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Identification of hypoxia-induced genes in a malignant glioma cell line (U-251) by cDNA microarray analysis

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Abstract

Overcoming the metabolic restrictions of hypoxia may allow the progression of lower-grade tumors to glioblastoma multiforme. Our findings of up-regulation of HIF-1α and its downstream targets VEGF, GLUT-1, and CAIX in higher-grade gliomas support this hypothesis. We compared the gene expression profiles of the U-251 malignant glioma cell line under normoxic and hypoxic conditions to discover future research targets. U-251 cells were grown to 75% confluence and exposed to either normoxic or hypoxic conditions for 24 h. RNA was extracted, amplified, and hybridized to a cDNA microarray chip containing ~8,800 universal cellular genes. A threefold increase in mRNA expression was used as a threshold value for differential expression. Identified genes were divided into cell cycle control, stress response, and “newly connected” genes. Hybridization identified 11 hypoxia-induced genes: 1 involved with cell cycle control (CCNG2), 6 in stress response (IGFBP3, SLC2A3, GSTT2, FOS, DDIT3, AKR1C3), and 2 newly connected genes (Depp, AKAP4). One stress-related gene (AKR1C3) encodes for an enzyme that synthesizes progesterone. Of newly connected genes, the gene decidual protein induced by progesterone (Depp) showed the highest expression (4.2-fold increase). Possible future targeting for “hypoxic” glioma cells includes the targets for the AP-1 transcription factor complex (FOS), as well as blockade of the enzyme AKR1C3 with nonsteroidal anti-inflammatory drugs. Possible functions of the highly expressed gene Depp include tumor vascularization. Future studies will focus on the hypothesis that Depp is up-regulated in an autocrine fashion by the AKR1C3 enzyme in U-251 glioma cells under hypoxic conditions.

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References

  1. Bates S, Rowan S, Vousden KH (1996) Characterisation of human cyclin G1 and G2: DNA damage inducible genes. Oncogene 13:1103–1109

    PubMed  CAS  Google Scholar 

  2. Berkman RA, Merrill MJ, Reinhold WC, Monacci WT, Saxena A, Clark WC, Robertson JT, Ali IU, Oldfield EH (1993) Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms. J Clin Invest 91:153–159

    Article  PubMed  CAS  Google Scholar 

  3. Bi M, Naczki C, Koritzinsky M, Fels D, Blais J, Hu N, Harding H, Novoa I, Varia M, Raleigh J, Scheuner D, Kaufman RJ, Bell J, Ron D, Wouters BG, Koumenis C (2005) ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth. EMBO J 24:3470–3481

    Article  PubMed  CAS  Google Scholar 

  4. Boado RJ, Black KL, Pardridge WM (1994) Gene expression of GLUT3 and GLUT1 glucose transporters in human brain tumors. Brain Res Mol Brain Res 27:51–57

    Article  PubMed  CAS  Google Scholar 

  5. Budanov AV, Shoshani T, Faerman A, Zelin E, Kamer I, Kalinski H, Gorodin S, Fishman A, Chajut A, Einat P, Skaliter R, Gudkov AV, Chumakov PM, Feinstein E (2002) Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability. Oncogene 21:6017–6031

    Article  PubMed  CAS  Google Scholar 

  6. Burger AM, Leyland-Jones B, Banerjee K, Spyropoulos DD, Seth AK (2005) Essential roles of IGFBP-3 and IGFBP-rP1 in breast cancer. Eur J Cancer 41:1515–1527

    Article  PubMed  CAS  Google Scholar 

  7. Carriere A, Carmona MC, Fernandez Y, Rigoulet M, Wenger RH, Penicaud L, Casteilla L (2004) Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect. J Biol Chem 279:40462–40469

    Article  PubMed  CAS  Google Scholar 

  8. Cavenee WK, Furnari FB, Nagane M, Huang H-JS, Newcomb EW, Bigner DD, Weller M, Berens ME, Plate KH, Israel MA, Nobel MD, Kleihues P (2000) Diffusely infiltrating astrocytomas. In: Kleihues P, Cavenee WK (eds) World Health Organization classification of tumours: pathology and genetics: tumours of the nervous system. Lyon, IARC Press, pp 10–21

    Google Scholar 

  9. Chan AS, Leung SY, Wong MP, Yuen ST, Cheung N, Fan YW, Chung LP (1998) Expression of vascular endothelial growth factor and its receptors in the anaplastic progression of astrocytoma, oligodendroglioma, and ependymoma. Am J Surg Pathol 22:816–826

    Article  PubMed  CAS  Google Scholar 

  10. Ciaccio PJ, Shen H, Jaiswal AK, Lyttle MH, Tew KD (1995) Modulation of detoxification gene expression in human colon HT29 cells by glutathione-S-transferase inhibitors. Mol Pharmacol 48:639–647

    PubMed  CAS  Google Scholar 

  11. Davare MA, Dong F, Rubin CS, Hell JW (1999) The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons. J Biol Chem 274:30280–30287

