""""""""Hypoxia-induced apoptosis of tumor cells in vivo requires wild type p53, and hypoxia is perhaps the most physiologic inducer of wtp53. However, the mechanism by which hypoxia induces p53 has remained elusive. Cells sense low oxygen tension via a heme-containing sensor protein, and the hypoxic state can thus be mimicked by both cobalt chloride and the iron chelator desferrioxamine. Like hypoxia, cobalt and DFX induce the novel transcription factor hypoxia-inducible factor 1 alpha (HIF-1alpha), which stimulates transcription of a number of hypoxia-related genes, including erythropoietin and vascular endothelial growth factor. We have shown in this study that cobalt and DFX both induce wtp53 via protein stabilization, without change in p53 mRNA level. Induction of p53 does not occur in a mutant hepatoma cell line unable to induce HIF-1alpha following exposure to hypoxic stimuli, nor in an embryonic stem cell line in which the HIF-1 gene has been knocked out. p53 immunoprecipitates from wtp53-expressing MCF7 cells exposed to DFX contain HIF-1alpha. Likewise, HA immunoprecipitates from p53-null PC3M cells co-transfected with HA-HIF-1alpha and wtp53 contain p53. These data suggest that HIF-1alpha and p53 proteins directly associate under hypoxic conditions. Finally, transfection of normoxic MCF7, but not PC3M, cells with HIF-1alpha alone stimulates the activity of a co-transfected p53-dependent reporter plasmid and elevates the steady-state level of endogenous p53 protein, supporting the hypothesis that hypoxic induction of wtp53 occurs via protein stabilization dependent on direct association with HIF-1alpha. Ongoing studies are examining mechanisms of HIF-1alpha stability in hypoxia and stimulation of nuclear entry of this protein. Additionally, a panel of p53 mutants are being studied to correlate inhibitory effects on HIF activity with p53 transcriptional activity and binding to HIF and/or p300. The mechanism by which hypoxia causes nuclear translocation of p53 is also being studied.""""""""
