Hotz M A, Traganos F, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10595.
Exp Cell Res. 1992 Jul;201(1):184-91. doi: 10.1016/0014-4827(92)90362-c.
The antitumor drug fostriecin (phosphotrienin, FST) has been reported to exert its cytostatic and cytotoxic effects via inhibition of DNA topoisomerase II. The sensitivity of human lymphocytic leukemic MOLT-4 and promyelocytic HL-60 leukemic cells to a wide range of FST concentrations was studied by analyzing the cell cycle-specific effects and changes in nuclear chromatin induced by this inhibitor. The latter was evaluated by assaying the sensitivity of DNA in situ to acid-induced denaturation cytofluorimetrically, with the use of the metachromatic fluorochrome acridine orange (AO), which differentially stains double-stranded and denatured DNA. The cytostatic effects were observed soon after addition of FST (at concentrations of 1-30 microM for MOLT-4 cultures and 1-5 microM for HL-60 cultures) as a perturbation of cell progression through S and G2 phases of the cell cycle. Cell progression through the cycle was halted at greater than 30 microM FST in MOLT-4 cultures and at greater than 5 microM in HL-60 cultures; the effect was instantaneous and affected all phases of the cycle, so that no changes in the cell cycle distribution were apparent with increasing length of exposure to the drug. Instead, at these high FST concentrations, immediate cytotoxic effects became evident, manifesting either as cell apoptosis or necrosis. Apoptosis was observed only in the case of HL-60 cells, at FST concentrations of 5-100 microM, and was characterized by markedly increased sensitivity of DNA to denaturation combined with a decrease in overall DNA stainability, either with the DNA-specific dye DAPI or with AO, indicative of the activation of endogenous nucleases. Necrotic cell death was observed at FST concentrations of 1 mM and at greater than 30 microM for HL-60 and MOLT-4 cells, respectively: in both cases the overall DNA stainability, with either DAPI or AO, was unchanged compared to the control, but their DNA was very sensitive to denaturation. Interestingly, DNA in G2 and late S phase MOLT-4 cells, which were undergoing necrotic death, was much more sensitive to denaturation than was DNA in G1 cells of this lineage. The data indicate that chromatin changes induced by DNA topoisomerase II inhibitors in cells that undergo apoptotic or necrotic death can be conveniently monitored by the assay of DNA in situ sensitivity to denaturation.
据报道，抗肿瘤药物福司曲星（磷三烯菌素，FST）通过抑制DNA拓扑异构酶II发挥其细胞生长抑制和细胞毒性作用。通过分析这种抑制剂诱导的细胞周期特异性效应和核染色质变化，研究了人淋巴细胞白血病MOLT-4细胞和早幼粒细胞白血病HL-60细胞对多种FST浓度的敏感性。通过使用异染荧光染料吖啶橙（AO），以细胞荧光分析法测定原位DNA对酸诱导变性的敏感性来评估后者，AO可对双链DNA和变性DNA进行差异染色。添加FST后很快就观察到了细胞生长抑制效应（对于MOLT-4培养物，FST浓度为1 - 30 microM；对于HL-60培养物，FST浓度为1 - 5 microM），表现为细胞周期S期和G2期进程受到干扰。在MOLT-4培养物中，FST浓度大于30 microM时细胞周期进程停止；在HL-60培养物中，FST浓度大于5 microM时细胞周期进程停止；这种效应是即时性的，影响细胞周期的所有阶段，因此随着药物暴露时间延长，细胞周期分布没有明显变化。相反，在这些高FST浓度下，立即出现细胞毒性效应，表现为细胞凋亡或坏死。仅在HL-60细胞中观察到凋亡，FST浓度为5 - 100 microM，其特征是DNA对变性的敏感性显著增加，同时用DNA特异性染料DAPI或AO检测时总体DNA染色性降低，这表明内源性核酸酶被激活。在FST浓度为1 mM时观察到坏死性细胞死亡，对于HL-60和MOLT-4细胞，分别在FST浓度大于30 microM时观察到坏死性细胞死亡：在这两种情况下，与对照相比，用DAPI或AO检测时总体DNA染色性没有变化，但它们的DNA对变性非常敏感。有趣的是，正在经历坏死性死亡的G2期和S期晚期MOLT-4细胞中的DNA比该谱系G1期细胞中的DNA对变性更敏感。数据表明，通过测定原位DNA对变性的敏感性，可以方便地监测DNA拓扑异构酶II抑制剂在经历凋亡或坏死性死亡的细胞中诱导的染色质变化。
