Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase.
Treatment of HL-60 cells with 0.5 mM-butyric acid resulted in morphological changes, including the formation of cytoplasmic granules, nuclear condensation and segmentation. These differentiated cells had an elevated phospholipase A2 activity and an increased capacity to synthesize a variety of eicosanoids, including both lipoxygenase and cyclooxygenase products. Phospholipase A2-mediated release of arachidonic acid is accompanied by an equimolar production of potentially cytotoxic lysophospholipid. In association with the differentiation process, there was a 2-3-fold increase in lysophospholipase activity. Subsequent studies were undertaken to identify and characterize the lysophospholipases in this cell system, with 1-[1-14C]palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine as substrate. Hydrophobic chromatography of both undifferentiated and differentiated cell extracts revealed three peaks of enzyme activity. Extracts of differentiated cells contained a dramatic increase in activity contained in peak 2. The increase in enzymic activity of peak 2 appeared to account for the increase in total lysophospholipase activity found in the differentiated cell homogenates. The lysophospholipases contained in peaks 2 and 3 were purified to homogeneity and were 20 and 22 kDa respectively, as determined by denaturing polyacrylamide-gel electrophoresis. Peaks 2 and 3 were similar on the basis of amino acid composition, but had distinctive C-terminal peptide amino acid sequences. Enzymic characterization of these proteins demonstrated that there was no detectable level of non-specific esterase, acyltransferase or transacylase activity associated with these proteins. We concluded that peak 2 lysophospholipase is regulated by differentiation in HL-60 cells and may play an important role in protecting these cells from the cytolytic effects of the lysophospholipids produced by the activation of phospholipase A2.
Garsetti
D
D
Schering-Plough Research, Bloomfield, NJ 07003.
Holtsberg
F
F
Steiner
M R
MR
Egan
R W
RW
Clark
M A
MA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003594:Cytoplasmic Granules / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology*; D007951:Leukemia, Myeloid / Q000201:enzymology* / Q000473:pathology; D008245:Lysophospholipase / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000378:metabolism / Q000502:physiology; D054467:Phospholipases A2; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000596:Amino Acids; D002087:Butyrates; D007527:Isoenzymes; D020148:Butyric Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase