PHysiological roles and action Mechanisms of programd cell death in the brain during the ontogeny

在程序性细胞死亡期间，新生小鼠大脑产生杀伤糖蛋白NBCF(62 kDa，等电点9.1)，它在细胞死亡的最初阶段诱导细胞聚集、固缩和细胞质收缩，并在人神经母细胞瘤NB1细胞中诱导核DNA双链断裂180-210bp，NBCF处理时间为6小时，对应于琼脂糖凝胶电泳法所示的不返回点。细胞溶解在加入NBCF后12-24小时继续进行，而细胞内DNA断裂程度在12小时停止增加，与细胞外DNA断裂程度相似，表明有限程度的核小体间DNA断裂是细胞溶解的原因。最初加入锌或TPCK可抑制DNA断裂，但最初加入放线菌酮或6小时后加入锌或TPCK则不能抑制DNA断裂，提示DNA断裂是通过激活预先存在的核酸内切酶(EN)前体而不是促进EN转录实现的。NB1细胞用人脑胶质母细胞瘤A172细胞来源的分化因子(36 kDa，pI 5.5)分化后，表现出许多突起的生长，随后对NBCF的敏感性降低，并同时分泌一种蛋白类的NBCF拮抗因子(43 kDa，基础)，该因子在NBCF处理的未分化NB1细胞的DNA断裂程度略有增加后，短暂地减弱DNA断裂程度，提示细胞凋亡是可逆的或耐受的。NBCF可单独或与NB1细胞的细胞匀浆或细胞膜一起使单个细胞核免于DNA断裂，但不能使整个细胞免于DNA断裂，说明完整细胞内的胞外因子对DNA断裂是必不可少的。

At a period of programd cell death, the neonatal mouse brain generates the killer glycoprotein NBCF(62 kDa, pI 9.1), which induced cell aggregation, pycnosis, and shrinkage of cytoplasm at the initial stage as typically observed for apoptosis, and double strand scission of nuclear DNA by 180-210 bp in human neuroblastoma NB1 cells by a NBCF-treatment time of 6 hr corresponding to a point of no return as shown by agarose gel electrophoresis. Cytolysis continued to proceed at 12-24 hr after NBCF addition, whereas degrees of intracelluar DNA scission ceased to increase at 12 hr and were similar to extracellular DNA scission degrees, suggesting that a limited degree of internucleosomal DNA scission is responsible for cytolysis. The DNA scission was inhibited with Zn or TPCK added at the initial stage, but not with cycloheximide initially added or with Zn or TPCK added after 6 hr, suggesting DNA scission by activation of preexisting endonuclease (EN) precursor but not by promotion of EN transcription. NB1 cells differentiated with a human glioblastoma A172 cell-derived defferentiation factor (36 kDa, pI 5.5) exhibited outgrowth of many neurites followed by reduced susceptibility to NBCF, and concurrently secreted a proteinic NBCF-antagonizing factor (43 kDa, basic), which transiently attenuated DNA scission degrees in undifferentiated NB1 cells treated with NBCF after a slight increase in DNA scission degrees initially observed, suggesting reversibility or tolerance in spoptosis. Isolated nuclei alone or together with cell homogenate or cell membrane of NB1 cells but not whole cells were saved from DNA scission by NBCF, showing that extracellular factors built in intact cells are essential for the DNA scission.