thromboxane-a2 and Leukemia--Erythroblastic--Acute

Effects of troglitazone, an antidiabetic thiazolidinedione that enhances insulin sensitivity, on thromboxane (TX) production were assessed in human erythroleukemia (HEL) cells and human platelets. Measurement of TX was performed by using the gas chromatography/selected ion monitoring (GC/SIM) method. We found that troglitazone reduced the TX production from HEL cells and human platelets. Furthermore, troglitazone also reduced arachidonic acid (AA)-induced TX production from HEL cell and thrombin-induced TX release from platelets. In addition, we compared the effect of troglitazone with that of alpha-tocopherol and BRL 49653. Other thiazolidinedione compound BRL 49653 had effects similar to troglitazone, but alpha-tocopherol had no effect on TX production. Our findings suggest that the thiazolidinedione group had an antithrombotic effect and was beneficial in preventing vascular complications often observed in diabetes mellitus.

Topics: Arachidonic Acid; Blood Platelets; Chromans; Diabetes Mellitus, Type 2; Fibrinolytic Agents; Gas Chromatography-Mass Spectrometry; Humans; Leukemia, Erythroblastic, Acute; Rosiglitazone; Thiazoles; Thiazolidinediones; Thromboxane A2; Troglitazone; Tumor Cells, Cultured; Vitamin E

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Sequence; Base Sequence; Blotting, Northern; Blotting, Western; Calcium; Dimethyl Sulfoxide; DNA Probes; Gene Expression; GTP-Binding Proteins; Humans; Lanthanum; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Nickel; Polymerase Chain Reaction; Prostaglandin Endoperoxides, Synthetic; Sequence Homology, Nucleic Acid; Signal Transduction; Thrombin; Thromboxane A2; Tumor Cells, Cultured

Thromboxane A2 (TxA2), a platelet aggregator and vasoconstrictor, has been implicated as a potential mediator of cardiovascular diseases. Abuse of androgenic steroids has been associated with thrombotic cardiovascular diseases. Human erythroleukemia (HEL) cells, a megakaryocyte-like cell line, express functional TxA2/prostaglandin H2 (PGH2) receptors with characteristics similar to those seen in platelets. This study characterized testosterone regulation of HEL cell TxA2/PGH2 receptors. TxA2/PGH2 receptor affinity (Kd) and density (Bmax) were determined via equilibrium binding experiments using the radiolabeled TxA2 mimetic (1S-[1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha])-7-(3-[3-hydroxy-4-(4'- iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]heptan-2-yl)-5-he ptenoic acid (125I-labeled BOP). Testosterone (200 nM) but not estradiol increased Bmax from 108 +/- 9 fmol/mg protein to 157 +/- 9 fmol/mg protein (n = 7 experiments; P < 0.01) without any significant change in Kd. Testosterone had no significant effect on alpha 2-adrenergic receptor density. The maximum increase in intracellular free calcium induced by the TxA2 agonists I-BOP or U-46619 was significantly (P < 0.005) greater in testosterone-treated cells compared with controls. Hydroxyflutamide (1 microM), an androgen-receptor antagonist, completely blocked the effect of testosterone (P < 0.01). Dihydrotestosterone, the active metabolite of testosterone, also increased Bmax in a concentration-dependent manner and was more potent than testosterone. The effect of testosterone to increase Bmax was significantly (P < 0.01) inhibited by coincubation with cycloheximide (0.1 microgram/ml) or actinomycin D (10 ng/ml). These results indicate that androgenic steroids regulate the expression of functional TxA2/PGH2 receptors in HEL cells. These findings may have relevance to cardiovascular disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 5-alpha Reductase Inhibitors; Azasteroids; Calcium; Cell Line; Cycloheximide; Dactinomycin; Dihydrotestosterone; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Testosterone; Thromboxane A2; Tumor Cells, Cultured; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds; Cell Membrane; Fatty Acids, Monounsaturated; Humans; Leukemia, Erythroblastic, Acute; Neoplasm Proteins; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Tetradecanoylphorbol Acetate; Thromboxane A2; Tumor Cells, Cultured

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.

