O(6)-methylguanine-DNA methyltransferase in glioma therapy: promise and problems

Abstract

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 O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-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.

Figure 1. Repair of O⁶-alkylguanine adducts by MGMT

MGMT mediates a stoichiometric reaction in which methyl and other alkyl groups bound to the O6 position of guanine in DNA are via a thioester linkage to a cysteine residue in the active site. Alkylation of MGMT leads to rapid degradation of the protein. Thus, the capacity of a cell to remove O⁶-alkylguanine from DNA reflects the rate at which adducts are produced by exogenous and endogenous agents and the rate of synthesis of new protein. The “suicide” reaction mechanism of MGMT is unique among human DNA repair enzymes, and suggests the use of substrate analog inhibitors of MGMT in order to increase sensitivity to chemotherapeutic alkylating agents. Image adapted from www.mgmt-agt.net/whatismgmt.htm.

Figure 2. Differential sensitivity of 5-methylcytosine and cytosine to bisulfite-mediated deamination

5-methylcytosine is insensitive to incubation with bisulfite at mildly acidic pH. In contrast, bisulfite treatment promotes the hydrolysis of cytosine, producing uracil and ammonia. This differential sensitivity has been exploited to detect 5-methylcytosine in DNA. Image adapted from www.imb-jena.de/~sweta/renzymes.

Figure 3. Methylation-specific PCR (MSP)

Bisulfite treatment converts cytosine in DNA to uracil (left) while leaving 5-methylcytosine unaltered (right). The uracil- and 5-methylcytosine-containing template sequences hybridize to unique primers to yield distinct products during PCR amplification.