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


CONCLUSIONS
P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.


CONCLUSIONS
RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.
Farrell
Andrew W
AW
School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia.
Gadeock
Safina
S
Pupovac
Aleta
A
Wang
Bin
B
Jalilian
Iman
I
Ranson
Marie
M
Sluyter
Ronald
R
D017209:Apoptosis; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004996:Ethidium / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D017455:Receptors, IgE / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C546468:P2RX7 protein, human; D012333:RNA, Messenger; D017455:Receptors, IgE; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D004996:Ethidium