Abstract
DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-beta (pol-beta) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-beta-deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference-mediated pol-beta suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-iota or pol-lambda did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-beta knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-beta coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.
Highlights
Melanoma and glioblastoma are significant causes of death due to malignancy in the United States
Pol-h/ alkyladenine DNA glycosylase (Aag) double-null cells were more resistant to temozolomide than polh null cells (P < 0. 008 for all doses), suggesting that the temozolomide-induced hypersensitivity of pol-h–deficient cells is dependent upon glycosylase-mediated initiation of repair (Fig. 1A) and that pol-h protects against accumulation of toxic base excision repair (BER) intermediates, such as the toxic 5Vdeoxyribose phosphate (5VdRP) BER intermediates [28,29,30]
Pol-h is the predominant polymerase participating in BER both in vitro and in vivo [22, 40], both polymerase E (pol-E) and pol-L encode a nucleotidyl transferase activity and a 5VdRP lyase activity, similar to that found for pol-h, suggesting that these polymerases may participate in BER [32,33,34]
Summary
Melanoma and glioblastoma are significant causes of death due to malignancy in the United States. The incidence of malignant melanoma has doubled in the past 10 years and is rising at a rate exceeding all other solid tumors [1]. Chemotherapy provides a response rate of 7% to 20% with rare complete remissions. 17,000 new cases of brain cancer are diagnosed annually, yet therapy remains inadequate and the mortality rate is high [2]. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
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