Appearance of an arachidonic acid 15-lipoxygenase pathway upon differentiation of the human promyelocytic cell-line HL-60 (original) (raw)
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Cancer Research, 1983
The arachidonic acid lipoxygenase products released into culture supernatants by the human myeloid cell line K562 were quantitated by high-performance liquid chromatography. Dur ing 2 hr of incubation, K562 cells spontaneously released a mean of 9 ng of 15-monohydroxyeicosatetraenoic acid, 35 ng of 5-monohydroxyeicosatetraenoic acid, and 87 ng of a par tially resolved mixture of 11-and 12-monohydroxyeicosatetraenoic acid per 107 cells. The addition of 50 fig of arachidonic acid per ml to the cell cultures increased the quantities of monohydroxyeicosatetraenoic acids generated after 2 hr of incubation by 10-to 100-fold; changes in the ratios of these lipoxygenase products occurred over 24 hr of incubation. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, at concentrations of 5 to 25 ng/ml increased arachidonic acid lipoxygenation 1-to 2-fold in cultures containing 50 fig of arachidonic acid per ml, and up to 20-fold in cultures not supplemented with arachidonic acid. The lipoxygenation of arachidonic acid was enhanced 2-to 9-fold for up to 24 hr after a 2-hr exposure of K562 cells to 12-O-tetradecanoylphorbol-13-acetate. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked the effects of 12-O-tetradecanoylphorbol-13acetate, suggesting that the monohydroxyeicosatetraenoic acids are the products of specific lipoxygenases rather than of nonenzymatic oxidative reactions. The capacities of phorbol esters to promote tumors in the mouse skin model corre sponded to their respective capacities to enhance the lipoxy genation of arachidonic acid to 15-and 5-monohydroxyeico satetraenoic acid but not to 11-and 12-monohydroxyeicosatetraenoic acid.
Biochemical and Biophysical Research Communications, 1988
The 15-lipoxygenase pathway is the predominant pathway for arachidonic acid metabolism in homogenates of human lung (l), in isolated human airway epithelial cells (2) and in human eosinophils (3,4), and keratinocytes(5). Although some functions of arachidonate18lipoxygenase metabolites have been described(6), little is known about the isolation or modulation of human 15-lipoxygenase. The 5lipoxygenase of human leukocytes has been purified to homogeneity(7), and has been separated from 15lipoxygenase using anion exchange chromatography(7,8). Our study provides new evidence that human leukocyte 15-lipoxygenase activity may be entirely accounted for by eosinophils. Using this observation, we have developed a method for purifying human 15-lipoxygenase with cation-exchange and hydrophobicinteraction chromatography using eosinophils obtained from patients treated with interleukin-2. The kinetic properties, pH dependence and divalent cation dependence of the 15-lipoxygenase are described. Our results indicate that human leukocyte 15-lipoxygenase differs from 5-lipoxygenase in both electrostatic and hydrophobic properties. In addition, we have found that phosphatidylcholine stimulates, and ATP inhibits, 15lipoxygenase activity. These observations suggest a regulatory role for both phosphatidylcholine and
Progress in Lipid Research, 1996
CONTENTS I. Introduction 203 II. Nomenclature of mammalian lipoxygenases 205 III. Is the 15-1ipoxygenase pathway involved in the arachidonic acid cascade? 205 IV. Tissue distribution and regulation of 15-1ipoxygenase expression 207 V. Biosynthesis of 15-H(p)ETE and 13-H(p)ODE 208 VI. Analysis of 15-H(p)ETE and 13-H(p)ODE 209 VII. Metabolization of 15-H(p)ETE and 13-H(p)ODE 211 VIII. Biological importance of 15-H(p)ETE and 13-H(p)ODE 213 A. Regulatory functions of 15-H(p)ETE and 13-H(p)ODE at the molecular level 213 B. 15..Lipoxygenase and 15-1ipoxygenase products in erythropoiesis 214 C. 15-.Lipoxygenase and 15-1ipoxygenase products in the cardiovascular system and 215 their role in atherogenesis D. 15..Lipoxygenase products in skin and cornea epithelium 217 E. 15..Lipoxygenase products in the respiratory system 218 F. 15..Lipoxygenase products in reproductive tissue 218 G. 15-.Lipoxygenase products and inflammation 219 H. 15-Lipoxygenase products and diabetis mellitus 220 I. Miscellaneous effects of 15-1ipoxygenase products 220 1. Cell proliferation and cell adhesion 220 2. 15-Lipoxygenase products and hormone synthesis 221 3. 15-Lipoxygenase and apoptosis 221 IX. Perspectives
Inflammation, 1984
The effects of nordihydroguairetic acid (NDGA), 3-amino-1-trifluoromethyl-)-phenyl-2-pyrazoline (BW755c), eicostatetraynoic acid (ETYA), phenidone, quercetin, and indomethacin (INDO) on the synthesis of 15-hydroxyeicosatatetraenoic acid (15-HETE) from soybean 15-lipoxygenase, leukotriene B4 (LTB4 from 5-lipoxygenase, and prostaglandin E2 (PGE2 from cyclooxygenase enzymes of rat neutrophils and mouse peritoneal macrophages were investigated. All of the drugs caused a dose-related inhibition of increased oxygen consumption by soybean 15-lipoxygenase in the presence of arachidonic acid and the rank order of potency was phenidone ≥ BW755c > ETYA > quercetin > NDGA > indomethacin. The reduction in oxygen consumption correlated with a reduction of 15-HETE formation as identified by high-performance liquid chromatography. Apart from indomethacin, these drugs were also effective against the rat neutrophil 5-lipoxygenase, although the rank order of potency did not correlate with that obtained with soybean 15-lipoxygenase. Furthermore, in both A23187-activated rat neutrophils and zymosan-activated mouse peritoneal macrophages the synthesis of prostaglandins was inhibited by all of these drugs. In the neutrophils, the rank order of potency was INDO > ETYA > BW755c > quercetin > NDGA > phenidone, whereas in mouse peritoneal macrophages, the order was INDO > ETYA > BW755c > NDGA > quercetin > phenidone. These results suggest that putative lipoxygenase inhibitors exhibit both qualitative and quantitative differences in their effects on both lipoxygenases and cyclooxygenases.
