The p53 signaling pathway is the most commonly subverted pathway identified in human tumors. In normal cells, p53 plays a critical role in maintaining normal physiology by coordinating the response of cells to a diverse range of environmental and endogenous stress. There is extensive evidence indicating that the ability of p53 to function as a sequence-specific transactivator is central to its role as a tumor suppressor. Recent results show that p53 is able to discriminate between consensus binding sites in downstream genes in vivo, presumably activating gene transcription that mediates the appropriate response in a given cellular environment and physiological situation. The overall goal of this proposal is to characterize the factors which dictate p53 DNA binding properties in vivo and the physiological relevance of select downstream target genes. To accomplish this goal, the following interrelated hypotheses are proposed: 1. The selectivity of transcriptional regulation by p53 is achieved by multiple factors working in concert, including: (a) the overall level of p53 in the cell, (b) the post-translational modification of p53 that may regulate stability or association with co-activators, and (c) the sequence heterogeneity of p53 binding sites in target genes that dictates p53 affinity for a site. 2. Cell fate after p53 activation is dependent on the differential, cell type-dependent transactivation of select genes, whose protein products regulate pathways of cell cycle arrest and survival. New technologies and reagents will be exploited, as well as classic molecular biology, genetic, and biochemical methods to test these hypotheses through the following Specific Aims: 1. To characterize p53 binding to target genes promoters in vivo in primary, normal human cell cultures. 2. To """"""""trap"""""""" p53 in vivo at consensus DNA binding sites in normal ceIIs after various forms of cell stress and create libraries of p53 DNA binding sites. These sites will be analyzed to determine: (a) the sequence of the sites to which p53 is bound and the extent of heterogeneity, (b) the affinity of p53 For a select number of the DNA sites to which it is bound under a given condition, and (c) the genetic loci for novel sites and adjacent coding regions. 3. To determine the role of select p53 target gene products in dictating cellular outcome after stress induction, with primary focus on p53-inducible gene 3 PIG3), a putative NAD(P)H oxidoreductase. The importance of understanding p53 signaling is underscored by the frequency of p53 mutations in tumors and the findings that modulating this pathway in cancer cells can induce cell cycle arrest, apoptosis, or chemosensitization depending upon cell type, mutations, and environment. Understanding how p53 mediates these outcomes is expected to translate into therapeutic benefit in the treatment of tumors, and thus would have major impact on human health.
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