Ku80 counters oxidative stress-induced DNA damage and cataract formation in the human lens

Smith, Andrew John Oliver, Ball, Simon Sidney Robert, Manzar, Kamal, Bowater, Richard Peter ORCID: https://orcid.org/0000-0002-2745-7807 and Wormstone, Ian Michael ORCID: https://orcid.org/0000-0002-6423-7766 (2015) Ku80 counters oxidative stress-induced DNA damage and cataract formation in the human lens. Investigative Ophthalmology and Visual Science, 56 (13). pp. 7868-7874. ISSN 0146-0404

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Abstract

PURPOSE: Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens.   METHODS: The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software.   RESULTS: Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls.   CONCLUSIONS: Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.

Item Type:	Article
Uncontrolled Keywords:	lens epithelium,lens opacity,cataractogenesis,oxidative damage,dna damage
Faculty \ School:	Faculty of Science > School of Biological Sciences Faculty of Science > School of Natural Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Cells and Tissues Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Biosciences Teaching and Education Research
Depositing User:	Pure Connector
Date Deposited:	22 Jan 2016 12:00
Last Modified:	04 Mar 2024 17:28
URI:	https://ueaeprints.uea.ac.uk/id/eprint/56750
DOI:	10.1167/iovs.15-18309

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