Boldogh Istvan, Roy Gargi, Lee Myung Soog, Bacsi Attila, Hazra Tapas K, Bhakat Kishor K, Das Gokul C, Mitra Sankar
Department of Microbiology and Immunology, Sealy Center for Molecular Sciences, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
Toxicology. 2003 Nov 15;193(1-2):137-52. doi: 10.1016/j.tox.2003.08.013.
Modulation of DNA repair represents a strategy to overcome acquired drug resistance of cells to genotoxic chemotherapeutic agents, including nitrogen mustards (NM). These agents induce DNA inter-strand cross-links, which in turn produce double strand breaks (dsbs). These breaks are primarily repaired via the nonhomologous end-joining (NHEJ) pathway. A DNA-dependent protein kinase (DNA-PK) complex plays an important role in NHEJ, and its increased level/activity is associated with acquired drug resistance of human tumors. We show in this report that the DNA-PK complex has comparable levels and kinase activity of DNA-PK catalytic subunit (DNA-PKcs) in a nearly isogenic pair of drug-sensitive (A2780) and resistant (A2780/100) cells; however, treatment with chlorambucil (Cbl), a NM-type of drug, induced differential effects in these cells. The kinase activity of DNA-PKcs was increased up to 2h after Cbl treatment in both cell types; however, it subsequently decreased only in sensitive cells, which is consistent with increased levels of DNA dsbs. The decreased kinase activity of DNA-PKcs was not due to a change in its amount or the levels of Ku70 and Ku86, their subcellular distribution, cell cycle progression or caspase-mediated degradation of DNA-PK. In addition to DNA cross-links, Cbl treatment of cells causes a 2.2-fold increase in the level of reactive oxygen species (ROS) in both cell types. However, the ROS in A2780/100 cells were reduced to the basal level after 3-4h, while sensitive cells continued to produce ROS and undergo apoptosis. Pre-treatment of A2780 cells with the glutathione (GSH) precursor, N-acetyl-L-cysteine prevented Cbl-induced increase in ROS, augmented the kinase activity of DNA-PKcs, decreased the levels of DNA dsbs and increased cell survival. Depletion in GSH from A2780/100 cells by L-buthionine sulfoximine (BSO) resulted in sustained production of ROS, lowered DNA-PKcs kinase activity, enhanced levels of DNA dsbs, and increased cell killing by Cbl. We propose that oxidative stress decreases repair of DNA dsbs via lowering kinase activity of DNA-PKcs and that induction of ROS could be the basis for adjuvant therapies for sensitizing tumor cells to nitrogen mustards and other DNA cross-linking drugs.
调节DNA修复是克服细胞对包括氮芥（NM）在内的基因毒性化疗药物获得性耐药的一种策略。这些药物会诱导DNA链间交联，进而产生双链断裂（dsb）。这些断裂主要通过非同源末端连接（NHEJ）途径进行修复。一种依赖DNA的蛋白激酶（DNA-PK）复合物在NHEJ中起重要作用，其水平/活性的增加与人类肿瘤的获得性耐药有关。我们在本报告中表明，在一对近乎同基因的药物敏感（A2780）和耐药（A2780/100）细胞中，DNA-PK复合物的DNA-PK催化亚基（DNA-PKcs）具有相当的水平和激酶活性；然而，用NM类药物苯丁酸氮芥（Cbl）处理在这些细胞中产生了不同的效应。在两种细胞类型中，Cbl处理后2小时内DNA-PKcs的激酶活性均增加；然而，随后仅在敏感细胞中下降，这与DNA双链断裂水平的增加一致。DNA-PKcs激酶活性的下降并非由于其数量、Ku70和Ku86的水平、它们的亚细胞分布、细胞周期进程或caspase介导的DNA-PK降解发生变化。除了DNA交联外，用Cbl处理细胞还会使两种细胞类型中的活性氧（ROS）水平增加2.2倍。然而，A2780/100细胞中的ROS在3-4小时后降至基础水平，而敏感细胞则继续产生活性氧并发生凋亡。用谷胱甘肽（GSH）前体N-乙酰-L-半胱氨酸预处理A2780细胞可防止Cbl诱导的ROS增加，增强DNA-PKcs的激酶活性，降低DNA双链断裂水平并提高细胞存活率。用L-丁硫氨酸亚砜胺（BSO）耗尽A2780/100细胞中的GSH会导致ROS持续产生，降低DNA-PKcs激酶活性，增强DNA双链断裂水平，并增加Cbl对细胞的杀伤作用。我们提出氧化应激通过降低DNA-PKcs的激酶活性来减少DNA双链断裂的修复，并且ROS的诱导可能是使肿瘤细胞对氮芥和其他DNA交联药物敏感的辅助治疗基础。
