P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

Abstract

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.

Fig. 1

KG-1 cells express P2X7. a RNA isolated from KG-1 and RPMI 8226 cells was amplified by RT-PCR using primers to P2X7 and products examined by agarose gel electrophoresis. b Whole cell lysates from KG-1 and RPMI 8226 cells were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-P2X7 (top panel) or anti-actin (bottom panel) pAb. c KG-1 and RPMI 8226 cells were labeled with Alexa Fluor 647-conjugated P2X7 (solid line) or isotype control (shaded) mAb, and the relative cell-surface P2X7 expression measured by flow cytometry. Representative results from (a,b) two or c three experiments shown

Fig. 2

ATP induces ethidium⁺ uptake into KG-1 cells. a KG-1 cells in sucrose medium or b RPMI 8226 cells in sucrose, KCl or NaCl medium were incubated with 25 μM ethidium⁺ in the presence of varying amounts of ATP (as indicated) at 37 °C for a 20 or b 5 min. Incubations were stopped by MgCl₂ solution and centrifugation, and ethidium⁺ uptake measured by flow cytometry. Ethidium⁺ uptake is expressed as percent maximum response compared to 1 mM ATP for each corresponding medium; results are mean ± SD (a, n = 3; b, n = 9)

Fig. 3

P2X7 activation mediates organic cation uptake into KG-1 cells. a–d KG-1 cells were suspended in sucrose medium. a Cells were incubated with 25 μM ethidium⁺ in the absence (Basal) or presence of 100 μM nucleotide (as indicated; αβMethATP, αβ-methylene ATP) at 37 °C for 20 min. Cells were pre-incubated b with DMSO or 100 nM AZ10606120 (left panel), in the absence (Control) or presence of 10 μM A-438079 (right panel), d in the absence (Control) or presence of 2 mM probenecid (left panel), or in the absence (Control) or presence of 20 μM carbenoxolone (right panel) at 37 °C for 15 min. Cells were then incubated with b,d 25 μM ethidium⁺, c 1 μM YO-PRO-1²⁺ or 25 μM propidium²⁺b–d in the absence (Basal) or presence of 100 μM ATP at 37 °C for 20 min. a–d Incubations were stopped by MgCl₂ solution and centrifugation, and cation uptake measured by flow cytometry. Results are mean ± SD (n = 3–6); *P < 0.05 or **P < 0.01 compared to corresponding basal, ^††P < 0.01 compared to corresponding ATP alone