Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0596263 (carcinogenesis)
 64,820 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of methylation of CpG dinucleotides in the regulation of O6-methylguanine-DNA methyltransferase (MGMT) gene expression has been investigated. A previous observation, that cell lines deficient in MGMT (Mer-) are methylated in a SmaI site in the MGMT gene promoter whereas MGMT-expressing cells (Mer+) are unmethylated in the same site, has been extended to a total of 30 cell lines, tumors and normal tissues. To examine further the association between methylation in the MGMT promoter and the Mer- phenotype we have treated Mer+ and Mer- cell lines with 5-azacytidine to inhibit DNA methylation. Reduced methylation in the SmaI site coincided with induction of MGMT expression for one of three Mer- cell lines. MGMT increased several-fold further upon continued culture of the induced cells in the absence of 5-azacytidine, coincident with an abrupt increase in methylation in the body of the MGMT gene even though the SmaI site remained demethylated. These results, and those of other previous studies, suggest that methylation of sequences within the MGMT gene promoter and methylation within the body of the gene have opposite effects.
Carcinogenesis 1994 Apr
PMID:Effect of 5-azacytidine on expression of the human DNA repair enzyme O6-methylguanine-DNA methyltransferase. 751 91

Transgenic mice expressing DNA-repair genes are an instructive model with which to study the protective role of DNA-repair pathways in both spontaneous and chemical carcinogenesis. Of particular interest in chemical carcinogenesis is the DNA-repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which repairs O6-alkylguanine-DNA adducts. Transgenic mice carrying expression constructs for the alkyltransferase gene--either the human MGMT cDNA or the bacterial ada gene--express increased levels of alkyltransferase and have increased capacity to remove O6-methylguanine-DNA adducts. Protection from the DNA damaging effects of N-nitroso compounds occurs specifically in the cells and tissues in which the alkyltransferase transgene is expressed. For instance, mice carrying the PEPCKada construct have increased alkyltransferase in the liver and more rapid removal of O6methylguanine-DNA adducts. The protective effect is noted in hepatocytes, which express PEPCK-linked genes, not in nonparenchymal cells of the liver, which do not. Other tissues that express the transgene in the various models include the thymus, spleen, testes, muscle, stomach and brain. Mice expressing the human alkyltransferase in the thymus have a reduced incidence of thymic lymphomas following exposure to methyl nitrosourea (MNU), evidence of a role for this DNA-repair protein in protection from carcinogenesis due to N-nitroso compounds. Protection has also been observed in the induction of hepatic tumors by N-nitroso-dimethylamine (NDMA). These models will be used to identify whether overexpression of a single DNA-repair gene can block the carcinogenic process of N-nitroso compounds in many different tissues.
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PMID:Alkyltransferase transgenic mice: probes of chemical carcinogenesis. 751 28

The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins.
Carcinogenesis 1995 Feb
PMID:A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases. 753 16

Previous studies have demonstrated that O6-methylguanine-DNA methyltransferase (MGMT) is a major contributor to tumor cellular resistance toward chloroethylnitrosoureas. To further clarify the effect of MGMT gene expression on cellular chemosensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)-3-(2-chloroethyl)-3-nitrosourea (ACNU) in human cells, a repair-deficient human tumor cell line (HeLa MR) was transfected with a human MGMT expression vector (pSV2MGMT-neo). Multiple unique transfectants were isolated which exhibited variable levels of MGMT mRNA by Northern hybridization analysis. Vector-transfected controls were generated simultaneously. Transfectants expressing high levels of MGMT activity showed an increased resistance to ACNU-induced cytotoxicity. Furthermore, the levels of the protective effect against ACNU correlated generally with the levels of introduced MGMT expression. This study further provided direct evidence of MGMT contribution to ACNU resistance in human tumor cells. Based on the results presented here, we also discussed the perspective of the clinical utility of MGMT cDNA transfer and expression.
Carcinogenesis 1995 Sep
PMID:Transfer and expression of human O6-methylguanine-DNA methyltransferase cDNA confers resistance of Mer- HeLa MR cells to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a. 755 90

