Abstract
Purpose:
Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein best known for its involvement in DNA repair processes. PARP-1 activity is also linked to the oxidative stress response, which involves pathways associated with cell fate. In the current study, we assessed the functional roles of PARP-1 within lens cells in response to oxidative stress.
Methods:
The human lens epithelial cell line FHL124, which has a 99.5% similarity in gene expression to native human lens tissue and human lenses obtained from donor eyes were used as the experimental models. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death assessed by LDH release. PARP-1 activity was suppressed by targeted siRNA and chemical inhibition. Immunocytochemistry and western blotting were used to assess PARP-1 expression. To determine DNA single and double strand break damage the alkaline comet assay was performed and quantified using Comet IV lite analysis software (Perceptive Instruments).
Results:
PARP-1 expression was demonstrated by immunocytochemistry in both the FHL124 cell line and whole human lenses; in both instances expression was predominantly nuclear. Chemical inhibition of PARP-1 rendered FHL124 cells more susceptible to H2O2 (30 μM)-induced DNA strand breaks. PARP-1 expression was also depleted with targeted siRNA resulting in a knockdown of greater than 80%; these cells were similarly more susceptible to H2O2-induced DNA strand breaks than control cells, treated with scrambled control siRNA. Interestingly, chemical inhibition of PARP-1 significantly inhibited H2O2 (100 μM)-induced cell death relative to control cells. Chemical inhibition of PARP-1 in whole human lenses resulted in a reduced level of opacity and cell death following exposure to 1 mM H2O2 relative to match pair controls without PARP-1 inhibition.
Conclusions:
PARP-1 is expressed in both FHL124 cells and whole human lenses. Inhibition of PARP-1 protects FHL124 cells against H2O2-induced cell death whilst rendering FHL124 cells more susceptible to DNA strand breaks. PARP-1 inhibition in whole human lenses protects them from H2O2-induced opacity and related cell death. PARP-1 can play a number of important roles in the fate of lens cells and further studies are required to elucidate the regulatory processes that give rise to these effects.
Keywords: 445 cataract •
634 oxidation/oxidative or free radical damage