Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line.
Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line.
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DOI：
10.1083/jcb.138.2.449
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发表时间：
1997-07-28
期刊：
影响因子：
--
通讯作者：
Hockenbery DM
中科院分区：
文献类型：
--
作者：
Mancini M;Anderson BO;Caldwell E;Sedghinasab M;Paty PB;Hockenbery DM
酪氨酸激酶抑制剂赫比霉素A可诱导低分化人结肠癌细胞系COLO-205细胞分化和延迟凋亡。结合DNA片段末端标记的细胞周期分析显示，G2期停滞先于细胞死亡。超微结构检查显示细胞上皮细胞分化的形态特征，包括形成微绒毛顶膜和侧方桥粒粘连。线粒体也有明显堆积。使用线粒体探针--壬基-吖啶橙和JC-1进行的荧光分析证实，线粒体质量逐渐增加。然而，这些细胞也表现出单位线粒体跨膜电位(ΔΨm)的进行性下降，这是由ΔΨm敏感的荧光探针罗丹明123和JC-1分析红色荧光所确定的。与这些线粒体变化相一致的是，经除草剂A处理的COLO-205细胞产生了更高水平的活性氧，这一点从二氯二氢荧光素二乙酸酯和二氢乙锭的氧化中得到了证明。用大鼠肝细胞核和24小时的Colo-205细胞提取物进行的无细胞凋亡检测表明，Colo-205裂解产物的凋亡活性需要线粒体的早期作用。在多聚ADP-核糖聚合酶裂解前的早期时间点观察到线粒体的形态和功能的变化。这些结果表明，分化的Colo-205细胞的凋亡涉及线粒体的不受抑制的增殖和进行性的膜功能障碍，这是一种新的凋亡机制。
Herbimycin A, a tyrosine kinase inhibitor, induces cellular differentiation and delayed apoptosis in Colo-205 cells, a poorly differentiated human colon carcinoma cell line. Cell cycle analysis in conjunction with end labeling of DNA fragments revealed that G2 arrest preceded apoptotic cell death. Ultrastructural examination of herbimycin-treated cells demonstrated morphologic features of epithelial differentiation, including formation of a microvillar apical membrane and lateral desmosome adhesions. A marked accumulation of mitochondria was also observed. Fluorometric analysis using the mitochondrial probes nonyl-acridine orange and JC-1 confirmed a progressive increase in mitochondrial mass. However these cells also demonstrated a progressive decline in unit mitochondrial transmembrane potential (ΔΨm) as determined by the ΔΨm-sensitive fluorescent probes rhodamine 123 and JC-1 analyzed for red fluorescence. In concert with these mitochondrial changes, Colo-205 cells treated with herbimycin A produced increased levels of reactive oxygen species as evidenced by oxidation of both dichlorodihydrofluorescein diacetate and dihydroethidium. Cell-free assays for apoptosis using rat-liver nuclei and extracts of Colo-205 cells at 24 h showed that apoptotic activity of Colo-205 lysates requires the early action of mitochondria. Morphological and functional mitochondrial changes were observed at early time points, preceding cleavage of poly (ADP-ribose) polymerase. These results suggest that apoptosis in differentiated Colo-205 cells involves unrestrained mitochondrial proliferation and progressive membrane dysfunction, a novel mechanism in apoptosis.