Gorczyca W, Melamed M R, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10523.
Toxicol Lett. 1993 Apr;67(1-3):249-58. doi: 10.1016/0378-4274(93)90060-b.
DNA topoisomerase I inhibitor camptothecin (CAM), topoisomerase II inhibitors teniposide (TN) and amsacrine (m-AMSA) induce apoptosis of HL-60 cells. One of the early events of apoptosis is DNA degradation, which occurs as a result of activation of the specific endonuclease. DNA strand breaks generated during this process were revealed, in the present study, by the in situ nick translation assay which was adapted to flow cytometry. In this assay, the incorporation of biotinylated dUTP by apoptotic cells was detected by the use of fluorescinated avidin, whereas simultaneous staining of DNA with propidium iodide made it possible to correlate the appearance of DNA strand breaks with cell position in the cell cycle. The breaks were detected as early as 90 min after the initial cell contact with CAM, and they were limited to cells in the S phase of the cell cycle. At that early stage of apoptosis DNA was not yet extractable from the cells; the loss of DNA from S-phase cells could not be seen, by flow cytometry, during the initial 2 h of incubation with CAM. DNA strand breaks induced by TN and m-AMSA also occurred preferentially in S-phase cells. The data indicate that DNA strand breaks resulting from activation of endonuclease in HL-60 cells treated with DNA topoisomerase I or II inhibitors can be conveniently measured using the in situ nick translation assay. This assay has certain advantages over other methods of identification of apoptotic cells by flow cytometry, such as providing direct evidence of DNA damage and offering the opportunity to correlate DNA damage with cell position in the cell cycle. The method may be of interest in clinical oncology where testing tumor response (by DNA degradation) to DNA topoisomerase inhibitors or other treatments may be of prognostic value.
DNA拓扑异构酶I抑制剂喜树碱（CAM）、拓扑异构酶II抑制剂替尼泊苷（TN）和安吖啶（m-AMSA）可诱导HL-60细胞凋亡。凋亡的早期事件之一是DNA降解，这是由特异性核酸内切酶激活所致。在本研究中，通过适用于流式细胞术的原位缺口平移试验揭示了在此过程中产生的DNA链断裂。在该试验中，通过使用荧光素化抗生物素蛋白检测凋亡细胞对生物素化dUTP的掺入，而同时用碘化丙啶对DNA进行染色使得将DNA链断裂的出现与细胞在细胞周期中的位置相关联成为可能。早在细胞最初接触CAM后90分钟就检测到了断裂，并且它们仅限于细胞周期S期的细胞。在凋亡的早期阶段，DNA尚未从细胞中提取出来；在与CAM孵育的最初2小时内，通过流式细胞术无法看到S期细胞中DNA的丢失。TN和m-AMSA诱导的DNA链断裂也优先发生在S期细胞中。数据表明，使用原位缺口平移试验可以方便地测量用DNA拓扑异构酶I或II抑制剂处理的HL-60细胞中由核酸内切酶激活导致的DNA链断裂。与通过流式细胞术鉴定凋亡细胞的其他方法相比，该试验具有某些优势，例如提供DNA损伤的直接证据，并提供将DNA损伤与细胞在细胞周期中的位置相关联的机会。该方法在临床肿瘤学中可能具有重要意义，在临床肿瘤学中，检测肿瘤对DNA拓扑异构酶抑制剂或其他治疗的反应（通过DNA降解）可能具有预后价值。
