Maeda S, Suzuki A, Lin K H, Inagaki H, Saito T
Department of Chemistry, National Industrial Research Institute of Nagoya.
J Biochem. 1995 Dec;118(6):1161-5. doi: 10.1093/oxfordjournals.jbchem.a125002.
We previously reported that internucleosomal DNA fragmentation, a biochemical feature of apoptosis, was induced spontaneously in high-cell-density culture of adult rat hepatocytes. To understand better the intracellular mechanism of the DNA fragmentation in this system, we have examined the effects of several inhibitors of specific intracellular functions on the DNA fragmentation. We found that the DNA fragmentation could be suppressed by treatment of the cells with inhibitors of mitochondrial respiration, KCN and CCCP, or a protein synthesis inhibitor, cycloheximide. We also demonstrated that calmodulin inhibitors, chlorpromazine and W-7, could suppress the DNA fragmentation. Together, these results lead us to conclude that the DNA fragmentation in hepatocytes cultured at high cell density is an active process dependent on energy availability, gene expression, and calmodulin, rather than a passive event resulting from necrosis. However, by analyzing the incidence of apoptotic morphology during culture, we found that only 2-3% of cells exhibited apoptotic morphology, while the incidence of DNA fragmentation was estimated to be much higher. Based on these results, we estimated that the DNA fragmentation may result from the incomplete progression of apoptosis or from the occurrence of another type of active cell death.
我们之前报道过，成年大鼠肝细胞在高细胞密度培养时会自发诱导出凋亡的生化特征——核小体间DNA片段化。为了更好地理解该系统中DNA片段化的细胞内机制，我们研究了几种特异性细胞内功能抑制剂对DNA片段化的影响。我们发现，用线粒体呼吸抑制剂氰化钾（KCN）和羰基氰-间氯苯腙（CCCP）或蛋白质合成抑制剂环己酰亚胺处理细胞，可以抑制DNA片段化。我们还证明，钙调蛋白抑制剂氯丙嗪和W-7也能抑制DNA片段化。这些结果共同使我们得出结论，高细胞密度培养的肝细胞中的DNA片段化是一个依赖于能量供应、基因表达和钙调蛋白的主动过程，而不是由坏死导致的被动事件。然而，通过分析培养过程中凋亡形态的发生率，我们发现只有2%-3%的细胞呈现凋亡形态，而DNA片段化的发生率估计要高得多。基于这些结果，我们推测DNA片段化可能是凋亡不完全进展或另一种类型的主动细胞死亡所致。
