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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

O6-Methylguanine is a substrate of the DNA repair enzyme O6-methylguanine-DNA methyltransferase, which is involved in the repair mechanism of DNA damage induced by chloroethylnitrosoureas such as 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). We tested the enhancement effect of O6-methylguanine pretreatment on ACNU cytotoxicity in ACNU-resistant brain tumors. Exposure to O6-methylguanine at various times ranging from 2 to 48 hours increased the cytotoxic effects of ACNU on C6-1 cells, and this effect was highest at higher concentrations 500 and 1,000 microM. Colorimetric cytotoxicity assay revealed at least a two-fold increase in ACNU cytotoxicity relative to controls without O6-methylguanine. Intraarterial ACNU after treatment with O6-methylguanine (two intravenous bolus injections of 80 and 40 mg/kg) significantly (P < 0.05 or P < 0.01) reduced the proliferation activity of transplanted C6-1 tumors for 96 hours after injection, whereas intravenous ACNU together with O6-methylguanine significantly (P < 0.05) reduced C6-1 activity for only 48 hours. Thus, pretreatment with O6-methylguanine prolonged the suppression effect of ACNU. The C6-1 tumors treated only with intravenous or intraarterial ACNU showed transient inhibition and rapid regrowth for 24 hours after treatment. These results indicate that O6-methylguanine increases ACNU cytotoxicity in an in vitro and in vivo brain tumor model.
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PMID:Enhancement of ACNU cytotoxicity by pretreatment with O6-methylguanine in ACNU-resistant brain tumors. 781 4

The purine analogues O6-methylguanine and O6-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O6-methylguanine or O6-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas. We studied the effect of O6-methylguanine or O6-benzylguanine pretreatment on cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O6-methylguanine at higher concentrations (500 microM, 1,000 microM) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O6-Benzylguanine at concentrations between 10 and 100 microM markedly enhanced the cytotoxic effect. The ACNU sensitivity of the tumour cels pretreated with O6-benzylguanine was 5-40 times that of the cells without O6-benzylguanine. Neither O6-methylguanine nor O6-benzylguanine appreciably enhanced ACNU cytotoxicity of 9 L cells, which were originally sensitive to ACNU. Intracarotid ACNU with O6-methylguanine or O6-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O6-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment. These results indicate that O6-methylguanine or O6-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.
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PMID:Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours. 784 29

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.
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PMID:Enhancing effect of O6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells. 807 57

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.
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PMID:In vitro methylation of the human O6-methylguanine-DNA methyltransferase promoter reduces transcription. 811 Aug 28

Activation of the Ha-ras oncogene in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors has been well documented. Such Ha-ras activation is thought to be brought about by direct action of carcinogens resulting in a G-->A transition at the second nucleotide of codon 12. However, a DNA repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), can specifically remove methyl groups from O6-methylguanine, which is a major mutagenic and carcinogenic DNA lesion leading to the G-->A transition. In this study, we compared the amount of MGMT mRNA in MNU-induced rat mammary tumors with and without such Ha-ras activation. A single injection of MNU into 82 female Sprague-Dawley rats induced 80 mammary carcinomas. RNase protection analysis and subsequent sequencing revealed that 42 of 65 randomly selected tumors contained Ha-ras oncogenes activated by the G-->A transition. The amount of MGMT mRNA was then measured by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) amplification and Southern hybridization. No obvious difference in the level of MGMT mRNA was detected between the two tumor groups. In addition, in the course of our experiment, five of 42 tumors classified as containing activated Ha-ras oncogenes proved to contain low percentages of tumor cells with the Ha-ras activation. These results suggest that Ha-ras activation in MNU-induced rat mammary tumors may not necessarily be influenced by differences in MGMT activity. They also raise the possibility that activation of other oncogenes and/or inactivation of unidentified tumor suppressor gene(s) may be involved in development of a certain proportion of tumors with activated Ha-ras oncogenes, as is suspected in the case of tumors without Ha-ras activation.
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PMID:Comparison of O6-methylguanine-DNA methyltransferase mRNA levels in Ha-ras mutated and non-mutated rat mammary tumors induced by N-methyl-N-nitrosourea. 811 29

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a 1 h exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed.
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PMID:Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts. 820 Jan 5

Chloroethylnitrosoureas (CENUs) alkylate DNA at specific sites and inhibit DNA replication in tumor cells. O6-Alkylguanine moieties resulting from alkylation of guanine bases are thought to be one of most lethal adducts in living cells. Effectiveness of CENUs is known to relate well with an enzymic activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which recognizes and removes O6-alkylguanine. To improve therapeutic results of CENUs, we have measured MGMT activity of human brain tumors and studied the relationship between MGMT activity and clinical responsiveness to I-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). Thirty-seven patients with brain tumors were entered into the study. The neoplasms included gliomas, non-glial tumors, and brain metastases. The MGMT activity of gliomas was significantly lower than that of non-glial tumors and brain metastases. No significant difference in the enzyme activity was noted between low- and high-grade gliomas. Out of the 22 gliomas 5 tumors indicated a value below 60 fmol/mg, suggestive of a methyl excision repair minus (Mer-) tumor. Two out of 3 evaluable patients with a Mer- tumor responded well to post-operative ACNU adjuvant chemotherapy. Our results suggest that brain tumors include a certain percentage of Mer- phenotype tumors, and that CENUs such as ACNU should be applied selectively on tumors with a low MGMT activity in order to increase the therapeutic effectiveness.
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PMID:Influence of O6-methylguanine-DNA methyltransferase activity on chloroethylnitrosourea chemotherapy in brain tumors. 839 42

