UV irradiation leads to transient changes in phosphorylation and stability of tumor suppressor protein p53

Tumor suppressor protein p53 is thought to play a crucial role in maintaining the integrity of the genome. DNA damage caused by genotoxic drugs, UV or gamma-irradiation leads to accumulation of p53 and activation of its DNA binding and transcriptional activities and subsequently to cell cycle arrest or apoptosis. We investigated whether the apparent activation of p53 might be due to post-translational modification. The rat fibroblast cell lines REF52, 208F, and rat1 were irradiated with W-A and the synthesis, stability and phosphorylation state of p53 were investigated by pulse chase experiments, SDS-PAGE and two-dimensional phosphopeptide mapping. The three cell lines exhibited different sensitivities and biological responses to UV irradiation, REF52 cells responded with a growth arrest whereas 208F and rat1 cells underwent apoptosis. The fate of p53 was similar in all cases. Both the stability of p53 and its phosphorylation increased instantaneously but transiently. However, the amount of p53 that accumulated after UV treatment was much higher in 208F and rat1 than in REF52 cells. Interestingly, p53 that was synthesized early after irradiation was stable for more than 14 h whereas molecules synthesized 8 or more hours post irradiation were increasingly susceptible to degradation. Moreover, between 14 and 20 h after treatment, the rate of synthesis of p53 decreased to a level lower than in untreated cells suggesting negative feed back control. The expression of different p53-responsive genes, waf1/cip1, Gadd45, and bax was investigated by protein analyses. Surprisingly, p21(waf1) was expressed only in REF52 cells but not in the others. Furthermore, UV irradiation led only to a moderate increase of p21(waf1) expression. Expression of Gadd45 and box was detectable in both cell types but its expression did not change significantly upon UV treatment. Our results suggest i) that both cell types share a common pathway which upon UV irradiation results in enhanced stability and phosphorylation of p53 and ii) that the decision whether a cell undergoes a growth arrest or apoptosis may be determined independent of p53 by the cellular environment i.e. the expression patterns of genes that mediate the response.