Quenching of DNA cross-link precursors of chloroethylnitrosoureas and attenuation of DNA interstrand cross-linking by glutathione.
Quenching of DNA cross-link precursors of chloroethylnitrosoureas and attenuation of DNA interstrand cross-linking by glutathione.
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作者：
F. Ali-Osman
链间DNA交联是氯乙基亚硝基脲（CENUs）抗肿瘤活性所必需的。关键的交联已经被提出涉及一个快速的O 6-鸟嘌呤氯乙基化的一条DNA链，然后由重排的O 6-（2-氯乙基）鸟嘌呤和缓慢的烷基化的第二条DNA链。鉴于谷胱甘肽（GSH）的相对细胞内丰度及其硫醇盐离子的亲核性，研究了GSH与和与β 2-羟甲基化DNA反应的能力以及这种相互作用降低CENUs的净DNA交联的可能性。将氯乙基化小牛胸腺DNA与GSH反应，沉淀并再溶解DNA，并测定随后的DNA链间交联。比较GSH处理和2-氯乙基化未处理DNA的DNA交联指数。同时，将Col E1质粒DNA氯乙基化并与GSH反应，通过琼脂糖凝胶电泳测定DNA链间交联的程度并与对照进行比较。结果表明，时间和GSH浓度依赖性淬灭氯乙基化的DNA，与相应的DNA交联指数下降。使用[甲基-3H]GSH，还证明了56%的总GSH与淬灭的2-氯乙基化Col E1 DNA结合，25%与淬灭的2-氯乙基化小牛胸腺DNA结合。GSH与交联DNA和天然DNA的结合是微不足道的。它的结论是，除了反应性细胞毒性CENU物种的直接失活，GSH也可以通过淬灭氯乙基化DNA来调节细胞对CENU的反应，从而减少潜在致命的DNA交联的形成。
Interstrand DNA cross-linking is essential for the antitumor activity of chloroethylnitrosoureas (CENUs). The critical cross-links have been proposed to involve a rapid O6-guanine chloroethylation on one DNA strand, followed by a rearrangement of the O6-(2-chloroethyl)guanine and slow alkylation of the second DNA strand. In view of the relative intracellular abundance of glutathione (GSH) and nucleophilicity of its thiolate ion, the ability of GSH to react with and to inactivate 2-chlorethylated DNA and the possibility that this interaction decreases net DNA cross-linking by CENUs were investigated. Chloroethylated calf thymus DNA was reacted with GSH, the DNA was precipitated and redissolved, and subsequent DNA interstrand cross-linking was determined. The DNA cross-link index was compared for both GSH-treated and 2-chloroethylated untreated DNA. Simultaneously, Col E1 plasmid DNA was chloroethylated and reacted with GSH, and the extent of DNA interstrand cross-linking was determined by agarose gel electrophoresis and compared with controls. The results show both a time- and GSH concentration-dependent quenching of chloroethylated DNA, with a corresponding decrease in the DNA cross-link index. Using [methyl-3H]GSH, it was also demonstrated that 56% of the total GSH was bound to quenched 2-chloroethylated Col E1 DNA and 25% to quenched 2-chloroethylated calf thymus DNA. GSH binding to cross-linked DNA and native DNA was insignificant. It is concluded that, in addition to direct inactivation of reactive cytotoxic CENU species, GSH may also modulate cellular response to CENUs by quenching chloroethylated DNA, thereby decreasing the formation of potentially lethal DNA cross-links.