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Simon Sidney Robert Ball, Andrew J. O. Smith, Richard Peter Bowater, Michael Wormstone; DNA repair proteins in the anatomical organisation and protection of the human lens. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2025.
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© ARVO (1962-2015); The Authors (2016-present)
DNA repair systems maintain genome stability in all cells, but these are a neglected area of research in the human lens. This study identifies the expression of the DNA repair proteins PARP-1, DNA-PK and Ku80, which are all involved in the repair of DNA double-strand breaks. The purpose of the current study was to evaluate the distribution of these proteins in the lens in relation to lens anatomy and mechanisms that defend against genotoxic stress.
Non-cultured whole human donor lenses obtained from the East Anglian Eye Bank were fixed in 4% v/v formaldehyde, paraffin embedded and sectioned at 6 µm. Samples were subjected to antigen retrieval before fluorescent immunohistochemistry using Alexa-488 secondary antibody and nuclear counterstaining with DAPI. Sections were visualised using a Zeiss Axioplan fluorescence microscope and digital camera. The lens epithelial cell line, FHL124 was also used as an experimental system with immunofluorescence techniques again being used to identify protein expression and DNA damage was assessed using the alkaline comet assay. Genotoxic stress was invoked in the cell line by treatments with hydrogen peroxide or neocarzinostatin (NCS). To examine the roles of specific proteins, PARP-1 and DNA-PK were inhibited by the pharmacological agents AG14361 and NU7026 respectively, while Ku80 was knocked down using targeted siRNA methods.
PARP-1, DNA-PK and Ku80 were present in the cell nucleus of the lens epithelial layer and the FHL124 cell line. Within fibre cells, newly laid cells also presented a predominantly nuclear expression before levels rapidly declined. This reduced expression appeared to precede changes in chromatin appearance that could be attributed to lens fibre cell de-nucleation. With respect to DNA damage, FHL124 cells were sensitive to both hydrogen peroxide and NCS, with significant levels observed following treatment. The scale of this damage was further increased in cases where PARP-1, DNA-PK and Ku80 were inhibited.
The DNA repair proteins, PARP-1, DNA-PK and Ku80 are present in human lens epithelial and cortical fibre cells. All three proteins appear to play a key role in the defence mechanisms of lens epithelial cells against genotoxic stress. Expression of these proteins within lens fibre cells appears to decline in fibre cells prior to de-nucleation and could play a role in this process.
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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