P53 protein subcellular localization and apoptosis in rodent corneal epithelium cell culture following ultraviolet irradiation

Abstract

The tumor-suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. p53 expression and function have been documented in malignancy, apoptosis and the aging processes. Recently, p53 has been mapped and characterized in the normal cornea across different species. In the present study, high levels of cytoplasmic p53 protein were noted in normal primary corneal epithelium cultures by immunohistochemistry and western blot analysis. Following ultraviolet(UV) irradiation, the level of cytoplasmic p53 protein expression was increased beginning from 30min and lasting until 6h post-irradiation and then returned close to control levels by 24h. Cytoplasmic p53 phosphorylation was detected from 30min following UV treatment until 6h post-irradiation. p53 protein became apparent in the nucleus in a fraction of these cultured cells beginning 30min following UV irradiation and was still present 24h later. We also found that p53 colocalized with mitochondria 2h following UV irradiation in some of the cells and remained there up to 24h. As the expression levels of p53 transcription following UV irradiation were not significantly altered, the increase in cytoplasmic p53 protein expression may be conditional only upon post-translational stabilization. We also observed that the apoptotic index increased following UV irradiation in the same time frame as the p53 nuclear transfer and was partially suppressed by pifithrin-α, which is a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription. The present study offers new evidence suggesting that cytoplasmic p53 in rodent corneal epithelium is functionally active.