Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that are involved in crucial aspects of cancer biology, including angiogenesis, cell survival, glucose metabolism and invasion. Intratumoral hypoxia and genetic alterations can lead to HIF-1alpha overexpression, which has been associated with increased patient mortality in several cancer types. In preclinical studies, inhibition of HIF-1 activity has marked effects on tumour growth. Efforts are underway to identify inhibitors of HIF-1 and to test their efficacy as anticancer therapeutics.

[Abstract unavailable

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced 02 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We (8,9).The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and, thus, blood 02-carrying capacity (1, 4). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3' flanking region, and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription: inducers of EPO expression (1% 02, CoC12, and desferrioxamine) also induced HIF-1 DNA-binding activity with similar kinetics, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity, and mutations in the EPO 3' flanking region that eliminated HIF-1 binding also eliminated enhancer function (4). These results also support the hypothesis that 02 tension is sensed by a hemoprotein (10) and suggest that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (4 Glycerol gradient sedimentation, UV crosslinking, and methylation interference studies suggested that HIF-1 was a heterodimer consisting of the 120-kDa HIF-la subunit complexed with a 91-to 94-kDa HIF-13 subunit and that both subunits contacted DNA in the major groove (11,13,14). Here we show that HIF-la and HIF-13 are basic-helix-loop-helix (bHLH)-PAS proteins whose expression is regulated by cellular 02 tension.t MATERIALS AND METHODSProtein Microsequence Analysis. Purified HIF-1 subunits were fractionated by SDS/PAGE, and the 120-and 94-kDa polypeptides were transferred to poly(vinylidene difluoride) membranes and individually digested with trypsin in situ, and peptides were fractionated by reverse-phase HPLC (13). Protein microsequence analysis was performed at the Wistar Protein Microchemistry Laboratory (Philadelphia) (15).cDNA Library Construction and Screening. Poly(A)+ RNA was isolated from Hep 3B human hepatoma cells cultured for 16 hr at 37°C in a chamber flushed with 1% 02/5% C02/94% N2. cDNA was synthesized by using oligo(dT) and random hexamer primers, and bacteriophage libraries were constructed in Agtl 1 and Uni-ZAP XR (Stratagene). cDNA libraries were screened with 32P-labeled cDNA fragments by plaque hybridization (16).PCR. Degenerate oligonucleotide primers were designed by using codon preference rules (17). aF1 (5'-ATCGGATCCAT-CACIGARCTSATGGGITATA-3') was based upon the amino terminus of HIF-la peptide 87-1 and used as a forward primer. Two nested reverse primers, aRl (5'-ATTAAGCTTT-TGGTSAGGTGGTCISWGTC-3') and aR2 (5'-ATTAAGCT-TGCATGGTAGTAYTCATAGAT-3'), were based upon the carboxyl terminus of peptide ...