title: Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens.
abstract: OBJECTIVE
  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.
authors.affiliation: School of Biological Sciences University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Andrew John Oliver
authors.initials: AJ
authors.lastname: Smith
authors.affiliation: School of Biological Sciences University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Simon Sidney Robert
authors.initials: SS
authors.lastname: Ball
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Kamal
authors.initials: K
authors.lastname: Manzar
authors.affiliation: School of Biological Sciences University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Richard Peter
authors.initials: RP
authors.lastname: Bowater
authors.affiliation: School of Biological Sciences University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Ian Michael
authors.initials: IM
authors.lastname: Wormstone
mesh_terms: D034961:Antigens, Nuclear / Q000096:biosynthesis / Q000235:genetics*; D002386:Cataract / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000235:genetics*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D000072200:Ku Autoantigen; D007908:Lens, Crystalline / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress / Q000235:genetics*
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen