Purpose: The aims of this study were to identify a robust apoptosis marker suitable for both quantification and back-to-back analyses of programmed cell death and to define specific upstream targets for apoptosis in corneal cells. Methods: Apoptotic cleavage of initiator caspases and their downstream targets such as lamins and poly-ADP ribose polymerase was investigated in human corneal endothelial cells (HCEC-12), keratocytes (HCK), epithelial cells (HCEp), and full-thickness corneas using Western blotting and confocal microscopy following apoptosis induction with staurosporine. We specifically focused on nuclear lamins, which have important structural and regulatory functions in the cell nucleus. Results: The cleavage of lamin A in HCEC-12 was significantly increased following apoptotic induction compared with HCK. More importantly, lamin A cleavage was detected in a dose-dependent manner in full-thickness corneal tissue by both Western blot analysis and fluorescence microscopy. Our study also demonstrates that HCEp show approximately three-fold increase in caspase 6 cleavage compared with endothelial cells or keratocytes. The presence of cleaved caspase 9 was lower in endothelial cells compared with epithelial cells and keratocytes. Conclusions: We successfully established lamin A cleavage as a quantifiable marker of apoptosis in both corneal cells and tissue. Quantification of lamin A cleavage by Western blotting followed by a back-to-back analysis with fluorescence microscopy was studied for the first time in the experimental (donor) corneal tissue. Screening of downstream apoptosis proteins and establishing cell type-specific protocols allowed us to identify possible targets (caspases, Apaf-1, etc.) for protective therapeutic approaches.
Purpose: The aims of this study were to identify a robust apoptosis marker suitable for both quantification and back-to-back analyses of programmed cell death and to define specific upstream targets for apoptosis in corneal cells. Methods: Apoptotic cleavage of initiator caspases and their downstream targets such as lamins and poly-ADP ribose polymerase was investigated in human corneal endothelial cells (HCEC-12), keratocytes (HCK), epithelial cells (HCEp), and full-thickness corneas using Western blotting and confocal microscopy following apoptosis induction with staurosporine. We specifically focused on nuclear lamins, which have important structural and regulatory functions in the cell nucleus. Results: The cleavage of lamin A in HCEC-12 was significantly increased following apoptotic induction compared with HCK. More importantly, lamin A cleavage was detected in a dose-dependent manner in full-thickness corneal tissue by both Western blot analysis and fluorescence microscopy. Our study also demonstrates that HCEp show approximately three-fold increase in caspase 6 cleavage compared with endothelial cells or keratocytes. The presence of cleaved caspase 9 was lower in endothelial cells compared with epithelial cells and keratocytes. Conclusions: We successfully established lamin A cleavage as a quantifiable marker of apoptosis in both corneal cells and tissue. Quantification of lamin A cleavage by Western blotting followed by a back-to-back analysis with fluorescence microscopy was studied for the first time in the experimental (donor) corneal tissue. Screening of downstream apoptosis proteins and establishing cell type-specific protocols allowed us to identify possible targets (caspases, Apaf-1, etc.) for protective therapeutic approaches.