Mammalian cells have an absolute requirement of oxygen and nutrients for their survival and are usually located within short proximity of blood vessels. Due to their high growth rate, rumors overgrow their vascular supply and are usually exposed to a hypoxic microenvironment. Hypoxia acts as a selective factor for mutations that increase survival and enhance metastatic capabilities. Furthermore, it increases the resistance of tumor cells to radiation and chemotherapeutic agents. Most of the adaptive responses of tumor cells to hypoxia are mediated by the transcriptional complex HIF-1, a master regulator of hypoxic-gene expression. These HIF-regulated genes include those that control angiogenesis, energy metabolism, and tumor invasiveness. The HEF-1 complex is composed by two sub-units: HIFIP that is constitutively expressed, and HIF-1alpha, whose levels and activity depend on oxygen concentration. Oxygen control of HEF1alpha stability depends on hydroxylation reactions and interaction with the VHL protein, which acts as an ubiquitin-ligase. Similarly, oxygen dependent hydroxylation determines the interaction of HIF1alpha with p300, its transcriptional co-activator. Notably, HIF-complexes are regulated not only by hypoxia but also by non-hypoxic signals like oncogenic mutations, cytokines and growth factor stimulations, suggesting a more ample role of HIF-1 in cell functions. Although the mechanisms by which these non-hypoxic signals control HIF-1 are not completely clear, current knowledge and our preliminary work indicate that a complex array of post-transcriptional modifications, including phosphorylation and acetylation determine the final activity of the HIF-1 complex, hi particular, acetylation appears to regulate both the stability of HIFla protein and its interactions with p300. It is the purpose of this proposal to characterize the role and mechanisms of action of the acetyl-transferases and deacetylases enzymes that act on HIF-1alpha and the HIF1alpha-p300 complex. Furthermore, we plan to investigate the possible role of the Von Hippel Lindau (VHL) protein in these effects. Deacetylase inhibitors are currently being tested as therapeutic agents in cancer therapy and the studies proposed herein will facilitate the understanding of its effects and help in the development of target-oriented inhibitors.
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