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Metabolic profiling of hypoxia-inducible factor-1β-deficient and wild type Hepa-1 cells: effects of hypoxia measured by 1H magnetic resonance spectroscopy

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Abstract

HIF-1 is upregulated across a broad range of cancers and modulates tumor progression through genes including those for glycolytic enzymes and glucose transporters. Hepa-1 cell lines that were either wild type (WT), or deficient in HIF-1β (c4) were studied by metabolic profiling. Western blots indicated that the HIF-1β deficient c4 cells failed to translocate the HIF-1α-β complex to the nucleus under hypoxia whereas both HIF-1α and HIF-1β accumulated in the nuclei of Hepa-1 WT cells after 18 h exposure to hypoxia (1% O2, 5% CO2, N2). Reporter gene assays showed that, compared to WT cells, c4 cells were unable to activate transcription from the PGK and LDH HRE regulated promoters. ELISA assays showed similar VEGF secretion by both cell types under normoxia and a 1.6-fold increase in WT cells under hypoxia. Metabolite profiles (by 1H MR spectroscopy) under normoxia (95% air, 5% CO2) showed similar lactate content and secretion whilst hypoxia caused lactate to increase ∼2.5-fold in both deficient and WT cells. In addition phosphocholine (PC), NTP (by 31P MRS) and glycine were significantly lower in c4 than WT cells under normoxia. Although the immunoblotting and transcription assays indicated loss of activation of HIF-1β, the c4 cells were still able to upregulate glycolysis in hypoxia (as shown by the lactate data). We conclude that there are mechanisms other than HIF-1β transcription that may up-regulate glycolysis in HIF-1 deficient Hepa-1 cells.

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References

  • Arsham A.M, Plas D.R, Thompson C.B, Simon M.C. (2004) Akt and Hypoxia-inducible-factor-1 independently enhance tumor growth and angiogenesis. Cancer Res. 64: 3500–3507

    Article  PubMed  Google Scholar 

  • Blouw B., Song H., Tihan T., Bosze J., Ferrara N., Gerber H.P., Johnson R.S., Bergers G. (2003) The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell 4:133–146

    Article  PubMed  Google Scholar 

  • Brown L.M., Williams K.J., Cowen R.L., Melillo G., Sausville E., Stratford I.J.(2003) The potential therapeutic application of targeting HIF-1 using gene therapy or small molecule approaches. Proc. AACR. 44: 4633

    Google Scholar 

  • Carmeliet P., Dor Y., Herbert J.M., Fukumura D., Brusselmans K., Dewerchin M., Neeman M., Bono F., Abramovitch R., Maxwell P., Koch C.J., Ratcliffe P., Moons L., Jain R.K., Collen D., Keshert E., Keshet E. (1998) Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature 394: 485–490

    Article  PubMed  Google Scholar 

  • Chilov D., Camenisch G., Kvietikova I., Ziegler U., Gassmann M., Wenger R.H. (1999) Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha. J. Cell Sci. 112: 1203–1212

    PubMed  Google Scholar 

  • Dang C.V., Semenza G.L. (1999). Oncogenic alterations of metabolism. Trends Biochem. Sci. 24: 68–72

    Article  PubMed  Google Scholar 

  • Dang C.V., Lewis B.C., Dolde C., Dang G., Shim H. (1997) Oncogenes in tumor metabolism, tumorigenesis, and apoptosis. J. Bioenergetics & Biomembranes 29: 345–354

    Article  PubMed  Google Scholar 

  • Danial N.N., Gramm C.F., Scorrano L., Zhang C.Y., Krauss S., Ranger A.M., Datta S.R., Greenberg M.E., Licklider L.J., Lowell B.B., Gygi S.P., Korsmeyer S.J.(2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424: 952–956

    Article  PubMed  Google Scholar 

  • Dignam J.D., Lebovitz R.M., Roeder R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475–1489

    PubMed  Google Scholar 

  • Discher D.J., Bishopric N.H., Wu X., Peterson C.A., Webster K.A. (1998) Hypoxia regulates beta-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element. J. Biol. Chem. 273: 26087–26093

