Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA.
Free Radic Biol Med. 2010 May 15;48(10):1435-43. doi: 10.1016/j.freeradbiomed.2010.02.033. Epub 2010 Mar 1.
DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by gamma-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24-72h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.
DNA 依赖性蛋白激酶（DNA-PK）是一种关键的非同源末端连接（NHEJ）核丝氨酸/苏氨酸蛋白激酶，参与多种 DNA 代谢和损伤信号通路，有助于维持基因组稳定性和预防癌症。为了研究 DNA-PK 在非双链断裂（DSB）聚集性 DNA 损伤处理中的作用，我们使用了三种 DNA-PK 缺乏模型，即通过新型抑制剂 IC86621 和 NU7026 化学失活其激酶活性、敲低和完全缺失人乳腺癌（MCF-7）和神经胶质瘤细胞系（MO59-J/K）中的蛋白。DNA-PK 修复途径受损导致 γ 射线诱导的聚集性 DNA 损伤累积。缺乏蛋白表达或激酶活性受到抑制的肿瘤细胞在使用脉冲场凝胶电泳或单细胞凝胶电泳（彗星分析）的改良版本测量处理氧化诱导的非 DSB 聚集性 DNA 损伤的能力显著下降。在所有情况下，DNA-PK 失活导致损伤持续存在的水平更高，即使在修复 24-72 小时后也是如此。我们提出了一个模型，其中 DNA-PK 缺乏首先通过损害碱基切除修复来影响这些簇的处理，其次由于 DNA-PK 无法从 DNA 末端解离，催化失活的 DNA-PK 抑制这些损伤的有效处理，从而影响这些簇的处理。该信息对于理解不仅在 NHEJ 缺乏的情况下癌症的发病机制，而且还对于基于诱导氧化应激和抑制簇修复的癌症治疗都非常重要。
