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J. C. O'Connor, D. M. Wallace, C. J. O'Brien, T. G. Cotter; The Tumor Suppressor Gene p53: Is It Neuroprotectant for the Retinal Ganglion Cell?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2071.
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Recent evidence has suggested that the tumour suppressor gene p53 may have a role as an antioxidant. The retina is an organ highly susceptible to oxidative stress, not only because of its high blood supply and oxygen rich environment, but also because it is subject to constant exposure to UV light in the atmosphere. Furthermore, there is evidence that oxidative stress is a factor in the precipitation of apoptosis of the retinal ganglion cell, the final common pathway in glaucoma, and a process which has been shown to be partially regulated by p53. With this in mind, we sought to demonstrate for the first time that p53 actually has a paradoxical second role as an antioxidant in the retinal ganglion cell.
We evaluated the expression level of p53, and the antioxidant enzymes GPX1, Sestrin 2, Catalase, SOD 1 and SOD 2 in the developing retina of the Sprague-Dawley rat by immunohistochemistry and Western blot. To further explore a putative link between p53 and the antioxidants in the retina, we examined the retina on a cellular level, utilising the retinal ganglion cell line RGC-5. SiRNA specific to p53 was used to knock down p53, and downstream effects on the antioxidants were observed by Western blot. We then performed chromatin immunoprecipitation (ChIP) assays on whole retina to demonstrate a direct link between p53 and antioxidants in the retina. Finally, the effect of p53 deficiency on basal reactive oxygen species levels within the cell, and on susceptibility to oxidative stress induced apoptosis was measured by flow cytometry.
We found that expression patterns of p53 and certain antioxidants, including Catalase, mirrored each other during development. At the cellular level p53 knockdown by siRNA resulted in a significant decrease in Catalase. By genomic analysis we identified p53 binding sites on the rat Catalase promoter, and by ChIP assay we affirmed an in vivo link between p53 and Catalase in the retina. We also found that p53 knockdown in the cell resulted in a corresponding increase in basal reactive oxygen species, and increased susceptibility to H2O2 induced cell death.
Our results demonstrate a regulating influence of p53 on the essential cellular antioxidant Catalase in the retina, and more specifically in the retinal ganglion cell. Because oxidative stress is thought to play a role in effecting death of the retinal ganglion cell in glaucoma, these findings will have implications into our understanding of the molecular mechanisms promoting survival of this cell type, and hence may ultimately provide another target avenue for neuroprotection.
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