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Catherine A. Taylor, Michelle Senchyna, John Flanagan, Elizabeth M. Joyce, Dominic O. Cliche, Adrienne N. Boone, Shelley Culp-Stewart, John E. Thompson; Role of eIF5A in TNF-α–Mediated Apoptosis of Lamina Cribrosa Cells. Invest. Ophthalmol. Vis. Sci. 2004;45(10):3568-3576. doi: https://doi.org/10.1167/iovs.03-1367.
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purpose. To determine the role of eukaryotic translation initiation factor 5A (eIF5A) in TNF-α–induced apoptosis of lamina cribrosa (LC) cells.
methods. LC cells were isolated from optic nerve heads of eyes of two human donors. The cells were treated with TNF-α and camptothecin, a TNF synergist, and the incidence of apoptosis was scored by Hoechst staining. Expression of eIF5A protein in response to camptothecin or a combination of camptothecin and TNF-α was determined by Western blot analysis. The ability of small inhibitory (si)RNAs directed against eIF5A to protect LC cells from TNF-α–induced apoptosis was determined by Hoechst and TUNEL staining of transfected LC cells.
results. TNF-α and camptothecin synergized to induce greater than two times more apoptosis in LC cells than when the cells were treated with TNF-α or camptothecin separately. Expression of eIF5A protein increased significantly after 8 hours of exposure to TNF-α and camptothecin, but not in response to camptothecin alone. siRNAs directed against eIF5A reduced apoptosis of LC cells in response to TNF-α and camptothecin by between 35% and 69%, as determined by Hoechst staining. An siRNA against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also reduced apoptosis of LC cells by 42%. TUNEL of transfected LC cells treated with TNF-α and camptothecin revealed an 80% reduction in apoptosis with siRNA against eIF5A.
conclusions. TNF-α, in synergy with camptothecin, induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF-α, and siRNAs against eIF5A protect LC cells from apoptosis. Thus, eIF5A appears to be a novel proapoptotic protein in the TNF pathway and a possible target for treatment of glaucoma.
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