Decreased stability of DNA in cells treated with alkylating agents.
Decreased stability of DNA in cells treated with alkylating agents.
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用烷化剂处理的细胞中 DNA 的稳定性降低。
DOI：
10.1016/0014-4827(90)90003-s
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发表时间：
1990
影响因子：
3.7
通讯作者：
Frankfurt,OS
中科院分区：
文献类型：
--
作者：
Frankfurt,OS
报道了一种改良的高灵敏度程序，用于评价用烷化剂处理的单个细胞中的DNA损伤。新的方法是基于在Mg 2+存在下加热烷基化DNA中单链的扩增。用氮芥（HN 2）处理人卵巢癌细胞A2780，在甲醇中固定，并用针对HN 2处理的DNA产生的单克隆抗体（MOAB）F7-26染色。通过流式细胞术和间接免疫荧光测量MOAB的结合。在含有1.25 mMMgCl 2的PBS中于100 °C加热5分钟后，观察到未处理和HN 2处理的细胞之间荧光的最大差异。高浓度MgCl 2抑制MOAB与HN 2处理细胞的结合，低浓度加热诱导MOAB与对照细胞的结合。在补充有MgCl 2的Tris缓冲液中加热后，观察到MOAB与对照和药物处理的细胞的强烈结合。因此，在加热过程中磷酸盐和MgCl 2的存在是必要的检测HN 2诱导的DNA稳定性的变化。用单链特异性S1核酸酶处理后，HN 2处理的细胞的荧光降低至背景水平。MOAB F7-26与DNA中的单链区域相互作用，并且不与dsDNA或其他细胞抗原结合。通过抗生物素蛋白-生物素ELISA确定MOAB F7-26与脱氧胞苷的特异性反应性。单链构象是MOAB与DNA分子上的脱氧胞苷结合所必需的。这表明鸟嘌呤的烷基化降低了DNA分子的稳定性，并增加了MOAB F7-26对相对DNA链上的脱氧胞苷的接近。
A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 °C for 5 min in PBS containing 1.25 mMMgCl2. Higher concentrations of MgCl2inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.