Chiu S M, Xue L Y, Friedman L R, Oleinick N L
Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Cancer Res. 1989 Feb 15;49(4):910-4.
The antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits topoisomerase II activity through the formation of a complex of DNA and covalently bound enzyme which, upon protein denaturation, yields DNA breaks (single strand breaks). In the present study, this complex served as a standard for analysis of radiation-induced DNA-protein cross-links (DPC). Following the treatment of exponentially growing mouse L929 cells with 0-100 ng/ml of m-AMSA for 1 h, a linear dose-dependent increase was found in the amount of DNA retained on nitrocellulose filters during subsequent analysis. This result indicates that the assay can detect DPC that have a single protein bound to each DNA fragment. The results of fractionation of nuclear DNA show that m-AMSA induces 20- to 45-fold more DPC in nuclear matrix-associated DNA than in the majority distal loop DNA, supporting the notion that topoisomerase II is located at the nuclear matrix. The frequency of single strand breaks induced by m-AMSA, which should be equal to the frequency of DPC, was determined by alkaline elution. Results of the alkaline elution assay could be correlated with the percentage of DNA retained on nitrocellulose filters; i.e., 1% DNA retention corresponded to 2560 DPC per log-phase L929 cell, which has been determined to have a DNA content of 22.25 pg. Using this standard curve, DPC induced by gamma-irradiation in air were estimated to be formed at a frequency of 133 DPC/cell/Gy, a frequency approximately 3% that of gamma-ray-induced single strand breaks. The radiation dose response for DPC production was unaffected by the high levels of DPC present in cells previously treated with m-AMSA. In addition, DPC induced by m-AMSA were rapidly reversed after the removal of the drug, in contrast to a slower removal of DPC induced by gamma-radiation. These observations suggest that although m-AMSA and gamma-radiation both preferentially induce DPC with matrix-attached DNA, they produce independent types of DPC.
抗肿瘤药物4'-(9-吖啶基氨基)甲磺基间茴香胺（m-AMSA）通过形成DNA与共价结合酶的复合物来抑制拓扑异构酶II的活性，该复合物在蛋白质变性时会产生DNA断裂（单链断裂）。在本研究中，这种复合物用作分析辐射诱导的DNA-蛋白质交联（DPC）的标准物。用0至100 ng/ml的m-AMSA处理指数生长的小鼠L929细胞1小时后，在随后的分析中发现，硝酸纤维素滤膜上保留的DNA量呈线性剂量依赖性增加。这一结果表明该检测方法能够检测到每个DNA片段上结合有单个蛋白质的DPC。核DNA分级分离的结果显示，m-AMSA在核基质相关DNA中诱导的DPC比大多数远端环DNA中多20至45倍，这支持了拓扑异构酶II位于核基质的观点。由m-AMSA诱导的单链断裂频率（应与DPC频率相等）通过碱性洗脱法测定。碱性洗脱检测的结果与硝酸纤维素滤膜上保留的DNA百分比相关；即，1%的DNA保留对应于每个对数期L929细胞2560个DPC，已确定该细胞的DNA含量为22.25 pg。利用该标准曲线，估计空气中γ射线照射诱导的DPC形成频率为133 DPC/细胞/戈瑞，该频率约为γ射线诱导的单链断裂频率的3%。DPC产生的辐射剂量反应不受先前用m-AMSA处理的细胞中存在的高水平DPC的影响。此外，与γ射线诱导的DPC去除较慢相反，m-AMSA诱导的DPC在药物去除后迅速逆转。这些观察结果表明，尽管m-AMSA和γ射线都优先诱导与基质附着DNA相关的DPC，但它们产生的是独立类型的DPC。
