Ling Y H, Priebe W, Perez-Soler R
Department of Head, Neck, and Thoracic Medical Oncology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Cancer Res. 1993 Apr 15;53(8):1845-52.
The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells. P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent. In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect. By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder. Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures. Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin. Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event. DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid. Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases. In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis. The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.
在小鼠白血病P388细胞中检测了拓扑异构酶II抑制剂阿霉素及其非交叉耐药类似物安那霉素对DNA降解和程序性细胞死亡的影响。暴露于不同浓度阿霉素和安那霉素24小时的P388亲本细胞呈现剂量依赖性DNA裂解：在1微摩尔时，阿霉素和安那霉素均能有效诱导DNA断裂，但在10微摩尔时，这种作用明显减弱或完全消失。在多药耐药的P388/Dox细胞中，阿霉素不会引起DNA裂解，而10微摩尔的安那霉素则有显著作用。通过琼脂糖凝胶分析，药物诱导的DNA片段化呈现出核小体间梯状的特征模式。形态学上，用1微摩尔阿霉素或安那霉素处理24小时的P388细胞显示细胞体积减小和核结构浓缩。在暴露于10微摩尔安那霉素24小时的P388/Dox细胞中观察到类似变化，但在暴露于10微摩尔阿霉素的细胞中未观察到。时间进程研究表明，与1微摩尔阿霉素或安那霉素孵育12小时后检测到DNA片段化，而膜完整性丧失出现在24小时，因此表明DNA降解是一个先行事件。阿霉素和安那霉素引起的DNA片段化受到RNA合成抑制剂放线菌素D、蛋白质合成抑制剂环己酰亚胺和核酸内切酶抑制剂金精三羧酸的抑制。环己酰亚胺和金精三羧酸部分阻止了药物诱导的细胞死亡，因此表明这些药物引起的凋亡过程需要基因表达、新蛋白质的合成以及内源性核酸酶的激活。相比之下，用1毫摩尔乙二醇双（2-氨基乙醚）-N,N,N',N'-四乙酸孵育细胞不会影响DNA裂解，因此表明细胞内钙耗竭不会影响蒽环类药物诱导的凋亡。所获得的结果表明，蒽环类药物的细胞杀伤作用至少部分是由凋亡诱导介导的。
