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Victoria Treboschi, Zhe Ma, Grace Tewkesbury, Ping Yang, Glenn J Jaffe; The Protective Effect of Bax Inhibitor 1 (BI-1) on Tunicamycin-induced Human RPE Cell Death. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6549.
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© ARVO (1962-2015); The Authors (2016-present)
In eyes with geographic atrophy (GA) associated with age-related macular degeneration (AMD), there is apoptotic and non-apoptotic RPE cell death. Bax Inhibitor 1 (BI-1) is an ER-associated protein that protects against BAX-mediated apoptosis. In the current study, we determined whether BI-1 is expressed in human retinal pigment epithelial (hRPE) cells and if it can protect against tunicamycin-induced cell death.
Endogenous BI-1 mRNA expression was evaluated by qPCR and protein expression by immunofluorescent staining of cultured confluent hRPE cells. Cell death in hRPE by tunicamycin, a known apoptosis inducer, was evaluated at different concentrations by Western Blot for caspase 3 cleavage. To achieve optimal BI-1 overexpression, hRPE cells were infected with LacZ or BI-1 adenovirus for 24 hours, and BI-1 protein levels were assessed by Western Blot at various times after infection. To test the effect of BI-1 transfection on RPE cell death, LacZ or BI-1 infected cells were treated at 5 days post-infection with 20-30 ug/mL tunicamycin for 0-24 hours in 1% FBS MEM. Cell morphology was assessed by light microscopy. Cell death was determined by DNA fragmentation and cell permeability ELISA assay.
Human RPE cells expressed endogenous BI-1 mRNA and RPE cell BI-1 protein was detected by immunofluorescence. Tunicamycin treatment induced RPE cell death in a dose-dependent manner. After cells were transfected with the BI-1 gene, BI-1 protein was detected by 3 days post-transfection and continued to increase 10-12 days post-transfection. BI-1-transfected RPE cells were resistant to the death inducing effect of tunicamycin; morphologically this BI-1 protective effect was evident after 4 hours of 30 ug/mL tunicamycin treatment and persisted 24 hours later. On DNA permeability ELISA assay, cell permeability was significantly lower in BI-1 than LacZ transfected cells after 24 hours of treatment with 30 ug/mL tunicamycin (p = .005), but DNA fragmentation was not significantly different after this treatment.
BI-1 is endogenously expressed in hRPE cells, and, when over-expressed, protects against tunicamycin-induced RPE cell death. BI-1 may serve as a native RPE survival protein that protects cells from endogenous and exogenous death-inducing stimuli. Strategies to up-regulate this molecule may be a novel approach to prevent RPE cell death in eyes with AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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