Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens.
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ABSTRACT:
SUBMITTER: Miller CT
PROVIDER: S-EPMC1165965 | biostudies-other | 1973 Oct
REPOSITORIES: biostudies-other
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Project description:Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O(6)-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O(6)-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma.
| S-EPMC4777656 | biostudies-literature
Project description:BackgroundO(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins.MethodsA total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status.ResultsMGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, P<0.001 and 105 vs 34 months, P=0.006) or MGMT promoter methylation assessed by pyrosequencing (respectively, 26.4 vs 10.8 months, P=0.002 and 77 vs 43 months, P=0.026).ConclusionsOur results suggest that MGMT status is associated with response to alkylant-based chemotherapy in NETs.
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Project description:DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant ‘chronic’ temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-022-01367-4.
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Project description:Trained immunity is the heightened state of innate immune memory that enhances immune response resulting in nonspecific protection. Epigenetic changes and metabolic reprogramming are critical steps that regulate trained immunity. In this study, we reported the involvement of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme of lesion induced by alkylating agents, in regulation the trained immunity induced by β-glucan (BG). Pharmacological inhibition or silencing of MGMT expression altered LPS stimulated pro-inflammatory cytokine productions in BG-trained bone marrow derived macrophages (BMMs). Targeted deletion of Mgmt in BMMs resulted in reduction of the trained responses both in vitro and in vivo models. The transcriptomic analysis revealed that the dampening trained immunity in MGMT KO BMMs is partially mediated by ATM/FXR/AMPK axis affecting the MAPK/mTOR/HIF1α pathways and the reduction in glycolysis function. Taken together, a failure to resolve a DNA damage may have consequences for innate immune memory.
2024-01-23 | GSE231419 | GEO
Project description:This study aimed to investigate the influence of miR-221-3p and O6-methylguanine-DNA methyltransferase (MGMT) interaction in human hepatocellular carcinoma (HCC), thereby revealing a novel molecular mechanism of hepatic carcinogenesis involving miR-221-3p and MGMT.Fluorescence qPCR and immunoblot assays were performed to determine the expression of RNA and protein in HCC tissues and cell lines. We also employed the firefly and Renilla luciferase assay to verify the target relationship between miR-221-3p and MGMT mRNA. Assessments including the MTT assay, wound-healing assay, transwell assay, colony foci formation experiment, and flow cytometric experiment were carried out to determine the viability, migration, invasion, proliferation, cell cycle progression, and apoptosis of SMMC-7721 and BEL-7404 cell lines with the modulated expression of miR-221-3p and MGMT. Compared to healthy tissues and cell line HL7702, miR-221-3p was significantly upregulated but MGMT was significantly downregulated in carcinomas and cancerous cell lines. Forced miR-221-3p overexpression was found to enhance the proliferation, migration, invasion, and clonogenicity of cell lines, but it suppressed cell apoptosis. Findings also revealed that forced miR-221-3p overexpression had little effect on cell cycle progression. After MGMT was confirmed to be atarget gene of miR-221-3p, it was found that the forced upregulation of miR-221-3p downregulated MGMT mRNA and protein levels significantly. MiR-221-3p was identified as an HCC promoting factor, and it specifically inhibited the expression of the MGMT. Besides, the upregulation of miR-221-3p had apositive influence on HCC pathogenesis by inhibiting MGMT expression.
| S-EPMC7644250 | biostudies-literature
Project description:The human DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O6-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O6-alkylguanines. One such ODN containing O6-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC50 of 0.1 nM.
| S-EPMC1435717 | biostudies-literature
Project description:Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to cell cycle arrest and apoptosis. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Characterizing genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT. O6-MeG signatures were highly similar to mutational signatures from patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes in TMZ-exposed cells and naked DNA proposing an indirect influence of transcription to O6-MeG formation. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and nucleotide sequence. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.
2024-10-30 | GSE279423 | GEO
Project description:Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to apoptosis due to mismatch-repair overload. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair mechanisms, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT transfection. O6-MeG signatures were highly similar to mutational signatures of patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes, suggesting an additional transcription-associated contribution to its repair. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and regulation. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.
2023-12-12 | GSE249155 | GEO
Project description:O(6)-Methylguanine (O(6)-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O(6)-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O(6)-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs O(6)-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O(6)-benzylguanine, ? 58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O(6)-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.
| S-EPMC3001077 | biostudies-literature
Project description:The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O(6)-CMG is not a substrate for the human version of the DNA damage reversal protein O(6)-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O(6)-alkylguanine lesions by removing alkyl groups from the O(6)-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O(6)-methylguanine (O(6)-MeG) or O(6)-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O(6)-alkyl group and its transfer to the active-site cysteine residue of MGMT. O(6)-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O(6)-CMG is a potential causative agent.
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