P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells.
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 (EC(50) 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 (EC(50) of ≈ 3 or ≈ 99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) 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-1(2+) and propidium(2+) 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.
School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia.
Safina
S
Gadeock
Aleta
A
Pupovac
Vanessa
V
Sluyter
Mari
M
Spildrejorde
Ronald
R
Sluyter
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D022423:Myeloid Cells / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology*; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*
D002412:Cations; C546468:P2RX7 protein, human; D058920:Purinergic P2X Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate