Smith, Andrew (2014) INVESTIGATIONS OF DNA DAMAGE AND REPAIR IN THE HUMAN LENS. Doctoral thesis, University of East Anglia.

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Purpose: Cataract is the leading cause of blindness worldwide, affecting millions each year. Not only is cataract a debilitating disease for the individual sufferer, it also places a huge burden on healthcare budgets. Developing a greater understanding of the underlying causes could lead to potential therapies or preventative treatments being elucidated. Oxidative stress is a key factor in the formation of cataract, of which one consequence is DNA damage. The current study investigates the role of DNA damage and repair in cataract formation. The present body of work has three main aims. Firstly, to develop a model to study the effects of oxidative stress in human lens cells; secondly, investigate the role of non-homologous end joining (NHEJ) in the lens in respect to oxidative stress and cataract formation; thirdly, investigate the role of poly(ADP-ribose) polymerase-1 (PARP-1) in human lens cell responses to oxidative stress and cataract.
Methods: The FHL124 human lens epithelial cell line and a whole human lens culture system were used as the experimental models. Oxidative stress was induced via exposure to hydrogen peroxide (H2O2). Oxidative stress-induced changes in FHL124 cell viability/cytotoxicity and apoptosis were assessed with the ApoTox-Glo Triplex Assay and DNA strand breaks determined by the alkaline comet assay. Lactate dehydrogenase levels in culture medium were assayed as a marker of cell death in both FHL124 cells whole human lenses. Targeted siRNA was used to deplete expression of the NHEJ protein Ku80 and PARP-1 with protein expression evaluated by western blot and immunocytochemistry. Chemical inhibition of PARP-1 activity was also undertaken.
Results: A 24 hour exposure to hydrogen peroxide induced a concentration-dependent decrease in cell viability and increase in cytotoxicity and apoptosis. A 24 hour exposure to 1mM H2O2 induced significant decreases in loss of visual quality and cataract in whole human lenses; these changes corresponded to increased cell death. Depletion of Ku80 rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks and whole human lenses to significant decreases in visual quality compared to match-paired controls. Chemical inhibition of PARP-1 and its depletion by siRNA rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Conversely, chemical inhibition of PARP-1 protected FHL124 cells from H2O2-induced cell death and protected whole human lenses from oxidative stress-induced decreases in visual quality and cataract.
Conclusions: The experimental model developed allows for the study of oxidative stress-induced changes in lens cell line and the whole organ system. The NHEJ protein Ku80 is required for efficient responses to oxidative stress-induced DNA strand breaks; thus, Ku80 expression is important to protecting the human lens against opacity formation induced with H2O2. PARP-1 has multiple roles in both cell death and survival, with its depletion or inhibition rendering FHL124 cells susceptible to DNA strand breaks whilst conversely protecting against cell death and formation of opacity in whole human lens cultures.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2259 not found.
Date Deposited: 09 Jul 2014 11:38
Last Modified: 09 Jul 2014 11:38


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