leukotriene-b4 and Leukemia--Myeloid--Acute

Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline-based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating the synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

Topics: Antineoplastic Agents; Cell Self Renewal; Daunorubicin; Drug Resistance, Neoplasm; Humans; Leukemia, Myeloid, Acute; Leukotriene B4; Neoplastic Stem Cells

Several reports have demonstrated an important role of leukotriene B(4) (LTB(4)) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB(4), 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M(0-2) and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB(4) in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A(2) suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB(4), 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB(4). However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB(4), 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB(4)-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Apoptosis; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blast Crisis; Calcium; Cell Proliferation; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Leukemia, Myeloid, Acute; Leukotriene B4; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Messenger; Thromboxane A2

Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in acute myeloid leukemia (AML) cells (16 clones) and G-CSF mobilized peripheral blood CD34+ cells. CD34+ cells from patients with non-myeloid malignancies expressed cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase but not 5-lipoxygenase (5-LO). The enzyme cPLA2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5-LO, FLAP, and LTA4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187-induced release of [14C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA2 protein. Biosynthesis of LTB4, upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1-(14)C] AA release nor 5-LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease.

Topics: Adult; Aged; Antigens, CD34; Arachidonate 5-Lipoxygenase; Calcimycin; Female; Gene Expression Regulation, Leukemic; Granulocyte Colony-Stimulating Factor; Humans; Ionophores; Leukemia, Myeloid, Acute; Leukotriene B4; Male; Middle Aged; Models, Biological; Phospholipases A2, Cytosolic

Exposure of a human leukemic cell line HL-60 to 1% dimethylsulfoxide (DMSO) for 4 days induced myeloid differentiation. DMSO-differentiated HL-60 cells displayed high and low-affinity binding sites for leukotriene B4 (LTB4). The pretreatment of myeloid differentiated HL-60 cells with 1-10 nM LTB4 enhanced superoxide production evoked by 100 nM formyl-methionyl-leucylphenylalanine (fMLP) to 127-137% of the controls stimulated by fMLP alone. A concentration eliciting a half maximal increase (EC50) of LTB4 for the enhancing effect on superoxide production evoked by fMLP was 0.32 nM. This was roughly similar to the dissociation constant (Kd) of high affinity receptors for LTB4 (0.23 nM). These results suggest that high affinity receptors transduce the enhancing effect of LTB4 on fMLP-induced superoxide production. Although it seems possible that enhancement of fMLP-induced superoxide production is associated with a substantial increase and/or an affinity alteration in receptors for fMLP, LTB4-pretreated cells failed to show significant changes in fMLP binding compared to non-pretreated ones. It seems likely that Ca2+ influx transduces enhancement of fMLP-induced superoxide production, because extracellular Ca2+ is necessary for an enhancing effect of fMLP-induced superoxide production. Also, EC50 of LTB4 for Ca2+ influx (0.78 nM) was similar to that of the enhancing effect of superoxide generation evoked by fMLP. Although pretreatment of LTB4 failed to enhance the maximal level of fMLP-induced intracellular Ca2+ rise, transient overshoot in intracellular Ca2+ evoked by fMLP declined more rqpidly after LTB4 pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcium; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Immunologic; Receptors, Leukotriene B4; Superoxides

The ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose-dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12-myristate 13-acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H-7. Preincubation of the cells with the synthetic chemotactic factor fMet-Leu-Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet-Leu-Phe and platelet-activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL-60 cells. However, the treatment of the HL-60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.

Topics: Calcium; Cell Line; Cholera Toxin; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid, Acute; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Propionates; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

Thrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well-known chemotactic agents such as fMet-Leu-Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL-60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP-alpha-thrombin, as well as the thrombin-derived chemotactic peptide CB67-129, elicits a dose-dependent chemotactic response in HL-60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL-60/mono), whereas no such response is evident in either undifferentiated HL-60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL-60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte-differentiated cells. In HL-60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15-20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL-60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL-60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL-60/mono (but HL-60/gran nor undifferentiated HL-60) are capable of significant specific 125-I-labeled alpha-thrombin-binding (ka approximately 20 nM), and possess an estimated 400,000 thrombin-binding sites per cell. Our findings further suggest that the thrombin response of HL-60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.

