Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

Abstract

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O(6)-methylguanine (O(6)-meG) mediates GBM cell killing. Moreover, low or absent expression of O(6)-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O(6)-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O(6)-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O(6)-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O(6)-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

Keywords: Ape1; DNA repair; alkyladenine-DNA glycosylase; apurinic endonuclease; predictive marker; treatment outcome.

FIGURE 1

Removal of 3-meA by base excision repair (BER). TMZ-induced 3-meA in DNA is recognized by AGG that excises the methylated base by cleaving the glycosylic linkage between the adducted base and deoxyribose. AAG remains bound to the resulting abasic site (ABS) until displaced by Ape1. The apurinic endonuclease activity of Ape1 incises the DNA phosphodiester backbone immediately 5′ to the abasic site, producing a single-strand break (red square) containing a 5′-deoxyribose phosphate (5′-dRP) terminus. Ape1 is replaced by DNA polymerase β(Pol β) that removes the 5′-dRP via an intrinsic lyase activity to produce a single-nucleotide gap (red line). Using the opposite DNA strand as a template, Pol βthen inserts a complementary nucleotide leaving a single-strand break (red square) that is subsequently sealed by DNA ligase. This process is closely coordinated to insure that the potentially lethal abasic sites, and single strand breaks and gaps do not persist. XRCC1 and PARP (not shown) facilitate repair by coordinating protein binding at damage sites. BER is also essential for repair of abasic sites that arise from spontaneous or methylation-enhanced base loss and single-strand breaks produced by ionizing radiation. Some radiation-induced strand breaks contain fragmented or 5′-oxidized deoxyribose moieties that cannot be excised by the lyase activity of Pol β. In this circumstance an alternative BER pathway employs FEN1 endonuclease to excise a two to eight nucleotide long single-strand containing the damage, and the resulting gap is filled by DNA polymerase δ or ε and sealed by DNA ligase (Robertson et al., 2009).