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Glioblastoma pp 111-123 | Cite as

Induction and Assessment of Hypoxia in Glioblastoma Cells In Vitro

  • Jean-Pierre Gagner
  • Mirna Lechpammer
  • David Zagzag
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1741)

Abstract

To simulate and study the hypoxic microenvironment associated with intracerebral glioma in vivo, simple and reproducible methods are described and discussed for inducing hypoxia or chemical pseudohypoxia in glioma cell cultures and assessing their effects on the expression and nuclear translocation of hypoxia-inducible factor (HIF)-1α, a key transcriptional factor of oxygen homeostasis, by Western blot analysis and immunocytochemistry.

Keywords

Glioblastoma Tumor hypoxia Prolyl hydroxylases Hypoxia-inducible factor-1α Hypoxia-regulated genes Glioma cell lines Hypoxia cell culture chamber Chemical pseudohypoxia Western blot analysis Immunocytochemistry 

Notes

Acknowledgments

This work was supported by the NIH/NINDS grant R21-NS074055 (D.Z.). We gratefully acknowledge Dr. Mine Esencay for performing the glioma cell immunofluorescence experiment and Scott Kamen for assisting with the manuscript editing and figure preparation.

References

  1. 1.
    McKeown SR (2014) Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response. Br J Radiol 87(1035):20130676. https://doi.org/10.1259/bjr.20130676 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lechpammer M, Tran YP, Wintermark P, Martinez-Cerdeno V, Krishnan VV, Ahmed W, Berman RF, Jensen FE, Nudler E, Zagzag D (2017) Upregulation of cystathione beta-synthase and p70S6K/S6 in neonatal hypoxic ischemic brain injury. Brain Pathol 27(4):449–458. https://doi.org/10.1111/bpa.12421 CrossRefPubMedGoogle Scholar
  3. 3.
    Hockel M, Vaupel P (2001) Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 93(4):266–276. https://doi.org/10.1093/jnci/93.4.266
  4. 4.
    Vartanian A, Singh SK, Agnihotri S, Jalali S, Burrell K, Aldape KD, Zadeh G (2014) GBM’s multifaceted landscape: highlighting regional and microenvironmental heterogeneity. Neuro-Oncology 16(9):1167–1175. https://doi.org/10.1093/neuonc/nou035 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gagner JP, Golfinos JG, Graber JJ, Zagzag D (2011) Molecular basis of glioma neovascularization and its therapeutic applications. In: Mehta M, Chang SM, Newton H, Guha A, Vogelbaum M (eds) Principles and practice of neuro-oncology: a multidisciplinary approach. Demos Medical Publishing, New York, NY, pp 122–144. i005–i007Google Scholar
  6. 6.
    Dings J, Meixensberger J, Jager A, Roosen K (1998) Clinical experience with 118 brain tissue oxygen partial pressure catheter probes. Neurosurgery 43(5):1082–1095CrossRefPubMedGoogle Scholar
  7. 7.
    Evans SM, Judy KD, Dunphy I, Jenkins WT, Nelson PT, Collins R, Wileyto EP, Jenkins K, Hahn SM, Stevens CW, Judkins AR, Phillips P, Geoerger B, Koch CJ (2004) Comparative measurements of hypoxia in human brain tumors using needle electrodes and EF5 binding. Cancer Res 64(5):1886–1892. https://doi.org/10.1158/0008-5472.CAN-03-2424
  8. 8.
    Lally BE, Rockwell S, Fischer DB, Collingridge DR, Piepmeier JM, Knisely JP (2006) The interactions of polarographic measurements of oxygen tension and histological grade in human glioma. Cancer J (Sudbury, MA) 12(6):461–466CrossRefGoogle Scholar
  9. 9.
    Koh MY, Powis G (2012) Passing the baton: the HIF switch. Trends Biochem Sci 37(9):364–372. https://doi.org/10.1016/j.tibs.2012.06.004 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kondo K, Kim WY, Lechpammer M, Kaelin WG Jr (2003) Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biol 1(3):E83. https://doi.org/10.1371/journal.pbio.0000083 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Semenza GL (2013) HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations. J Clin Invest 123(9):3664–3671. https://doi.org/10.1172/jci67230 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Koivunen P, Hirsila M, Kivirikko KI, Myllyharju J (2006) The length of peptide substrates has a marked effect on hydroxylation by the hypoxia-inducible factor prolyl 4-hydroxylases. J Biol Chem 281(39):28712–28720. https://doi.org/10.1074/jbc.M604628200 CrossRefPubMedGoogle Scholar
  13. 13.
    Fong GH, Takeda K (2008) Role and regulation of prolyl hydroxylase domain proteins. Cell Death Differ 15(4):635–641. https://doi.org/10.1038/cdd.2008.10 CrossRefPubMedGoogle Scholar
  14. 14.
    Zagzag D, Zhong H, Scalzitti JM, Laughner E, Simons JW, Semenza GL (2000) Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression. Cancer 88(11):2606–2618. https://doi.org/10.1002/1097- 0142(20000601)88:11<2606::AID-CNCR25>3.0.CO;2-WGoogle Scholar
  15. 15.
    Strickland M, Stoll EA (2017) Metabolic reprogramming in glioma. Front Cell Dev Biol 5:43. https://doi.org/10.3389/fcell.2017.00043 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nakazawa MS, Keith B, Simon MC (2016) Oxygen availability and metabolic adaptations. Nat Rev Cancer 16(10):663–673. https://doi.org/10.1038/nrc.2016.84 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Salnikow K, Donald SP, Bruick RK, Zhitkovich A, Phang JM, Kasprzak KS (2004) Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress. J Biol Chem 279(39):40337–40344. https://doi.org/10.1074/jbc.M403057200 CrossRefPubMedGoogle Scholar
