changes in methylation of cytosine in DNA occurred concurrently with changes in developmental potential, and that the presence of 5-azacytosine in DNA interfered with the action of DNA methyltransferase. Since DNA methylation is believed to be involved at some level in the regulation of gene expression, the hypothesis was developed that changes in methylation allowed the expression of new genes whose activity initiated new pathways of differentiation. The characterization of this drug-induced system of differentiation has therefore opened the way to identifying genes directly involved in the initiation or modification of pathways of differentiation. The first of these was MyoD, a member of a family of myogenic determination genes. Expression of MyoD in myogenic cell lines has been correlated with loss of methylation at specific sites in the genome, but the critical events leading to expression of MyoD and muscle differentiation are poorly understood. Recent developments in understanding this mechanism are discussed.
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PMID:5-Aza-2'-deoxycytidine: cell differentiation and DNA methylation. 768 53

5-Azacytidine inhibits DNA synthesis and to a lesser proportion RNA synthesis in S. antibioticus. The biosynthesis of proteins is not affected. The main inhibitory effect of 5-azacytidine on DNA and RNA synthesis is probably caused by its incorporation into newly synthesized DNA or RNA and the formation of covalent complexes between cytosine-specific methyltransferases and the modified DNA or RNA templates. To analyze whether such effects could occur at the oriC region of S. antibioticus we analyzed the methylation status of this region using the bisulphite assisted genomic sequencing method. One of the cytosine residues found to be partially methylated was contained within an unique NaeI sequence (GCCGGC) in oriC. Subsequent analysis shows chromosomal DNA from S. antibioticus to be resistant to R.NaeI restriction indicating that this strain contains a NaeI-specific cytosine C5-methyltransferase activity. Following 5-azacytidine treatment the NaeI site within the oriC region becomes partially demethylated. Our results suggest that some of the 5-azacytidine effects on DNA and RNA synthesis might indeed be related to the complex formation and inhibition of a cytosine-specific DNA methyltransferase.
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PMID:Analysis of DNA methylation processes related to the inhibition of DNA synthesis by 5-azacytidine in Streptomyces antibioticus ETH 7451. 962 53

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.
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PMID:Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector. 1157 73

Accumulation of genetic changes characterizes the progression of cells, initiated by carcinogens, to full malignancy. Various epigenetic mechanisms, such as high polyamine synthesis, aberrant DNA methylation, and production of reactive oxygen species, may favor this process by stimulating growth and inducing DNA damage. We observed a decrease in S-adenosyl-L-methionine (SAM) content in the liver, associated with DNA hypomethylation in rat liver, during the development of preneoplastic foci, and in neoplastic nodules and hepatocellular carcinomas, induced in diethylnitrosamine-initiated rats by "resistant hepatocyte" (RH) protocol. Reconstitution of the methyl donor level in the liver by SAM administration inhibits growth and induces phenotypic reversion and apoptosis of preneoplastic cells. A 6-month SAM treatment results in a sharp and persistent decrease in development of neoplastic nodules, suggesting a long duration of SAM chemopreventive effect. Various observations support the suggestion of a role of DNA methylation in chemoprevention by SAM: (1) Exogenous SAM reconstitutes the SAM pool in preneoplastic and neoplastic liver lesions. (2) DNA methylation is positively correlated with SAM:S-adenosylhomocysteine (SAH) ratio in these lesions. (3) 5-Azacytidine, a DNA methyltransferase inhibitor, inhibits chemoprevention by SAM. (4) c-Ha-ras, c-Ki-ras, and c-myc are hypomethylated and overexpressed in preneoplastic liver. Their expression is inversely correlated with SAM:SAH ratio in SAM-treated rats. (5) S-Adenosyl-L-methionine treatment results in overall DNA methylation and partial methylation of these genes. Other possible mechanisms of SAM treatment include inhibition of polyamine synthesis, linked to partial transformation of SAM into 5'-methylthioadenosine (MTA), and antioxidant and antifibrogenic activities of both SAM and MTA.
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PMID:Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine. 1216 49


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