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)

We initiated this study to determine whether three structurally related bifunctional alkylating agents could induce the expression of a presumptive human DNA repair gene. The gene chosen for this study is known to encode the ribosomal phosphoprotein PO, but ironically may also share functions related to DNA repair. We now show by Northern analysis that PO is induced by L-phenylalanine mustard, 4-hydroperoxycyclophosphamide and mechlorethamine, which are DNA-damaging agents commonly used as chemotherapeutic antitumor agents. In further support of its involvement in DNA repair is the finding of a 30- to 50-fold constitutive overexpression of the PO gene in human tumor cell lines that are Mer-, cells which lack O6-methylguanine methyltransferase activity, when compared to Mer+ cell lines. This constitutively elevated level of PO in Mer- cell lines, which are thus DNA repair defective for O6-alkyguanine lesions, was not observed for other genes tested, including the human ribosomal gene S17 whose mRNA steady-state levels were uniformly the same in both Mer- and Mer+ cells. Taking these data together, it appears that increased levels of PO are somehow linked to DNA repair, and increased expression of PO may compensate for the decreased O6-methylguanine DNA methyltransferase activity in Mer- cells. Furthermore, the PO gene has also been shown to be overexpressed in colorectal tumors and polyps and the sera of some systemic lupus erythematosus patients contain antibodies against PO. The titer of the anti-PO antibodies rises significantly during lupus psychosis.
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PMID:Expression of ribosomal phosphoprotein PO is induced by antitumor agents and increased in Mer- human tumor cell lines. 174 17

O-6-Alkylguanine is a mutagenic and carcinogenic DNA lesion induced by a variety of alkylating agents, including the chloroethylnitrosoureas. The lesion is repaired by the alkyl-accepting suicide enzyme O-6-methylguanine DNA methyltransferase (MGMT). Approximately 25% of cell lines derived from human tumors are phenotypically deficient in this enzyme and are described as Mer-. Recent cloning of the human MGMT cDNA (Tano, K.; Shiota, S.; Collier, J.; Foote, R.S.; Mitra, S. Proc. Natl. Acad. Sci. USA 87:686-690; 1990) has allowed for a more detailed analysis of the basis of the Mer- phenotype in human Mer- tumor cell lines. Using the polymerase chain reaction (PCR) technique, an MGMT cDNA probe based on the published sequence was generated. The probe and the PCR technique were then used to analyze the presence and expression of the MGMT gene in two Mer+ and four Mer- lines, including one SV40-transformed Mer- line and three Mer- human tumor cell lines. The data demonstrate that while all six cell lines contained a relatively nonamplified, nonrearranged MGMT gene, Mer- lines contained levels of MGMT mRNA detectable only by PCR analysis. Of the three Mer- tumor cell lines examined, two (COLO 320 HSR, A1235) contained MGMT mRNA levels that were four to five orders of magnitude lower than that of the prototype Mer+ tumor line (HT-29), while one (BE) contained no consistently detectable MGMT mRNA. These results suggest that in the human Mer- tumor lines tested, the Mer- phenotype was mediated by a severe reduction in MGMT mRNA levels, despite the presence of the MGMT gene.
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PMID:Comparison of O-6-methylguanine DNA methyltransferase (MGMT) mRNA levels in Mer+ and Mer- human tumor cell lines containing the MGMT gene by the polymerase chain reaction technique. 236 49

The Mex- (Mer-) phenotype of human cells is characterised by a sensitivity to agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). The hypersensitivity of Mex- cells is a consequence of their failure to express the DNA-repair enzyme m6-Gua-DNA methyltransferase. Resistance to MNNG and MNU may be acquired by Mex- cells either by reexpression of a methyltransferase function or by an ill-defined process of tolerance in which the cytotoxic potential of m6-Gua is circumvented without the altered base being removed from DNA. It has been suggested that tolerance might involve an altered mismatch correcting function. We have investigated proteins which recognise and bind specifically to DNA fragments containing single-base mismatches. Cell-free extracts of a Burkitt's lymphoma cell line (Raji) contain two such mismatch binding activities. Neither protein appears to have a high affinity for m6-Gua-containing base pairs. The data indicate that m6-Gua-containing base pairs might be poor substrates for mismatch repair processes in human cells.
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PMID:Mismatch binding proteins and tolerance to alkylating agents in human cells. 239 14

