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R. H. Nagaraj, D. Smith, S. Howell, L. Reneker, M. Mailankot; Cytotoxic Effects of Kynurenines in Mouse Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1904.
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Indoleamine 2,3-dioxygenase (IDO) is present in lens epithelial cells. It is the first enzyme of tryptophan metabolism that produces kynurenines. Kynurenines are cytotoxic and cause cell death. They also react with cellular proteins and alter their structure. In this study we have investigated the effects of kynurenines produced in situ in mouse lens epithelial cells.
Lens epithelial cells from wild type (Wt) and heterozygous transgenic for IDO (hetTg) were isolated and cultured. Cells from homozygous transgenic animals could not be cultured as they did not attach or proliferate under the conditions employed for cell culture. IDO was detected by immunostaining and IDO activity was measured by HPLC. Kynurenines and kynurenine-modifications in proteins were measured by HPLC and a direct ELISA, respectively in epithelial cells incubated with or without 1mM L-tryptophan (for 2 hrs). Cell cycle was assessed by flow cytometry and cell proliferation was measured by the MTT assay.
IDO was activity was 0.14 ± 0.02 units /mg protein in epithelial cells of hetTg and was undetectable in epithelial cells of Wt. Addition of 0.1 micromolar 1-methyl tryptophan in the culture media (for 3 days) completely inhibited the IDO activity. IDO immunoreactivity was distributed throughout cytoplasm of cells. Kynurenine and kynurenine-modified proteins were detected in cells from hetTg but not in cells from Wt. Immunohistochemistry confirmed kynurenine-modified proteins in hetTg cells. Cell proliferation was severely blunted in hetTg cells and cell cycle analysis revealed that approximately 30% of the cells were arrested in G2/M phase in hetTG cells. Incubation of Wt cells with 50 micromolar kynurenine in the culture media for 3 days also resulted in approximately 30% cells arrested in G2/M phase.
These results imply that kynurenines formed within cells can cause defects in the cell cycle and lead to defects in cell proliferation, and suggest that IDO activity is tightly regulated in the lens to prevent kynurenine-mediated cytotoxicity.
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