Topics: Actins; Calcium; Cell Differentiation; Cell Line; Chemotaxis; Chemotaxis, Leukocyte; Complement C5; Complement C5a; Cytoskeleton; Humans; Leukemia, Myeloid, Acute; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Peptide Fragments; Prothrombin; Thrombin

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.

Topics: Arachidonic Acids; Calcimycin; Calcium; Cell Differentiation; Cell Line; Chromatography, High Pressure Liquid; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Leukotriene B4; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; SRS-A

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.

Topics: Calcitriol; Calcium; Cell Differentiation; Cell Line; Cytosol; Humans; Leukemia, Myeloid, Acute; Leukotriene B4; Receptors, Cell Surface; Receptors, Leukotriene; Receptors, Prostaglandin

Exposure of HL-60 cells for 6 days to a combination of 1.25% (v/v) dimethyl sulfoxide (DMSO) and 10 microM dexamethasone (DEX) induces myeloid differentiation which results in a cell with many of the characteristics of a mature granulocyte. At 4 degrees C myeloid differentiated, but not undifferentiated, monocytic differentiated or eosinophilic differentiated HL-60 cells display marked specific leukotriene B4 binding. Leukotriene B4 binding at 4 degrees C reaches a maximum within 10 min, is readily reversed by unlabelled leukotriene B4, and is stereospecific. Only molecules with structural and biological similarity to leukotriene B4 can competitively inhibit leukotriene B4 binding. Scatchard analysis at 4 degrees C in differentiated cells shows two classes of binding sites. The high affinity sites have a Kd of 0.27 nM and a Bmax of 14.8 fmoles/10(7) cells; the low affinity sites have a Kd of 0.58 microM and a Bmax of 2453 fmoles/10(7) cells. The appearance of specific leukotriene B4 binding sites in the myeloid differentiated cells correlates with their ability to chemotax in response to leukotriene B4. Undifferentiated cells do not chemotax to leukotriene B4. At 37 degrees C leukotriene B4 is incorporated into phospholipid and triglyceride species in both undifferentiated and myeloid differentiated HL-60 cells making binding studies at 37 degrees C in intact cells impossible. Interestingly, incubation of the cells with cyclooxygenase inhibitors during differentiation enhances receptor density. No evidence of omega-hydroxylase activity was found in HL-60 cells. These data suggest that the HL-60 cell may be an excellent model system for the study of leukotriene B4 receptor binding, processing and gene expression.

Topics: Binding, Competitive; Cell Differentiation; Cell Line; Chemotaxis; Dexamethasone; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid, Acute; Leukotriene B4; Receptors, Immunologic; Receptors, Leukotriene B4

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukemia, Myeloid, Acute; Leukotriene B4; Lipoxygenase; Prostaglandin-Endoperoxide Synthases

We have begun to characterize the development of the excitation-response coupling sequence in the human promyelocytic leukemia cell line HL60. Using the recently developed fluorescent calcium probe quin-2, it was found that DMSO induced myeloid differentiation of the HL60 cells is accompanied by the development of a calcium response to the addition of the chemotactic factors fMet-Leu-Phe and leukotriene B4. The characteristics (time course, concentration dependence, stereospecificity, and metabolic dependence) of the calcium response are extremely similar to those previously described in human neutrophils. These results imply that functional receptors for leukotriene B4 appear in HL60 cells upon the induction of differentiation and also lend strong support to the use of these HL60 cells as a model of human myeloid differentiation. We have also characterized the emergence of a secretory response to fMet-Leu-Phe and leukotriene B4 in cytochalasin B treated HL60 cells. In addition, it is found that differentiation was required for the calcium ionophore A23187 to express its secretory activity toward the HL60 cells. This last set of results implies that differentiation is accompanied by the coordinated appearance of surface receptors and cytoplasmic factors required for the expression of cellular responsiveness.

Topics: Aminoquinolines; Calcium; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Fluorescent Dyes; Humans; Kinetics; Leukemia, Myeloid, Acute; Leukotriene B4