Human Lens Epithelium in Cataract: Expression of Bcl-2, BAX and Caspase-3 in Fresh and Cultured Epithelium

Abstract

Purpose: : Irreversible cellular stress induced by UV-irradiation and oxidative damage, and initiation of apoptosis, are pathogenetic factors in the development of age related human cataract. In the present study we examine freshly removed human lens epithelium for changes in expression of Bcl-2, Bax and Caspase-3 induced by transfer to a cell culture system.

Methods: : Through a clear corneal incision viscoelastic material was introduced and the anterior capsule obtained. The samples were either fixed in 4% formalin (n=6) and processed for immunohistochemical detection of Bcl-2, Bax and Caspase-3, or processed for RT-PCR for evaluation of expression of the Bcl-2, Bax and Caspase-3 genes (n=6). Other samples were immersed in Dulbecco’s Modified Eagle Medium / F12 (DMEM/F12) with 15% foetal bovine serum (FBS) and 100 U/l Penicillin-Streptomycin (n=12) for culturing in an incubator in a humid atmosphere at 37°C in 5% CO₂ prior to the analytical procedures.

Results: : Transfer of the lens epithelium to cell culture induced a significant up-regulation of Bcl-2 and Bax in the epithelium. Expression of Bcl-2 increased 10 fold, while there was a 15 fold increase in expression of Bax. However, in both fresh and cultured epithelium the Bcl-2/Bax ratio was approximately 1. There was virtually no change in the expression of Caspase-3 in the cultured epithelium.

Conclusions: : We here demonstrate that transfer of cataractous human lens epithelium to a cell culture system induce a significant up-regulation of Bcl-2 and Bax gene expression. However, cell culture did not induce any significant shift in the Bcl-2/Bax ratio. The transfer to a cell culture system does therefore not increase the apoptotic suceptibility mediated by a relative increase in expression of Bax.An equilibrium between anti- and pro-apoptotic signals is further indicated by the similar expression of Caspase-3 in both fresh and cultured epithelium.Our results support the usefulness of ex vivo systems in assays aimed at exploring mechanisms related to lens epithelial cell- and molecular damage and repair.

Keywords: cataract • apoptosis/cell death • gene/expression