P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells
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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.
KeywordsPurinergic receptor Extracellular ATP Acute myelogenous leukaemia Cation channel Single nucleotide polymorphism
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.
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