Leukemia (2000) 14, 2118–2127 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Modifications in phospholipase D activity and isoform expression occur upon maturation and differentiation in vivo and in vitro in human myeloid cells M El Marjou1, V Montalescot1, A Buzyn2 and B Geny1 1INSERM U332, Laboratoire de Signalisation, Inflammation et Transformation Cellulaire, ICGM, Paris; and 2INSERM U445, CHU Cochin- Port-Royal, Paris, France Activation of phospholipase D (PLD) occurs in response to process of maturation and differentiation is important for the various stimuli and results from the activity of two isozymes, development of AML therapies. hPLD1 and hPLD2. PLD activity appears to be involved in sev- eral myeloid cell processes during their development and acti- Over the past two decades, phospholipase D (PLD) has vation, including proliferation of myeloblasts in the bone mar- been well documented to play an essential role in signal trans- row and secretion, phagocytosis and NADPH oxidase duction in a large variety of mammalian cells.4 Its activation activation, essential functions of differentiated neutrophils. The leads to hydrolysis of phosphatidylcholine, producing choline present work studies PLD characteristics, activity and both iso- and phosphatidic acid (PA), a phospholipid recognized as a zyme expression during maturation and differentiation of second messenger (see review Ref. 5). Two PLD isoforms have myeloid cells by using three different systems: leukemic mye- been cloned in human cells6,7 and were shown to be differ- loblasts at different stages of maturation, terminally differen- 8,9 tiated neutrophils ex vivo and four human myeloid cell lines, ently expressed in most mammalian cell types and tissues. NB4, HL-60, PLB 985 and U937, induced to differentiate with all- During the past few years, many studies have been focused trans retinoic acid (ATRA), a cyclic adenosine monophosphate on PLD activity which indicate that this enzyme activity might (cAMP) analogue or both agents together. HL-60, a bipotential be involved in numerous physiological processes of myeloid cell line has also been differentiated along the granulocytic cells, such as proliferation, differentiation, phagocytosis, pathway with DMSO and the monocytic pathway with 1,25-dihy- droxy vitamin D3. In all these systems, PLD activity increases secretion and NADPH oxidase activation (see review Ref. 4). with maturation and differentiation whatever the inducer used Moreover, in mammals, myeloid cells appear to be amongst and the granulocytic or monocytic pathways. Increase in basal the richest in both PLD isozyme mRNAs.8 Despite major inter- activity which reflects the expression during development of est in the understanding of the relative participation of each both hPLD1 and hPLD2 appears to be mainly related to the for- PLD isoform in physiological processes, their respective roles mer isozyme expression. Association of PLD characteristic in myeloid cell functions are still unknown. changes with maturation and differentiation was also con- firmed using two NB4 clones resistant to these processes. To get an insight on PLD changes during myeloid matu- Comparison between PLD characteristics in myeloblasts dur- ration and differentiation, the levels of PLD activity and PLD ing maturation and differentiation ex vivo and in vitro in the isoform expression were measured during these processes. different cell lines demonstrated that NB4 induced to differen- This study was performed in ex vivo circulating human leu- tiate with ATRA represents the best model for further studies kemic myeloblasts at different maturation and differentiation on the specific roles of each PLD isoform in various functions and in four different myeloid cell lines established in vitro of differentiated myeloid cells. Leukemia (2000) 14, 2118–2127. Keywords: myeloid cells; maturation; differentiation; phospho- from patients sufferring from different AML subtypes. HL-60 lipase D; PLD isoform expression cells now recognized to be established from an AML stage M2 (M2)10 and NB4 from a M3 AML carrying a t(15;17) translo- cation11 can be induced to differentiate towards neutrophil- Introduction like cells in the presence of all-trans retinoic acid (ATRA) and/or a cyclic AMP analogue. PLB 985 (M4) and U937 Early myeloid cells are able to proliferate along the pathway (histiocytic), already irreversibly induced toward the mono- of maturation. Upon maturation, they lose this capacity and cytic lineage, differentiate towards monocytes in the presence differentiate mainly into neutrophils, and to a lesser extent into of ATRA. monocytes. Fully differentiated myeloid cells possess several In the present work, PLD activity and PLD isoform properties linked to their final role: prevent infection. Indeed, expression were studied in parallel upon maturation and dif- they are able to ingest and destroy microorganisms.1 ferentiation in ex vivo human leukemic myeloblasts and in Acute myeloblastic leukemia (AML) is due to abnormal pro- several myeloid cell lines induced to mature and