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

abstract

  • 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.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.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 ~116 μM, 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.P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

authors

  • Farrell, Andrew W
  • Gadeock, Safina
  • Pupovac, Aleta
  • Wang, Bin
  • Jalilian, Iman
  • Ranson, Marie
  • Sluyter, Ronald

publication date

  • November 2010