Stimulation of quiescent corneal endothelial cells by direct delivery of the SV40 large T-antigen protein

Purpose. To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothel ial cells. Methods. Liposome-mediated delivery of proteins into bovine corneal endothe lial cells (BCEC) was utilized in this study. Initially, beta-galactosidase was used as a marker protein for cell delivery and cells were assayed colo rimetrically for beta-galactosidase activity. Subsequently, SV40 large T-an tigen protein was introduced into BCEC and positive cells were identified b y immunohistochemistry 24 hours after liposome-protein treatment. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the SV40 large T-antigen was detected by standard immunohistochemical methods. Results. Beta-galactosidase or SV40 large T antigen were introduced into BC ECs using liposome transfer methods. The transfer efficiency of beta-galact osidase was > 30% of the cells. SV40 large T antigen was successfully intro duced and was localized to the nuclei of BCECs. The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation. Co-lab eling confirmed that only cells containing SV40 large T antigen were positi ve for de novo DNA synthesis. Conclusions. This study demonstrates that proteins can be inserted directly into corneal endothelial cells. In the case of the SV40 large T-antigen, t he protein localized to the nucleus and maintained its bioactivity by induc ing DNA synthesis. This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endo thelial cell proliferation.