Trivedi Ram N, Almeida Karen H, Fornsaglio Jamie L, Schamus Sandra, Sobol Robert W
Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213-1863, USA.
Cancer Res. 2005 Jul 15;65(14):6394-400. doi: 10.1158/0008-5472.CAN-05-0715.
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.
DNA烷化剂在许多人类肿瘤的治愈性治疗中起着核心作用；然而，对这些药物的耐药性限制了它们的有效性。烷化剂替莫唑胺的疗效归因于O6-甲基鸟嘌呤（O6-MeG）的诱导，O6-MeG是一种由O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）修复的DNA损伤。对替莫唑胺的耐药性归因于MGMT水平升高和/或错配修复减少。然而，替莫唑胺诱导的DNA损伤中>80%是N-甲基化碱基，这些碱基由DNA糖基化酶识别而不是由MGMT识别，因此对替莫唑胺的耐药性也可能部分归因于强大的碱基切除修复（BER）。我们使用缺乏BER酶DNA聚合酶β（pol-β）和烷基腺嘌呤DNA糖基化酶（Aag）的同基因细胞来确定BER在替莫唑胺细胞毒性作用中的作用。与野生型或Aag缺陷型细胞相比，pol-β缺陷型细胞对替莫唑胺杀伤的敏感性显著更高，这种超敏反应可能是由BER中间体的积累引起的。RNA干扰介导的pol-β抑制足以提高替莫唑胺的疗效，而pol-ι或pol-λ的缺陷并没有增加替莫唑胺介导的细胞毒性。Aag（起始BER酶）的过表达引发了替莫唑胺诱导的细胞毒性的进一步增加。增强的Aag表达与pol-β敲低相结合，使替莫唑胺的疗效提高了4倍。此外，pol-β的缺失与替莫唑胺治疗相结合引发了H2AX的磷酸化，表明由于未修复的损伤而激活了DNA损伤反应途径。因此，BER途径是细胞对替莫唑胺耐药的主要原因，其疗效取决于特定的BER基因表达和活性。
