Regulation of glut1 mRNA by Hypoxia-inducible Factor-1

Abstract

Oncogenic transformation and hypoxia both induceglut1 mRNA. We studied the interaction between theras oncogene and hypoxia in up-regulating glut1mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1mRNA levels under normoxic conditions and additively increasedglut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of theglut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-α protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1α protein levels in these cells. Collectively these results indicate that H-Ras up-regulates theglut1 promoter, at least in part, by increasing HIF-1α protein levels leading to transactivation of promoter through the HIF-1 binding site. Oncogenic transformation and hypoxia both induceglut1 mRNA. We studied the interaction between theras oncogene and hypoxia in up-regulating glut1mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1mRNA levels under normoxic conditions and additively increasedglut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of theglut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-α protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1α protein levels in these cells. Collectively these results indicate that H-Ras up-regulates theglut1 promoter, at least in part, by increasing HIF-1α protein levels leading to transactivation of promoter through the HIF-1 binding site. hypoxia-inducible factor-1 serum-responsive element phosphatidylinositol 3-kinase Dulbecco's modified Eagle's medium base pair(s) kilobase pair(s) polymerase chain reaction mitogen-activated protein vascular epidermal growth factor Oncogenic transformation of mammalian cells is associated with many alterations in metabolism (see Ref. 1Dang C.V. Semenza G.L. Trends Biochem. Sci. 1999; 24: 68-72Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar for review). An increased rate of glucose transport is among the most characteristic biochemical markers of the transformed phenotype. The Glut1 glucose transporter is one of the proteins responsible (reviewed in Ref. 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (958) Google Scholar). A number of oncogenes, including fps, src, and rashave been shown to increase glucose transport and to up-regulateglut1 mRNA and protein levels (3Flier J.S. Mueckler M.M. Usher P. Lodish H.F. Science. 1987; 235: 1492-1495Crossref PubMed Scopus (692) Google Scholar, 4Birnbaum M.J. Haspel H.C. Rosen O.M. Science. 1987; 235: 1495-1498Crossref PubMed Scopus (245) Google Scholar, 5Racker E. Resnick R.J. Feldman R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3535-3538Crossref PubMed Scopus (97) Google Scholar). glut1gene expression and glucose transport are also stimulated in a variety of cells under hypoxic conditions, a response that is mediated by the transcription factor HIF-1.1 HIF-1 binds to acis-acting binding sites located within the 5′- flanking region of the glut1 gene (8Murakami T. Nishiyama T. Shirotani T. Shinohara Y. Kan M. Ishii K. Kanai F. Nakazuru S. Ebina Y. J. Biol. Chem. 1992; 267: 9300-9306Abstract Full Text PDF PubMed Google Scholar, 9Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Because hypoxia and oncogenic mutations are both commonly present in tumors, we set out to examine the interaction between the two in up-regulating glut1 mRNA levels. Mutations in Ras are seen in a third of human cancers (6Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar); therefore, as our model system we used Rat1 fibroblasts transformed with H-ras. Transformation with H-ras led to a substantial increase in glut1mRNA levels under normoxic conditions and additively increasedglut1 mRNA levels in concert with hypoxia. Our results indicate that a HIF-1 binding site located in the glut1promoter is important in its regulation by Ras. We further show that oncogenic H-Ras leads to increased promoter activity by up-regulating the steady-state level of HIF-1α protein and that this involves the PI3K pathway, known to be downstream of Ras (7Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). Rat1 cells were maintained in culture in DMEM supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals) and cultured in a humidified 95% air/5% CO2 incubator at 37 °C. To establish stable cell lines, Rat1 cells were transfected with an H-ras plasmid using Fugene (Roche Molecular Biochemicals) according to the manufacturer's instructions. Briefly, Rat1 cells were plated into a 100-mm dish 24 h before transfection. Cells were transfected using 6 μl of Fugene