Suppressed expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), characterized as the Mer- phenotype, occurs only in malignant or transformed cell lines. To investigate the relationship between the transformation process and loss of MGMT expression, we derived 20 cloned lines of IMR90 normal fibroblasts transfected with the plasmid pSV3neo expressing the SV40 large-T antigen. Of the five lines that were grown until crisis phase, four emerged as continuously proliferating immortal lines. Of these, only one retained MGMT, the other three having become Mer-. In every case the loss of MGMT coincided with the final phase of immortalization following crisis. Because these were cloned cell lines it is clear that the phenotypic change to Mer- is not merely due to selection of a Mer- cell from the initial population, but must involve a cellular change in MGMT regulation. It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression. In addition, we have shown that, consistent with previous observations, both hypermethylation in promoter sequences and hypomethylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression. These studies also illustrate the utility of these new cloned cell lines for characterizing molecular events associated with transformation and immortalization.
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PMID:Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts. 862 42

Repair of alkylated bases in DNA is performed by O6-methylguanine-DNA methyltransferase (MGMT) and a set of enzymes of the base excision repair pathway involving N-methylpurine-DNA glycosylase (MPG), apurinic endonuclease (APE), DNA polymerase beta (Pol beta) and DNA ligase. The level of expression of these enzymes may exert a profound effect on resistance of cells towards alkylating drugs. We have comparatively analyzed the expression of MGMT and the different base excision repair genes in rat hepatoma cells (line H4IIE) after exposure to alkylating agents, X-rays and the glucocorticoid hormone dexamethasone. Furthermore, the effect of these agents on the activity of the cloned human MGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ionizing radiation increased MGMT mRNA levels up to 4.5-fold. Under the same conditions of treatment, exerting only a weak toxic effect, MPG and DNA ligase I mRNA levels were not enhanced, whereas the amounts of APE and Pol beta mRNA transiently increased by approximately 2-fold after X-ray and MNNG treatment, respectively. Dexamethasone induced both MGMT, APE and Pol beta mRNA and the induction paralleled the increase in mRNA of the glucocorticoid-dependent gene tyrosine aminotransferase. The observed increase in MGMT mRNA was due to promoter activation, which was shown in transient transfection assays with MGMT promoter-CAT reporter constructs in H4IIE cells. In these assays, the human MGMT promoter was found to be induced by methylating agents (MNNG and methyl methanesulfonate), ionizing radiation and dexamethasone. Weak induction of the promoter was observed after UV irradiation. Treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was ineffective in promoter activation. The transfected MGMT promoter was not inducible by mutagens in HeLa S3 cells, which do not respond with induction of the endogenous MGMT gene. This is the first report showing hormone induction of a DNA repair gene (MGMT). The induction of MGMT and other genes encoding enzymes involved in DNA alkylation damage repair may be relevant in cancer therapy by causing resistance of tumor cells to alkylating drugs.
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PMID:Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. 896 45

Nitrosoureas are antitumor alkylating agents widely used in the chemotherapy of malignant brain tumors. However, the effectiveness of adjuvant nitrosourea chemotherapy has proved inadequate, failing to provide any significant prolongation of survival time. One of the reasons for the poor results is a drug resistance system in the form of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). O6-alkylguanine derivatives are well known to be inhibitors of MGMT, and inactivation of MGMT by these derivatives leads to increased tumor cell sensitivity to nitrosoureas. In this study, the authors tested the ability of O6-benzylguanine, O6-(4-, 3- and 2-fluorobenzyl) guanines, O6-(4-, 3- and 2-trifluoromethylbenzyl) guanines, O6-(4-, 3- and 2-pyridylmethyl) guanines and O6-(2- and 1-naphthylmethyl) guanines to reduce MGMT activity in SF-188 cell-free extract by using [3H] methylated substrate DNA and analyzed their enhancing effect on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) by using a calorimetric cytotoxicity assay. The MGMT activity in the SF-188 cell-free extract was 944 +/- 43 fmol/mg protein (Mean +/- SD, n = 5). O6-(4- and 3-fluorobenzyl) guanines were found to be more effective in inactivating MGMT than O6-benzylguanine. O6-(4-trifluoromethylbenzyl) guanine considerably reduced MGMT activity as did O6-benzylguanine. O6-(3-trifluoromethylbenzyl) guanine, O6-(4- and 3-pyridylmethyl) guanines, and O6-(2-naphthylmethyl) guanine were intermediately effective, but O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine were less effective. ACNU cytotoxicity in SF-188 cells was strongly enhanced by pretreatment with O6-(4- and 3-fluorobenzyl) guanines and O6-(4-trifluoromethylbenzyl) guanine and moderately enhanced by O6-(3- trifluoromethylbenzyl) guanine and O6-(4- and 3-pyridylmethyl) guanines, but not enhanced by O6-(2-fluorobenzyl) guanine, O6-(2-trifluoromethylbenzyl) guanine and O6-(1-naphthylmethyl) guanine. The test compounds were not cytotoxic at concentrations between 0.5 and 5.0 microM. The enhancing effects on ACNU cytotoxicity were consistent with the inhibition of MGMT activity after two-hour pretreatment with O6-arylmethylguanine derivatives. These results indicate that the 2-position of the O6-benzyl group plays an important role in the inactivation of the MGMT activity and the potentiation of ACNU cytotoxicity.
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PMID:[Study on potentiation of nitrosourea-cytotoxicity by DNA repair enzyme inhibitors in human brain tumor cells]. 919 92


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