INDUCTION OF CELLULAR P53 ACTIVITY BY DNA-DAMAGING AGENTS AND GROWTH ARREST

The tumor suppressor p53 can function as a sequence-specific transcrip tion factor and is required for activation by ionizing radiation (IR) of one or more downstream effector genes, such as the human GADD45 gen e. One important consequence of IR that is probably mediated by these downstream effector genes is activation of the p53-mediated G1 cell cy cle checkpoint. While the induction of reporter constructs containing p53-binding sites has already been demonstrated with p53 expression ve ctors, we have now demonstrated the direct activation of such a constr uct after treatment of the human RKO line, which has a normal p53 phen otype, with various types of DNA-damaging agents and also after growth arrest produced by medium depletion (starvation). IR, UV radiation, a nd methylmethane sulfonate were found to induce p53 activity when a st ably integrated reporter construct containing functional p53-binding s ites was used and also in mobility shift assays with a p53-binding sit e from the GADD45 gene, and IR-inducible gene previously associated wi th growth arrest. The same cell treatments that induced this p53 activ ity also caused an increase in cellular p53 protein levels. The respon se in cells lacking normal p53 or in RKO cells expressing a dominant n egative mutant p53 was markedly reduced. Interestingly, the spectrum o f effective inducing agents for the above-described experiments was si milar to that which induces GADD45 either in cells with a normal p53 s tatus or, with the exception of IR, in cells lacking normal p53. These results indicate a role for p53 in the IR pathway, which is completel y p53 dependent, and in other genotoxic stress responses, in which p53 has a cooperative effect but is not required.