  18. 18.
    Wang GL, Semenza GL (1993) Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82(12):3610–3615PubMedGoogle Scholar
  19. 19.
    Yuan Y, Hilliard G, Ferguson T, Millhorn DE (2003) Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278(18):15911–15916. https://doi.org/10.1074/jbc.M300463200 CrossRefPubMedGoogle Scholar
  20. 20.
    Wu D, Yotnda P (2011) Induction and testing of hypoxia in cell culture. J Vis Exp 54. https://doi.org/10.3791/2899
  21. 21.
    Byrne MB, Leslie MT, Gaskins HR, Kenis PJ (2014) Methods to study the tumor microenvironment under controlled oxygen conditions. Trends Biotechnol 32(11):556–563. https://doi.org/10.1016/j.tibtech.2014.09.006 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Mendez O, Yee H, Voura EB, Newcomb EW (2006) Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion. Lab Invest 86(12):1221–1232. https://doi.org/10.1038/labinvest.3700482 CrossRefPubMedGoogle Scholar
  23. 23.
    Zagzag D, Esencay M, Mendez O, Yee H, Smirnova I, Huang Y, Chiriboga L, Lukyanov E, Liu M, Newcomb EW (2008) Hypoxia- and vascular endothelial growth factor-induced stromal cell-derived factor-1alpha/CXCR4 expression in glioblastomas: one plausible explanation of Scherer's structures. Am J Pathol 173(2):545–560. https://doi.org/10.2353/ajpath.2008.071197 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zagzag D, Nomura M, Friedlander DR, Blanco CY, Gagner JP, Nomura N, Newcomb EW (2003) Geldanamycin inhibits migration of glioma cells in vitro: a potential role for hypoxia-inducible factor (HIF-1alpha) in glioma cell invasion. J Cell Physiol 196(2):394–402. https://doi.org/10.1002/jcp.10306 CrossRefPubMedGoogle Scholar
  25. 25.
    Moroz E, Carlin S, Dyomina K, Burke S, Thaler HT, Blasberg R, Serganova I (2009) Real-time imaging of HIF-1alpha stabilization and degradation. PLoS One 4(4):e5077. https://doi.org/10.1371/journal.pone.0005077 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Allen M, Bjerke M, Edlund H, Nelander S, Westermark B (2016) Origin of the U87MG glioma cell line: good news and bad news. Sci Transl Med 8(354):354re353. https://doi.org/10.1126/scitranslmed.aaf6853 CrossRefGoogle Scholar
  27. 27.
    Timerman D, Yeung CM (2014) Identity confusion of glioma cell lines. Gene 536(1):221–222. https://doi.org/10.1016/j.gene.2013.11.096 CrossRefPubMedGoogle Scholar
  28. 28.
    Stepanenko AA, Kavsan VM (2014) Karyotypically distinct U251, U373, and SNB19 glioma cell lines are of the same origin but have different drug treatment sensitivities. Gene 540(2):263–265. https://doi.org/10.1016/j.gene.2014.02.053 CrossRefPubMedGoogle Scholar
  29. 29.
  30. 30.
    Rosenberg S, Verreault M, Schmitt C, Guegan J, Guehennec J, Levasseur C, Marie Y, Bielle F, Mokhtari K, Hoang-Xuan K, Ligon K, Sanson M, Delattre JY, Idbaih A (2017) Multi-omics analysis of primary glioblastoma cell lines shows recapitulation of pivotal molecular features of parental tumors. Neuro-Oncology 19(2):219–228. https://doi.org/10.1093/neuonc/now160 PubMedGoogle Scholar
  31. 31.
    Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M, Squire JA, Smith A, Dirks P (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4(6):568–580. https://doi.org/10.1016/j.stem.2009.03.014 CrossRefPubMedGoogle Scholar
  32. 32.
    Mathupala SP, Kiousis S, Szerlip NJ (2016) A lab assembled microcontroller-based sensor module for continuous oxygen measurement in portable hypoxia chambers. PLoS One 11(2):e0148923. https://doi.org/10.1371/journal.pone.0148923 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Wang R, Jin F, Zhong H (2014) A novel experimental hypoxia chamber for cell culture. Am J Cancer Res 4(1):53–60PubMedPubMedCentralGoogle Scholar
  34. 34.
    Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C (2011) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 15(6):1239–1253. https://doi.org/10.1111/j.1582-4934.2011.01258.x CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Dimova EY, Kietzmann T (2010) Hypoxia-inducible factors: post-translational crosstalk of signaling pathways. Methods Mol Biol (Clifton, NJ) 647:215–236. https://doi.org/10.1007/978-1-60761-738-9_13 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Jean-Pierre Gagner
    • 1
    • 2
  • Mirna Lechpammer
    • 3
  • David Zagzag
    • 1
    • 2
    • 4
    • 5
  1. 1.Microvascular and Molecular Neuro-Oncology Laboratory, Department of PathologyNYU Langone Medical CenterNew YorkUSA
  2. 2.Department of PathologyNYU Langone Medical CenterNew YorkUSA
  3. 3.Department of Pathology and Laboratory Medicine, Division of Neuropathology, Medical CenterUniversity of California, DavisSacramentoUSA
  4. 4.Division of Neuropathology, Department of NeurosurgeryNYU Langone Medical CenterNew YorkUSA
  5. 5.NYU Langone Laura and Isaac Perlmutter Cancer CenterNew YorkUSA

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