Cellular responses to DNA damaging agents influence both the likelihood of tumor development and the responses of tumor cells to chemotherapy and radiation therapy. After DNA damage, both cell cycle perturbations and apoptosis are modulated by the ability of the cell to increase p53 protein levels and activity, which are mediated by a multi-step signal transduction pathway. This proposal will investigate the molecular mechanisms involved in p53 induction and activation by DNA damaging agents. Experiments proposed include: 1) Investigation of irradiation-associated regulation of p53 translation and mapping of the domains of p53 mRNA which influence translation rates, by utilizing both in vitro translation systems and reporter constructs to evaluate the role of the 5' and 3' untranslated regions of the p53 mRNA in this levels of regulation. 2) Determine whether exposure to DNA damaging agents causes alterations in the """"""""activity"""""""" of p53 and elucidation of the nature of these changes, with particular emphasis in specific changes in phosphorylation patterns of p53, identification of the site(s) of altered phosphorylation, and the kinase or phosphatase responsible for the alteration; the potential effects of cellular redox modulations caused by the Ref-1 gene product on this process will be evaluated also. 3) Evaluation of the role of Gadd45 in cell cycle modulations, apoptosis an cellular differentiation by using tetracycline inducible vectors. 4) A neuroblastoma model system in which irradiation-induced differentiation is dependent on p53 will be used to clarify the roles of Gadd45, N-myc, Mad-3 and cdc25 in this process.