To clarify the involvement of O6-methylguanine (O6-MeG) in mutagenesis, we have been trying to isolate Mer+ cells from a HeLa S3 Mer- cell line, and to compare the mutation frequencies between the cell lines. We previously isolated the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR10-1, from HeLa S3 Mer- cells. However, the MR10-1 cells still had only a little O6-MeG-DNA methyltransferase (MT) activity. In the present study, we have isolated two 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr41 and ACr42, from the MR10-1 cells. The two ACr cells had increased MT activities. The ACr cells were also significantly more resistant to 1-(2-chloroethyl)-1-nitrosourea and slightly more resistant to MNNG than the MR10-1 cells. When the mutation frequencies were tested at the hypoxanthine-guanine phosphoribosyl transferase and ouabain loci in these cell lines, the two ACr cells were more resistant to the mutagenic effect of MNNG than the MR10-1 cells. These results show the linkage between the resistance to the cytotoxic effect of nitrosourea compounds and MT activity, and strongly support the hypothesis that O6-MeG is the main pre-mutagenic lesion induced by MNNG.
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PMID:Increased O6-methylguanine-DNA methyltransferase activity and reduced mutability in 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2- chloroethyl)-3-nitrosourea-resistant HeLa S3 cells. 316 31

To clarify the involvement of O6-methylguanine (O6-MeG) in mutagenesis, we isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR10-1 from HeLa S3 mer- cells. MR10-1 cells were 40 times more resistant to MNNG than the parental cells. MR10-1 cells were also significantly more resistant to N-methyl-N-nitrosourea and slightly more resistant to methyl methanesulfonate and dimethyl sulfate than parental cells. However, we found that MR10-1 cells had still little O6-MeG-DNA methyltransferase activity and were sensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl- 3-(2-chloroethyl)-3-nitrosourea hydrochloride, like HeLa mer- cells, thereby showing that MR10-1 cells are still mer-. When induced 6-thioguanine (6TG)-resistant colonies were plotted as a function of the corresponding percentage survival, the resistant colonies of MR10-1 cells were induced much more frequently than in the case of HeLa mer- cells. However, induction of 6TG-resistant cells in both cell lines did not differ significantly in terms of mutant cells per 0.1 microM MNNG. On the contrary, MR10-1 cells (mer-) and two HeLa S3 mer+ cells lines differed in the induction of mutation as a function of MNNG concentration. The HeLa mer+ cell lines were not mutable, while MR10-1 cells were highly mutable. These above results clearly show that the HeLa mer- cell has at least two defects in the repair of the alkylated adducts which are related to cell killing and mutation, and also suggest that O6-MeG is involved in the induction of mutation.
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PMID:N-methyl-N'-nitro-N-nitrosoguanidine-resistant HeLa S3 cells still have little O6-methylguanine-DNA methyltransferase activity and are hypermutable by alkylating agents. 360 93

Mer- human cells, which lack O6-methylguanine DNA methyltransferase activity, are extremely sensitive to alkylation induced killing, mutation and sister chromatid exchange. We have analyzed a Mer+, a Mer-, and a Mer- revertant HeLa cell line and found that the methyltransferase deficiency correlates with increased levels of mutation and sister chromatid exchange, but does not correlate with increased killing of Mer- HeLa cells by alkylating agents. Furthermore, we show that HeLa Mer- cells repair N-3-methylguanine and N-3-methyladenine just as efficiently as HeLa Mer+ cells.
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PMID:DNA alkylation repair and the induction of cell death and sister chromatid exchange in human cells. 380 5

The repair of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-produced O6-methylguanine (O6-MeG) in DNA and its correlation with MNNG-produced cell-killing and sister chromatid exchange (SCE) induction were compared in mouse and reference human tumor cell strains. As a result, mouse cell strains were divided into three groups: (i) cells proficient in O6-MeG-repair and insensitive to MNNG similar to human Mer+ Rem+ strains; (ii) cells deficient in O6-MeG-removal and sensitive to MNNG similar to human Mer-Rem- strains; (iii) cells deficient in O6-MeG-removal but insensitive to MNNG similar to some SV40-transformed human strains. Attempts at correlating lack of capacity for O6-MeG-removal, MNNG-sensitivity and high SCE induction showed that O6-MeG in DNA may be a lesion common to cell-killing and SCE induction only in mouse cells of groups i and ii. Levels of O6-MeG-DNA methyltransferase activity in mouse cells were measured and the enzyme had the same molecular weight as that in human cells.
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PMID:Comparison of repair of O6-methylguanine produced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse and human cells. 672 79

