thromboxane-b2 and Leukemia--Erythroblastic--Acute

We investigated the ability of different hydroperoxides generated by lipoxygenase isozymes to induce programmed cell death (PCD) in human cells. Erythroleukemia K562 and neuroblastoma CHP100 cells were used, because they showed high basal activity of lipoxygenase. The hydroperoxides generated by 5-, 12-, or 15-lipoxygenases from linoleate, linolenate, or arachidonate, and the corresponding hydroxides, were able to induce PCD in both cell types, in a concentration- and time-dependent manner. After 24 h, K562 and CHP100 cells showed 2.5- to 3.5-fold more apoptotic bodies than the untreated controls. PCD elicited by lipoxygenase products was independent of intracellular glutathione concentration, and did not require mRNA transcription or protein synthesis. On the other hand, lipoxygenase products evoked an immediate and sustained rise in cytoplasmic calcium (within seconds), followed by mitochondrial uncoupling (within hours). Unlike the hydro(pero)xides, the terminal products of the arachidonate cascade (i.e., leukotrienes, prostaglandins and thromboxane) were not cytotoxic.

Topics: alpha-Linolenic Acid; Apoptosis; Arachidonic Acid; Calcium; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Glutathione; Humans; Isoenzymes; K562 Cells; Leukemia, Erythroblastic, Acute; Leukotrienes; Linoleic Acid; Lipid Peroxides; Lipoxygenase; Mitochondria; Neuroblastoma; Prostaglandins; Thromboxane B2; Time Factors; Tumor Cells, Cultured; Uncoupling Agents

We devised a simple and effective purification for the microdetermination of thromboxane B3 (TXB3), a hydrolysis product of TXA3- [18O2]TXB3 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O]water, to obtain an internal standard (IS) for the gas chromatography/selected ion monitoring (GC/SIM) of TXB3. The methyl ester (ME)-methoxime (MO)-dimethylisopropylsilyl (DMIPS) ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 668 for TXB3 and that at m/z 672 for IS. A good linear response over the range of 10 pg approximately 10 ng was demonstrated. We were able to detect the levels of TXB3 in the medium of human erythroleukemia (HEL) cell cultured with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). This method can be used to determine 3-series thromboxane in biological samples.

Topics: Calibration; Chromatography, Gas; Culture Media, Conditioned; Docosahexaenoic Acids; Eicosapentaenoic Acid; Humans; Isotope Labeling; Leukemia, Erythroblastic, Acute; Microchemistry; Oxygen Isotopes; Reproducibility of Results; Thromboxane B2; Thromboxanes; Tumor Cells, Cultured

NSAIDs inhibit the conversion of arachidonic acid into Prostaglandin G2 and Prostaglandin H2 which is catalyzed by the enzyme cyclooxygenase (COX). Two genetically distinct isoforms have been discovered, COX-1 and COX-2. While COX-1 is thought to account for homeostatic amounts of eicosanoids, COX-2 is induced during inflammation leading to pathologic amounts of eicosanoids. Since NSAIDs inhibit both COX isoforms, antiinflammatory drug research has refocused to discovering COX-2 inhibitors that do not inhibit COX-1. For this purpose, we have developed a whole cell assay system using the human erythroleukemic cell line HEL as a source for COX-1 and the human monocytic cell line Mono Mac 6 as a source for COX-2. Mono Mac 6 cells express high amounts of COX-2 upon stimulation with lipopolysaccharide (LPS) in the absence of any detectable COX-1 protein. On the other hand, we find HEL cells to naturally express COX-1 protein, but not COX-2. Testing of a panel of NSAIDs as well as some COX-2 specific inhibitors showed that this assay system is suitable for identifying compounds that selectively inhibit either COX-1 or COX-2. This test system offers the advantage of assessing COX-1 and COX-2 inhibitors within the human species, within a similar test set-up, and circumvents the need for tedious purification of either platelets or peripheral blood monocytes.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Blotting, Western; Cell Line; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Humans; Isoenzymes; Leukemia, Erythroblastic, Acute; Lipopolysaccharides; Membrane Proteins; Monocytes; Prostaglandin-Endoperoxide Synthases; Thromboxane B2; 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