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R Sexton, P Sassalos, ML McDermott, M Kurpakus-Wheater; Hypoxia Induces Caspase-9 Activation in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1669.
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Purpose:To investigate initiator caspase activation and soluble FAS (sFAS) secretion in cultured human corneal epithelial cells. To test the hypothesis that hypoxia induces caspase 9, not caspase 8, in corneal epithelium. Additionally, to test the hypothesis that one mechanism by which the caspase 8 response is inhibited in hypoxic cells is by increased secretion of sFAS. Methods: Tertiary cultures of human corneal epithelial cells (HCEC) were maintained in serum-free medium. The expression of caspase 8 and caspase 9 by HCEC was confirmed by Western blot analysis of whole cell lysates. Cells were cultured under hypoxic (2% oxygen) or normoxic (20% oxygen) conditions for times between 6 hours and 7 days. The relative levels of caspase 8 and caspase 9 activity in HCEC were determined by two types of activity assays, one colorimetric and one fluorometric. Gene microarray analysis was performed on total RNA preparations to compare relative caspase mRNA levels in normoxic and hypoxic HCEC. sFAS concentration (in ng/ml) in conditioned medium was determined using a commercial ELISA kit. Results: Western blot analysis confirmed the presence of both caspase 8 and caspase 9 proteins in HCEC cells. Compared to normoxic controls, hypoxic HCEC demonstrated significantly increased caspase 9 activity. The relative levels of caspase 9 activity in hypoxic HCEC compared to control levels increased with time of exposure to hypoxia. In contrast, increased caspase 8 activation in hypoxic HCEC compared to normoxic controls could not be detected at any of the culture times assayed. Caspase 9 gene expression was upregulated in hypoxic HCEC, however no corresponding upregulation of caspase 8 gene was observed. Although hypoxic HCEC secreted more sFAS compared to normoxic cells, the concentration difference became significant only after prolonged exposure to hypoxia. Conclusion: These results support our hypothesis that caspase 9 is the initiator caspase for human corneal epithelial cells under hypoxic stress. Caspase 8 activation in hypoxic cells may be inhibited, at least in part, by increased endogenous secretion of sFAS.
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