Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated high-proliferative capacity.
OBJECTIVE
Human corneal endothelial cells are thought to have limited capacity for proliferation. Little is known about the mechanisms that regulate the proliferation of these cells. The authors introduced oncogenes into human corneal endothelial cells to modulate proliferation. In 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 oncoproteins E6 and E7. Early-passage cells were also stably transfected by electroporation with the pMTV-D305 plasmid vector, in which SV40 large T antigen (SV40 LTAg) mRNA expression is positively regulated by the mouse mammary tumor virus promoter. Expression of E6/E7 mRNA or SV40 LTAg mRNA in cell lines was monitored with the polymerase chain reaction. SV40 LTAg protein expression was detected by immunocytology and Western blot analysis.


RESULTS
Human corneal endothelial cells were efficiently infected with disabled retrovirus coding for E6/E7, and seven strains of cells have continued active proliferation for more than 50 population 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. Transfection with pMTV-D305 is less efficient, and only a single strain was developed. pMTV-D305-transfected endothelial cells (dexamethasone induced) proliferated at a lower rate than E6/E7-transduced cells or cells transfected with a vector (pSV3neo) in which SV40 LTAg is constitutively regulated. In the absence of dexamethasone, the proliferation of pMTV-D305-transfected cells was even slower, but cells continued to produce SV40 LTAg mRNA and protein. The latter results indicated that SV40 LTAg mRNA continued to be synthesized at significant levels in pMTV-D305-transfected cells in the absence of the inducer dexamethasone.


CONCLUSIONS
This study suggests that human corneal endothelial cells have 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 as the SV40 large T antigen, specifically bind to and interfere with the activity of the retinoblastoma (RB) and p53 tumor suppressor proteins, our results suggest that these proteins have critical roles in regulating the proliferation of human corneal endothelial cells.
Wilson
S E
SE
Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas 75235.
Weng
J
J
Blair
S
S
He
Y G
YG
Lloyd
S
S
D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D002472:Cell Transformation, Viral; D002675:Child, Preschool; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D009857:Oncogenes; D027383:Papillomaviridae / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000032:analysis; D012097:Repressor Proteins; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16