and 2 μg of the plasmid pAL8-H-ras, which contains the H-ras genomic DNA and a neomycin-resistance gene. Transfected cells were selected in DMEM supplemented with 400 μg/ml G418 (Life Technologies, Inc., Gaithersburg, MD) for 1 week. Colonies were isolated using cloning cylinders, trypsinized, then expanded as cell lines. The expression of the H-ras gene was confirmed by Western blotting with an anti-H-Ras monoclonal antibody. Twelve G418-resistant colonies were expanded, all of which expressed high levels of H-Ras protein compared with the Rat1 parental cells. We used clone 12, which expressed approximately 10-fold the level of H-Ras as the parental Rat1 cells (data not shown) for most of our experiments. Rat1-ras cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 400 μg/ml G418. PD098059 and LY294002 (from Alexis Biochemicals, San Diego, CA) were dissolved in Me2SO at stock concentrations of 100 mm and 40 mm, respectively. Total RNA was isolated with TRIzol (Life Technologies Inc.) following the manufacturer's instructions. 5–10 μg of total RNA was run on a 0.9% agarose gel containing formaldehyde, transferred to a Duralon-UV membrane (Stratagene, La Jolla, CA), and UV cross-linked prior to hybridization. Labeling of radioactive DNA probes was performed using [32P]dCTP and a Prime-It kit (Stratagene). Hybridization was carried out at 65 °C, after which the membranes were washed to a stringency of 0.1 × SSC, 0.1% SDS at 65 °C. Autoradiography was carried out at −80 °C with intensifying screens. A 400-bp mouse glut1cDNA fragment (from M. Birnbaum, University of Pennsylvania) was used to make radioactive probes. For protein isolation, cells were washed once with cold phosphate-buffered saline containing 1 mm EDTA, then solubilized by adding lysis buffer (1% Triton X-100, 20 mm Tris, pH 7.6, 150 mm NaCl, 2 mm EDTA, 10% glycerol, 1 mm dithiothreitol, 1 mm orthovanadate, 2 mm phenylmethylsulfonyl fluoride) directly on the cells. Lysates were transferred into 1.5-ml Eppendorf tubes and centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatants were transferred to a fresh tube and frozen on dry ice. Protein concentrations were determined using a BCA Protein Assay kit (Pierce, Rockford, IL). For Western blotting, an equal amount of total protein was separated by SDS-polyacrylamide gel electrophoresis on a 6% polyacrylamide gel. After completion of gel electrophoresis, protein was transferred onto a Transblot nitrocellulose membrane (Bio-Rad, Hercules, CA) using a blotting apparatus. For detection of H-Ras protein, we used a anti-human H-Ras monoclonal antibody (Quality Biotech, Camden, NJ) at a dilution of 1:1000 followed by a secondary goat anti-mouse antibody (Bio-Rad) at a dilution of 1:2000. For detection of the HIF-1α protein, we used an anti-HIF-1α antibody (clone H1α67, Novus Biologicals, at a dilution of 1:1000 followed by a secondary goat anti-mouse antibody binding was by using an kit Biotech, For hypoxia × cells were plated on 1 into and maintained in a CO2 was used of its high to the of the before hypoxia before the of was with containing and 10 mm to were then into The was decreased by a of the total within a with 95% CO2 using a The was to with one to with a then with an to The were then on an in a maintained at 37 °C. A of containing the region of the glut1 gene were and are (8Murakami T. Nishiyama T. Shirotani T. Shinohara Y. Kan M. Ishii K. Kanai F. Nakazuru S. Ebina Y. J. Biol. Chem. 1992; 267: 9300-9306Abstract Full Text PDF PubMed Google Scholar). shown in (see and and of the region of the glut1 gene. contains a 480-base pair the of the glut1 promoter located at to to the transcription site number contains a HIF-1 binding site at to was by in the following The pair was used for using construct as a contains of theglut1 promoter located at approximately to to the transcription site (from to number were to the to which been shown to a in of the glut1 promoter (8Murakami T. Nishiyama T. Shirotani T. Shinohara Y. Kan M. Ishii K. Kanai F. Nakazuru S. Ebina Y. J. Biol. Chem. 1992; 267: 9300-9306Abstract Full Text PDF PubMed Google the HIF-1 binding site to within a site and an the could be with these two and into the sites of the reporter plasmid contains its promoter, which was the fragment that was the glut1 and transcription site. was using a In two pairs of were used in and in one and and in the and the HIF-1 binding site but a 4-bp which After the with these two of in the was then using and to a fragment containing the which was and into the The is shown in 