Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cisacting sequences that convey these responses. An enhancer lying 5' to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides 5' to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus, rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves activation of a serum response element.
Production of the glycoprotein hormone erythropoietin (Epo) Erythropoietin (Epo) is the hormone which regulates the oxygen-carrying capacity of the blood by controlling erythropoiesis: hypoxia stimulates production by a mechanism which involves increased Epo gene transcription (1). Transient-transfection studies in cultured cells have defined cisacting control sequences responsible for this effect, the most powerful of which is an oxygen-regulated transcriptional enhancer located 3' to the poly(A)-addition site of the gene (2-6). Extensive screening of tissue culture cell lines has revealed only two (the hepatoma lines Hep 3B and Hep G2) that produce Epo in an oxygen-regulated manner (7), yet the Epo 3' enhancer, when coupled to broadly active promoters, confers responsiveness to hypoxia in most (possibly all) cultured cells (8, 9). Furthermore, hypoxia-inducible factor 1 (HIF-1), which binds to a functionally critical region of the Epo 3' enhancer, has been found both in cells which produce Epo and in those that do not (10, 11). These findings suggest that oxygen sensing, signal transduction, and gene activation mechanisms very similar, if not identical, to those present in Epo-producing cells must be widespread. The implication is that these mechanisms are involved in the regulation of other genes in cells which do not produce Epo.We report here that the human phosphoglycerate kinase 1 (PGK-1) and mouse lactate dehydrogenase A (LDH-A) genes are regulated by hypoxia in a manner which is strikingly similar to Epo gene regulation. Using transient transfection, we have located cis-acting control sequences responsible for hypoxia-inducible expression in the 5' flanking region ofeach gene. For the PGK-1 gene we have characterized an 18-bp element which is necessary for hypoxia-inducible expression and which has sequence similarity to a region within the Epo 3' enhancer. Oligonucleotides containing the functionally active PGK-1 and Epo sequences cross-compete for a hypoxia-inducible factor (or factors) in electrophoretic mobility-shift assays. MATERIALS AND METHODSCell Lines and Culture Conditions. The cell lines used were Hep G2 (human hepatoma), HeLa (human cervical carcinoma), and L cells (mouse fibroblast) grown to %700% confluence. The medium was then replaced, and cells were subjected to the following conditions for [14][15][16] hr: (i) normoxia (20% 02/5% C02/75% N2); (ii) hypoxia (1% 02/5% C02/94% N2 in a Napco 7100 incubator); (iii) hypoxia with cycloheximide (100 LAM); (iv) normoxia with cobaltous chloride (50 pM); (v) normoxia with cyanide (100 KM); or (vi) hypoxia with cyanide (100 pM).Translent Transfection and RNA Analysis. In all experiments the test plasmid (10-100 pg), encoding either human al-globin or human growth hormone (GH) as a reporter, was cotransfected with a control plasmid (10-50 ,g) by electroporation (4). After electroporation, transfected cells were split equally and incubated in parallel for 14-16 hr under normoxic or hypoxic conditions or were exposed to chemical agents, as ...
Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.
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