Gorczyca W, Gong J, Ardelt B, Traganos F, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10595.
Cancer Res. 1993 Jul 1;53(13):3186-92.
The studies were aimed to detect the cell cycle-associated differences in the susceptibility of HL-60 cells to apoptosis induced by diverse agents. Exponentially growing HL-60 cells were treated with the DNA topoisomerase I inhibitor camptothecin; the DNA topoisomerase II inhibitors teniposide, m-AMSA, Mitoxantrone, or Fostriecin; the presumed tyrosine kinase inhibitor genistein; a serine/threonine kinase inhibitor H7; the protein synthesis inhibitor cycloheximide; the DNA replication inhibitor hydroxyurea; the nucleoside antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-azacytidine; and the alkylating agent nitrogen mustard, cisplatin, hyperthermia, and gamma irradiation. Endonucleolysis, which accompanied apoptosis induced by these agents, was assessed by two different flow cytometric methods, one based on DNA content measurements following extraction of low molecular weight DNA, and another using exogenous terminal deoxynucleotidyl transferase to label in situ DNA strand breaks. Each method allowed for both identification of apoptotic cells and analysis of the cell cycle distribution of the unaffected cell population; the method using terminal transferase also allowed for identification of the cell cycle position of apoptotic cells. Confirmed by analysis of DNA degradation by gel electrophoresis and changes in cell morphology, apoptosis was observed as early as 3 h after administration of most drugs and for some drugs was cell cycle phase specific. Cells progressing through S phase were selectively susceptible when treated with camptothecin, teniposide, m-AMSA, Mitoxantrone, H7, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine. Cells in G2-M preferentially underwent apoptosis in cultures treated with H7 or with gamma-irradiation. Cells in G1 phase were preferentially affected by 5-azacytidine, nitrogen mustard, and hyperthermia. No significant cell cycle specificity was observed in the case of Fostriecin, genistein, cycloheximide, or cisplatin. The cell cycle related difference in susceptibility to apoptosis may be a reflection of both the severity of the lesion induced by a given drug and the ability of the cells to repair that lesion; both can vary depending on the cell cycle phase.
这些研究旨在检测HL-60细胞对不同药物诱导凋亡的易感性中与细胞周期相关的差异。对数生长期的HL-60细胞用DNA拓扑异构酶I抑制剂喜树碱、DNA拓扑异构酶II抑制剂替尼泊苷、m-AMSA、米托蒽醌或福司曲星、假定的酪氨酸激酶抑制剂染料木黄酮、丝氨酸/苏氨酸激酶抑制剂H7、蛋白质合成抑制剂环己酰亚胺、DNA复制抑制剂羟基脲、核苷抗代谢物1-β-D-阿拉伯呋喃糖基胞嘧啶和5-氮杂胞苷以及烷化剂氮芥、顺铂、热疗和γ射线照射处理。通过两种不同的流式细胞术方法评估伴随这些药物诱导凋亡的内源性核酸酶解，一种基于提取低分子量DNA后测量DNA含量进行，另一种使用外源性末端脱氧核苷酸转移酶原位标记DNA链断裂。每种方法都能鉴定凋亡细胞并分析未受影响细胞群体的细胞周期分布；使用末端转移酶的方法还能鉴定凋亡细胞的细胞周期位置。通过凝胶电泳分析DNA降解和细胞形态变化得到证实，在大多数药物给药后3小时就观察到凋亡，并且对于某些药物，凋亡具有细胞周期阶段特异性。用喜树碱、替尼泊苷、m-AMSA、米托蒽醌、H7、羟基脲和1-β-D-阿拉伯呋喃糖基胞嘧啶处理时，处于S期进展的细胞选择性敏感。在H7或γ射线照射处理的培养物中，G2-M期的细胞优先发生凋亡。G1期的细胞优先受到5-氮杂胞苷、氮芥和热疗的影响。在福司曲星、染料木黄酮、环己酰亚胺或顺铂的情况下未观察到明显的细胞周期特异性。细胞周期对凋亡易感性的相关差异可能反映了给定药物诱导损伤的严重程度以及细胞修复该损伤的能力；两者都可能因细胞周期阶段而异。
