adenosine-5--o-(3-thiotriphosphate) and Leukemia--Promyelocytic--Acute

ATP and UTP cause mobilization of Ca2+ from the intracellular stores with similar potency in several cell types including both undifferentiated and differentiated HL60 cells. We show here that, in HL60 cells with Ca2+ stores that had been fully and irreversibly emptied using the endomembrane Ca(2+)-ATPase inhibitor thapsigargin, both nucleotides produced a biphasic effect on Ca2+ entry, first rapid inhibition and then delayed (about 15 s) activation. ATP was more effective at producing the initial inhibition of Ca2+ entry, whereas UTP was more effective at activating the delayed Ca2+ entry. Previous incubation with UTP desensitized the Ca2+ mobilization and the delayed activation of Ca2+ entry induced by ATP but not the inhibition of Ca2+ entry. The ATP analogue 2-methylthioATP (2-MeSATP) barely mobilized stored Ca2+ but inhibited Ca2+ entry. These results could be explained by the presence of two receptors: (i) a P2u receptor sensitive to ATP and UTP, responsible for activation of phospholipase C and Ca2+ mobilization, early inhibition of Ca2+ entry and delayed activation of Ca2+ entry and (ii) a P2y-like receptor sensitive to ATP and 2-MeSATP which produces only inhibition of Ca2+ entry. The inhibition of Ca2+ entry by nucleotides increased greatly during differentiation. Given that Ca2+ mobilization by nucleotides is not modified by differentiation, this suggests that a component of the mechanism of inhibition of Ca2+ entry is gradually expressed during differentiation of HL60 cells.

Topics: Adenosine Triphosphate; Calcium; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Manganese; Terpenes; Thapsigargin; Thionucleotides; Tumor Cells, Cultured; Uridine Triphosphate

The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps: DEAE-Sepharose, casein-agarose, cibacron-blue--agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin II phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP[gamma s] and autophosphorylation of the enzyme in the presence of [gamma-32P]ATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 microM and the Vmax 3.1 nmol.min-1.mg-1 using angiotensin II as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.

Topics: Adenosine Triphosphate; Angiotensin II; Binding, Competitive; Blotting, Western; Caseins; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Magnesium; Manganese; Metals; Osmolar Concentration; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil/monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATP gamma S (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear/cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATP gamma S, HL-60 cells showed no obvious changes in morphology, viability, or the levels of beta-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a greater than 90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.

Topics: Actins; Adenosine; Adenosine Triphosphate; Calcium; Cell Differentiation; Cell Division; Cell Line; Genes, myc; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; N-Formylmethionine Leucyl-Phenylalanine; Phagocytes; Phenotype; Receptors, Purinergic; RNA, Messenger; Superoxides; Tetradecanoylphorbol Acetate; Uridine Triphosphate