O6-Methylguanine-DNA methyltransferase (MGMT) plays an important role in protecting cells from the mutagenic potency of alkylating agents. This study addresses the role of DNA methylation in the expression of the human MGMT gene. Southern blot analysis of DNA from human Mer+ (MGMT proficient) and Mer- (MGMT deficient) cell lines demonstrated that the methylation state of a unique SmaI site in the MGMT gene promoter, previously shown by others to be invariably unmethylated in Mer+ cells and methylated in Mer- cells, did not correlate with the Mer phenotype. Neither was there any significant difference in the density of CpG methylation in the MGMT gene 5'-flanking sequences between Mer+ and Mer- cells. On the other hand, the body of the MGMT gene was less methylated in most Mer- cells relative to Mer+ cells, and in three of six Mer- cell lines the gene was essentially methylation-free. Interestingly, the Mer- cells that were hypomethylated in the MGMT gene also tended to be less methylated at other loci. Widespread hypomethylation is a frequent trait in carcinogenesis, and may be involved in development of the frequently found Mer- phenotype.
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PMID:The methylation status of the gene for O6-methylguanine-DNA methyltransferase in human Mer+ and Mer- cells. 763 15

O6-Methylguanine-DNA methyltransferase plays an important role in cellular defence against mutagens and carcinogens with alkylating activity. Certain tumor-derived cell lines, termed Mer-, are defective in the enzyme activity and have an increased sensitivity to alkylating agents. We cloned the genomic sequence coding for the human O6-methylguanine-DNA methyltransferase and elucidated the structure. The gene consisted of 5 exons and spanned more than 170 kb, while mRNA for the enzyme was 950 nucleotides long. No or only little mRNA for the enzyme was formed in Mer- cells, though there was no gross difference in the coding and promoter regions of the gene between Mer+ and Mer- cells. The putative promoter region, derived from Mer+ cells, was placed upstream of the chloramphenicol acetyltransferase reporter gene and the constructs were introduced into Mer+ and Mer- cells. In Mer- cells, a lowered level of transient expression of the gene was observed as compared with Mer+ cells, but this difference alone does not account for the in vivo difference of expression of the gene in the two types of cells; there might be difference in cis-acting elements. The DNA sequence in the 5' upstream region of the gene was extremely GC-rich and there were no consensus sequences, such as the TATA and CAAT boxes. There were lower levels of methylation in the putative promoter of various Mer- cells, as compared with findings in Mer+ cells. Methylation in this region may be involved in regulating expression of the gene.
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PMID:Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase. 767 40

Common Mer- cell lines deficient in O6-methylguanine DNA methyltransferase (MTase) activity probably result from the down-regulation of, rather than mutations in, the MGMT gene. However, the down-regulation of other unrelated genes was also observed in some of these cell lines, making it difficult to determine the precise functions of the MGMT MTase gene. To study the biological function of human MGMT MTase, we seek to utilize a newly created yeast mgt1 mutant deficient in the DNA repair MTase activity. The human MGMT cDNA was cloned into yeast expression vectors so that the MGMT gene is under the control of either an inducible GAL1 promoter or a constitutive ADH1 promoter. Upon galactose induction, the PGAL1-MGMT transformant had about 40-fold MTase activity compared to the wild-type strain. MGMT overexpression protected the yeast mgt1 mutant against alkylation-induced killing and mutation. Limited expression of the MGMT gene in the mgt1 mutant still provides significant alkylation resistance, albeit at a reduced level. The yeast mgt1 mutants increase spontaneous mutation rate, whereas constitutive expression of the MGMT gene lowered the spontaneous mutation rate in the mgt1 mutant to the wild-type level. We suggest that MGMT MTase may play the same role in human cells as the MGT1 MTase in yeast cells. Thus our results demonstrate that the human MGMT gene functionally complements the yeast MTase-deficient mutant in the protection against exogenous and endogenous DNA alkylation damage, which provides a useful tool for the study of in vivo mammalian MTase functions.
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PMID:Expression of the human MGMT O6-methylguanine DNA methyltransferase gene in a yeast alkylation-sensitive mutant: its effects on both exogenous and endogenous DNA alkylation damage. 788 84


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