Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models
Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models
复制标题
DOI：
10.1111/j.1474-9726.2005.00163.x
复制
发表时间：
2005-08-01
期刊：
影响因子：
7.8
通讯作者：
Campisi, J
中科院分区：
文献类型：
--
作者：
Goldstein, JC;Rodier, F;Campisi, J
与年龄相关的组织功能丧失和几种慢性疾病可能部分源于凋亡细胞死亡水平细微变化的累积效应。由于细胞凋亡是快速的，一旦完成就无法检测到，因此即使在控制良好的细胞培养中，其发生率的微小变化也很难检测到。我们描述了一种新的细胞凋亡测定，它提供了比传统测定更大的灵敏度，因为它测量了凋亡细胞的积累。利用化学抑制剂Q-VD-OPH （QVD）抑制caspase活性，将诱导凋亡的人、小鼠成纤维细胞和人乳腺上皮细胞保存3天。细胞色素c从健康细胞的线粒体重新分布到死亡细胞的细胞质中，通过免疫染色对悬浮在凋亡过程中的细胞进行评分。当放线菌素D诱导细胞凋亡时，这种不依赖caspase的细胞色素c释放（CICR）检测方法比几种传统的检测方法更敏感，并且可以检测到3天间隔内细胞凋亡的累积背景水平。通过该实验，我们发现正常成纤维细胞在x射线照射下很少发生凋亡，表明这种细胞类型的衰老反应占主导地位。此外，当人乳腺上皮细胞和皮肤成纤维细胞进入危象时，细胞凋亡的增加虽微小但可测量，这表明危象中的细胞死亡在很大程度上是非凋亡的。
Age-associated loss of tissue function and several chronic diseases may derive in part from the cumulative effects of subtle changes in the level of apoptotic cell death. Because apoptosis is rapid and undetectable once complete, small changes in its incidence are difficult to detect, even in well-controlled cell cultures. We describe a new apoptosis assay that provides greater sensitivity than conventional assays because it measures the accumulation of apoptotic cells. Human and mouse fibroblasts and human mammary epithelial cells that initiated apoptosis were preserved for 3 days by inhibiting caspase activity using the chemical inhibitor Q-VD-OPH (QVD). Cells suspended in the process of apoptosis were scored by immunostaining for cytochrome c, which redistributed from mitochondria in healthy cells to the cytoplasm in dying cells. This caspase-independent cytochrome c release (CICR) assay was more sensitive than several conventional assays when apoptosis was induced by actinomycin D, and detected cumulative background levels of apoptosis over a 3-day interval. Using this assay, we show that normal fibroblasts undergo very little apoptosis upon X-irradiation, indicating dominance of the senescence response in this cell type. Further, apoptosis increased subtly but measurably when human mammary epithelial and skin fibroblast cells entered crisis, indicating that cell death during crisis is largely non-apoptotic.