Ferlini C, Di Cesare S, Rainaldi G, Malorni W, Samoggia P, Biselli R, Fattorossi A
Laboratory of Immunology, D.A.S.R.S., Pratica di Mare, Rome, Italy.
Cytometry. 1996 Jun 1;24(2):106-15. doi: 10.1002/(SICI)1097-0320(19960601)24:2<106::AID-CYTO2>3.0.CO;2-H.
The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.
在新鲜分离的受辐照人淋巴细胞、生长因子剥夺培养的人淋巴细胞以及地塞米松（DEX）处理的小鼠胸腺细胞中，研究了凋亡过程中质膜、染色质和线粒体水平上发生的早期事件。在完整、未固定的细胞中，通过评估光散射特性和用溴化乙锭（EB）检测DNA可染性，能够识别出提示初始凋亡的细胞亚群。在受辐照人淋巴细胞模型中通过细胞分选证实了这些细胞的凋亡性质。碘化丙啶不能替代EB，这表明所使用的DNA探针的性质对于检测初始凋亡变化至关重要。由于线粒体被认为是凋亡过程中的主要靶点，我们在测量EB摄取的同时，测量了线粒体跨膜电位敏感探针（罗丹明123）和不敏感探针（10-壬基吖啶橙）的摄取情况。开始凋亡的细胞对两种线粒体染料的摄取均增加，这与EB一起识别出了几个细胞亚群。这表明在凋亡早期线粒体结构和功能发生了复杂改变。为了研究在凋亡相似阶段染色质水平上发生的现象，将受辐照的人淋巴细胞和DEX处理的小鼠胸腺细胞破碎，并在核悬液中评估DNA可染性。在凋亡早期，伴随着光散射特性的特定改变，观察到DNA可染性短暂增加，即在人模型中出现明显的超二倍体峰，在小鼠模型中G0/1峰普遍上移。这些发现表明染色质结构在凋亡早期发生改变，并影响DNA可染性。
