This Project has two goals: 1. To identify and characterize the single nucleotide polymorphisms (SNPs) that are found in the human genes which compose the p53 pathway in cells. 2. To expand and utilize the algorithm we have previously designed to detect the p53 responsive elements (P53 REs, the DNA sequences that permit a gene to be regulated by p53) and identify the genes regulated by p53 in the mouse and human genomes. To date we have identified over 1,300 SNPs in 82 genes in the human genome that function in the p53 network and we are creating a web site to share these data. We have developed methods to identify which SNPs have the best chance of altering the molecular, cellular and clinical phenotypes of the p53 pathway. We have focused upon several SNPs (the lead SNPs are in HDM-2, Perp and AKT), which result in an altered phenotype at the molecular (changed levels and activity), cellular (an altered frequency of apoptosis) and clinical (a younger age of onset of cancers and increased number of cancers in humans) levels of analysis. We have characterized 120 EBV-transformed lymphoblastoid cell lines from normal individuals for their apoptotic index (the percentage of cells that undergo apoptosis after 5 Gy of radiation, a reproducible p53-dependent function) and typed these lines for our SNPs providing statistically significant correlations for the impact of a SNP. These cell lines will become a resource for the field that wishes to analyze apoptosis and the p53 pathway. For example SNP 309 in the promoter of the HDM-2 gene, creates a new SP-1 site, increases the levels of HDM-2 protein in a cell, lowers the p53 transcriptional response, lowers the apoptotic index and reduces the age of onset of cancers in humans by 12-15 years. We will validate and expand new p53 targets (eg, PTEN, TSC-2, Huntingtin and LINE-1) and determine when and how they are regulated by p53. Collaborative studies with Prives, Lowe and Cordon-Cardo are planned to carry out these aims.
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