Characterization of HIF-1 alpha overexpressing HeLa cells and implications for gene therapy.

Abstract

Upon exposing mammalian tissues to hypoxia, expression of a number of physiologically important genes such as erythropoietin and vascular endothelial growth factor (VEGF) increases. The key regulator for this oxygen-dependent gene expression is the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of an alpha and a beta subunit. Both HIF-1 subunits are widely expressed in the cells and tissue of vertebrates, flies, fishes, worms and probably most other species. The beta subunit (also termed ARNT, aryl hydrocarbon receptor nuclear translocator) is abundantly expressed in an oxygen-independent manner. On the other hand, HIF-1alpha cannot be detected above a critical partial pressure of oxygen when it is subjected to rapid ubiquitinylation and proteasomal degradation. Hypoxic exposure leads to stabilization of HIF-1alpha protein and subsequent activation of HIF-1-dependent target genes. HIF-1 is not only a master regulator of oxygen homeostasis, it also appears to play a key role in tumor development as well as cardiovascular and ischemic diseases. Genetic modulation of HIF-1alpha activity in vivo may therefore represent a novel therapeutic approach to these disorders. In this overview, we report on the generation of HIF-1alpha overexpressing HeLa cell lines and demonstrate the feasibility of normoxic HIF-1 gene transfer in vitro and in vivo thereby identifying the limiting steps for full activation of HIF-1.