2 (see For reporter gene were performed using (Life Technologies Inc.) according to the manufacturer's instructions. Briefly, cells were plated into that 24 h were Cells in dish were transfected using μl of 2 μg of reporter and 1 μg of to for 24 h cells were by the with phosphate-buffered and directly adding 100 μl of lysis buffer this an was used for luciferase and for and were performed using the kit and the Assay respectively. were performed on a and To examine oncogenic transformation by human H-ras expression of the glut1 we transfected Rat1 cells with an H-ras expression We compared the expression of glut1 mRNA in Rat1 cells to Rat1-ras cells. Rat1 cells expressed levels of glut1 mRNA but a increase in glut1 mRNA levels after 6 h of hypoxia 1 In Rat1-ras cells increased levels of glut1 mRNA under normoxic conditions Rat1 2 and showed an after 6 h of hypoxia 4 H-ras both the level mRNA and with hypoxia to additively increase the level of this results were using a H-ras transformed (data not To the increased expression of glut1mRNA by H-ras and hypoxia was to increased promoter we transfected a glut1 construct in into Rat1-ras and Rat1 cells. 24 h after the were to hypoxia for 6 h and were maintained in cells all the were and for luciferase In Rat1 cells was a of the glut1 promoter by hypoxia Rat1-ras cells showed a higher luciferase activity than Rat1 cells under normoxic conditions and a further increase to hypoxia To that theglut1 promoter activity in Rat1-ras cells was to the effect of we a construct dominant negative into these cells, which in a in glut1 promoter activity the activity of the glut1 promoter is increased in Rat1-ras cells compared with Rat1 cells, and be further increased by hypoxia. To the in the region of the gene to a of of the promoter was used in in both Rat1 and Rat1-ras cells under normoxia and hypoxia glut1 promoter activity was in the Rat1 parental cells with all of the was approximately a of promoter activity by hypoxia with and but a with construct which a HIF-1 binding site. to results shown in in Rat1-ras cells, the level of glut1 promoter activity with construct A was than in Rat1 cells, and hypoxia led to a of activity compared with normoxia in Rat1-ras cells. a higher level of expression in Rat1-ras cells under normoxia hypoxia that construct A to the that construct contains a of the glut1 and transcription site in A and The level of expression of construct was higher in Rat1-ras cells than Rat1 the for this is as was of the glut1 promoter activity by hypoxia with construct in Rat1-ras cells. promoter is known to a HIF-1 binding which is for its transactivation by hypoxia through the transcription factor HIF-1 B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). To this site is important for transactivation of the promoter by H-Ras under normoxic conditions we a pair of reporter construct which contains of the promoter, including the HIF-1 binding and construct which is for a 4-bp which the HIF-1 binding site construct but not construct was by hypoxia. We performed of both into Rat1-ras cells with and cotransfection of a dominant negative expression that the promoter activity using construct which a HIF-1 binding was than that of construct which an intact HIF-1 binding site. In two we the promoter activity of construct to be and than the activity of construct (data not also that promoter activity is by in cells with construct and RasN17, effect on luciferase expression in cells transfected with construct M. results the that H-Ras a effect on the glut1 promoter transcription through the HIF-1 binding site. that the HIF-1 binding site in theglut1 promoter was important for its regulation by we that H-Ras might increase the level of the HIF-1α We that Rat1-ras cells higher levels of the protein under both normoxic and hypoxic conditions than Rat1 cells also that which is a hypoxia leads to higher steady-state levels of HIF-1α in Rat1-ras cells than Rat1 cells. the PI3K and been show to be by Ras (7Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). to the through which expression of Glut1 is by we transfected cells with construct A then with a PI3K inhibitor an inhibitor of under normoxic hypoxic conditions before cells for luciferase PD098059 and LY294002 both down-regulated glut1 promoter activity using construct A in cells under normoxic conditions, and both the of glut1 mRNA by hypoxia results were using construct both PI3K and a negative effect on the glut1 To an effect on glut1 mRNA Rat1-ras cells were with inhibitor under normoxic