The tumor microenvironment influences both therapeutic outcome and malignant progression. Previous studies from my laboratory have indicated that hypoxia induces apoptosis in oncogenically transformed cells in vitro, can act as a selective pressure for the expansion of transformed cells possessing diminished apoptotic potential, and co-localizes with apoptotic regions in tumors. The tumor suppressor protein p53 induces rapid apoptosis in response to oxygen concentrations that induce an S-phase arrest. Hypoxia-induced p53 is nuclear and associates with p53-response elements in target genes, such as mdm2 and p21. The cellular decision to use p53 transactivation or transrepression is mediated after the binding of p53 to the promoter by the stress, and is determined by the presence of co-activators or co-repressors. In contrast to p53-induced by DNAdamaging agents, hypoxia-induced p53 has primarily transrepression activity. Using extensive microarray analysis, we identified families of repressed targets of p53 that are involved in cell signaling, DNA repair, cell-cycle control and differentiation. Mutation analysis has determined that loss of transrepressor activity of p53 results in diminished apoptosis under hypoxic conditions. Mutation of residues 25,26 or 53,54 in the amino terminus of p53, that have previously been shown to inactivate p53-dependent transactivation, could still signal apoptosis under hypoxic conditions. However, mutation of all four residues completely abolished p53 dependent apoptosis as well as p53 dependent transrepression. This study defines a new role for the 53,54 residues of p53 regulating transrepression and suggests that 25,26 and 53,53 work in the same pathway to induce apoptosis through gene repression. Furthermore, p53 induced by both genotoxic as well as nongenotoxic stress is able to bind to the same promoters, but it is the stress that determines whether apoptosis is mediated by transcriptionally dependent or independent pathways. The critical hypothesis that we will be testing is that genotoxic and non-genotoxic stresses modulate gene activation and repression via p53's association with chromatin. In this proposal, we will demonstrate the importance of gene repression by hypoxia in apoptosis and differentiation using in vivo derived models. We will investigate the importance of protein interactions with the amino terminus of p53 in regulating gene repression by hypoxia. We will test these hypotheses through a combination of genetic and biochemical approaches in cell lines and in mice. ? ?
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