Human bcl-2 Gene Attenuates the Ability of Rabbit Lens Epithelial Cells against H₂O₂-induced Apoptosis through Down-regulation of the αB-crystallin Gene

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H₂O₂ revealed that bcl-2-transfected cells were less capable of detoxifying H ₂O₂ than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H₂O₂-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H ₂O₂-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the αB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of αB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H₂O ₂-induced apoptosis. Introduction of a mouse αB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of αB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, αB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the αB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H ₂O₂-induced apoptosis.