Advertisement

Purinergic Signalling

, Volume 8, Issue 4, pp 669–676 | Cite as

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

  • Safina Gadeock
  • Aleta Pupovac
  • Vanessa Sluyter
  • Mari Spildrejorde
  • Ronald SluyterEmail author
Brief Communication

Abstract

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium+ uptake into KG-l cells suspended in sucrose medium (EC50 of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium+ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC50 of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC50 of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium+ uptake into KG-1 cells. ATP-induced ethidium+ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium+ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-12+ and propidium2+ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.

Keywords

Purinergic receptor Extracellular ATP Acute myelogenous leukaemia Cation channel Single nucleotide polymorphism 

Notes

Acknowledgments

This work was kindly supported by Cure Cancer Australia and the University of Wollongong. We gratefully acknowledged helpful advice from Marie Ranson, Mark Dowton and Simon Cook (all University of Wollongong), and excellent technical assistance by Margaret Phillips (University of Wollongong) and the staff of the Illawarra Health and Medical Research Institute. The authors have no conflicts of interest.

References

  1. 1.
    Wiley JS, Sluyter R, Gu BJ, Stokes L, Fuller SJ (2011) The human P2X7 receptor and its role in innate immunity. Tissue Antigens 78:321–332PubMedCrossRefGoogle Scholar
  2. 2.
    Lenertz LY, Gavala ML, Zhu Y, Bertics PJ (2011) Transcriptional control mechanisms associated with the nucleotide receptor P2X7, a critical regulator of immunologic, osteogenic, and neurologic functions. Immunol Res 50:22–38PubMedCrossRefGoogle Scholar
  3. 3.
    Sluyter R, Stokes L (2011) Significance of P2X7 receptor variants to human health and disease. Recent Pat DNA Gene Seq 5:41–54PubMedGoogle Scholar
  4. 4.
    Gorodeski GI (2009) P2X7-mediated chemoprevention of epithelial cancers. Expert Opin Ther Targets 13:1313–1332PubMedCrossRefGoogle Scholar
  5. 5.
    Roger S, Pelegrin P (2011) P2X7 receptor antagonism in the treatment of cancers. Expert Opin Investig Drugs 20:875–880PubMedCrossRefGoogle Scholar
  6. 6.
    Feng YH, Li X, Zeng R, Gorodeski GI (2006) Endogenously expressed truncated P2X7 receptor lacking the C-terminus is preferentially upregulated in epithelial cancer cells and fails to mediate ligand-induced pore formation and apoptosis. Nucleosides Nucleotides Nucleic Acids 25:1271–1276PubMedCrossRefGoogle Scholar
  7. 7.
    Di Virgilio F, Ferrari D, Adinolfi E (2009) P2X7: a growth-promoting receptor—implications for cancer. Purinergic Sig 5:251–256CrossRefGoogle Scholar
  8. 8.
    Adinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, Bianchi G, Kroemer G, Pistoia V, Di Virgilio F (2012) Expression of the P2X7 receptor increases in vivo tumor growth. Cancer Res. doi: 10.1158/0008-5472.CAN-11-1947
  9. 9.
    Ren S, Zhang Y, Wang Y, Lui Y, Wei W, Huang X, Mao W, Zuo Y (2010) Targeting P2X7 receptor inhibits the metastasis of murine P388D1 lymphoid neoplasm cells to lymph nodes. Cell Biol Int 34:1205–1211PubMedCrossRefGoogle Scholar
  10. 10.
    Gu BJ, Wiley JS (2006) Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor. Blood 107:4946–4953PubMedCrossRefGoogle Scholar
  11. 11.
    Jelassi B, Chantôme A, Alcaraz-Pérez F, Baroja-Mazo A, Cayuela ML, Pelegrin P, Surprenant A, Roger S (2011) P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness. Oncogene 30:2108–2122PubMedCrossRefGoogle Scholar
  12. 12.
