Expression of P2X7 in human hematopoietic cell lines and leukemia patients

Abstract 

The P2X7 nucleotide receptor is an adenosine 5′-triphosphate (ATP) -gated ion channel, which is widely expressed in cells of hematopoietic origin and functions as a non-selective cation channel permeable to Na⁺, Ca²⁺, etc upon stimulation. Here, we investigated P2X7 expression in 11 human hematopoietic cell lines, representing different lineages, as well as bone marrow mononuclear cells (BMMC) samples from 87 leukemia and 10 myelodysplastic syndrome (MDS) patients. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry results showed that both P2X7 mRNA and protein were detected in eight cell lines with a non-lineage-specific manner. Samples from 69 leukemia and 9 MDS patients were P2X7 positive at mRNA level. Moreover, both positive rates and relative expression levels were significantly higher in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and MDS groups than that in normal donor group. The expression levels varied among AML subtypes with higher levels being observed in M4, M5, and M6 groups but not in M1 or M2 group. Furthermore, after one course of standard induction therapies, the remission rate in high P2X7 expression group was lower than that in either P2X7 negative group or low P2X7 expression group. Cytoplasmic free calcium increase was detected in five of eight P2X7⁺ cell lines as well as P2X7⁺ normal donor and patient samples tested, but not in three Epstein–Barr virus (EBV) positive cell lines (J6-1, Namalwa, and LCL-H) in Locke’s solution upon stimulation by extracellular ATP or the more potent and specific agonist, 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP). The possible mechanisms causing the loss of P2X7 function were discussed.

Abbreviations:  ALL, acute lymphoblastic leukemia, AML, acute myelogenous leukemia, ATP, adenosine 5′-triphosphate, BzATP, 2′,3′-O-(4-benzoyl)benzoyl-ATP, BM, bone marrow, BMMC, bone marrow mononuclear cells, BSA, bovine serum albumin, CLL, chronic lymphocytic leukemia, CML, chronic myelogenous leukemia, CR, complete remission, DEPC, diethylpyrocaronate, EBV, Epstein–Barr virus, ERK, extracellular signal-regulated kinase, FCS, fetal calf serum, Fura-2/AM, fura 2-acetoxymethyl ester, HHV-6, human herpesvirus 6, MDS, myelodysplastic syndrome, NFAT, nuclear factor of activated T cells, PBMC, peripheral blood mononuclear cell, PR, partial remission, RI, relative index, RT-PCR, reverse transcription polymerase chain reaction

Keywords:  P2X7, Leukemic cell, Adenosine 5′-triphosphate, Intracellular calcium, Epstein–Barr virus