Takauji R, Yoshida A, Iwasaki H, Tohyama K, Ueda T, Nakamura T
First Department of Internal Medicine, Fukui Medical School.
Jpn J Cancer Res. 1995 Jul;86(7):677-84. doi: 10.1111/j.1349-7006.1995.tb02452.x.
Internucleosomal DNA fragmentation and morphological changes in nuclei typical of apoptosis were observed in L1210 cells incubated with 1.0 micrograms/ml of 1-beta-D-arabinofuranosylcytosine (ara-C). To investigate the mechanisms involved, we examined the activities of endogenous endonucleases in nuclei and cytoplasm. Both fractions of control cells contained Ca(2+)-dependent endonuclease which was capable of mediating internucleosomal DNA fragmentation. The assay system using two kinds of target substrates, i.e., nuclear chromatin of CCRF-CEM cells and naked DNA purified from the same cells, revealed that the activity of Ca(2+)-dependent endonuclease was enhanced in the crude nuclear extracts of cells treated with 1.0 microgram/ml of ara-C for 24 h or 48 h. The activity was extracted more easily from ara-C-treated cells than control cells without sonication of the nuclear fraction. On the other hand, in the cytoplasmic fraction of the cells, the activity towards naked DNA was unchanged, whereas that towards nuclear chromatin was clearly enhanced. These results suggest that internucleosomal DNA fragmentation induced by ara-C treatment is associated with enhancement and activation of constitutively expressed Ca(2+)-dependent endonuclease in L1210 cells.
在用1.0微克/毫升的1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C）孵育的L1210细胞中，观察到了典型凋亡的核小体间DNA片段化和细胞核形态变化。为了研究其中涉及的机制，我们检测了细胞核和细胞质中内源性核酸内切酶的活性。对照细胞的两个组分均含有能介导核小体间DNA片段化的Ca(2+)依赖性核酸内切酶。使用两种靶底物（即CCRF-CEM细胞的核染色质和从同一细胞中纯化的裸DNA）的检测系统显示，在用1.0微克/毫升ara-C处理24小时或48小时的细胞的粗核提取物中，Ca(2+)依赖性核酸内切酶的活性增强。与未对核组分进行超声处理的对照细胞相比，ara-C处理的细胞更容易提取出该活性。另一方面，在细胞的细胞质组分中，对裸DNA的活性未改变，而对核染色质的活性明显增强。这些结果表明，ara-C处理诱导的核小体间DNA片段化与L1210细胞中组成性表达的Ca(2+)依赖性核酸内切酶的增强和激活有关。
