13-hydroxy-9-11-octadecadienoic-acid and Leukemia--Basophilic--Acute

The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)- and 15(S)-HETE and 13(S)-HODE (12- and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL-1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [3H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F2alpha. In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETE-protein complexes as this compound both inhibited the 5-lipoxygenase and interfered with the binding of [3H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE > 5(S)-HETE approximately equal to 15(S)-HETE > > stearic acid approximately equal to arachidonic acid approximately equal to 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Biotinylation; Carrier Proteins; Cytosol; Dinoprost; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Ligands; Linoleic Acid; Linoleic Acids; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

Apart from the generation of potent inflammatory mediators, the effects of fatty acid oxygenase activation, per se, on the host cell have not been well-delineated. Fatty acid oxygenases were activated in rat basophilic leukemia cells (RBL-1) by incubating them for 2-4 hr with 33-300 microM of arachidonic acid (AA) or linoleic acid (LA). As a control, the cells were incubated with one of two analogs of these fatty acids which are not oxygenase substrates: eicosatetraynoic acid or linoelaidic acid. Effects of oxygenase activation on cell viability were monitored by an assay for mitochondrial function. Cytotoxicity occurred in incubations with exogenous AA or LA in direct proportion to the substrate concentration but was not found in the control incubations or in incubations with the principal monohydroxylated AA products, 5-, 15-, and 12-HETE. Nordihydroguaiaretic acid (80 microM) and alpha-tocopherol (100 microM) significantly decreased the cell death observed during incubations with AA or LA. It is concluded that extensive oxygenase activation can result in cell death from intermediates produced proximal to the stable monohydroxylated derivatives.

Topics: Animals; Arachidonic Acid; Cell Survival; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Linoleic Acid; Linoleic Acids; Lipoxygenase; Masoprocol; Rats; Tumor Cells, Cultured; Vitamin E