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N. Awasthi, B.J. Wagner; Interferon-gamma Induces Apoptosis in Alpha-TN4 Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1222.
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Purpose: Although production of IFN-γ is suppressed in the normal eye, its presence has been demonstrated in inflammatory intraocular disorders including viral infections, retinopathay and uveitis. Lens-targeted ectopic expression of IFN-γ disrupts the normal lens differentiation program, suggesting its role in ocular pathology including cataractogenesis [Egwuagu, et al., Devel.Biol. 1994]. We previously measured up-regulation of the immunoproteasome in the lens of IFN-γ expressing transgenic mice, demonstrating a mechanism by which IFN-γ could affect the lens [Singh, et al., Arch.Bioch.Bioph. 2002]. IFN-γ induces apoptosis in several cell types and proteasome function has been related to apoptosis and lens differentiation. We tested the hypothesis that IFN-γ induces apoptosis in lens epithelial cells. Methods: The murine lens epithelial cell line, α-TN4-1, was grown in DMEM, treated with 100 u/ml of IFN-γ for 12 h, and apoptosis was measured using Annexin V/ PI staining and DNA fragmentation. mRNA expression of STAT-1, IRF-1 and caspase-1, Bax and Bcl-2 were examined by RT-PCR in IFN-γ treated and untreated cells. Caspase-1, -3 and -8 activities were measured using colorimetric substrates. Results: IFN-γ treatment at concentrations that induce immunoproteasome expression causes ~ 20% increase in early apoptotic cells as observed by Annexin V/ PI staining. A dose-dependent increase in DNA fragments (1.5-3 fold) of ~1900, 1600 and 700 bp was observed in IFN-γ treated cells compare to untreated cells. In most cell types, IFN-γ induced apoptosis involves up-regulation of STAT-1, IRF-1 and caspase-1. In our study a dramatic increase in STAT-1, >2-fold increase in IRF-1 and 1.7-2 fold increase in caspase-1 mRNA expression was observed in IFN-γ treated cells. In addition, a 2.4-3.0 fold decrease in Bcl-2 mRNA (antiapoptotic) expression was observed whereas Bax mRNA (proapoptotic) levels remained the same in IFN-γ treated cells compare to untreated cells. Caspase-1, caspase-3 and caspase-8 activities measured with colorimetric substrates were 2.9, 3.1 and 2.7 fold higher, respectively, in IFN-γ treated cells. Conclusions: IFN-γ causes apoptosis of α-TN4-1 cells and the apoptotic process is accompanied by the up-regulation of all known specific effectors of IFN-γ induced apoptosis. IFN-γ induced apoptosis also involves Bcl-2 family proteins and specific apoptotic caspases. Thus IFN-γ induced apoptosis of lens epithelial cells may cause disruption of lens differentiation and cataractogenesis.
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