Sailer B L, Valdez J G, Steinkamp J A, Crissman H A
Los Alamos National Laboratory, Life Sciences Division, New Mexico 87545, USA.
Cytometry. 1998 Mar 1;31(3):208-16.
Fluorescence lifetime analysis was used in combination with conventional flow cytometric analysis to monitor changes in residual chromatin in apoptotic HL-60 cell populations following treatment with camptothecin, cycloheximide, genistein, H7, and gamma radiation. Data presented show that all of these metabolic inhibitors, which act through different signaling cascades, produce apoptotic subpopulations with decreased but different lifetimes for DNA-bound ethidium bromide (EB). Additionally, treatment with certain agents reduced the fluorescence lifetime in the apoptotic cells prior to extensive endonuclease degradation of DNA and the appearance of the typical sub-G0/G1 peak in the DNA histogram. A lifetime value of 21.15 +/- 0.12 ns was obtained for EB bound to nonapoptotic cells, while values for EB bound to the apoptotic subpopulations following treatment with the different agents were: camptothecin, 19.87 +/- 0.08 ns; cycloheximide, 19.39 +- 0.02 ns; H7, 19.77 +/- 0.03 ns; genistein, 20.04 +/- 0.04 ns; and gamma radiation, 19.67 +/- 0.03 ns. Traditional methods of analysis, including gel electrophoresis or morphology assessment, revealed no significant differences among apoptotic subpopulations induced by treatment with these agents. Our data suggest that the mode of action of the various agents induces structural changes in chromatin organization that differentially alter accessibility of DNA to endonuclease digestion. Subsequent fluorescence lifetime analysis appears sensitive to the resulting differences in the residual chromatin in apoptotic cells following DNA cleavage. Results presented indicate that lifetime analysis, used in conjunction with conventional flow cytometry, can be useful for early detection of apoptosis-induced chromatin changes and may also potentially provide new information on the effects of different apoptosis-inducing agents.
荧光寿命分析与传统流式细胞术分析相结合，用于监测喜树碱、环己酰亚胺、染料木黄酮、H7和γ射线处理后凋亡HL-60细胞群体中残留染色质的变化。呈现的数据表明，所有这些通过不同信号级联起作用的代谢抑制剂，都会产生凋亡亚群，其与DNA结合的溴化乙锭（EB）的寿命缩短但各不相同。此外，用某些试剂处理会在DNA被广泛核酸内切酶降解和DNA直方图中出现典型的亚G0/G1峰之前，降低凋亡细胞中的荧光寿命。与非凋亡细胞结合的EB的寿命值为21.15±0.12纳秒，而用不同试剂处理后与凋亡亚群结合的EB的寿命值分别为：喜树碱，19.87±0.08纳秒；环己酰亚胺，19.39±0.02纳秒；H7，19.77±0.03纳秒；染料木黄酮，20.04±0.04纳秒；γ射线，19.67±0.03纳秒。传统的分析方法，包括凝胶电泳或形态学评估，在这些试剂处理诱导的凋亡亚群之间未发现显著差异。我们的数据表明，各种试剂的作用方式会诱导染色质组织的结构变化，从而不同程度地改变DNA对核酸内切酶消化的可及性。随后的荧光寿命分析似乎对DNA切割后凋亡细胞中残留染色质的差异敏感。呈现的结果表明，与传统流式细胞术结合使用的寿命分析，可用于早期检测凋亡诱导的染色质变化，也可能潜在地提供有关不同凋亡诱导剂作用的新信息。
