ATP inhibits AML cell proliferation by repressing the overexpression of P2X7R. Freshly isolated leukemic blasts from 7 AML patients and highly purified CD34⁺ cells from 4 healthy donors were seeded at a density of 1 × 10⁶/mL, primed with a cocktail of cytokines for 24 hours, and then treated or not with extracellular nucleotides. (Ai) Cell proliferation was measured by CellTiter 96 Aqueous One Solution (Promega) as described in “Methods.” The inhibitory activity of ATP was reverted by the blocking agent apyrase and restored when denaturated apyrase was used. The percentage of proliferating cells in control samples was 60% ± 23%. Conversely, CD34⁺ cell proliferation was not inhibited by ATP. Shown are the results of cell-cycle analysis of 4 AML samples (ii) and CD34⁺ cells from 4 healthy donors (iii). Results are expressed as the percentage of cells in different phases of the cell cycle. (B) Leukemic blasts from 10 patients were incubated for 24 hours in the presence or absence of 1mM ATP. The expression of the P2X7R was studied by real-time PCR using untreated cells as a control (i) and by flow cytometry (ii). (iii) Pretreatment with KN-62, a specific P2X7R antagonist, counteracted the inhibitory effect of ATP on AML cells proliferation. (C) Leukemic cells from 15 patients and CD34⁺ cells from 7 healthy donors were plated in methylcellulose in the presence of cytokines and increasing concentrations of ATP. The mean number of colonies in AML and healthy control samples was 103 ± 36 and 306 ± 68, respectively. *P < .05 comparing ATP-treated samples with control samples; **P < .05 comparing leukemic cells with normal CD34⁺ cells.