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.

Propensity of red blood cells to undergo P2X7 receptor–mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging

“…To determine if adenine can impair P2X7 activation, RPMI 8226 cells were pre--incubated with either 485 or 910 µM adenine, followed by incubation with increasing ATP concentrations and ethidium + uptake was assessed as above. As repeatedly observed with this cell line, 15,21 incubation with ATP alone induced ethidium + uptake in a concentration--dependent manner with a half maximal effective concentration (EC50) of 104 ± 7 µM and near--maximal uptake at 0.3 mM (Fig. 4).…”

Human P2X7 receptor activation induces the rapid shedding of CXCL16

“…P2X7-induced pore formation was assessed by flow cytometric measurements of ATP-induced ethidium + uptake into cells suspended in NaCl medium as described [13].…”

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

“…We have previously shown that human RPMI 8226 B cells express P2X7, and that activation of P2X7 on these cells induces pore formation and the shedding of CD23 [17,28,29]. During our preliminary investigations of the possible intracellular signalling enzymes involved in P2X7-induced CD23 shedding from RPMI 8226 cells we observed that the 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one analogue and specific PLD1 inhibitor, CAY10593 [30], significantly impaired P2X7-induced CD23 shedding.…”

“…RPMI 8226 cells have been previously shown to express endogenous P2X7 [17,28,29]. Therefore, to test these specific P2X7 antagonists on endogenously expressed P2X7, RPMI 8226 cells were pre-incubated in the absence or presence of increasing concentrations of AZ10606120, AZ11645373 or A-438079, and the ATPinduced ethidium + uptake (pore formation) was measured by flow cytometry.…”

“…Therefore, to further characterise the effect of the above three PLD antagonists on P2X7 activation, cells were pre-incubated in the presence of increasing concentrations of each antagonist and the ATPinduced ethidium + uptake measured, using 120 μM ATP which is approximate to the EC 50 for ATP in this process [17,28]. CAY10593 inhibited ATP-induced ethidium + uptake in a concentration-dependent manner, with maximal inhibition occurring near 10 μM and an IC 50 of 2.0±0.5 μM (Fig.…”