O 6 methylguanine DNA methyltransferase

Function and mechanism

Although alkylating mutagens preferentially modify the guanine base at the N7 position, O⁶-alkyl-guanine is a major carcinogenic lesion in DNA. This DNA adduct is removed by the repair protein O⁶-alkylguanine DNA alkyltransferase through an S_N2 mechanism. This protein is not a true enzyme since it removes the alkyl group from the lesion in a stoichiometric reaction and the active enzyme is not regenerated after it is alkylated (referred to as a suicide enzyme). The methyl-acceptor residue in the protein is a cysteine.

  → M G M T

Clinical significance

Methylation of the gene's promoter may play a significant role in carcinogenesis. In patients with glioblastoma multiforme, a severe type of brain tumor, the methylation state of the MGMT gene determined whether tumor cells would be responsive to temozolomide; if the promoter was methylated, temozolomide was more effective. On a clinical level, this translates into a prolonged survival of glioblastoma patients with a methylated MGMT promoter. In addition, MGMT methylation can be used to predict patient survival in clinical prediction models. For testing of the MGMT promoter methylation status in the clinical setting, DNA-based methods such as methylation-specific polymerase chain reaction (MS-PCR) or pyrosequencing are preferred over immunohistochemical or RNA- based assays.

MGMT has also been shown to be a useful tool increasing gene therapy efficiency. By using a two component vector consisting of a transgene of interest and MGMT, in vivo drug selection can be utilized to select for successfully transduced cells.

Mutagens in the environment, in tobacco smoke, food, as well as endogenous metabolic products generate reactive electrophilic species that alkylate or specifically methylate DNA, generating 6-O-Methylguanine (m6G).

In 1985 Yarosh summarized the early work that established m6G as the alkylated base in DNA that was the most mutagenic and carcinogenic. In 1994 Rasouli-Nia et al. showed that about one mutation was induced for every eight unrepaired m6Gs in DNA. Mutations can cause progression to cancer by a process of natural selection.

Epigenetic repression

Only a minority of sporadic cancers with a DNA repair deficiency have a mutation in a DNA repair gene. However, a majority of sporadic cancers with a DNA repair deficiency do have one or more epigenetic alterations that reduce or silence DNA repair gene expression. For example, in a study of 113 sequential colorectal cancers, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration).

MGMT can be epigenetically repressed in a number of ways. When MGMT expression is repressed in cancers, this is often due to methylation of its promoter region. However, expression can also be repressed by di-methylation of lysine 9 of histone 3 or by over-expression of a number of microRNAs including miR-181d, miR-767-3p and miR-603.

Deficiency in field defects

A field defect is an area or "field" of epithelium that has been preconditioned by epigenetic changes and/or mutations so as to predispose it towards development of cancer. A field defect is illustrated in the photo and diagram shown of a colon segment having a colon cancer and four small polyps within the same area as well. As pointed out by Rubin, "The vast majority of studies in cancer research has been done on well-defined tumors in vivo, or on discrete neoplastic foci in vitro. Yet there is evidence that more than 80% of the somatic mutations found in mutator phenotype human colorectal tumors occur before the onset of terminal clonal expansion." Similarly, Vogelstein et al. point out that more than half of somatic mutations identified in tumors occurred in a pre-neoplastic phase (in a field defect), during growth of apparently normal cells.

In the Table above, MGMT deficiencies were noted in the field defects (histologically normal tissues) surrounding most of the cancers. If MGMT is epigenetically reduced or silenced, it would not likely confer a selective advantage upon a stem cell. However, reduced or absent expression of MGMT would cause increased rates of mutation, and one or more of the mutated genes may provide the cell with a selective advantage. The expression-deficient MGMT gene could then be carried along as a selectively neutral or only slightly deleterious passenger (hitch-hiker) gene when the mutated stem cell generates an expanded clone. The continued presence of a clone with an epigenetically repressed MGMT would continue to generate further mutations, some of which could produce a tumor.

Deficiency with exogenous damage

MGMT deficiency alone may not be sufficient to cause progression to cancer. Mice with a homozygous mutation in MGMT did not develop more cancers than wild-type mice when grown without stress. However, stressful treatment of mice with azoxymethane and dextran sulphate caused more than four colonic tumors per MGMT mutant mouse, but less than one tumor per wild-type mouse.

Repression in coordination with other DNA repair genes

In a cancer, multiple DNA repair genes are often found to be simultaneously repressed. In one example, involving MGMT, Jiang et al. conducted a study where they evaluated the mRNA expression of 27 DNA repair genes in 40 astrocytomas compared to normal brain tissues from non-astrocytoma individuals. Among the 27 DNA repair genes evaluated, 13 DNA repair genes, MGMT, NTHL1, OGG1, SMUG1, ERCC1, ERCC2, ERCC3, ERCC4, MLH1, MLH3, RAD50, XRCC4 and XRCC5 were all significantly down-regulated in all three grades (II, III and IV) of astrocytomas. The repression of these 13 genes in lower grade as well as in higher grade astrocytomas suggested that they may be important in early as well as in later stages of astrocytoma. In another example, Kitajima et al. found that immunoreactivity for MGMT and MLH1 expression was closely correlated in 135 specimens of gastric cancer and loss of MGMT and hMLH1 appeared to be synchronously accelerated during tumor progression.

Deficient expression of multiple DNA repair genes are often found in cancers, and may contribute to the thousands of mutations usually found in cancers (see Mutation frequencies in cancers).

Interactions

O⁶-methylguanine-DNA methyltransferase has been shown to interact with estrogen receptor alpha.