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P. Yang, B.S. McKay, J.B. Allen, G.J. Jaffe; Resistance to TNF--Induced Cell Death by Overexpressing Mutant IB in Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2946.
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Purpose: In many cell types, TNF-α-induced apoptotic cell death is prevented by parallel TNF-α-induced antiapoptotic protein production, a process mediated by the nuclear transcription factor-ΚB (NF-ΚB). TNF-α is widely expressed in PVR membranes and is present in the vitreous of eyes with PVR. Unwanted RPE cell proliferation contributes to membrane formation in PVR. Therefore, it is important to understand mechanisms responsible for RPE cell survival and death in this disease. Herein we determined whether specific NF-ΚB blockade by mutant inhibitory ΚB (IΚB) overexpression affects TNF-α-induced cell death. Methods: Cultured human RPE cells and T-98G glioma cells were infected with adenovirus encoding either ß-galactosidase (LacZ) or mutant IΚB and then treated with or without TNF-α (22ng/ml) for varying times. The optimal multiplicity of infection (MOI) for each cell type was examined by X-gal assay and Western blot. Mutant and endogenous IΚB protein production was examined by Western blot. Cell viability was evaluated by trypan blue exclusion assay. Apoptosis was measured with an ELISA to detect caspase-3 activity, and a TUNEL assay to identify DNA fragmentation. Results: More than 90% of the cells were transduced with LacZ adenovirus vector at a MOI of 10 and 300 in human RPE cells and T-98G cells, respectively. Stimulation of RPE cells and T-98G cells with TNF-α for 30 minutes caused rapid degradation of endogenous, but not mutant, IΚB protein. Marked cell death was observed in T-98G cells expressing mutant IΚB at 12h, 24h, and 48h following TNF-α exposure. In contrast, cell death was not observed in uninfected T-98G cells, LacZ-infected T-98G cells or mutant IΚB-expressing RPE cells at any of these times following TNF-α exposure. Caspase-3 activity was significantly increased in T-98G cells expressing mutant IΚB at 8h following TNF-α exposure (P<0.0007), but not in uninfected T-98G cells, LacZ-infected T-98G cells or mutant IΚB-expressing RPE cells. Similarly, DNA fragmentation was observed in T-98G cells expressing mutant IΚB as early as 8h after TNF-α exposure, but was not seen in uninfected T-98G cells, LacZ-infected T-98G cells or mutant IΚB-expressing RPE cells. Conclusions: In contrast to many other cell types, RPE cells are resistant to TNF-α-induced cell death, even after NF-ΚB activation is specifically blocked. RPE cell resistance to apoptotic signals present in eyes with PVR may help to explain unwanted and unchecked cell proliferation in this disease.
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