甲基鸟嘌呤甲基转移酶表达调节在肿瘤发生和治疗中的作用

甲基鸟嘌呤甲基转移酶（O⁶-methylguanine-DNA methyltransferase,MGMT）是从细菌到哺乳类机体中存在的一种独特的DNA修复蛋白,其作用是在DNA损伤的修复过程,催化DNA分子鸟嘌呤O⁶位上的烷基从鸟嘌呤碱基转移至MGMT蛋白的半胱氨酸残基上,而使DNA分子鸟嘌呤复原.因此,机体中MGMT适当的表达有利于修复由烷化剂诱导而形成的O⁶烷基鸟嘌呤DNA加合物.MGMT蛋白的含量和活性不但在基因水平受到各种因素的调控,并且与某些药物的直接作用有关.调节MGMT在细胞内的活性,对于防御肿瘤的发生及某些肿瘤的治疗过程中克服肿瘤耐药性和克服骨髓毒性具有重要的意义.     

O-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity,promoter methylation and immunohistochemistry

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O⁶-methylguanine. It also repairs larger adducts on the O⁶-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O⁶-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O⁶-alkylating drugs are still lacking.     

Activation of gene expression of the O<Superscript>6</Superscript>-methylguanine-DNA-transferase repair enzyme upon the influence of EMAP II cytokine in human cells in vitro

The aim of the study is to evaluate the effect of recombinant EMAP II cytokine (endothelial and monocyte-activating polypeptide II) on the level of MGMT gene expression; this gene encodes the O⁶-methylguanine-DNA-methyltransferase (MGMT) repair enzyme in the cell culture of humans. An investigation into the EMAP II effect on the proliferation of cells was carried out using the standard MTT test. The MGMT protein in a cell extract was identified by Western blot analysis. The following cell lines were investigated: A102 (fibroblasts), CB-1 (umbilical cord blood stromal cells), and 4BL6 (cells obtained from peripheral blood). It was shown in these experiments that the EMAP II cytokine induces MGMT expression in human cells of the investigated lines. There was observed a decrease in the quantity of cells in the presence of a high concentration of this cytokine. The level of expression of the MGMT repair enzyme was established to increase in human cells in vitro in a serum-free culture medium with the EMAP II cytokine.     

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth)

O⁶-methylguanine (O⁶meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O⁶ position of guanine to a cysteine residue in its active center. Because O⁶meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O⁶meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O⁶meG is not repaired, and during replication, O⁶meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O⁶meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O⁶meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O⁶meG toxicity by removing the methyl residue from the guanine. Although removal of O⁶meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O⁶meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O⁶meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O⁶meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O⁶meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.     

O-Methylguanine-DNA methyltransferase in glioma therapy: Promise and problems

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O⁶-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.     

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O⁶-methylguanine (O⁶MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O⁶-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O⁶-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O⁶-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O⁶MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O⁶MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.     

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O⁶-alkylguanine adducts in DNA are repaired by O⁶-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1⁺, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O⁶-methylguanine in [³H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O⁶-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O⁶-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O⁶-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.     

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT)

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.     

DNA repair in personalized brain cancer therapy with temozolomide and nitrosoureas

Alkylating agents have been used since the 60ties in brain cancer chemotherapy. Their target is the DNA and, although the DNA of normal and cancer cells is damaged unselectively, they exert tumor-specific killing effects because of downregulation of some DNA repair activities in cancer cells. Agents exhibiting methylating properties (temozolomide, procarbazine, dacarbazine, streptozotocine) induce at least 12 different DNA lesions. These are repaired by damage reversal mechanisms involving the alkyltransferase MGMT and the alkB homologous protein ALKBH2, and through base excision repair (BER). There is a strong correlation between the MGMT expression level and therapeutic response in high-grade malignant glioma, supporting the notion that O⁶-methylguanine and, for nitrosoureas, O⁶-chloroethylguanine are the most relevant toxic damages at therapeutically relevant doses. Since MGMT has a significant impact on the outcome of anti-cancer therapy, it is a predictive marker of the effectiveness of methylating anticancer drugs, and clinical trials are underway aimed at assessing the influence of MGMT inhibition on the therapeutic success. Other DNA repair factors involved in methylating drug resistance are mismatch repair, DNA double-strand break (DSB) repair by homologous recombination (HR) and DSB signaling. Base excision repair and ALKBH2 might also contribute to alkylating drug resistance and their downregulation may have an impact on drug sensitivity notably in cells expressing a high amount of MGMT and at high doses of temozolomide, but the importance in a therapeutic setting remains to be shown. MGMT is frequently downregulated in cancer cells (up to 40% in glioblastomas), which is due to CpG promoter methylation. Astrocytoma (grade III) are frequently mutated in isocitrate dehydrogenase (IDH1). These tumors show a surprisingly good therapeutic response. IDH1 mutation has an impact on ALKBH2 activity thus influencing DNA repair. A master switch between survival and death is p53, which often retains transactivation activity (wildtype) in malignant glioma. The role of p53 in regulating survival via DNA repair and the routes of death are discussed and conclusions as to cancer therapeutic options were drawn.     

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme

The enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O⁶-methylguanine (O⁶-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [³H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90°?C for at least 30?min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (Δada, Δogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine.     

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.     

Plant Impact on CO<Subscript>2</Subscript> Consumption by Silicate Weathering: The Role of Bamboo

CO₂ consumption by silicate weathering has exerted a major control on atmospheric CO₂ over geologic time. In order to assess plant impact on this process, the study compared water geochemistry and CO₂ consumption rates by silicate weathering in watersheds covered by bamboos and other forests. Our study showed that SiO₂ concentrations (80?~?150 μmol/L, average 105 μmol/L) in water from pure bamboo forest watersheds were higher than that (15?~?85 μmol/L, average 60 μmol/L) from other watersheds. Si/(Na_silicate?+?K_silicate) ratios in water draining from bamboo watersheds (2.0?~?4.0, average 2.9) were higher than that from other watersheds ?>(0.7?~?2.7, average 2.2). CO₂ consumption rates by silicate weathering in bamboo watersheds (1.8?~?3.4 10⁵ mol/km²/yr, average 2.5 10⁵ mol/km²/yr) were higher than that in other watersheds (1.5?~?2.6 10⁵ mol/km²/yr, average 2.0 10⁵ mol/km²/yr). Therefore, bamboo-enhanced silicate weathering is a potential biogeochemical remediation approach for atmospheric CO₂.     

KDM6B promotes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O⁶-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.     

RANTES-mediated control of excitatory amino acid release in mouse spinal cord

O⁶‐methylguanine‐DNA methyltransferase (MGMT) is a DNA‐repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth‐depressing transfection of a T98G glioma cell line with liver‐type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA‐transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non‐transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100–1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.     

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.     

A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O⁶-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.     

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

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 O⁶-carboxymethylguanine (O⁶-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 O⁶-CMG is not a substrate for the human version of the DNA damage reversal protein O⁶-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O⁶-alkylguanine lesions by removing alkyl groups from the O⁶-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O⁶-methylguanine (O⁶-MeG) or O⁶-CMG effectively inactivate MGMT in vitro (IC₅₀ 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O⁶-alkyl group and its transfer to the active-site cysteine residue of MGMT. O⁶-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O⁶-CMG is a potential causative agent.