Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Burnstock G (2004) Introduction: P2. Curr Top Med Chem 4:793–803
Chan K, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2+-ATPase activity. Anal Biochem 157:375–380
Czajkowski R, Banachewicz W, Unytska O, Drobot LB, Baranska J (2004) Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidilinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells. Br J Pharmacol 141:497–507
Deininger MH, Seid K, Engel S, Meyermann R, Schuesener HJ (2000) Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas. Acta Neurophatol 100:673–680
Di Virgilio F (2000) Dr. Jekyll/Mr. Hyde: the dual role of extracellular ATP. J Auton Nerv Syst 81:59–63
Fields RD, Burnstock G (2006) Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci 7:423–436
Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021
Ghods AJ, Irvin D, Liu G, Yuan X, Abdulkadir IR, Tunici P, Konda B, Wachsmann-Hogiu S, Black KL, Yu JS (2007) Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells. Stem Cells 25:1645–1653
Giard DJ, Aaronson SA, Todaro GJ (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51:1417–1423
Giese A, Bjerkvig R, Berens ME, Westphal M (2003) Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol 21:1624–1636
Guo L, Trautmann K, Schluesener HJ (2004) Expression of P2X4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia. J Neuroimmunology 152:67–72
Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W, Julius D (2006) The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 9:1512–1519
Holland EC (2001) Gliomagenesis: genetic alterations and mouse models. Nat Rev Genet 2:120–129
Jain RK, Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT (2007) Angiogenesis in brain tumours. Nat Rev Neurosci 8:610–622
Khakh B, Kennedy C (1998) Adenosine and ATP: progress in their receptors structures and functions. Trends Pharmacol Sci 19:39–41
Kim SG, Gao ZG, Soltysiak KA, Chang TS, Brodie C, Jacobson KA (2003) P2Y6 nucleotide receptor activates PKC to protect 1321N1 astrocytoma cells against tumor necrosis factor-induced apoptosis. Cell Mol Neurobiol 23:401–418
Korkov J, Raimundo LN, Du X, Sims SM, Dixon SJ (2005) P2Y6 nucleotide receptors activate NF-kappaB and increase survival of osteoclasts. J Biol Chem 280:16909–16915
Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, Park JK, Fine HA (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391–403
Lemoli RM, Ferrari D, Fogli M, Rossi L, Pizzirani C, Forchap S, Chiozzi P, Vaselli D, Bertoli F, Foutz T, Aluigi M, Baccarani M, Di Virgilio F (2004) Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo. Blood 104:1662–1670
Lendahlet U, Zimmerman LB, McKay RDG (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585–595
Lenz G, Gottfried C, Luo Z, Avruch J, Rodnight R, Nie WJ, Kang Y, Neary JT (2000) P2Y purinergic receptor subunits recruit different Mek activators in astrocytes. Br J Pharmacol 129:927–936
Morrone FB, Jacques-Silva MC, Horn AP, Bernardi A, Schwartsmann G, Rodnight R, Lenz G (2003) Extracellular nucleotides and nucleosides induce proliferation and increase nucleoside transport in human glioma cell line. J Neurooncol 64:211–218
Morrone FB, Horn AP, Stella J, Spiller F, Sarkis JJF, Salbego C, Lenz G, Battastini AMO (2005) Increased resistence of glioma cell lines to extracellular ATP cytotoxicity. J Neurooncol 71:135–140
Morrone FB, Oliveira DL, Gamermann P, Stella J, Wofchuk S, Wink MR, Meurer L, Edelweiss MIA, Lenz G, Battastini AMO (2006) In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model. BMC Cancer 23:226
Neary JT, Kang Y, Willoughby KA, Ellis EF (2003) Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. J Neurosci 23:2348–2356
Plesner L (1995) Ecto-ATPases: identities and functions. Int Rev Cytol 158:141–214
Ponten J, Macintyre EH (1968) Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:465–486
Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Rathbone MP, Middlemiss PJ, Gysbers JW, Andrew A, Herman M, Reed JK, Ciccarelli R, Di Iorio P, Caciagli F (1999) Trophic effects of purines in neurons and glial cells. Prog Neurobiol 59:663–690
Robson SC, Sévigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationship and pathophysiological significance. Purinergic Signalling 2:409–430
Sanai N, Alvarez-Buylla A, Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353:811–822
Sak K, Illes P (2005) Neuronal and glial cell lines as model systems for studying P2Y receptor pharmacology. Neurochem Int 47:401–412
Stupp R, Mason WP, Bent MJ van den, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996
Takano T, Lin JHC, Arcuino G, Gao Q, Yang J, Nedergaard M (2001) Glutamate release promotes growth of malignant gliomas. Nat Med 7:1010–1015
Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, Farkas DL, Black KL, Yu JS (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23:9392–9400
White N, Burnstock G (2006) P2 receptors and cancer. Trends Pharmacol Sci 27:211–217
White N, Ryten M, Clayton E, Butler P, Burnstock G (2005) P2Y purinergic receptors regulate the growth of human melanomas. Cancer Lett 224:81–91
Wink MR, Lenz G, Braganhol E, Tamajusuku ASK, Schwartsmann G, Sarkis JJF, Battastini AMO (2003) Altered extracellular ATP, ADP and AMP catabolism in glioma cell lines. Cancer Lett 198:211–218
Zimmermann H (2001) Ectonucleotidases: some recent developments and a note on nomenclature. Drug Dev Res 52:44–56
We are grateful to N. Copetti, J.A. Lopes and L.R. Blazina for their excellent technical assistance and to F. Bahrami for comments on and corrections to the manuscript.
This study was supported by grants from the Brazilian agencies CNPq, FAPERGS and Fundo de Incentivo à Pesquisa e Eventos (HCPA). E. Braganhol and D. Huppes were recipients of Brazilian CNPq fellowships; A. Bernardi was the recipient of a CAPES fellowship.
About this article
Cite this article
Braganhol, E., Huppes, D., Bernardi, A. et al. A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 ex vivo glioma model. Cell Tissue Res 335, 331–340 (2009). https://doi.org/10.1007/s00441-008-0723-4
- C6 glioma
- Purinergic receptors
- Cell line culture (Rat)
- Glioma ex vivo culture (Rat)