Didenko Vladimir V
Departments of Neurosurgery and Molecular & Cellular Biology, Baylor College of Medicine, and Michael E. DeBakey VA Medical Center, Houston, TX, USA.
Methods Mol Biol. 2011;682:77-87. doi: 10.1007/978-1-60327-409-8_7.
Recently, the concept of apoptotic cell elimination was expanded and programed cell death is no longer viewed as an individual cellular event. The complete description of the apoptotic process now includes two phases: the self-driven cell disassembly and the externally-controlled elimination of apoptotic cell corpses by phagocytizing cells. The second, phagocytic phase is essential, highly conserved, and is even more important than the internal phase of cell disassembly. This is because it ensures the complete degradation of the dying cell's DNA, preventing the release of pathological, viral and tumor DNA, and self-immunization. In different cells and species from mammals to flies, a single conserved enzyme--DNase II is responsible for the elimination of cellular DNA in the second "mopping up" phase of apoptosis. Here, we present an assay for the selective detection of the phagocytic phase of apoptosis. The technology capitalizes on the fact that phagocytic DNase II produces identifiable signature DNA breaks, which can be labeled by vaccinia topoisomerase. The assay permits labeling of the previously underestimated phase of apoptotic execution and is a useful tool in the apoptosis detection arsenal.
最近，凋亡细胞清除的概念得到了扩展，程序性细胞死亡不再被视为单个细胞事件。现在，对凋亡过程的完整描述包括两个阶段：自我驱动的细胞解体以及吞噬细胞对凋亡细胞尸体的外部控制清除。第二个阶段，即吞噬阶段至关重要，高度保守，甚至比细胞解体的内部阶段更重要。这是因为它确保了垂死细胞DNA的完全降解，防止病理性、病毒和肿瘤DNA的释放以及自身免疫。在从哺乳动物到果蝇的不同细胞和物种中，一种单一的保守酶——DNase II负责在凋亡的第二个“清理”阶段清除细胞DNA。在此，我们提出一种用于选择性检测凋亡吞噬阶段的测定方法。该技术利用了吞噬性DNase II产生可识别的标志性DNA断裂这一事实，这些断裂可由痘苗拓扑异构酶标记。该测定方法允许对先前被低估的凋亡执行阶段进行标记，是凋亡检测方法中的一个有用工具。
