Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor

Hypoxia-inducible factor (HIF) mediates a widespread transcriptional respon se to hypoxia through binding to cis-acting DNA sequences termed hypoxia re sponse elements (HREs). Activity of the transcriptional complex is suppress ed in the presence of oxygen by processes that include the targeting of HIF -alpha subunits for ubiquitin-mediated proteolysis. To provide further insi ghts into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antige ns and used these cells in genetic screens for mutants that demonstrated co nstitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increase d HIF-1 alpha protein levels in normoxic culture. Transfection and cell fus ion studies suggested that these cells possess recessive defects that affec t one or more pathways involved in HIF-alpha proteolysis. Two lines were de monstrated to harbor truncating mutations in the von Hippel-Lindau VHL) tum or suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1 alpha in vitro could be complemented by wild type pVHL, and re- expression of a wild type VHL gene restored a normal pattern of HIF/ HRE ac tivity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIEF-1 alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processi ng of HIF-alpha subunits.