Polzar B, Peitsch M C, Loos R, Tschopp J, Mannherz H G
Institut für Zytobiologie und Zytopathologie, Philipps-Universität, Marburg, Germany.
Eur J Cell Biol. 1993 Dec;62(2):397-405.
COS-cells were transiently transfected with the pSG5 plasmid containing the cDNA of rat parotid deoxyribonuclease I (DNase I) either in right or inverse orientation. Expression of DNase I in transfected cells was only observed when the plasmid contained the cDNA in the right orientation. Expression of DNase I was monitored by measuring the DNase I specific DNA-degrading activity present in the conditioned cell culture medium and in cell homogenates. The expressed DNase I activity could be inhibited by monospecific polyclonal antibodies and by G-actin. Immunofluorescence indicated that approximately 20% of the COS-cells transfected with the DNase I-cDNA in right orientation expressed DNase I. These transfected cells contained large amounts of DNase I, which was found to be localized within the rough endoplasmic reticulum, the Golgi-complex and finally concentrated in a perinuclear location. Occasionally cells were observed which contained the DNase I in small apparently secretory transport vesicles. Transfected cells with perinuclear concentration of DNase I exhibited progressive nuclear destruction, i.e., pyknosis and cytoplasmic shrinkage. Solely the DNA extracted from isolated nuclei of cells transfected with the DNase I-cDNA in correct orientation revealed an internucleosomal DNA-degradation (ladder formation) typical for apoptosis after incubation in the presence of CaCl2 and MgCl2. Only the conditioned medium of COS-cells transfected with the right-oriented DNase I-cDNA contained the nucleolytic activity able to internucleosomally degrade the chromatin of substrate nuclei. Thus, these results indicate that overexpression of DNase I alone is sufficient to induce the morphological and biochemical changes observed during apoptosis.
将含有大鼠腮腺脱氧核糖核酸酶I（DNase I）cDNA的pSG5质粒以正向或反向瞬时转染COS细胞。仅当质粒含有正向cDNA时，才观察到转染细胞中DNase I的表达。通过测量条件细胞培养基和细胞匀浆中存在的DNase I特异性DNA降解活性来监测DNase I的表达。表达的DNase I活性可被单特异性多克隆抗体和G-肌动蛋白抑制。免疫荧光表明，约20%正向转染DNase I-cDNA的COS细胞表达DNase I。这些转染细胞含有大量的DNase I，发现其定位于粗面内质网、高尔基体复合物中，最终集中在核周位置。偶尔观察到细胞含有少量明显的分泌运输小泡中的DNase I。核周集中有DNase I的转染细胞表现出渐进性核破坏，即核固缩和细胞质收缩。仅从正向转染DNase I-cDNA的细胞分离核中提取的DNA在CaCl2和MgCl2存在下孵育后显示出典型的凋亡核小体间DNA降解（梯状形成）。只有正向转染DNase I-cDNA的COS细胞的条件培养基含有能够核小体间降解底物核染色质的核酸酶活性。因此，这些结果表明，单独过表达DNase I足以诱导凋亡过程中观察到的形态和生化变化。
