Non-Homologous End Joining Repair in the Lens

Abstract

Purpose: DNA damage to the lens epithelium is likely to contribute to cataract formation. Extensive oxidative stress can lead to DNA strand breaks. Here, we investigate links between systems that repair DNA strand breaks, such as non-homologous end joining (NHEJ), and cataract.

Methods: The human lens epithelial cell line FHL124 and human lenses obtained from donor eyes were used. Oxidative stress was induced through exposure to hydrogen peroxide (H₂O₂). Immunocytochemistry and western blotting were employed to assess expression of NHEJ proteins (Ku80, DNA-PK, Lig IV). ApoTox-Glo Triplex Assay was employed to measure cell viability, cytotoxicity and apoptosis in the FHL124 cell line. To determine DNA strand breaks the alkaline comet assay was performed and quantified using Comet Assay IV software. To suppress expression of the NHEJ protein Ku80 in the cell line and whole lenses a targeted siRNA approach was adopted.

Results: Expression of NHEJ proteins Ku80, DNA-PK and LigIV was demonstrated in both FHL124 cells and whole human lenses. The expression of these proteins was predominately in the cell nucleus. Exposure of FHL124 cells to H₂O₂ (0-100µM) produced a dose-response effect that resulted in decreasing viability, increasing cytotoxicity and elevated rate of apoptosis. Treatment of FHL124 cells with siRNA targeted against Ku80 resulted in a knockdown (>80%) in expression. Using the alkaline comet assay, application of 30 μM H₂O₂ to control cells lead to an increase in DNA strand breaks with a peak at 30 minutes post-treatment, before returning to baseline levels. Interestingly, in Ku80 knockdown cells the level of DNA strand breaks detected was greater than in control cells. Expression of Ku80 in whole human lenses was also depleted using targeted siRNA. Upon exposure to 1 mM H₂O₂ greater opacities were observed in Ku80 knockdown lenses than match-paired controls.

Conclusions: NHEJ proteins are present within human lens epithelial cells. Depletion of Ku80 expression is likely to give rise to greater levels of DNA damage resulting from oxidative stress. It is therefore possible that the NHEJ DNA repair system could play a role in cataract formation. Elucidating the factors that control expression and function will be important lines of investigation in the future.

Keywords: 634 oxidation/oxidative or free radical damage • 656 protective mechanisms