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L. Lu, L. Wang; UV–induced p53 activation involving membrane and nuclear effects in corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3813.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Recent studies from our lab indicate that UV irradiation induces Kv channel activation and subsequently activates JNK signaling pathway resulting in corneal epithelial cell apoptosis. The present study is to investigate p53 activation as downstream events in response to UV irradiation in corneal epithelial cells. Methods: Rabbit corneal epithelial cells were cultured in DMEM/F–12 medium containing 10% FBS. Western blot and immunocomplex kinase assay were used to determine JNK kinase activation and levels of protein expression. Protein–protein interaction was determined by immunocoprecipitation. Site–specific anti–phospho–p53 antibodies were used to determine activity of p53. Results: 1) UV irradiation elicited an early response to activate the JNK signaling pathway and p53 in these cells. The effect of continuous exposure of cells to UV irradiation within 15 min on JNK and p53 activations was markedly inhibited by suppression of Kv channel activity. 2) Suppression of Kv channel activity could not prevent a continuous exposure of cell to UV irradiation after 15 min that induced later response causing JNK and p53 activation, suggesting that UV–induced DNA damage may play a role in the later response. 3) UV irradiation–induced later activation of JNK and p53 could be mimicked by treatment of cells with melphalan, a DNA damage agent. 4) Western blot analysis indicated that total amounts of intracellular JNK and p53 proteins were not changed during UV irradiation induction. 5) In corneal epithelial cells, UV irradiation– and DNA damage–induced events characterized by phosphorylation of JNK and by direct protein–protein interaction between JNK and p53 resulting in p53 phosphorylation. Conclusions: Our results indicate that in corneal epithelial cells UV irradiation induces earlier and later intracellular events involving activations of JNK signaling pathway and p53. The early event in response to UV irradiation is initiated by activating Kv channels in the cell membrane. However, the later event is properly due to UV irradiation–caused DNA damage.
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