Biochemistry, 2009
Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k cat /K M and k cat for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k cat /K M KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k cat /K M SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K M [O 2 ] for AA to 15 μM but increases the K M [O 2 ] for linoleic acid (LA) to 22 μM, such that the k cat /K M [O 2 ] values become similar for both substrates (∼0.3 s -1 μM -1 ). Considering that the oxygen concentration in cancerous tissue can be less than 5 μM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.
Arachidonic acid metabolism in HEL/30 murine epidermal Cell Line
Archives of Dermatological Research, 1988
The established mouse epidermis-derived cell line HEL/30 was incubated in the presence of 3H arachidonic acid (AA) for 1 h. After medium removal, cells were reincubated with fresh medium in the presence or absence of the calcium ionophore A23187 and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The AA metabolites formed were extracted from cell-free medium and analyzed using TLC and HPLC. The distribution of the recovered radioactivity showed PGE2, 15-hydroxy-eicosatetraenoic acid (15-HETE), and leukotriene B4 (LTB4), as major products of AA metabolism. The presence of calcium ionophore A23187 increased the release of radioactivity, without affecting the profile of metabolites present in the medium. TPA elicited a preferential increase of cycloxygenase metabolism, this effect being reversed by indomethacin. 5,8,11,14-eicosatetraynoic acid (ETYA) almost completely inhibited LT and HETE formation in A23187 and TPA-treated cells. The results show that HEL/30 cells are able to metabolize AA via both cyclo-and lipoxygenase pathways and that these activities can be modified by chemical means. This cell line might be a suitable tool for studying the involvement of arachidonic acid cascade in cell response to exogenous stimuli.
Deacylation of cellular lipids and arachidonic acid metabolism
Progress in Lipid Research, 1981
The products of cellular arachidonic acid metabolism depend, both quantitatively and qualitatively, on the relative activities of enzymes such as acylhydrolases; CoA-acyltransferases; cyclooxygenases; prostacyclin, thromboxane, and the prostaglandin synthases; lipoxygenases; glutathione S-transferases; ~,-glutamyl transpeptidases; and the metabolizing enzymes, 15-hydroxydehydrogenases and A13 reductases. Each cell has a unique profile of arachidonate metabolic products. Stimulation of cellular arachidonic acid metabolism by several compounds results in levels of products in the culture media in excess not 0nly of that found free in the cells but also in excess of that synthesized from the nonesterified arachidonic acid found in cells. Some deacylation of esterified arachidonic acid must occur. In this presentation, we will describe some aspects of the relationship between acylhydrolase activity and arachidonic acid metabolism. BIOSYNTHESIS OF CYCLOOXYGENASE PRODUCTS BY CELLS IN CULTURE Many, but not all, mammalian cells have the enzyme4s) that synthesize endoperoxides from arachidonic acid. As mentioned above, the capacity to generate thromboxanes, prostacyclin, and the prostaglandins from endogenous endoperoxides may vary considerably among cells. We have determined such variation in six different cell cultures (Table 1)."a~ In order to stimulate the cells to synthesize more endogenous endoperoxides, the cells were incubated either with melittin ~6~ or 12-0-tetradecanoyiphorbol-13acetate, TPA. (x9~ The generated endoperoxides were then metabolized by the thromboxane, prostacyclin and prostaglandin synthases of each cell. Greater than 90% of cyclooxygenase products of endothelial cells derived from bovine aorta was prostacyclin. A similar percentage of prostacyclin was found with three additional isolates of bovine aorta endothelial cells, while 80% of the products of endothelial cells derived from bovine adrenal was prostacyclin, and only 26% of the cyclooxygenase metabolites from endothelial cells derived from a human umbilicus vein was prostacyclin. Previously, about TAI~LE 1. Arachidonic Acid Metabolism by Cells in Culture: Analyses by Immunochromatography (HPLC-RIAI and Radioimmunoassay IRIA) Is
Chemical Research in Toxicology, 2007
Rat intestinal epithelial cells that permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 µM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E 2 , 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE 2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 µM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-(S)-HETE concentrations were approximately twice that of PGE 2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE 2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE 2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudofirst-order rate constants of 0.0121 and 0.0073 min-1 , respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.