Wride M A, Parker E, Sanders E J
Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Dev Biol. 1999 Sep 1;213(1):142-56. doi: 10.1006/dbio.1999.9375.
The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis.
在发育过程中，通过程序性地从晶状体纤维细胞中去除细胞核和其他细胞器，确保了晶状体的光学清晰度。退化细胞核的形态与细胞凋亡过程中观察到的相似，并伴有DNA片段化。bcl-2原癌基因家族编码的蛋白质在促进或抑制细胞凋亡中起重要作用，而半胱天冬酶参与下游的蛋白水解事件。在此，通过免疫细胞化学、蛋白质印迹法以及使用生物素化的半胱天冬酶抑制剂对半胱天冬酶进行亲和标记，研究了bcl-2家族成员（bcl-2、bax、bad和bcl-x(s/l)）以及半胱天冬酶-1、-2、-3、-4和-6在鸡晶状体发育的一系列阶段中的表达。使用分化的晶状体上皮细胞培养物，结果表明，通过图像分析评估，向培养物中添加半胱天冬酶-1、-2、-4、-6和-9的合成肽抑制剂后，每单位培养面积中退化细胞核的数量减少了50-70%。这些效果与向培养物中添加半胱天冬酶的通用抑制剂时观察到的效果相当。另一方面，半胱天冬酶-3和-8的抑制剂在显著减少TUNEL标记细胞核的数量方面无效。在发育中的晶状体中也观察到了半胱天冬酶底物聚（ADP-核糖）聚合酶（PARP）和DNA片段化因子45 kDa亚基（DFF 45）的表达。对添加了半胱天冬酶抑制剂的培养物进行的蛋白质印迹显示，PARP的切割模式发生了改变，但DFF的切割模式没有改变。这些结果证明了bcl-2家族成员和半胱天冬酶在鸡晶状体次级纤维发育过程中晶状体纤维细胞核退化中的作用，并支持了这一过程与细胞凋亡有许多共同特征的观点。
