Oliveri M, Daga A, Cantoni C, Lunardi C, Millo R, Puccetti A
Department of Experimental Medicine, University of Genova, Genova, Italy.
Eur J Immunol. 2001 Mar;31(3):743-51. doi: 10.1002/1521-4141(200103)31:3<743::aid-immu743>3.0.co;2-9.
Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.
核酸内切酶激活后出现的核小体间DNA片段化是细胞凋亡的常见终点。DNase I是一种在哺乳动物组织中普遍表达的依赖Ca(2+) / Mg(2+)的核酸内切酶，被认为在这一过程中发挥作用。为了分析该酶在人类细胞中的体内功能，我们构建了一个DNase I基因靶向缺失的细胞系，以及几个过表达DNase I基因的稳定细胞系。在对药物刺激诱导的凋亡性细胞死亡高度敏感的Jurkat T细胞克隆JA3中实现了人类DNase I基因的失活。DNase I基因破坏后的JA3细胞对凋亡刺激产生了抗性。DNase I在人类细胞系JA3、K562（红白血病）、M 14（黑色素瘤）和CEM（T细胞淋巴瘤）中过表达。值得注意的是，DNase I基因的稳定过表达导致JA3细胞凋亡加速，并在K562、CEM和M14细胞系中诱导凋亡，否则这些细胞系在药物刺激后对核小体间DNA降解具有抗性。我们的研究提供了首个体内证据，表明DNase I介导了药物诱导凋亡的人类细胞中的核小体间DNA降解。
