EXPRESSION OF E6 E7 OR SV40 LARGE T-ANTIGEN-CODING ONCOGENES IN HUMANCORNEAL ENDOTHELIAL-CELLS INDICATES REGULATED HIGH-PROLIFERATIVE/

Purpose. Human corneal endothelial cells are thought to have limited c apacity for proliferation. Little is known about the mechanisms that r egulate the proliferation of these cells. The authors introduced oncog enes into human corneal endothelial cells to modulate proliferation. I n addition, they sought to establish cell lines to facilitate study of human corneal endothelial cells. Methods, Early-passage human corneal endothelial cells were transduced with disabled retrovirus (pLXSN16E6 /E7) coding for the human papilloma virus type 16 transforming oncopro teins E6 and E7. Early-passage cells were also stably transfected by e lectroporation with the pMTV-D305 plasmid vector, in which SV40 large T antigen (SV40 LTAg) mRNA expression is positively regulated by the m ouse mammary tumor virus promoter. Expression of E6/E7 mRNA or SV40 LT Ag mRNA in cell lines was monitored with the polymerase chain reaction , SV40 LTAg protein expression was detected by immunocytology and West ern blot analysis. Results, Human corneal endothelial cells were effic iently infected with disabled retrovirus coding for E6/E7, and seven s trains of cells have continued active proliferation for more than 50 p opulation doublings (PD) (<8 control PD). E6/E7 mRNA was expressed by each cell strain. E6/E7 transformed cells proliferate rapidly and form a monolayer of cells with a high degree of contact inhibition. Transf ection with pMTV-D305 is less efficient, and only a single strain was developed. pMTV-D305-transfected endothelial cells (dexamethasone indu ced) proliferated at a slower rate than E6/E7-transduced cells or cell s transfected with a vector (pSV3neo) in which SV40 LTAg is constituti vely regulated. In the absence of dexamethasone, the proliferation of pMTV-D305-transfected cells was even slower, but cells continued to pr oduce SV40 LTAg mRNA and protein. The latter results indicated that SV 40 LTAg mRNA continued to be synthesized at significant levels in pMTV -D305-transfected cells in the absence of the inducer dexamethasone. C onclusions, This study suggests that human corneal endothelial cells h ave a high capacity for proliferation. Thus, cell division is normally controlled in human corneal endothelial cells by poorly characterized , but efficient, mechanisms. Because the E6 and E7 proteins, as well a s the SV40 large T antigen, specifically bind to and interfere with th e activity of the retinoblastoma (RE) and p53 tumor suppressor protein s, our results suggest that these proteins have critical roles in regu lating the proliferation of human corneal endothelial cells.