The p53 tumor suppressor gene is a frequently inactivated gene in human cancer cells. In response to cell stressors p53 induces cell cycle arrest or programmed cell death. We have discovered that a small thiol-containing agent (pyrrolidine dithiocarbamate) inhibits some aspects of p53 activity in cultured cells and increase our understanding of the mechanisms by which p53 activity is modulated as such mechanisms may alter its ability to respond to genotoxic agents and perform its tumor suppressor activities. Dithiocarbamates are commonly used as herbicides, fungicides, and insecticides world-wide. Environmental toxins that act on p53 in a fashion similar to pyrrolidine dithiocarbamate may contribute to some types of diseases. We developed a novel technique to measure protein cysteine oxidation reactions in cultured cells. Evidence from our laboratory and others suggest that the redox state of p53 modulates its ability to specifically bind its DNA promoter targets, activate transcription of its effector genes, and suppress cell cycle progression. The hypothesis to be tested are: 1) p53 protein undergoes cysteine residue, oxidation after cellular treatment with some oxidizing reagents; 2) p53 oxidation alters its ability to bind its consensus sequence with DNA promoters and insertion/deletion mismatch DNA sequences.
The specific aims are: 1. Identify the sites of cysteine residue oxidation on p53 by site-directed mutagenesis and mass spectroscopy. 2. Determine if Ref-1 is required for maintaining a low p53 redox state in the presence of dithiocarbamate-based pesticides and hydrogen peroxide. 3. Determine the redox potential of p53 as a function of oxidation, binding to p53 consensus sequence DNA and binding to insertion/deletion mismatch DNA. Successful completion of this project will uncover new insight into this novel p53 modulation mechanism.
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