Peitsch M C, Polzar B, Stephan H, Crompton T, MacDonald H R, Mannherz H G, Tschopp J
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
EMBO J. 1993 Jan;12(1):371-7. doi: 10.1002/j.1460-2075.1993.tb05666.x.
Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.
凋亡引起的细胞死亡发生在多种生理事件中，包括淋巴细胞的库选择以及体内免疫反应过程中。凋亡的一个标志是核小体间DNA降解，为此假定存在一种Ca2+、Mg(2+)依赖性核酸内切酶。这种核酸酶活性是从大鼠胸腺细胞和淋巴结细胞核中提取的。当与仅具有有限量内源性核酸酶活性的细胞核一起孵育时，可诱导出凋亡特有的DNA片段梯形模式。这种可提取的核酸olytic活性用针对大鼠脱氧核糖核酸酶I（DNase I）的特异性抗体进行免疫沉淀，并被与凝溶胶蛋白片段1结合的肌动蛋白抑制，强烈表明核提取物中存在DNase I型酶。用大鼠腮腺DNase I的cDNA瞬时转染的COS细胞表达了该酶，并且它们的细胞核能够将其DNA降解为寡聚体大小的片段。对从胸腺、淋巴结细胞和肾脏分离的mRNA进行PCR分析，得到的产物大小与大鼠腮腺DNase I的产物相同。用抗大鼠DNase I抗体进行免疫组织化学染色证实了胸腺细胞和淋巴结细胞中存在DNase I抗原。因此，DNase I的组织分布扩展到没有消化功能的组织以及已知易发生凋亡的细胞。我们提出，在凋亡过程中，由于内质网和核膜的破裂，一种与DNase I无法区分的核酸内切酶进入细胞核。
