2--3--dialdehyde-atp and Leukemia--Lymphocytic--Chronic--B-Cell

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Calcium; Cell Line; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Ethidium; Humans; Intracellular Fluid; K562 Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Macrophages; Mice; Molecular Sequence Data; Oxidation-Reduction; Polymyxin B; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Transfection

CD23 is a transmembrane protein expressed on the surface of B-lymphocytes that binds IgE, CD21, CD11b, and CD11c. High concentrations of soluble CD23 and L-selectin are found in the serum of patients with B-chronic lymphocytic leukemia (B-CLL). Because extracellular adenosine triphosphate (ATP) causes shedding of L-selectin via activation of P2Z/P2X7 receptors expressed on B-CLL lymphocytes, we studied the effect of ATP on shedding of CD23. ATP-induced shedding of CD23 at an initial rate of 12% of that for L-selectin, whereas the EC50 for ATP was identical (35 micromol/L) for shedding of both molecules. Furthermore, benzoylbenzoyl ATP also produced shedding of CD23 and L-selectin with the same agonist EC50 values for both (10 micromol/L). Inactivation of the P2Z/P2X7 receptor by preincubation with oxidized ATP abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2Z/P2X7 receptor, inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nmol/L). Ro 31-9790, a membrane permeant zinc chelator that inhibits the phorbol-ester-stimulated shedding of L-selectin, also inhibited shedding of CD23 from B-CLL lymphocytes. However, the IC50 for this inhibition by Ro31-9790 was different for L-selectin and CD23 (83 v 6 micromol/L, respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester, CD23 was not lost under these conditions. The data show that extracellular ATP induces shedding of L-selectin and CD23 from B-CLL lymphocytes by an action mediated by the P2Z/P2X7 receptor. However, different membrane metalloproteases seem to mediate the shedding of L-selectin and CD23.

Topics: Adenosine Triphosphate; B-Lymphocytes; Biomarkers, Tumor; Calcium; Humans; Hydroxamic Acids; L-Selectin; Leukemia, Lymphocytic, Chronic, B-Cell; Metalloendopeptidases; Neoplasm Proteins; Neoplastic Stem Cells; Protease Inhibitors; Receptors, IgE; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Solubility; Tetradecanoylphorbol Acetate

To investigate the role of purinergic P2z receptors in human leukemic lymphocyte apoptosis induced by extracellular adenosine triphosphate (ATP).. Eleven B-CLL patients were studied. Leukemic lymphocytes with (n = 8) or without (n = 3) P2z receptors were exposed in vitro to ATP, benzoyl-benzoic-ATP (BzATP), 2-methylthio-ATP(2MeSATP), adenosine-5'-[gamma-thio] triphosphate (ATP-gamma S), and other nucleosides for 8 h. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and quantitative TdT assay.. Apoptosis was detected only in leukemic lymphocytes with P2z receptors. By using the quantitative assay, ATP-inducing DNA strand breaks were found to occur specifically for BzATP, ATP and 2MeSATP, but not for ATP-gamma S and other nucleosides. Meanwhile, ATP-inducing DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2z receptors. Ca2+/Calmodulin complex played a role in the regulation of the CLL cell apoptosis induced by ATP, because an antagonist of this complex, 1-[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), was found to inhibit the ATP-inducing apoptosis.. P2z receptors on lymphocytes play an important role in the apoptosis induced by ATP in vitro.

Topics: Adenosine Triphosphate; Apoptosis; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Tumor Cells, Cultured

Lymphocytes from normal subjects or patients with chronic lymphocytic leukemia are known to possess receptors for extracellular ATP termed P2Z purinoceptors whose physiological role is undefined. Addition of extracellular ATP (50-500 microM) to both normal and leukemic lymphocytes caused loss of binding of monoclonal antibodies to L-selectin (CD62L) on the cell surface. UTP, ADP, and adenosine (all at 500 microM) had no effect on L-selectin expression. Several features of the ATP-induced loss of L selectin indicate that this effect is mediated by lymphocyte P2Z purinoceptors. First the loss was attenuated in isotonic NaCl medium compared to 150 mM KCl medium. Second the loss of L-selectin was immediately halted by addition of Mg2+ ions in molar excess of ATP. The most potent nucleotide causing L-selectin loss was benzoylbenzoic ATP (> 10 microM) which is also the most potent agonist for the P2Z purinoceptor. Finally preincubation of lymphocytes with oxidized ATP, an irreversible inhibitor of P2Z purinoceptors, also inhibited ATP induced loss of L-selectin. Extracellular ATP is known to open an ion channel associated with the P2Z purinoceptor on B-lymphocytes which allows influx of Ca2+. However, ATP-induced loss of L-selectin did not require extracellular Ca2+. Moreover addition of the calcium ionophore, ionomycin, had minimal effect on L-selectin expression. Staurosporine (500 nM), an inhibitor of protein kinase C, inhibited only 10% of ATP induced loss of L-selectin but completely inhibited the loss of L-selectin caused by 50 nM PMA. Thus extracellular ATP interacts with lymphocyte P2Z purinoceptors which leads to shedding of L-selectin via a pathway which requires neither Ca2+ influx nor activation of protein kinase C. ATP may have a physiological role in the loss of L-selectin which occurs during the interactions of lymphocytes with other cells.

Topics: Adenosine Triphosphate; Alkaloids; Calcium; Cells, Cultured; Down-Regulation; Humans; L-Selectin; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Magnesium; Protein Kinase C; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured