Biosynthesis of leukotriene B4 in hematological malignancies

University dissertation from Stockholm : Karolinska Institutet, Department of Medicine

Abstract: Leukotrienes (LT) are biologically active metabolites of the fatty acid arachidonic acid (AA). After liberation of AA by phospholipase A 2 (PLA 2), this fatty acid can be converted to leukotrienes, lipoxins, prostaglandins or thromboxane. The conversion of AA to LTA 4 is catalyzed by five-lipoxygenase (5-LO). LTA 4 can then be further metabolized to LTB 4 or LTC 4 by the catalytic action of LTA 4 hydrolase or LTC 4 synthase, respectively. Cellular leukotriene synthesis is dependent on 5-LO activating protein (FLAP), a membrane protein that binds AA and facilitates the 5-LO reaction. LTB 4 is produced in myeloid cells and B-lymphocytes. Besides a role in various immunological and inflammatory reactions, several reports indicate that LTB 4 may have a role in the proliferation of myeloid and lymphoid cells. Cells from patients with precursor B-acute lymphoblastic leukemia (B-ALL) were studied. All eight investigated clones expressed the genes for FLAP, LTA 4 hydrolase and cyclooxygenase-1 (COX-1). Seven out of eight clones expressed COX-2. Four of the more mature cell clones expressed 5-LO but not cPLA 2 and the cells also produced LTB 4. The remaining four clones expressed cPLA 2 but not 5-LO and hence produced no leukotrienes. On the basis of the expression of cPLA 2 and 5-LO, two biologically different subsets of B-ALL were identified. This finding may be of clinical relevance in future treatment of B-ALL. Splice variants of the cytosolic calcium independent PLA 2 (iPLA 2) have been observed in myeloid and lymphoid cells and are suggested to regulate enzyme activity. The expression of iPLA 2 and its role in leukotriene synthesis was studied in immature myeloid cells and granulocytes. One additional splice variant, believed to function as a negative regulator of enzyme activity, was expressed in acute myeloid leukemia (AML) and HL-60 cells but not in granulocytes. Results obtained with inhibitors, suggest that iPLA 2 is involved in leukotriene synthesis in granulocytes. The majority of studied AML clones were found to express 5-LO, FLAP and LTA 4 hydrolase proteins. Only three of 16 clones produced similar amounts of leukotrienes as granulocytes upon calcium ionophore A23187 stimulation. Addition of exogenous AA and/or diamide, a redox active substance , resulted in activation of leukotriene synthesis. The AA release in AML cells was two to ten times less than that observed in granulocytes after calcium ionophore activation. The expression of cPLA 2 was high in all investigated clones. However, cPLA 2 may be activated by other mechanisms than calcium influx in AML cells, and contribute to proliferation in AML, and thus be a putative target in this disease. B-cell chronic lymphocytic leukemia (B-CLL) cells produced low amounts of LTB 4 after stimulation with A23187 and AA but similar amounts in homogenates as granulocytes. B-CLL cells expressed the high affinity LTB 4 receptor BLT1. Cultivation of B-CLL cells with CD40-ligand-transfected fibroblasts stimulated DNA synthesis and the expression of the activation markers CD23, CD150 and the adhesion molecule ICAM-1 (CD54). The specific leukotriene biosynthesis inhibitors MK886 and BWA4C counteracted this stimulation. Addition of exogenous LTB 4 (150 nM) almost completely reversed the effect of the inhibitors. In summary, these studies indicate that there are several enzymes and receptors in the arachidonic acid cascade that might be putative drug targets. Such drugs may have a therapeutic role in certain malignant hematological diseases.

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