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Originally published In Press as doi:10.1074/jbc.M102195200 on September 6, 2001

J. Biol. Chem., Vol. 276, Issue 46, 43435-43445, November 16, 2001
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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*

Ying-Wei MaoDagger §, Hua XiangDagger §, Juan WangDagger , Stanley Korsmeyer, John Reddan||, and David Wan-Cheng LiDagger **

From the Dagger  Department of Molecular Biology, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, Stratford, New Jersey 08084, the  Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, and the || Department of Biological Sciences, Oakland University, Rochester, Michigan 48309

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 H2O2 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 H2O2-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alpha B-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 alpha B-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H2O2-induced apoptosis. Introduction of a mouse alpha B-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alpha B-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alpha B-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 alpha B-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis.

* This work was supported by National Institutes of Health grants and start-up funds from University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this paper.

** To whom correspondence should be addressed: Dept. of Molecular Biology, Science Center, Rm. 347, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, Two Medical Center Dr., Stratford, NJ 08084. Fax: 856-566-6291; E-mail: DWL168@att.net.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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