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.

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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 ...

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposedUnder normal physiologic conditions, each of the approximately 10 14 cells in the adult human body is provided with an adequate supply of O 2 to meet its metabolic demands through the concerted function of the pulmonary, hematopoietic, and cardiovascular systems. O 2 is transported through the circulation by erythrocytes, the production of which is controlled by the glycoprotein hormone/growth factor erythropoietin (EPO) (reviewed in references 20, 23, and 43). Cells in the liver and kidney that produce EPO are able to sense O 2 concentration and respond to systemic hypoxia with increased EPO gene transcription (8,15,46). A hypoxia-inducible enhancer element was identified in the 3Ј-flanking region of the human and mouse EPO genes (2,3,33,42,48,50). Hypoxia-inducible factor 1 (HIF-1) was detected in nuclear extracts of hypoxic Hep3B cells (exposed to 1% O 2 for 4 h) and was undetectable in extracts from nonhypoxic cells (maintained at 20% O 2 ). HIF-1 bound to the EPO enhancer, and mutations that eliminated HIF-1 binding also eliminated enhancer function (50). Exposure of hypoxic cells to inhibitors of protein synthesis (cycloheximide) or phosphorylation (2-aminopurine) inhibited the induction of both EPO mRNA and HIF-1 DNA-binding activity, and other inducers of EPO expression (CoCl 2 and desferrioxamine) also induced HIF-1 activity (50,58,60). Methylation interference analysis revealed that HIF-1 bound to the EPO enhancer sequence 5Ј-TACGTGCT-3Ј by making major groove contacts with both guanine residues on each strand (59).Protein purification indicated that HIF-1 was a heterodimeric protein (61). Peptide and nucleic acid sequence analysis demonstrated that both subunits were basic helixloop-helix (bHLH) proteins (57). HIF-1␣ was a novel 826-amino-acid polypeptide, whereas HIF-1␤ was identical to the 774-and 789-amino-acid products of the ARNT (aryl hydrocarbon receptor nuclear translocator) gene previously shown to heterodimerize with the aryl hydrocarbon receptor (AHR) (57). HIF-1␣, HIF-1␤ (ARNT), and AHR are all members of a subfamily of bHLH proteins that contain a conserved PAS domain following the bHLH motif (4,18,57). In all three polypeptides, the basic domain is required for DNA binding following heterodimerization mediated by the HLH and PAS domains, and the C terminus contains one or more transactivation domains (6,21,29,32,44,63). Forced expression of HIF-1␣ and HIF-1␤ (ARNT) in cultured cells transfected with a reporter plasmid containing the EPO enhancer resulted in significantly higher levels of transcription, both at 1% and at 20% O 2 , than in cells transfected with the reporter plasmid alone, demonstrating that transcriptional activation via the EPO enhancer is mediated by HIF-1 (21).In contrast to systemic hypoxia, which elicits increased EPO synthesis, hypoxia can also be restricted to cells within a localized region of a specific organ, usually as a result of insufficient perfusion, as in the case of myocard...

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.

Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes.

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a −/− embryonic stem cells that did not express the O 2 -regulated HIF-1␣ subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a −/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1␣ resulted in developmental arrest and lethality by E11 of Hif1a −/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a +/+ embryos, HIF-1␣ expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a −/− embryos. These results demonstrate that HIF-1␣ is a master regulator of cellular and developmental O 2 homeostasis.

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) Erythropoietin (EPO) is the glycoprotein hormone which regulates mammalian erythrocyte production and, as a result, tissue oxygen delivery. EPO RNA levels increase several hundredfold in rodent liver and kidney in response to hypoxia (8,23,24) or anemia (3,4,18). Human EPO RNA levels show similar increases in transgenic mouse liver and kidney (17,25,26,28). Hypoxia also induces EPO RNA expression in Hep3B human hepatoma cells (10, 12), demonstrating that the same cell type can sense hypoxia and respond by increasing EPO RNA levels. The 50-fold increase in steady-state EPO RNA in hypoxic Hep3B cells requires new protein synthesis and is accounted for by an approximately 10-fold increase in the rate of transcription, with the remaining increase due to posttranscriptional mechanisms (10, 11). Nuclear extracts prepared from hypoxic Hep3B cells support a higher level of EPO gene transcription in vitro than extracts from nonhypoxic cells (5).We have previously demonstrated that a 256-nucleotide (nt) EPO gene 3'-flanking sequence element functions as a hypoxia-inducible enhancer when cloned 3' to a simian virus 40 (SV40) early-region promoter-chloramphenicol acetyltransferase (CAT) reporter gene and transiently expressed in Hep3B cells (27 We have now performed functional studies and binding assays to further characterize the hypoxia-inducible enhancer. These studies indicate that hypoxia-inducible EPO gene transcription is mediated by a 50-nt element that contains at least three different transcription factor binding sites. One of these sites binds a factor in Hep3B nuclear extracts which is induced by hypoxia. MATERIALS AND METHODSPlasmid constructs. The pSVcat reporter plasmid (pCATPromoter; Promega) contained SV40 early-region promoter (excluding the 72-bp repeat enhancer), bacterial CAT coding sequence, SV40 splice site, and polyadenylation signal.