    Article  PubMed  CAS  Google Scholar 

  12. Deal C, Ma J, Wilkin F, Paquette J, Rozen F, Ge B, Hudson T, Stampfer M, Pollak M (2001) Novel promoter polymorphism in insulin-like growth factor-binding protein-3: correlation with serum levels and interaction with known regulators. J Clin Endocrinol Metab 86:1274–1280

    Article  PubMed  CAS  Google Scholar 

  13. Eddy EM, Toshimori K, O’Brien DA (2003) Fibrous sheath of mammalian spermatozoa. Microsc Res Tech 61:103–115

    Article  PubMed  CAS  Google Scholar 

  14. Elmlinger MW, Deininger MH, Schuett BS, Meyermann R, Duffner F, Grote EH, Ranke MB (2001) In vivo expression of insulin-like growth factor-binding protein-2 in human gliomas increases with the tumor grade. Endocrinology 142:1652–1658

    Article  PubMed  CAS  Google Scholar 

  15. Ferry RJ Jr, Cerri RW, Cohen P (1999) Insulin-like growth factor binding proteins: new proteins, new functions. Horm Res 51:53–67

    Article  PubMed  CAS  Google Scholar 

  16. Fujimoto M, Sheridan PJ, Sharp ZD, Weaker FJ, Kagan-Hallet S, Story JL (1989) Proto-oncogene analyses in brain tumors. J Neurosurg 70:910–915

    PubMed  CAS  Google Scholar 

  17. Fujimoto M, Weaker FJ, Herbert DC, Sharp ZD, Sheridan PJ, Story JL (1988) Expression of three viral oncogenes (v-sis, v-myc, v-fos) in primary human brain tumors of neuroectodermal origin. Neurology 38:289–293

    PubMed  CAS  Google Scholar 

  18. Fung KM, Samara EN, Wong C, Metwalli A, Krlin R, Bane B, Liu CZ, Yang JT, Pitha JV, Culkin DJ, Kropp BP, Penning TM, Lin HK (2006) Increased expression of type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) and its relationship with androgen receptor in prostate carcinoma. Endocr Relat Cancer 13:169–180

    Article  PubMed  CAS  Google Scholar 

  19. Gonzalez-Aguero G, Ondarza R, Gamboa-Dominguez A, Cerbon MA, Camacho-Arroyo I (2001) Progesterone receptor isoforms expression pattern in human astrocytomas. Brain Res Bull 56:43–48

    Article  PubMed  CAS  Google Scholar 

  20. Inoue T, Akahira J, Suzuki T, Darnel AD, Kaneko C, Takahashi K, Hatori M, Shirane R, Kumabe T, Kurokawa Y, Satomi S, Sasano H (2002) Progesterone production and actions in the human central nervous system and neurogenic tumors. J Clin Endocrinol Metab 87:5325–5331

    Article  PubMed  CAS  Google Scholar 

  21. Jensen RL (2006) Hypoxia in the tumorigenesis of gliomas and as a potential target for therapeutic measures. Neurosurg Focus 20(4):E24

    Article  PubMed  Google Scholar 

  22. Jensen RL, Ragel BT, Whang K, Gillespie D (2006) Inhibition of hypoxia inducible factor-1α (HIF-1α) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas. J Neurooncol 78:233–247

    Article  PubMed  CAS  Google Scholar 

  23. Kaur B, Khwaja FW, Severson EA, Matheny SL, Brat DJ, Van Meir EG (2005) Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis. Neuro-oncol 7:134–153

    Article  PubMed  CAS  Google Scholar 

  24. Khanna M, Qin KN, Wang RW, Cheng KC (1995) Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3 α-hydroxysteroid dehydrogenases. J Biol Chem 270:20162–20168

    Article  PubMed  CAS  Google Scholar 

  25. Koch M, Korf HW (2002) Distribution of regulatory subunits of protein kinase A and A kinase anchor proteins (AKAP 95, 150) in rat pinealocytes. Cell Tissue Res 310:331–338

    Article  PubMed  CAS  Google Scholar 

  26. Landi S (2000) Mammalian class theta GST and differential susceptibility to carcinogens: a review. Mutat Res 463:247–283

    Article  PubMed  CAS  Google Scholar 

  27. Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R, Nagata K, Harding HP, Ron D (2004) CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev 18:3066–3077

    Article  PubMed  CAS  Google Scholar 

  28. Martinez-Gac L, Marques M, Garcia Z, Campanero MR, Carrera AC (2004) Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead. Mol Cell Biol 24:2181–2189

    Article  PubMed  CAS  Google Scholar 

  29. Matsuura K, Shiraishi H, Hara A, Sato K, Deyashiki Y, Ninomiya M, Sakai S (1998) Identification of a principal mRNA species for human 3 α-hydroxysteroid dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D2 11-ketoreductase activity. J Biochem (Tokyo) 124:940–946

