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G Thuret, P Gain, S Herrag, JM Dumollard, J Bednarz, K Engelmann, L Campos; Staurosporine-induced Apoptosis In Human Cornea Endothelial Cells In Vitro . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3182.
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Purpose: Apoptosis is very likely to have a critical role in the endothelial cell loss during corneal storage in organ culture. Staurosporine is a nonspecific serine-threonine kinase inhibitor and known to induce apoptosis in several cell types. We investigated the apoptotic response of human cornea endothelial cells (HCEC) to staurosporine to get insights into the intracellular modulators that participate in and regulate endothelial cell death. Methods: Using a model of immortalized human corneal endothelial cells we investigated different apoptotic markers. HCEC were studied at 3, 6, 12 and 24 hours of incubation with 0.2 µM staurosporine. HCEC desquamation was monitored. Cell lysis was assessed by lacticodehydrogenase release assay. For detection of nuclear alterations consistent with apoptosis, chromatin condensation was assessed with Hoechst 33342 fluorescent DNA staining. Caspase 3 and 9 active forms were assessed using immunocytochemistry confirmed by western blot and proteolytic activity detection. Morphological features of apoptosis were studied using transmitted electron microscopy (TEM) as a gold standard. Results: Specific apoptotic nature of the staurosporine induced HCEC death was confirmed by TEM. HCEC desquamation, DNA condensation increased with time. Caspase 3 and 9 activity were detected as early as 3 hours after exposure with 0.2 µM staurosporine. Conclusion: Our data strongly suggest that the pattern of HCEC cell death induced by staurosporine is apoptotic in nature. Caspase activation is an early phenomenon. The main consequence of HCEC apoptosis is desquamation as it may occur during HCEC death caused by corneal storage. Staurosporine induced apoptosis of immortalized HCECs could be a useful model to study different strategy of cell death inhibition.
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