    Article  PubMed  Google Scholar 

  • Downward J. (2003) Metabolism meets cell death. Nature 424:896–897

    Article  PubMed  Google Scholar 

  • Eigenbrodt E., Fister P., Reinacher M. (1985). New Perspectives on Carbohydrate Metabolism in tumor cells. In: Beitner R. (eds) Regulation of Carbohydrate Metabolism. CRC Press, Boca Raton, Florida pp. 142–178

    Google Scholar 

  • Elstrom R.L., Bauer D.E., Buzzai M., Karnauskas R., Harris M.H., Plas D.R., Zhuang H., Cinalli R.M., Alavi A., Rudin C.M., Thompson C.B. (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 64: 3892–389

    Article  PubMed  Google Scholar 

  • Griffiths J.R., McSheehy P.M., Robinson S.P., Troy H., Chung Y.L., Leek R.D., Williams K.J., Stratford I.J., Harris A.L. and Stubbs M. (2002). Metabolic changes detected by in vivo Magnetic Resonace studies of HEPA-1 wild-type and tumors deficient in hypoxia-inducible factor-1β (HIF-1β): evidence of an anabolic role for the HIF-1 pathway. Cancer Res. 62: 688–695

    PubMed  Google Scholar 

  • Harrigan G.C., Goodacre R. (2003) Introduction. In: Harrigan GC, Goodacre R. (eds) Metabolic Profiling, Its role in biomarker discovery and gene function analysis. Kluwer Academic Publishers, Boston, pp. 1–8

    Google Scholar 

  • Harris A.L. (2002). Hypoxia-a key regulatory factor in tumor growth. Nature Rev. Cancer 2: 38–47

    Article  Google Scholar 

  • Isaacs J., Jung Y.J., Lee S., Torres-Cabala C., Merino M., Trepel J., Zbar B., Toro J., Linehan M., Neckers L. (2004) Novel Role of fumarate in antagonizing VHL function. Euro. J. Cancer Suppl. 8: 104

    Article  Google Scholar 

  • Jaakkola P., Mole D.R., Tian Y.M., Wilson M.I., Gielbert J., Gaskell S.J., Kriegsheim A.v., Hebestreit H.F., Mukherji M., Schofield C.J., Maxwell P.H., Pugh C.W. and Ratcliffe P.J. (2001) Targeting of HIF- alpha to the von-Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472

    PubMed  Google Scholar 

  • Leek R.D., Stratford I.J. and Harris A.L. (2005) The Role of Hypoxia-Inducible Factor-1 in Three-dimensional Tumor Growth, Apoptosis, and Regulation by the Insulin-Signaling Pathway. Cancer Res. 65: 4147–4152

    Article  PubMed  Google Scholar 

  • Matteucci E., Modora S., Simone M., Desiderio M.A. (2003) Hepatocyte growth factor induces apoptosis through the extrinsic pathway in hepatoma cells: favouring role of hypoxia-inducible factor-1 deficiency. Oncogene 22: 4062–4073

    Article  PubMed  Google Scholar 

  • Maxwell P.H., Dachs G.U., Gleadle J.M., Nicholls L.G., Harris A.L., Stratford I.J., Hankinson O., Pugh C.W. and Ratcliffe P.J. (1997) Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 94: 8104–8109

    Article  PubMed  Google Scholar 

  • Maxwell P.H., Pugh C.W. and Ratcliffe P.J. (2001) Activation of the HIF pathway in cancer. Curr. Opin. Genetics. Develop. 11: 293–299

    Article  Google Scholar 

  • Mayr M., Siow R., Chung Y.L., Mayr U., Griffiths J.R., Xu Q. (2004) Proteomic and metabolomic analysis of vascular smooth muscle cells: role of PKCdelta. Cir.c Res. 94: 87–96

    Article  Google Scholar 

  • Mazure N.M., Brahimi-Horn M.C., Berta M.A., Benizri E., Bilton R.L., Dayan F., Ginouves A., Berra E., Pouyssegur J. (2004) HIF-1: master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs. Biochem. Pharmacol. 68: 971–980

    Article  PubMed  Google Scholar 

  • Moreno A., Lopez L.A., Fabra A., Arus C. (1998) 1H MRS markers of tumor growth in intrasplenic tumors and liver metastasis induced by injection of HT-29 cells in nude mice spleen. NMR Biomed. 11: 93–106

