Mutation of the p53 tumor suppressor gene is the most prevalent genetic change associated with human cancer. It is well established that accumulation of p53 following genotoxic stress results in cell cycle arrest and/or apoptosis. Both cell cycle arrest and apoptosis contribute to p53 tumor suppression. Genetic studies have shown a strong correlation between its ability to act as a transcriptional regulator and its cell cycle arrest function. However, it is less clear how p53 induces apoptosis. Recently the investigators have used a tetracycline-regulated promoter to generate a number of stable cell lines that inducibly express p53. The results show that p53 levels, functional domains and DNA damage determine the extent of the apoptotic response of tumor cells. In addition, they show that the p53 transcription-dependent and transcription-independent pathways of apoptosis are genetically separable and synergistically cooperate in inducting apoptosis. Further understanding of the mechanism of p53-dependent apoptosis will be vital to its application in cancer therapy.
The specific aims are: (1) To delineate the functional domains of p53 that are required for apoptosis and its cooperation with various therapeutic drugs in inducing apoptosis. By generating and subsequently expressing various deletion and point mutants of p53 in the activation domain, the hinge region between the activation and sequence specific DNA binding domains, the nuclear localization signal, the oligomerization and carboxyl-terminal nonspecific DNA binding domains, the roles of these domains in p53-dependent apoptosis and cell cycle arrest will be analyzed in cells. By using cell lines inducibly expressing either wild-type or various mutated forms of p53, the domain(s) of p53 that cooperate with various therapeutic drugs in inducing apoptosis will be determined. (2) To identify and characterize potential mediators of p53-dependent apoptosis. By using differential display analysis, they have identified four novel potential p53 target genes. These genes will be characterized for their ability to confer p53-dependent apoptosis and cell cycle arrest. Additional p53 target genes will also be identified. Furthermore, by using cell lines that inducibly express the influenza hemagglutinin (HA)-tagged p53 protein, both immunoprecipitation and affinity chromatography will be performed to identify new potential p53-interacting proteins that may mediate p53 transcription-dependent apoptosis.
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