O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repair of alkylating agent-induced DNA damage. Among the alkylation products of DNA, O6-methylguanine is one of the most critical lesions leading to the induction of mutations. The enzyme MGMT transfers the methyl group from O6-methylguanine of DNA to its own cysteine residue. Although mutations of other DNA repair genes involved in nucleotide excision repair and mismatch repair have been proven to be related to human tumorigenesis, the question of whether MGMT gene mutation might play a role in human carcinogenesis has hitherto not been elucidated. If there is a population with decreased enzyme activity due to defective MGMT gene, the affected individuals should be at risk of developing cancer early in life because of an increased susceptibility to alkylating agents. To test this hypothesis, germ line mutations of the MGMT gene were investigated in 12 young patients with adult type cancers (mean, 16.7 years old, 8 hepatocellular carcinomas, 3 gastric cancers, 1 cholangiocellular carcinoma) and 28 elderly patients who died of non-cancer diseases as controls (mean, 66 years old). A point mutation at codon 160 in exon 5, GGA to AGA, converting glycine to arginine was found in three of the young patients (25%), while the same mutation was found in three out of 28 (10.7%) in the control group. The mutated codon was located 15 codons from a functional cysteine residue toward the carboxyl terminal. Investigation of enzyme function, even in cases of bi-allelic mutation, revealed comparable activities for both mutated and wild type MGMT. Thus, we conclude the mutation is a normal polymorphism of the MGMT gene, present in approximately 15% of the population, although this does not rule out a possible influence in other tissues.
Carcinogenesis 1995 Oct
PMID:A polymorphism at codon 160 of human O6-methylguanine-DNA methyltransferase gene in young patients with adult type cancers and functional assay. 758 49

Transgenic mice over-expressing MGMT, which codes for the human protein O6-alkylguanine-DNA alkyltransferase, are protected from methylating agent-induced thymic lymphomas. In this study we evaluated the ability of transgenic overexpression of MGMT in the colon to protect mice from the development of azoxymethane(AOM)-induced aberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferase in MGMT+ transgenic mice was > 5-fold higher than in nontransgenics: 10.5 +/- 1.1 vs 2.2 +/- 1.1 fmol/micrograms DNA, P = < 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15 mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons were examined for ACF. A significant protective effect of MGMT was seen in mice given single dose of 20 mg AOM/kg. The incidence of ACF/colon was lower in MGMT+ mice (2.0 +/- 1.2) than in nontransgenic mice (3.9 +/- 1.8, P = 0.02). G to A mutations in codon 12 of K-ras were detected by PCR-RFLP in ACF and in random samples of normal appearing mucosa. The incidence of ACF with mutant K-ras in MGMT transgenic mice (0.6 +/- 0.7/colon) was significantly reduced compared to nontransgenic mice (2.3 +/- 1.7/colon, P = 0.02). We propose that AOM induces at least two overlapping but not identical premalignant lesions (aberrant crypt foci and K-ras mutations) which can be prevented by over-expression of MGMT. Thus, MGMT may protect colonic mucosa from carcinogenesis involving methylating agents such as AOM.
Carcinogenesis 1995 Mar
PMID:Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon. 769 97

