Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-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 H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-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 alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-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 alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.
Mao
Y W
YW
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, Stratford, New Jersey 08084, USA.
Xiang
H
H
Wang
J
J
Korsmeyer
S
S
Reddan
J
J
Li
D W
DW
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003459:Crystallins / Q000096:biosynthesis* / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D019254:Genes, bcl-2; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D018384:Oxidative Stress; D011233:Precipitin Tests; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014867:Water / Q000378:metabolism*
D003459:Crystallins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D014867:Water; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D019311:Staurosporine; D002166:Camptothecin