    CAS  Google Scholar 

  30. Mechaly I, Bourane S, Piquemal D, Al-Jumaily M, Venteo S, Puech S, Scamps F, Valmier J, Carroll P (2006) Gene profiling during development and after a peripheral nerve traumatism reveals genes specifically induced by injury in dorsal root ganglia. Mol Cell Neurosci 32:217–229

    Article  PubMed  CAS  Google Scholar 

  31. Nagamatsu S, Sawa H, Wakizaka A, Hoshino T (1993) Expression of facilitative glucose transporter isoforms in human brain tumors. J Neurochem 61:2048–2053

    Article  PubMed  CAS  Google Scholar 

  32. Nishioka T, Oda Y, Seino Y, Yamamoto T, Inagaki N, Yano H, Imura H, Shigemoto R, Kikuchi H (1992) Distribution of the glucose transporters in human brain tumors. Cancer Res 52:3972–3979

    PubMed  CAS  Google Scholar 

  33. Ozanne BW, Spence HJ, McGarry LC, Hennigan RF (2006) Invasion is a genetic program regulated by transcription factors. Curr Opin Genet Dev 16:65–70

    Article  PubMed  CAS  Google Scholar 

  34. Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H, Moore M, Palackal N, Ratnam K (2000) Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem J 351:67–77

    Article  PubMed  CAS  Google Scholar 

  35. Shin D, Anderson DJ (2005) Isolation of arterial-specific genes by subtractive hybridization reveals molecular heterogeneity among arterial endothelial cells. Dev Dyn 233:1589–1604

    Article  PubMed  CAS  Google Scholar 

  36. Shweiki D, Itin A, Soffer D, Keshet E (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359:843–845

    Article  PubMed  CAS  Google Scholar 

  37. Sik A, Gulacsi A, Lai Y, Doyle WK, Pacia S, Mody I, Freund TF (2000) Localization of the A kinase anchoring protein AKAP79 in the human hippocampus. Eur J Neurosci 12:1155–1164

    Article  PubMed  CAS  Google Scholar 

  38. Su Y, Balice-Gordon RJ, Hess DM, Landsman DS, Minarcik J, Golden J, Hurwitz I, Liebhaber SA, Cooke NE (2004) Neurobeachin is essential for neuromuscular synaptic transmission. J Neurosci 24:3627–3636

    Article  PubMed  CAS  Google Scholar 

  39. Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, Ushio Y, Mori M (2004) Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ 11:403–415

    Article  PubMed  CAS  Google Scholar 

  40. Tajiri S, Yano S, Morioka M, Kuratsu J, Mori M, Gotoh T (2006) CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation. FEBS Lett 580:3462–3468

    Article  PubMed  CAS  Google Scholar 

  41. Watanabe H, Nonoguchi K, Sakurai T, Masuda T, Itoh K, Fujita J (2005) A novel protein Depp, which is induced by progesterone in human endometrial stromal cells activates Elk-1 transcription factor. Mol Hum Reprod 11:471–476

    Article  PubMed  CAS  Google Scholar 

  42. Zhang JZ, Behrooz A, Ismail-Beigi F (1999) Regulation of glucose transport by hypoxia. Am J Kidney Dis 34:189–202

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Kristin Kraus, M.S., for her editorial assistance in preparing this manuscript, as well as Brett Milash, M.S., for his expertise in analyzing microarray data.

This work was supported in part by a grant from the American Association of Neurological Surgeons Neurosurgery Research and Education Foundation to Brian Ragel.

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  1. Department of Neurosurgery, University of Utah, 175 N. Medical Drive East, Salt Lake City, UT, 84132, USA

    Brian T. Ragel, William T. Couldwell, David L. Gillespie & Randy L. Jensen

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  1. Brian T. Ragel
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  2. William T. Couldwell
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  3. David L. Gillespie
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  4. Randy L. Jensen
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Correspondence to Randy L. Jensen.

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Comments

Hidetoshi Kasuya, Tokyo

In this article the authors used hypoxic conditions in a commercially available malignant glioma cell line (U-251) and found 11 up-regulated genes by cDNA microarray analysis. They detected the up-regulated genes that are more than threefold differentially expressed by comparing a set of normoxic and hypoxic samples.

For future studies we are hoping for confirmation of these expression results by RT-PCR and also by checking the protein amount through Western blot. Furthermore, it would also be interesting to know whether these differentially expressed genes could be found in other cell lines under the same conditions.

Ultimately, gene expression results of the diverse clinical samples of glioblastoma multiforme and various other forms of glioma are the ones that may also play a crucial role in finding new therapeutic approaches.

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Ragel, B.T., Couldwell, W.T., Gillespie, D.L. et al. Identification of hypoxia-induced genes in a malignant glioma cell line (U-251) by cDNA microarray analysis. Neurosurg Rev 30, 181–187 (2007). https://doi.org/10.1007/s10143-007-0070-z

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  • DOI: https://doi.org/10.1007/s10143-007-0070-z

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