Research proposed in this competitive renewal will utilize genetically modified mice to explore the roles of the Mdm2 proto-oncogene in development, cell growth and death, and in cancer. Two mouse models have been generated. The first model contains a conditional allele of Mdm2. Our preliminary evidence indicates that Mdm2 governs both p53-mediated cell cycle arrest and apoptosis in early development, and can also influence cell differentiation. We will use embryonic fibroblasts derived from Mdm2-conditional mice to analyze Mdm2 regulation of p53 functions, to document a role for Mdm2 in regulation of other p53 family members, and to document requirement of the cyclin-dependent kinase inhibitor p21 in Mdm2-induced cell arrest and cell death. We will also explore a role for Mdm2 regulating the development of multiple tissues during gestation, and examine the role of Mdm2 in regulating growth, maintenance, and DNA damage response of skin to determine if Mdm2 functions in terminally differentiated tissue. These experiments will also confirm a link between upregulation of p53 in various tissues and in rapid aging phenotyopes in mice. A second mouse model involves a site-specific modification we have generated in the p53 gene. Preliminary evidence presented in this proposal indicates that phosphorylation of this site specifically regulates p53 induced apoptosis following DNA damage. These mice and cells derived from these mice will be used to determine if post-translational modification of this p53 residue alters Mdm2-mediated inhibition of p53 or whether phosphorylation of this site impacts p53 functions and stability via an Mdm2-independent mechanism. We will also explore the importance of this site to other p53 modifications and we will determine the role of this p53 modification in oncogene-induced activation of p53. Collectively, these proposed studies should lead to a finer understanding of the role of Mdm2 in regulating cell growth and apoptosis in development and in adult tissue, and the effects of p53 phosphorylation on Mdm2-p53 regulation and on p53 functions.
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