    Jamieson GP, Snook MB, Thurlow PJ, Wiley JS (1996) Extracellular ATP causes of loss of L-selectin from human lymphocytes via occupancy of P2Z purinocepters. J Cell Physiol 166:637–642PubMedCrossRefGoogle Scholar
  13. 13.
    Lin C, Ren S, Zhang L, Jin H, Sun J, Zuo Y (2011) Extracellular ATP induces CD44 shedding from macrophage-like P388D1 cells via the P2X7 receptor. Hematol Oncol. doi: 10.1002/hon.1008
  14. 14.
    Farrell AW, Gadeock S, Pupovac A, Wang B, Jalilian I, Ranson M, Sluyter R (2010) P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells. Biochim Biophys Acta 1800:1173–1182PubMedCrossRefGoogle Scholar
  15. 15.
    Deli T, Csernoch L (2008) Extracellular ATP and cancer—an overview with special reference to P2 purinergic receptors. Pathol Oncol Res 14:219–231PubMedCrossRefGoogle Scholar
  16. 16.
    White N, Burnstock G (2006) P2 receptors and cancer. Trends Pharmacol Sci 27:211–217PubMedCrossRefGoogle Scholar
  17. 17.
    Koeffler HP, Golde DW (1978) Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science 200:1153–1154PubMedCrossRefGoogle Scholar
  18. 18.
    Gadeock S, Tran JN, Georgiou JG, Jalilian I, Taylor RM, Wiley JS, Sluyter R (2010) TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes. Biochim Biophys Acta 1798:2058–2066PubMedCrossRefGoogle Scholar
  19. 19.
    Michel AD, Chessell IP, Humphrey PP (1999) Ionic effects on human recombinant P2X7 receptor function. Naunyn Schmiedebergs Arch Pharmacol 359:102–109PubMedCrossRefGoogle Scholar
  20. 20.
    Jursik C, Sluyter R, Georgiou JG, Fuller SJ, Wiley JS, Gu BJ (2007) A quantitative method for routine measurement of cell surface P2X7 receptor function in leucocyte subsets by two-colour time-resolved flow cytometry. J Immunol Methods 325:67–77PubMedCrossRefGoogle Scholar
  21. 21.
    Pelegrin P, Surprenant A (2006) Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J 25:5071–5082PubMedCrossRefGoogle Scholar
  22. 22.
    Silverman WR, de Rivero Vaccari JP, Locovei S, Qiu F, Carlsson SK, Scemes E, Keane RW, Dahl G (2009) The pannexin 1 channel activates the inflammasome in neurons and astrocytes. J Biol Chem 284:18143–18151PubMedCrossRefGoogle Scholar
  23. 23.
    Shemon AN, Sluyter R, Fernando SL, Clarke AL, Dao-Ung LP, Skarratt KK, Saunders BM, Tan KS, Gu BJ, Fuller SJ, Britton WJ, Petrou S, Wiley JS (2006) A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and function impairs ATP-induced mycobacterial killing by macrophages. J Biol Chem 281:2079–2086PubMedCrossRefGoogle Scholar
  24. 24.
    Cabrini G, Falzoni S, Forchap SL, Pellegatti P, Balboni A, Agostini P, Cuneo A, Castoldi G, Baricordi OR, Di Virgilio F (2005) A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes. J Immunol 175:82–89PubMedGoogle Scholar
  25. 25.
    Roger S, Mei ZZ, Baldwin JM, Dong L, Bradley H, Baldwin SA, Surprenant A, Jiang LH (2010) Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions. J Psychiatr Res 44:347–355PubMedCrossRefGoogle Scholar
  26. 26.
    Stokes L, Fuller SJ, Sluyter R, Skarratt KK, Gu BJ, Wiley JS (2010) Two haplotypes of the P2X7 receptor containing the Ala-348 to Thr polymorphism exhibit gain-of-function effect and enhanced interleukin-1β secretion. FASEB J 24:2916–2927PubMedCrossRefGoogle Scholar
  27. 27.
    Constantinescu P, Wang B, Kovacevic K, Jalilian I, Bosman GJ, Wiley JS, Sluyter R (2010) P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells. Biochim Biophys Acta 1798:1797–1804PubMedCrossRefGoogle Scholar
  28. 28.
    Smart ML, Gu B, Panchel RG, Wiley JS, Cromer B, Williams DA, Petrou S (2003) P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region. J Biol Chem 278:8853–8860PubMedCrossRefGoogle Scholar
  29. 29.
    Hattori M, Gouaux E (2012) Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485:207–212PubMedCrossRefGoogle Scholar
  30. 30.
