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 ␣B-crystallin gene was distinctly downregulated 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 ␣B-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H 2 O 2 -induced apoptosis. Introduction of a mouse ␣B-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of ␣B-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, ␣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 ␣B-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H 2 O 2 -induced apoptosis.

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERTtransfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerasenegative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.

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