EXTENDED LIFE OF HUMAN CORNEAL ENDOTHELIAL-CELLS TRANSFECTED WITH THESV40 LARGE T-ANTIGEN

Purpose. To transfect human corneal endothelial cells with a plasmid v ector coding for the SV40 large T antigen to extend the life of the ce lls in culture. Methods. Human corneal endothelial cells were transfec ted with the SV40 large T antigen-coding plasmid pSV3neo using the ele ctroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluoresc ence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Pol ymerase chain reaction and hot blotting were used to demonstrate messe nger RNA coding for several growth factors and receptors in transfecte d and control cells. Results. The transfected cells continued to proli ferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not co ntrol cells, expressed messenger RNA coding for the Simian virus 40 la rge T antigen. Similarly, immunofluorescent staining with monoclonal a ntibodies demonstrated that the Simian virus 40 large T antigen protei n was present in the nucleus of the transfected cells. Transfected cel ls were shown to produce messenger RNA coding for epidermal growth fac tor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interle ukin-1 receptor, transforming growth factor beta-1, and the glucocorti coid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal e ndothelial cells. Conclusions. In culture, the life of human corneal e ndothelial cells transfected with a plasmid vector coding for the Simi an virus 40 large T antigen is extended. This study suggests that huma n corneal endothelial cells have the capacity for extensive proliferat ion, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.