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Gadeock S.,University of Wollongong | Gadeock S.,Institute of Illawarra Health and Medical Research Institute | Pupovac A.,University of Wollongong | Pupovac A.,Institute of Illawarra Health and Medical Research Institute | And 6 more authors.
Purinergic Signalling | Year: 2012

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. © 2012 Springer Science+Business Media B.V.

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