    Article  PubMed  Google Scholar 

  • Nikiforov M.A., Chandriani S., O’Connell B., Petrenko O., Kotenko I., Beavis A., Sedivy J.M, Cole M.D. (2002) A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism. Mol. Cell. Bio. 22: 5793–5800

    Article  PubMed  Google Scholar 

  • Ratcliffe P.J., O’Rourke J.F., Maxwell P.H., Pugh C.W. (1998) Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. .J. Exper. Bio. 201: 1153–1162

    PubMed  Google Scholar 

  • Seagroves T.N., Ryan H.E., Lu H., Wouters B.G., Knapp M., Thibault P., Laderoute K., and Johnson R.S. (2001) The transcription factor HIF-1 is a necessary mediator of the Pasteur effect in mammalian cells. Mol. Cell. Bio. 21:3436–3444

    Article  PubMed  Google Scholar 

  • Semenza G.L. (2000) Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem. Pharmacol. 59: 47–53

    Article  PubMed  Google Scholar 

  • Semenza G.L. (2001) Hypoxia-inducible-factor 1:oxygen homeostasis and disease pathophysiology. Trends Mol. Med. 7: 345–350

    Article  PubMed  Google Scholar 

  • Semenza G.L., Roth P.H., Fang H,M. and Wang G.L. (1994). Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269: 23757–23763

    PubMed  Google Scholar 

  • Stubbs M., Bashford C.L., Griffiths J.R. (2003) Understanding the tumor metabolic phenotype in the genomic era. Curr. Mol. Med. 3: 49–59

    Article  PubMed  Google Scholar 

  • Troy, H., Chung, Y.L., Stubbs, M., Griffiths, J.R.G., Mayr, M., Ly, L., Stratford, I., Williams, K., Latigo, J., Perumal, M. and Aboagye, E. (2004). Glucose uptake in HIF-1β deficient Hepa-1 tumors and wild type. Abstract ISMRM workshop on Advances in Experimental and Clinical Cancer Research, pp. 163–164

  • Webster KA. (2003) Evolution of the coordinate regulation of glycolytic enzyme genes by hypoxia. J. Exper. Bio. 206: 2911–2922

    Article  PubMed  Google Scholar 

  • Williams K.J., Telfer B.A., Airley RA., Peters J.P.W., Sheridan M.R., van der Kogel A.J., Harris A.L. and Stratford IJ. (2002) A protective role for HIF-1 in response to redox manipulation and glucose deprivation: implications for tumorigensis. Oncogene 21: 282–290

    Article  PubMed  Google Scholar 

  • Wood S.M., Gleadle J.M., Pugh C.W., Hankinson O., Ratcliffe P.J. (1996) The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells. J. Biol. Chem. 271:15117–23

    Article  PubMed  Google Scholar 

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Acknowledgments

Dr. Louisa Brown (Maternal and Foetal Health Research Centre, University of Manchester, St Mary’s Hospital, Manchester, M13 0JH, UK) for the initial development of the dominant negative construct. The Medical Biomics Centre at St George’s Hospital Medical School, London, for the use of their 600 MHz NMR Spectrometer.

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Authors and Affiliations

  1. CR UK Biomedical MR Research Group, Department of Basic Medical Sciences, St. George’s, University of London, London, SW17 ORE, UK

    Helen Troy, Yuen-Li Chung, John Griffiths & Marion Stubbs

  2. Cardiac and Vascular Sciences, St. George’s, University of London, London, SW17 ORE, UK

    Manuel Mayr & Lucy Ly

  3. School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Manchester, M13 9PL, UK

    Kaye Williams & Ian Stratford

  4. Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, UK

    Adrian Harris

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  1. Helen Troy
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  2. Yuen-Li Chung
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  3. Manuel Mayr
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  4. Lucy Ly
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  7. Adrian Harris
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  8. John Griffiths
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  9. Marion Stubbs
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Correspondence to Marion Stubbs.

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This work was supported by CR UK Grant No. C12/A1209 and A1212

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Troy, H., Chung, YL., Mayr, M. et al. Metabolic profiling of hypoxia-inducible factor-1β-deficient and wild type Hepa-1 cells: effects of hypoxia measured by 1H magnetic resonance spectroscopy. Metabolomics 1, 293–303 (2005). https://doi.org/10.1007/s11306-005-0009-8

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  • DOI: https://doi.org/10.1007/s11306-005-0009-8

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