hypoxic conditions before cells for normoxic conditions, with inhibitor led to a in glut1 mRNA levels and 4 with and with both led to a in the level with We also Rat1-ras cells with a protein and that this effect on mRNA levels (data not We then Rat1-ras cells with the prior to to hypoxia. a effect on glut1 level with using both glut1 mRNA levels by 10 with To the effect of PD098059 and LY294002 in mRNA levels in normoxia might be mediated through we HIF-1α levels in Rat1-ras cells after that with LY294002 led to a in the level of with PD098059 confirmed this (data not For been known that compared with cells. increased metabolism even under conditions, an effect after The of and Scholar). To with this increased be an increased of the been shown to increased glucose in compared with (see Ref. E. E. U. M. Cancer Res. 1995; Google Scholar and of the proteins responsible for the of glucose into cells is which was the of a of glucose to be M. M. Lodish H.F. Science. 1985; PubMed Scopus Google Scholar). Glut1 is expressed in most and is responsible for glucose been shown to glut1 mRNA including oncogenes, growth and (reviewed in 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (958) Google Scholar and R. Cell. PubMed Scopus Google Scholar). a was that transformation of fibroblasts by M.J. Haspel H.C. Rosen O.M. Science. 1987; 235: 1495-1498Crossref PubMed Scopus (245) Google by the (3Flier J.S. Mueckler M.M. Usher P. Lodish H.F. Science. 1987; 235: 1492-1495Crossref PubMed Scopus (692) Google in a increase in glucose which was by an increase in Glut1 protein and mRNA In the of was shown that the increase mRNA at the level of transcription M.J. Haspel H.C. Rosen O.M. Science. 1987; 235: 1495-1498Crossref PubMed Scopus (245) Google Scholar). Glut1 levels been to be increased in a variety of human cancers M. M. J. Cancer Res. Google Scholar, T. Y. Y. K. T. Biochem. Res. PubMed Scopus Google Scholar). that Glut1 expression increased with increasing of in human cancers and was associated with a higher of M. M. J. Cancer Res. Google Scholar). Cells to hypoxia to the expression of many to for this to (reviewed in G.L. J. 2000; PubMed Scopus Google Scholar and H.F. 1999; PubMed Scopus Google Scholar). of this to hypoxia involves of that the for cells to to this of metabolism hypoxic conditions is also a increase in glucose which is by of Glut1 is a for glut1 mRNA in a variety of including cells J.D. J. S. J. 1992; PubMed Google cells A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google cell lines B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google and cells A. A. J. Biol. 1999; PubMed Scopus Google Scholar). The of glut1 by hypoxia is and is by and by of A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The former be by and the latter by The element in the glut1promoter been and is to the mouse B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). through this element in the promoter is mediated by HIF-1 B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google a transcription factor that binds to a DNA in the of many hypoxia-inducible Ref. G.L. J. 2000; PubMed Scopus Google Scholar). The HIF-1 transcription factor of two HIF-1α and both of which are is a of transcription and its protein level is not significantly by hypoxia. In HIF-1α protein, which is to the HIF-1 is in cells by the J. M. H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). HIF-1α protein is by hypoxia in a response on the Semenza G.L. J. PubMed Google Scholar). the by which hypoxia glut1mRNA expression through HIF-1 is is as Ras increase glut1 mRNA levels. The which protein is known to increase glut1 mRNA levels Y. Rosen O.M. M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar); Glut1 by growth and as a protein Y. Rosen O.M. M.J. J. Biol. Chem. Full Text PDF PubMed Google Y. M.J. 1989; PubMed Scopus Google Scholar). We were in a led to increased glut1 mRNA levels and in its interaction with hypoxia in For our model system we used Rat1 cells transformed with this We that H-ras transformation increased the level of glut1 mRNA under normoxia compared with Rat1 cells, with using (3Flier J.S. Mueckler M.M. Usher P. Lodish H.F. Science. 1987; 235: 1492-1495Crossref PubMed Scopus (692) Google Scholar, 4Birnbaum M.J. Haspel H.C. Rosen O.M. Science. 