Stimulation of quiescent corneal endothelial cells by direct delivery of the SV40 large T-antigen protein.
OBJECTIVE
To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells.


METHODS
Liposome-mediated delivery of proteins into bovine corneal endothelial cells (BCEC) was utilized in this study. Initially, beta-galactosidase was used as a marker protein for cell delivery and cells were assayed colorimetrically for beta-galactosidase activity. Subsequently, SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by 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 BCECs using liposome transfer methods. The transfer efficiency of beta-galactosidase was > 30% of the cells. SV40 large T antigen was successfully introduced and was localized to the nuclei of BCECs. The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation. Co-labeling confirmed that only cells containing SV40 large T antigen were positive 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, the protein localized to the nucleus and maintained its bioactivity by inducing DNA synthesis. This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.
Cho
K S
KS
Catholic University, Seoul, South Korea.
Joo
C K
CK
Williams
J S
JS
Ambroziak
B S
BS
Bassnett
S S
SS
Pepose
J S
JS
Fleming
T P
TP
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000493:pharmacokinetics; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004337:Drug Carriers; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D008081:Liposomes; D001616:beta-Galactosidase / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D004337:Drug Carriers; D008081:Liposomes; D004247:DNA; D001616:beta-Galactosidase; D001973:Bromodeoxyuridine