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M.R. Fernando, V.N. Gladyshev, M.F. Lou; MITOCHONDRIAL THIOLTRANSFERASE (Grx2) PROTECTS HUMAN LENS EPHITHELIAL CELLS FROM H2O2–INDUCED APOPTOSIS . Invest. Ophthalmol. Vis. Sci. 2004;45(13):398.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have undertaken this study to investigate the physiological functions of mitochondrial thioltransferase in human lens epithelial cells. Methods: Mouse Grx2 cDNA full length including mitochondrial signal was cloned to pcDNA3.1(+) vector and HLE–B3 cells were transfected with this recombinant vector to get Grx2 over–expressing cells. Transfected cells and normal cells were homogenized using a hand homogenizer and mitochondrial fraction was separated using centrifugation. Grx2 activity was assayed in mitochondria separated from normal cell homogenate and transfected homogenate. Normal HLE–B3 cells and transfected cells were incubated in serum free MEM medium with or without 100 µM H2O2 for 6 hr at 37O C in a 5% CO2 humidified atmosphere. After incubation cells were washed with PBS and genomic DNA was extracted. Then the concentration of DNA was determined and DNA samples (1µg) were loaded on 2% agarose gels containing ethidium bromide. After electrophoresis gels were photographed under UV illumination. Results: There was a two–fold increase in Grx2 activity in transfected cells as compared to normal cells. When normal HLE–B3 cells were treated with 100 µM H2O2 for 6 hr, characteristic DNA laddering indicating apoptosis was observed. No such laddering was observed in normal untreated cells. Cells transfected with Grx2 showed no DNA laddering even when the cells were treated with H2O2 under the same conditions. Conclusions:These results demonstrate that the Grx2 plays a role in protecting lens epithelial cells against oxidant– induced apoptosis. Supported by NIH grant EY 10590. None Reviewing code: 199 Keywords: 425, 441, 505
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