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Vidhi Mishra, Shravan K Chintala, Caroline Cencer, Daniel Feldmann, Mirna Awrow, Nahrain Putris, Mason Geno, Victor Leverenz, Frank Joseph Giblin; Role of poly (ADP-ribose) polymerase-1 in UVB challenged human lens epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2026.
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© ARVO (1962-2015); The Authors (2016-present)
Little is known about the function of poly (ADP-ribose) polymerase-1 (PARP-1) in repairing DNA damage in the lens epithelium. Upon binding to damaged DNA, PARP-1 is known to catalyze the synthesis of poly (ADP-ribose) (PAR). Poly (ADP-ribosyl)tion (PARsylation) of PARP-1 is crucial for many cellular processes such as transcriptional regulation, cell division and cell death. Here, we studied the abundance and localization of PARP-1 and PAR and its relation to apoptosis in UVB-irradiated human lens epithelial cells (HLECs).
HLECs (SRA 01/04) were cultured in DMEM medium with 15% fetal bovine serum at 37 °C until the cells reached confluency. The cells were exposed to UVB light for 2.5 minutes with a peak emission wavelength of 312 nm and 0.9 mW/cm2 of intensity and incubated for various times at 37 °C. To assess the level of DNA damage and repair, the comet assay was employed. Expression of PARP-1 protein, synthesis of PAR, and localization of these biomolecules were monitored by immunocytochemistry and Western blot analysis. To measure the production of reactive oxygen species (ROS), a CellROX® fluorescence ROS detection kit was used.
The comet assay results were as follows: DNA fragmentation was detected immediately after UVB-irradiation; comets disappeared at 30 min indicating complete DNA repair; DNA fragmentation reappeared at 90 min indicating biphasic DNA damage; and no DNA damage was found at 120 min. Immunocytochemistry and Western Blot analysis of PARP-1 showed the presence of enzyme in the nucleus for all time intervals at the same level as controls. However, analysis of PAR indicated a biphasic burst of PAR in the cells, first at 5 min and second at 90 min. Furthermore, detection of ROS was not apparent until 90 min, whereas DNA strand breaks occurred at 5 min and 90 min after the UVB exposure.
This study is the first report of the role of PARP-1/PAR in lens epithelial DNA repair and associated cellular processes leading to cell death in UVB-challenged HLECs. The results suggest biphasic DNA damage followed by PARP-1/PAR complex-assisted repair. In addition, the results indicate oxidative stress generation at the second phase of DNA damage (90 min) accompanied with cell commitment to apoptosis. Further, ongoing experiments will aid in developing a better understanding of observed PARP-1/PAR cellular events.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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