differentiate liferation of myeloid cells blocked at a specific stage of matu- in vitro. ration and differentiation classified from M0 to M5 according to the FAB leukemia subtype nomenclature. AML is a frequent disease which still has a serious prognosis. For several years, Materials and methods patients suffering from M3 promyelocytic leukemia have been treated with a high rate of remission with all-trans retinoic RPMI-1640 and medium 199 were purchased from Gibco BRL acid (ATRA), a differentiating agent which forces leukemic (Paisley, UK). Fetal bovine serum was from Dutcher (Vilmorin, cells to differentiate, acquire new functions and stop prolifer- France). Alkyl-sn-glyceryl-3-phosphorylcholine, 1-0-[alkyl- Ј Ј 3 ation.2,3 1 2 - H]-, (30–60 Ci/mmol) (lyso-PAF), phosphatidylcholine Understanding the events taking place during the complex were obtained from NEN (Boston, MA, USA). fMLP, phorbol 12-myristate 13-acetate (PMA), dibutyryladenosine3Ј:5Ј-cyc- lic monophosphate (dibutyryl-cAMP), dibutyryl-cAM8-(4- Ј Ј Correspondence: B Geny, Unite´ Inserm 332, ICGM, 22 rue Me´chain, chlorophenylthio)adenosine cyclic 3 ,5 -monophosphate (8- 75014 Paris, France; Fax: 33 01 40 51 64 54 CPT-cAMP), dimethyl sulfoxide (DMSO), all-trans retinoic Received 31 March 3000; accepted 16 August 2000 acid (ATRA), 1,25-dihydrocholecalciferol (1␣,25-dihydroxy Modifications in phospholipase D activity and isoform expression M El Marjou et al 2119 vitamin D3), cytochrome C, nitro-blue tetrazolium (NBT), NBT reduction Hank’s and HEPES buffers were purchased from Sigma (St Louis, MO, USA). Ficoll–Hypaque Plus was from Pharmacia Cell maturation was estimated by nitroblue tetrazolium (NBT) Biotech (Upsala, Sweden). RNase protection kit, protease and reduction and by morphological changes after cellular stain- fatty acid-free bovine serum albumine (BSA), protease inhibi- ing with May–Gru¨nwald-Giemsa. Determination of NBT tor cocktail (Complete) was purchased from Boehringer- reduction was performed by incubation of 106 cells in 1 ml Mannheim (Mannheim, Germany). Transfer PVDF membranes fresh culture medium for 20 min at 37°C with 0.1% NBT and were purchased from Amersham (UK). Silica gel 60 plates for 0.3 ␮M PMA. The percentage of cells containing intracellular thin layer chromatography (TLC) analysis were obtained from blue-black formazan deposits was then determined. At least Whatman (Maidstone, UK). Scintillant liquid, Optiphase ‘HiS- 200 cells were examined. afe’ 3, was from EG&G Wallack (Turku, Finland). Superoxide ion production assay Purification of human neutrophils, monocytes and NADPH oxidase activity was determined by using the leukemic cells − reduction of cytochrome C by O2 as indicator. Reduction of cytochrome C was measured as the change in absorption at Protocol on human cells were approved by the CCPPRB 550 nm over 20 min at 20 s intervals in a spectrophotometer (Comite´ Consultatif pour la Promotion des Recherches en (Perkin Elmer Lambda 4 apparatus; Perkin Elmer, Norwalk, Biologie). Informed consent was obtained from all blood CT, USA). Cells (2 × 106) were resuspended in PBS containing donors and patients. 30 mM glucose, 1 mM MgCl2 and 0.5 mM CaCl2 and then pre- Neutrophils were isolated from venous blood of five healthy incubated with 50 ␮M of cytochrome C for 5 min at 37°C. blood donors. The platelet-rich plasma was first removed. Superoxide ion formation was initiated by the addition of N- Neutrophils and monocytes were isolated by dextran sedi- formyl Met-Leu-Phe (fMLP) (1 ␮M) or PMA (1 ␮M). mentation and Ficoll–Hypaque centrifugation. Neutrophils present in the pellet were further purified by elimination of contaminating erythrocytes by hypotonic shock. Purified neu- trophils were resuspended in HBSS containing 20 mM HEPES, Determination of phospholipase D activity in intact pH 7.4, 0.1% fatty acid-free BSA, and 5.6 mM glucose. Cell cells viability was measured by trypan blue exclusion and was greater than 95%. Peripheral blood monocytes present with PEt measurement: PLD activity was measured in intact other monocytic cells at the interface of the gradient were sep- cells as previously described.14 Briefly, after washing, cells arated from lymphocytes by adhesion to plastic for 2–3 h and were labelled for 30 min at 37°C with 3H-lyso-PAF (2 ␮Ci/ml) recovered for further processes by a 10-min incubation with in an isotonic medium buffered with Hepes, pH 7.4. After 10 EDTA. min preincubation in the same medium containing 1.5% etha- Peripheral blood mononuclear cells (PBMC) of 12 leukemic nol, PLD activity measurement was performed using 106 cells patients were collected between November 1995 and March (100 ␮l aliquots). The enzymatic reaction was stopped after 1998 before the initiation of chemotherapy. The PBMC were 10 min incubation at 37°C by addition of 500 ␮lof separated from total blood samples of patients with hyperleu- chloroform/methanol (1:1, v/v) and samples were processed kocytosis (WBC Ͼ10 × 109/l) by Ficoll–Hypaque gradient as previously reported.14 (Pharmacia Biotech) and frozen in 10% DMSO, 10% human AB serum.