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Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase.

ABSTRACT: 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.

SUBMITTER: Garsetti D

PROVIDER: S-EPMC1131962 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundThe aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts.MethodsUsing flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots.ResultsWe demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRβ, a marker associated with retinoic acid syndrome was also increased.ConclusionsOur data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.

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Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase.

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