Barry M A, Reynolds J E, Eastman A
Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Cancer Res. 1993 May 15;53(10 Suppl):2349-57.
Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both physiological stimuli and cytotoxic treatment with many anticancer agents. This digestion has generally been considered to be mediated by a Ca2+/Mg(2+)-dependent endonuclease that is activated by increases in intracellular Ca2+. However, we suggest that an alternate endonuclease, DNase II, may be a more likely candidate. In these studies, apoptosis was induced in human HL-60 cells by a 30-min incubation with the topoisomerase II inhibitor etoposide. DNA digestion characteristic of apoptosis began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells underwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progressed to the G2 phase and arrested there for at least 48 h. Intracellular Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of up to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on the basis of intracellular pH and only the acidic cells showed the morphology and DNA digestion characteristic of apoptosis. These results demonstrate the involvement of DNase II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis as regulators of apoptosis.
细胞凋亡是一种细胞死亡途径，其特征是基因组DNA发生核小体间消化。这种DNA消化可由生理刺激和多种抗癌药物的细胞毒性处理诱导。这种消化通常被认为是由一种Ca2+/Mg(2+)依赖性核酸内切酶介导的，该酶通过细胞内Ca2+的增加而被激活。然而，我们认为另一种核酸内切酶DNase II可能是更合适的候选者。在这些研究中，通过将人HL-60细胞与拓扑异构酶II抑制剂依托泊苷孵育30分钟来诱导细胞凋亡。在去除依托泊苷后3小时内开始出现细胞凋亡特有的DNA消化。同时观察到细胞凋亡的形态学迹象。此时只有约20%的细胞发生凋亡；这些细胞在依托泊苷处理时似乎处于S期。其余细胞进入G2期并在那里停滞至少48小时。通过流式细胞术测量单个细胞内的Ca2+和pH值。未观察到细胞内Ca2+的变化，但约15%的细胞发生了高达1个pH单位的酸化，且与DNA消化出现的时间进程相关。根据细胞内pH值对细胞进行分选，只有酸性细胞表现出细胞凋亡特有的形态和DNA消化。这些结果证明了DNase II参与凋亡性DNA消化，并提示pH稳态作为细胞凋亡调节因子的机制。
