During the growth of a tumor, the need for oxygen and nutrients is provided by the development of new vascular beds. This process, named angiogenesis, is indispensable for tumor growth and metastasis development. Angiogenesis in tumors is induced by the release of growth factors like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) which are released by the tumor cells. These factors, especially VEGF, have been shown to be highly upregulated in tumors and play a pivotal role in the proliferation and migration of endothelial cells into the tumor. Hypoxia is a common feature of human and animal tumors. The vast majority of human cancers have a median P02, well below the level of their tissue origin. As the tumor grows, the center of the tumor became more and more hypoxic, sometimes producing central necrosis. Pioneer observations by Keshet and colleagues. established that VEGF was highly expressed in the most hypoxic and ischemic areas of the tumors, suggesting that the lack of oxygen was an important stimuli for VEGF production. Indeed, work by others as well as our group have shown that the transcription of the VEGF gene is strongly stimulated by hypoxia. The mechanism of hypoxic regulation of VEGF is similar to the one originally described for the erythropoietin gene, and involves the HIF-1 (hypoxia inducible factor-1) complex. HIF-1 complex is a heterodimer composed of two basic helix-loop-helix proteins, HIF-1alpha, and hypoxia regulated protein, and HIF1alpha, which is constitutively expressed. HIF-1alpha is continuously synthesized but rapidly degraded by the ubiquitin- proteasome system under normoxic conditions. Hypoxia, transition metals and iron chelators inhibit HIFI a degradation and allow the formation of the active HIF- I complex. Recent work from our laboratory as well as other laboratories have shown that the VHL protein is involved in the degradation of HIF-1alpha. Tumors lacking VHL are highly vascularized and express high levels of VEGF mRNA and protein. In these tumor cells, HIF-1alpha protein is expressed in normoxic conditions due to a low degradation rate. The mechanisms by which the VHL protein induces normoxic degradation of HIF- 1alpha will be studied using a two-hybrid system in yeast. These will allow the identification and cloning of the protease involved in HIF-1alpha degradation.
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