Frankfurt O S
Oncology Laboratory, Cedars Medical Center, Miami, Florida 33136.
Exp Cell Res. 1990 Dec;191(2):181-5. doi: 10.1016/0014-4827(90)90003-s.
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 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited 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 MgCl2 during 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 S1 nuclease. 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.
本文报道了一种经过改进的高灵敏度方法，用于评估经烷基化剂处理的单个细胞中的DNA损伤。新方法基于在Mg2+存在下加热使烷基化DNA中的单链性得以扩增。人卵巢癌细胞A2780用氮芥（HN2）处理，用甲醇固定，并用针对HN2处理的DNA产生的单克隆抗体（MOAB）F7-26进行染色。通过间接免疫荧光流式细胞术测量MOAB的结合情况。在含有1.25 mM MgCl2的PBS中于100℃加热5分钟后，观察到未处理细胞和HN2处理细胞之间荧光的最大差异。较高浓度的MgCl2会抑制MOAB与HN2处理细胞的结合，而较低浓度下加热会诱导其与对照细胞结合。在补充有MgCl2的Tris缓冲液中加热后，观察到MOAB与对照细胞和药物处理细胞的强烈结合。因此，加热过程中磷酸盐和MgCl2的存在对于检测HN2诱导的DNA稳定性变化是必要的。用单链特异性S1核酸酶处理后，HN2处理细胞的荧光降至背景水平。MOAB F7-26与DNA中的单链区域相互作用，不与双链DNA或其他细胞抗原结合。通过抗生物素蛋白-生物素ELISA确定了MOAB F7-26与脱氧胞苷的特异性反应性。单链构象对于MOAB与DNA分子上的脱氧胞苷结合是必要的。有人提出，鸟嘌呤的烷基化降低了DNA分子的稳定性，并增加了MOAB F7-26与相对DNA链上的脱氧胞苷的接触。