    Alqallaf SM, Evans BAJ, Kidd EJ (2009) Atypical P2X7 receptor pharmacology in two human ostoblast-like cell lines. Br J Pharmacol 156:1124–1135PubMedCrossRefGoogle Scholar
  31. 31.
    Pojoga LH, Haghiac ML, Moose JE, Hilderman RH (2004) Determination of ATP impurity in adenine dinucleotides. Nucleosides Nucleotides Nucleic Acids 23:581–598PubMedCrossRefGoogle Scholar
  32. 32.
    Sun C, Chu J, Singh S, Salter RD (2010) Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function. Purinergic Sig 6:31–45CrossRefGoogle Scholar
  33. 33.
    Chong JH, Zheng GG, Ma YY, Zhang HY, Nie K, Lin YM, Wu KF (2010) The hyposensitive N187D P2X7 mutant promotes malignant progression in nude mice. J Biol Chem 285:36179–36187PubMedCrossRefGoogle Scholar
  34. 34.
    Wiley JS, Chen R, Jamieson GP (1993) The ATP4- receptor-operated channel P2Z of human lymphocytes allows Ba2+ and ethidium+ uptake—inhibition of fluxes by suramin. Arch Biochem Biophys 305:54–60PubMedCrossRefGoogle Scholar
  35. 35.
    Sluyter R, Wiley JS (2002) Extracellular adenosine 5′-triphosphate induces a loss of CD23 from human dendritic cells via activation of P2X7 receptors. Int Immunol 14:1415–1421PubMedCrossRefGoogle Scholar
  36. 36.
    Panupinthu N, Rogers JT, Zhao L, Solano-Flores LP, Possmayer F, Sims SM, Dixon SJ (2008) P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis. J Cell Biol 181:859–871PubMedCrossRefGoogle Scholar
  37. 37.
    Milius D, Gröger-Arndt H, Stanchev D, Lange-Dohna C, Rossner S, Sperlagh B, Wirkner K, Illes P (2007) Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells. Toxicology 238:60–69PubMedCrossRefGoogle Scholar
  38. 38.
    Di Virgilio F, Steinberg TH, Silverstein SC (1990) Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11:57–62PubMedCrossRefGoogle Scholar
  39. 39.
    Qu Y, Misaghi S, Newton K, Gilmour LL, Louie S, Cupp JE, Dubyak GR, Hackos D, Dixit VM (2011) Pannexin-1 is required for ATP release during apoptosis but not for inflammasome activation. J Immunol Methods 186:6553–6561Google Scholar
  40. 40.
    Adinolfi E, Melchiorni L, Falzoni S, Chiozzi P, Morelli A, Tieghi A, Cuneo A, Castoldi G, Di Virgilio F, Baricordi OR (2002) P2X7 receptor expression in evolutive and indolent forms of chronic B lymphocytic leukemia. Blood 99:706–708PubMedCrossRefGoogle Scholar
  41. 41.
    Chong JH, Zheng GG, Zhu XF, Guo Y, Wang L, Ma CH, Liu SY, Xu LL, Lin YM, Wu KF (2010) Abnormal expression of P2X family receptors in Chinese pediatric acute leukemias. Biochem Biophys Res Commun 391:498–504PubMedCrossRefGoogle Scholar
  42. 42.
    Shemon AN, Sluyter R, Wiley JS (2007) Rottlerin inhibits P2X7 receptor stimulated phospholipase D activity in chronic lymphocytic leukaemia B-lymphocytes. Immunol Cell Biol 85:68–72PubMedCrossRefGoogle Scholar
  43. 43.
    Wiley JS, Dubyak GR (1989) Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemia lymphocytes. Blood 73:1316–1323PubMedGoogle Scholar
  44. 44.
    Zhang XJ, Zheng GG, Ma XT, Yang YH, Li G, Rao Q, Nie K, Wu KF (2004) Expression of P2X7 in human hematopoietic cell lines and leukemia patients. Leuk Res 28:1313–1322PubMedCrossRefGoogle Scholar
  45. 45.
    Janiak M, Hashmi HR, Janowska-Wieczorek A (1994) Use of the Matrigel-based assay to measure the invasiveness of leukemic cells. Exp Hematol 22:559–565PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Safina Gadeock
    • 1
    • 2
  • Aleta Pupovac
    • 1
    • 2
  • Vanessa Sluyter
    • 1
    • 2
  • Mari Spildrejorde
    • 1
    • 2
  • Ronald Sluyter
    • 1
    • 2
    Email author
  1. 1.School of Biological SciencesUniversity of WollongongWollongongAustralia
  2. 2.Institute of Illawarra Health and Medical Research InstituteWollongongAustralia

Personalised recommendations