Susin S A, Zamzami N, Larochette N, Dallaporta B, Marzo I, Brenner C, Hirsch T, Petit P X, Geuskens M, Kroemer G
Unité Propre de Recherche 420, Centre National de la Recherche Scientifique, Villejuif, France.
Exp Cell Res. 1997 Nov 1;236(2):397-403. doi: 10.1006/excr.1997.3733.
Purified nuclei exposed to apoptogenic factors in vitro undergo morphological and biochemical changes in chromatin organization. Most cell-free models of nuclear apoptosis are based on the quantitation of endonuclease-mediated DNA fragmentation on agarose gels or on the changes of nuclear morphology revealed by the DNA-intercalating fluorochrome 4'-6-diamidino-2-phenylindole dihydrochloride. In this work we develop a cytofluorometric system for the accurate quantitation of nuclear DNA loss. This system has been used to determine the conditions of nuclear apoptosis induced by apoptosis-inducing factor (AIF) contained in the supernatant of mitochondria induced to undergo permeability transition. AIF can provoke significant nuclear DNA loss in < or = 5 min, acts over a wide pH range (pH 6 to 9), and resists cysteine protease inhibitors such as iodoacetamide and N-ethylmaleimide. Moreover, we applied this system to the question of how the proapoptotic second messenger ceramide would induce apoptosis in vitro: via a direct effect on nuclei, a direct effect on mitochondria, or via indirect mechanisms? Our data indicate that ceramide has to activate yet unknown cytosolic effectors that, in the presence of mitochondria, can induce nuclear apoptosis in vitro.
体外暴露于凋亡诱导因子的纯化细胞核会在染色质组织上发生形态和生化变化。大多数细胞核凋亡的无细胞模型是基于琼脂糖凝胶上核酸内切酶介导的DNA片段化定量分析，或者基于DNA嵌入荧光染料4'-6-二脒基-2-苯基吲哚二盐酸盐所揭示的核形态变化。在这项工作中，我们开发了一种细胞荧光分析系统，用于准确量化细胞核DNA的损失。该系统已被用于确定由经历通透性转变的线粒体上清液中所含的凋亡诱导因子（AIF）诱导细胞核凋亡的条件。AIF可在≤5分钟内引发显著的细胞核DNA损失，在较宽的pH范围（pH 6至9）内起作用，并且对诸如碘乙酰胺和N-乙基马来酰亚胺等半胱氨酸蛋白酶抑制剂具有抗性。此外，我们将该系统应用于促凋亡第二信使神经酰胺如何在体外诱导凋亡这一问题：是通过对细胞核的直接作用、对线粒体的直接作用，还是通过间接机制？我们的数据表明，神经酰胺必须激活未知的胞质效应物，这些效应物在线粒体存在的情况下可在体外诱导细胞核凋亡。
