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C Zhang, N Strunnikova, J Baffi, S Cousins, K Csaky; Ratio of Caspase-8 to C-Flip Determines the Susceptibility of Human Retinal Pigment Epithelium (hRPE) to Tumor Necrosis Factor – a (TNF-a) Induced Cell Death . Invest. Ophthalmol. Vis. Sci. 2002;43(13):686.
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Purpose: Macrophages have been shown to be recruited to the choroid in age-related macular degeneration (AMD), secrete TNF-a and therefore may be involved in hRPE cell death. However, evidence suggests that death of the hRPE is a late occurrence in AMD and that hRPE are resistant to TNF-a induced cell death. The following study examines the possible molecular pathways involved in TNF-a signaling in hRPE cells. Methods: The molecular components of the TNF-a signaling pathway were examined using immunohistochemistry, western blot analysis and RT-PCR in hRPE cells and in a control U937 cells known to be sensitive to TNF-a cell death. Generation of a caspase-8 construct under the control of an inducible promoter was used to examine caspase-8 toxicity. Cell viability was determined by XTT assay. Mitochondria potential was monitored by mitotracker fluorescence, while ectopic expression of mitochondria pro-apoptotic factors was examined by immunobloting of organelle fractions and immunohistochemistry of intact cells. Results: All components of the TNF-a death inducing signaling complex (DISC) were present in both hRPE and U937 cells. However, U937 cells displayed higher levels of caspase-8 and a high ratio of caspase-8/ c-Flip (3:1) while hRPE showed low levels of caspase-8 and a reduced caspase-8/c-Flip ratio (1:3). While U937 cells displayed typical events of TNF-a cell death activity including cytochrome c translocation and caspase-3 cleavage, these events did not occur in hRPE cells. Introduction of caspase-8 by gene transfer sensitized hRPE cells to TNF-a toxicity. Conclusion: HRPE maintain a high ratio of survival factors/toxic factors and is more resistant to TNF-a induced cell death. Low level of caspase-8 expression or high levels of c-FLIP may be important in determining hRPE cell survival when exposed to TNF-a.
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