1987; 235: 1495-1498Crossref PubMed Scopus (245) Google Scholar). We also that H-ras additively with hypoxia to increase mRNA expression the effect of Ras on glut1 mRNA levels is mediated at the level of the promoter, the promoter activity is in Rat1-ras cells than in Rat1 cells In the region of the glut1 gene using in we that construct which contains a HIF-1 binding was to mediate of promoter activity by H-ras. construct contains a 480-base pair the of the glut1 promoter located between and to the transcription site. In to a HIF-1 binding site to this contains sites for transcription including binding sites to and to and an to which been in the regulation of this promoter (8Murakami T. Nishiyama T. Shirotani T. Shinohara Y. Kan M. Ishii K. Kanai F. Nakazuru S. Ebina Y. J. Biol. Chem. 1992; 267: 9300-9306Abstract Full Text PDF PubMed Google Scholar). to the importance of the HIF-1 binding we reporter and of which the of theglut1 promoter located approximately to to the transcription site (from to number and are that the former contains a pair mutation of the HIF-1 binding site. as construct was by but not construct M. showed activity in Rat1-ras cells under normoxic conditions, but a is that the activity of construct but not construct could be down-regulated by this the HIF-1 binding site is important in regulation by also lower luciferase activity in Rat1-ras cells than construct between and We that the that the is not is both of these and are by an promoter than the to theglut1 therefore, is a high level of The for the glut1 to the region of the gene that was by T. Nishiyama T. Shirotani T. Shinohara Y. Kan M. Ishii K. Kanai F. Nakazuru S. Ebina Y. J. Biol. Chem. 1992; 267: 9300-9306Abstract Full Text PDF PubMed Google Scholar). that the mouse glut1 was by showed that the and two sites within this were important in promoter not examine the that these sites in regulation by Ras. that these were for activity of the HIF-1 binding site within the mouse glut1 was B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Our indicate that this HIF-1 binding site is important in the regulation of the glut1 promoter by of the mouse glut1 promoter by in was shown to both the and the binding sites in but was of the A. R. Ebina Y. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). H-Ras activity of the HIF-1 binding site in theglut1 is by increasing the level of HIF-1α Rat1-ras cells higher levels of the protein than Rat1 cells under both hypoxic and normoxic conditions. HIF-1α was as a transcription which was under normoxic conditions and under hypoxia G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google is that is also important in gene regulation under normoxic cells been to higher levels of HIF-1α under normoxic conditions than parental cells F. A. Semenza G.L. Cancer Res. Google Scholar). HIF-1α been to be in cell lines by a variety of and growth including epidermal growth growth and E. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar, K. E. M.M. Semenza G.L. Cancer Res. 2000; Google Scholar, E. Y. M. J. PubMed Scopus Google Scholar, F. Semenza G.L. Cancer Res. 1999; Google Scholar). of the and the PI3K led to decreased glut1 promoter activity and glut1mRNA levels under In by to the of glut1 mRNA by the two an between the of these under normoxia and hypoxia might be by the that the of glut1 mRNA by hypoxia involves mRNA in to transactivation of the promoter A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in using promoter we are transcriptional in the the results are by the that RNA which be of PI3K and also a we that both and PI3K mRNA promoter the latter led to a in HIF-1α protein levels. A for the PI3K in HIF-1α protein levels been in the of expression in cells A. F. E. 2000; PubMed Google and epidermal growth factor in cells K. E. M.M. Semenza G.L. Cancer Res. 2000; Google Scholar). In to both and been shown to HIF-1α in and to HIF-1α transcription activity the level of the protein E. E. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the PD098059 might mRNA levels and promoter activity through an effect on HIF-1α the level of the protein Our results a between H-ras HIF-1α and glut1 mRNA In the we to this of to H-Ras leads to HIF-1α we the expression of a as a downstream of is that the of hypoxia-inducible as by a A number of shown that Ras leads to increased levels in cell S. K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Cancer Res. Google Scholar, J. Y. J. S. T. Cancer Res. 1995; Google Scholar). In cells, H-Ras been to increase reporter expression an intact HIF-1 binding site is present PubMed Google Scholar). mutations in H-ras not to transformation but also cells in a hypoxic by increasing the expression of through of We are to for and for with We and for the and We are to for of the and