N-methylnitrosourea (MNU) induces thymic lymphoma in a high proportion of susceptible C57BL/6xSJL (C57/SJL) mice. Expression of the human DNA repair gene, MGMT cDNA, which encodes O6-methylguanine-DNA-methyltransferase, in transgenic mice effectively prevents MNU-induced thymic lymphomas. In this study, we determined the phenotype of thymocytes expressing the transgene and defined whether the target cell population for MNU induced lymphomas were actually those that expressed the transgene. Transgene expression was characterized by in situ hybridization for MGMT mRNA and immunohistochemistry for the human alkyltransferase protein and was compared to the phenotype of the MNU induced lymphomas. The MGMT transgene was expressed uniformly in immature cortical thymocytes that were CD4+CD8+J11d+ and to a lesser extent in the medullary thymocyte. Lymphomas were induced by single [50 or 80 mg/kg] or multiple doses [30 mg/kg x 5] of MNU to evaluate the dose response of tumor induction and protection by the MGMT-CD2 transgene. Forty-seven of the 108 treated mice developed lymphomas: 38 of 58 nontransgenic and 9 of 50 MGMT+ mice. The T-cell phenotype of thymic lymphomas was established by immunohistochemistry and FACS analysis. Most of the lymphomas were J11d+ (98%), 70% of the tumors were CD4+CD8+, 21% were CD4-CD8+, 9% were CD4-CD8-, and none were CD4-CD8-. All lymphomas in MGMT+ transgenic mice were CD4+CD8+. Since the main phenotype of MNU induced lymphomas in these mice, CD4+CD8+J11d+, is also the cell phenotype which expresses the MGMT-CD2 transgene at high levels, it appears that MGMT-induced protection has occurred in the cell target for MNU induced transformation.
Carcinogenesis 1995 May
PMID:The immature thymocyte is protected from N-methylnitrosourea-induced lymphoma by the human MGMT-CD2 transgene. 776 63

Adducts of O6-alkylguanine in DNA that are induced by cytotoxic, carcinogenic or mutagenic alkylating agents can be removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Human tumor cell lines that do not express this enzyme (Mer-) are hypersensitive to the effects of such alkylating agents, although the molecular basis of MGMT gene suppression is not yet understood. Previous studies suggested that Mer- cells deficient in this enzyme lack neither the gene nor the trans-acting factors necessary for normal transcription. Methylation of CpG dinucleotides is an attractive mechanism to account for suppression of the MGMT gene; however, there have been reports of both direct and inverse correlations between methylation and MGMT expression. We previously demonstrated an inverse correlation between methylation at a single SmaI site in the human MGMT promoter and gene expression. To substantiate this observation, we examined additional CpGs in the promoters of three Mer+ and three Mer- cell lines, using rare methylation-sensitive restriction sites, and then sought to identify the region where methylation correlated with gene expression. Six CpGs in the region from -245 bp to +225 bp (relative to the transcription start site) were completely unmethylated in all Mer+ cells, whereas in Mer- cells were at least partially methylated. The methylation status of CpGs further upstream did not correlate with MGMT expression. We conclude, therefore, that the association between CpG methylation and suppressed MGMT gene activity extends to sites other than SmaI but is limited to a core region of the promoter.
Carcinogenesis 1995 Jun
PMID:Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression. 778 59

Alcohol has clearly been associated with an increase of cancers in numerous tissue, including the respiratory tract, colon, rectum, liver, but especially the esophagus, larynx, pharynx, and mouth. Alcohol alone has not been shown to be a mutagen until it is converted to acetaldehyde and, therefore, alcohol presumably acts as a cocarcinogen. Previous data has shown that alcohol concentrations of 2% or greater inhibits DNA repair, and in light of the widespread consumption of alcoholic beverages with alcohol contents ranging from 4 to 5% (beer and wine coolers) to 50% (whiskey), interest in determining the mechanism(s) responsible for alcohol-induced carcinogenesis has heightened. Although previous studies, in intact rats, have investigated the effects of chronic alcohol exposure on some aspects of DNA repair, we have begun to address the effects of acute or "binge" alcohol exposure on mammalian DNA repair. Toward this end, we report the inhibition of O6-methylguanine-DNA methyltransferase (MGMT) by a single intraperitoneal injection of 30% ethanol in adult male castrated rats. This inhibition lasted for at least 24 hr. We also observed a dose-response effect of ethanol on MGMT activity, again only in the castrated rats. The finding of ethanol's effect on MGMT activity in castrated and not intact rats implies a hormonal component of MGMT DNA repair response, which has only been alluded to in past research.
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PMID:Acute ethanol exposure suppresses the repair of O6-methylguanine DNA lesions in castrated adult male rats. 784 18

To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (> 50% of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5% and 0.8% of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.
Carcinogenesis 1994 Sep
PMID:The role of two conserved amino acids, glutamine 90 and asparagine 137, in O6-methylguanine-DNA methyltransferase stability, activity and substrate specificity. 792 83


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