P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells

Andrew W. Farrell, Safina Gadeock, Aleta Pupovac, Ben Wang, Iman Jalilian, Marie Ranson, Ronald Sluyter

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Background: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. Methods: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. Results: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116μ and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. Conclusions: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. General significance: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

LanguageEnglish
Pages1173-1182
Number of pages10
JournalBiochimica et Biophysica Acta - General Subjects
Volume1800
Issue number11
DOIs
Publication statusPublished - 1 Nov 2010

Keywords

  • B-lymphocyte
  • IgE receptor
  • Multiple myeloma
  • P2X receptor
  • Plasma cell
  • Purinergic receptor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Farrell, Andrew W. ; Gadeock, Safina ; Pupovac, Aleta ; Wang, Ben ; Jalilian, Iman ; Ranson, Marie ; Sluyter, Ronald. / P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells. In: Biochimica et Biophysica Acta - General Subjects. 2010 ; Vol. 1800, No. 11. pp. 1173-1182.
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P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells. / Farrell, Andrew W.; Gadeock, Safina; Pupovac, Aleta; Wang, Ben; Jalilian, Iman; Ranson, Marie; Sluyter, Ronald.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1800, No. 11, 01.11.2010, p. 1173-1182.

Research output: Contribution to journalArticle

TY - JOUR

T1 - P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells

AU - Farrell, Andrew W.

AU - Gadeock, Safina

AU - Pupovac, Aleta

AU - Wang, Ben

AU - Jalilian, Iman

AU - Ranson, Marie

AU - Sluyter, Ronald

PY - 2010/11/1

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N2 - Background: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. Methods: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. Results: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116μ and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. Conclusions: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. General significance: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

AB - Background: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. Methods: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. Results: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116μ and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. Conclusions: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. General significance: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

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KW - IgE receptor

KW - Multiple myeloma

KW - P2X receptor

KW - Plasma cell

KW - Purinergic receptor

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