Topics: Amino Acid Sequence; Arachidonic Acid; Base Sequence; Calcimycin; Electrophoresis, Polyacrylamide Gel; Gene Expression; Humans; Leukemia, Erythroblastic, Acute; Molecular Sequence Data; Polymerase Chain Reaction; Prostaglandins H; RNA, Messenger; Sequence Homology, Nucleic Acid; Thrombin; Thromboxane A2; Thromboxane-A Synthase; Tumor Cells, Cultured

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.

Topics: Acetylcholinesterase; Activins; Animals; Autoradiography; Cell Differentiation; Chromatography, Thin Layer; Cycloheximide; Hemoglobins; Humans; Inhibins; Leukemia, Erythroblastic, Acute; Mice; Thromboxane A2; Thromboxane-A Synthase; Tumor Cells, Cultured

Using [125I]I-S-145-OH, a radiolabeled derivative of a thromboxane (TX) A2 receptor antagonist, we have studied the expression of the TXA2 receptor in several lines of cultured leukemia cells. Specific binding of the ligand was observed in cells of two human erythroleukemia cell lines, K562 and HEL. However, only negligible binding was seen in HL-60 human promyelocytic leukemia cells and L-1210 murine leukemia cells. Scatchard analyses revealed a curvilinear plot which indicated the existence of two classes of binding sites in these cells. The Kd and Bmax values of the high and low affinity bindings in HEL cells were 2.4 nM and 24 fmol/10(6) cells, and 58 nM and 360 fmol/10(6) cells, respectively. These values in K562 cells were 2.8 nM and 16 fmol/10(6) cells, and 18 nM and 46 fmol/10(6) cells, respectively. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the cultures of K562 and HEL cells caused a concentration- and time-dependent increase in the binding activity. TPA at 10(-8) M increased the Bmax values of both high and low affinity bindings approximately 3 times without significant change in their Kd values and these increases were inhibited by the addition of actinomycin D. Both classes of the binding in cells of each cell line were specifically displaced by several TXA2/prostaglandin (PG) H2 analogues, although the relative specificities to the analogues were different in the two classes. These results suggest that both HEL and K562 cells express the TXA2 receptor on their cell surface and TPA strongly induces this expression in these cells.

Topics: Animals; Bridged Bicyclo Compounds; Dactinomycin; Fatty Acids, Monounsaturated; Humans; Iodine Radioisotopes; Kinetics; Leukemia; Leukemia, Erythroblastic, Acute; Mice; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Tetradecanoylphorbol Acetate; Thromboxane A2; Tumor Cells, Cultured

The human erythroleukemia (HEL) cell line is a cultured hematopoietic cell line reported to express platelet membrane glycoproteins and alpha-2 adrenergic receptors. The present studies were designed to determine if functional thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptors exist in HEL cells. Radioligand binding assays were performed using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist. Scatchard analysis revealed one class of binding sites for 1-PTA-OH with a Kd = 122 +/- 18 nM and maximum binding = 1.7 +/- 0.3 x 10(5) sites/cell. Competition for [125I]PTA-OH binding with the TXA2/PGH2 receptor agonists SQ26655 and U46619 revealed one class of binding sites for SQ26655 with a Kd = 17 nM and two classes of binding sites for for U46619 with a Kd = 45 nM for the high-affinity site and a Kd = 450 nM for the low-affinity site. Competition for [125I]PTA-OH by the steroisomeric TXA2/PGH2 receptor antagonists L657925 and L657926 revealed two classes of binding sites for the more potent L657925 with a Kd = 8 nM for the high-affinity site and a Kd = 400 nM for the low-affinity site whereas L657926 bound to one class of sites with a Kd = 380 nM. Stimulation of the TXA2/PGH2 receptor by SQ26655 and U46619 resulted in concentration-dependent increases in [Ca++], as measured by FURA-2 fluorescence, with EC50 values of 28 +/- 2 and 149 +/- 32 nM, respectively. I-PTA-OH, L657925 and L657926 antagonized this response to U46619 with IC50 values similar in rank potency to that seen in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcium; Cell Differentiation; Dimethyl Sulfoxide; Humans; Leukemia, Erythroblastic, Acute; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Radioligand Assay; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2; Thromboxane B2; Tumor Cells, Cultured