MUTATIONAL AND FUNCTIONAL ANALYSIS OF THE P53 TUMOR SUPPRESSOR GENE

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The carboxy-terminus of p53 can bind to damaged DNA, reanneal complementary single strands of nucleic acids and lead to tetramerization of the p53 protein. Because our data indicate that the carboxy-terminus p53 is also involved in apoptosis, we are using the following complimentary strategies to define a "apoptotic domain" in this region of p53: (a) microinjection of C-terminal p53 synthetic polypeptides (aa 319-393, 361-393, 319-360) into normal, Li-Fraumeni (041; p53-/-) or p53 mutant (missense or p53 null) human tumor cells and determining the frequency of apoptotic cells by DAPI staining and TUNEL assays; (b) penetratin coupling or pegylated tagged synthetic p53 polypeptides listed above, exposure of the cell types listed above and scoring apoptotic cells by DAPI, TUNEL and FACS, i.e., subG1 DNA content assays; (c) microinjection of CMV-driven expression vectors containing human cancer-derived p53 missense and frameshift mutants located in the carboxy-terminus of p53; (d) microinjection of CMV-driven expression vectors containing synthetic p53 mutants of putative phosphorylation sites (366ser, 376ser and 378ser) or deletion mutants (aa1-353, 1-356, 1-363 and 1-370) in the carboxy-terminus of p53. Our preliminary data have narrowed down one important region to be aa363 to aa370. Because Levine and Walker (p53 Workshop, Dundee, 1996) have shown that deletion of the five PXXP, SH3 motif, located in aa62 to aa91 of p53 inhibits its apoptotic activity whereas p53 transcription transaction is not affected, we are microinjecting the aa62 to aa91 polypeptide in normal human fibroblastic and mammary epithelial cells. If the putative cellular protein(s) binding to the SH3 region of p53 is upstream of p53 in its apoptotic pathway, then the polypeptide may block DNA damage induced apoptosis. If the putative p53 binding protein(s) is downstream of p53, the microinjected polypeptide might initiate apoptosis. We also have found that somatic mutations in the C-terminus of p53 from human cancers lose apoptotic activity and retain transcriptional activity. We have shown that p53 binds to DNA helicases, e.g., XPD, XPB, and Rad54, and are currently investigating p53 modulation of BLM and WRN helicase activities.