sq-23377 and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by the oncogenic activity of the Bcr-Abl protein, a deregulated tyrosine kinase. Calcium may act directly on cellular enzymes and in conjunction with other cellular metabolites, such as cyclic nucleotides, to regulate cell functions. Alteration in the ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation as well as the production of reactive oxygen species (ROS) has been correlates with normal cell proliferation through activation of growth-related signaling pathways. In this study we evaluated in peripheral blood leukocytes from CML patients the role of the balance between intracellular calcium and oxidative stress in CML disease in order to identify possible therapeutic targets in patients affected by this pathology. Our results demonstrated that peripheral blood mononuclear cells derived from CML patients displayed decreased intracellular calcium [Ca(2+)](i) fluxes both after InsP(3) as well as ATP and ionomycin (IONO) administration. CML cells showed lower levels of superoxide dismutase (SOD) activity and significantly higher malondialdehyde levels (MDA) than peripheral blood mononuclear cells derived from control patients. Finally we showed that resveratrol is able to down-regulate InsP3 and ATP effects on intracellular calcium [Ca(2+)](i) fluxes as well as the effects of ATP and IONO on oxidative stress in CML cells.

Topics: Adenosine Triphosphate; Adult; Calcium; Female; Homeostasis; Humans; Intracellular Space; Ionomycin; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Malondialdehyde; Middle Aged; Nitric Oxide; Oxidative Stress; Resveratrol; Stilbenes; Superoxide Dismutase

NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.

Topics: Animals; Calcium; Cell Differentiation; Cell Line; Cytotoxicity, Immunologic; DNA, Complementary; Dogs; Glycosylation; Humans; Ionomycin; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Membrane Proteins; Neoplasm Proteins; NK Cell Lectin-Like Receptor Subfamily C; Nucleopolyhedroviruses; Receptors, Immunologic; Receptors, Natural Killer Cell; Recombinant Fusion Proteins; Spodoptera; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; Collagen; Epinephrine; Humans; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Megakaryocytes; Rats; Receptors, Purinergic; Thrombin; Tumor Cells, Cultured