INCREASE IN P53 PROTEIN HALF-LIFE IN MOUSE KERATINOCYTES FOLLOWING UV-B IRRADIATION

Exposure of mammalian cells to UV radiation and other DNA-damaging age nts triggers a response known as the UV response. This induction respo nse involves a large number of genes including c-jun, cell-cycle regul atory proteins, specific repair enzymes, and the tumor suppressor gene p53. Altered expression of these genes following DNA damage is hypoth esized to result in G(1) arrest, thereby allowing cells to repair DNA damage prior to cell division. In the present study, we investigated e xpression of the p53 gene in mouse keratinocyte cell line 308 after ex posure to UV-B light at a biologically relevant dose. Irradiation of 3 08 cells with 40 J/m(2) UV-B resulted in a 4- to 10-fold induction in the level of p53 protein, peaking at 5 h post-irradiation. Northern bl ot analysis of RNA from UV-B irradiated cells showed no change in the steady-state level of p53 mRNA following irradiation. However, the hal f-life of p53 protein in UV-B irradiated 308 cells was extended simila r to 7-fold, from 30 to 200 min. Additional studies were performed wit h specific anti-p53 monoclonal antibodies to establish whether UV-B ir radiation induced a conformational change in p53 protein in irradiated cells. Metabolic labeling with S-35-methionine followed by immunoprec ipitation with p53 monoclonal antibody PAb246, which recognizes the wi ld-type murine p53 protein, demonstrated that the p53 protein present in 308 cells possessed the wild-type conformation both before and afte r UV-B irradiation. In contrast, p53 antibody PAb240, which recognizes a 'conformation-dependent ' epitope, was not reactive with the p53 pr otein present in 308 cells. Therefore, we conclude that the induction of p53 protein in mouse keratinocytes following UV-B irradiation occur red post-transcriptionally, and was due to a significant increase in p 53 protein half-life.