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 main taining the integrity of the genome. DNA damage caused by genotoxic dr ugs, UV or gamma-irradiation leads to accumulation of p53 and activati on of its DNA binding and transcriptional activities and subsequently to cell cycle arrest or apoptosis. We investigated whether the apparen t activation of p53 might be due to post-translational modification. T he rat fibroblast cell lines REF52, 208F, and rat1 were irradiated wit h W-A and the synthesis, stability and phosphorylation state of p53 we re investigated by pulse chase experiments, SDS-PAGE and two-dimension al phosphopeptide mapping. The three cell lines exhibited different se nsitivities and biological responses to UV irradiation, REF52 cells re sponded with a growth arrest whereas 208F and rat1 cells underwent apo ptosis. The fate of p53 was similar in all cases. Both the stability o f p53 and its phosphorylation increased instantaneously but transientl y. However, the amount of p53 that accumulated after UV treatment was much higher in 208F and rat1 than in REF52 cells. Interestingly, p53 t hat was synthesized early after irradiation was stable for more than 1 4 h whereas molecules synthesized 8 or more hours post irradiation wer e 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. T he expression of different p53-responsive genes, waf1/cip1, Gadd45, an d bax was investigated by protein analyses. Surprisingly, p21(waf1) wa s 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 result s 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 environ ment i.e. the expression patterns of genes that mediate the response.