Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors

Regulatory Peptides 68 (1997) 91–97

Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors a

a

a

a

Marie-Noelle Mathieu , Daniel Vittet , Marie-France Laliberte´ , Franc¸ois Laliberte´ , a a b a, Isabelle Nonotte , Dalil Hamroun , Jean-Marie Launay , Claude Chevillard * a

b

INSERM U 300, Faculte´ de Pharmacie, Montpellier, France Laboratoire de Biologie Cellulaire, Faculte´ de Pharmacie, Paris, France

Received 30 May 1996; revised 18 October 1996; accepted 18 November 1996

Abstract The aim of the present investigation was to determine whether endothelin (ET) could be expressed in and released from the human leukemia megakaryoblastic cell lines HEL, MEG-01, DAMI and the normal human platelet progenitors. Using the reverse transcriptase– polymerase chain reaction (RT–PCR) on total RNA isolated from the cells, we amplified a cDNA of the expected size (453 bp). Southern-blotting hybridization revealed that RT–PCR products from the cell lines were specific of ET-1 mRNA. Immunocytochemical analyses highlighted immunoreactive ET-1 in the cytoplasm of these cells which also released the mature peptide. ET-1 release from the three cell lines was increased by thrombin exposure. Although MEG-01 cells express ET receptors, ET-1, the selective ETB agonist sarafotoxin 6C and the non-selective ET-receptor antagonist PD 142893 showed no proliferative or antiproliferative action in basal or stimulating medium. This indicated a lack of autocrine ET-mediated effect on growth. These results demonstrate for the first time that human megakaryoblastic leukemia cell lines and normal bone marrow platelet precursors express ET-1 mRNA and release the mature peptide.  1997 Elsevier Science B.V. Keywords: Endothelin; MEG-01; HEL; DAMI cell lines; Glycoprotein IIb / IIIa immunodetection; RT–PCR

1. Introduction Endothelins (ETs) are a family of peptides (ET-1, ET-2 and ET-3) which display high homology, but whose precursors are encoded by different genes. ET-1 was originally identified in the culture medium of porcine endothelial cells [1], but ET-1 and / or other isoforms were subsequently found in other cell types (see [2] and [3] for reviews). Many cancer cells have been shown to produce ETs which could display autocrine / paracrine effects on cell growth. In human cancer cell lines, ETs have been detected in cell extracts and / or spent medium of many cells of different origin [4–10]. *Corresponding author. Tel: (33-04) 6752-4692; Fax: (33-04) 67042140. 0167-0115 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0167-0115( 96 )02108-8

Among these studies, Kusuhara et al. [5] showed that the frequencies of ET-1 production by cancer cell lines were high in mammary, pancreatic and colon carcinoma cell lines, whereas they did not detect any ET-1 in 10 cell lines derived from promyelocytic, T-cell and myelocytic leukemia or Burkitt’s lymphoma. However, no investigations have been performed to date to search for ET in human megakaryoblastic leukemia cell lines. We therefore analysed the expression of ET-1 mRNA in three of such cell lines: HEL [11], MEG-01 [12] and DAMI [13], and the presence of ET-1 in their culture media. In addition, since MEG-01 cells have been shown, contrary to HEL and DAMI, to express ET receptors [14], and as ETs have been shown to display autocrine / paracrine mitogenic actions on cancer cells [6,8], we investigated the effects of ET-1 and of ET-receptor blockade on MEG-01 cell growth.

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Since all three cell lines were found to express ET-1, it was of interest to determine whether this property was related to their malignant or to their megakaryoblast-like nature. Consequently, we also analysed the expression of ET-1 in human normal bone marrow cells sorted with respect to their positivity to glycoprotein (GP)IIb / IIIa, a specific marker of the megakaryocyte / platelet lineage.

2. Materials and methods

2.1. Cell culture of human leukemia cell lines HEL, MEG-01 and DAMI HEL cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine and 10% fetal calf serum (FCS). MEG-01 and DAMI cells were grown in RPMI 1640 medium supplemented with 2 mM glutamine, 1 mM Na-pyruvate, 0.4% non-essential amino-acids and 10% FCS (MEG-01) or horse serum (DAMI). Cells were maintained at 378C in a humidified atmosphere of 95% air–5% CO 2 .

2.2. Purification of GPIIb /IIIa positive cells from human bone marrow GPIIb / IIIa positive cells were purified from human normal marrow according to the method described by Miyazaki et al. [15]. This three-step procedure included in the following order: separation of marrow cells on a discontinuous Percoll gradient, adhesion to plastic culture dishes and specific immunoadsorption of non-adherent cells to dishes coated with a monoclonal antibody to human GPIIb / IIIa. After rinsing twice with phosphate buffered saline (PBS), adherent cells were removed from the dishes by vigorous pipetting.

2.3. Reverse transcriptase–polymerase chain reaction ( RT–PCR) and southern blotting Total RNA was extracted from the cells using a guanidinium thiocyanate cesium chloride method [16,17]. Oligonucleotides specific to the human ET-1 sequence [18]: forward primer 59-TCGTCCCTGATGGATAAAGAGTGTGTC (bp 166–192), backward primer 59-GGTCACATAACGCTCTCTGGAGGGCTT (bp 618–592) were used. The sequence between and including the two primers was 453 bp long and comprised three introns. First strand cDNA was synthesized using 1.6 mg of total RNA, 100 U MMLV reverse transcriptase (Gibco, France), 17.5 U human placental ribonuclease inhibitor (Amersham, France), 40 pmol backward primer, 0.5X buffer for MMLV, 20 mM dithiothreitol, 0.8 mM of each dNTP in a 25 ml final volume, for 1 h incubation at 378C. The reaction was stopped by heating at 958C for 5 min. cDNA amplification was carried out in a 50 ml final

volume. 4 ml of reverse transcriptase reaction and 4 U Taq DNA polymerase (Promega, France) were added to PCR buffer [67 mM Tris–HCl (pH 8.8), 16.6 mM (NH4) 2 SO 4 , 2 mM MgCl 2 , 50 mM KCl, 5% dimethylsulfoxide, 8 mg BSA, 10 mM b-mercaptoethanol], containing 0.25 mM dNTP. Samples were overlaid with mineral oil and heated in a PHC2 thermal cycler (Techne, UK) to 928C for 5 min, then 648C for 5 min. At this temperature, 25 pmol of each oligonucleotide were added (hot start). 30 cycles were conducted in three temperature steps: 3 min at 758C, 1 min at 928C and 1 min at 648C. Finally, the sample was incubated at 758C to achieve all primer extension. A ‘‘blank’’ was prepared without cDNA template. An aliquot of each PCR reaction mixture was submitted to electrophoresis on 1.5% agarose gel in Tris borate / EDTA buffer containing ethidium bromide for visualisation. After denaturation, DNA was blotted onto Hybond N membrane (Amersham, France) by electrophoretic transfer (Hoefer Scientific Instruments, USA) in 25 mM sodium phosphate for Southern blotting. An oligonucleotide probe (59-GACCCAAATGATGTCCAGGTG, bp 222–202) specific to an ET-1 sequence occurring between the two PCR primers, was end-labelled with (30 pmol, 90 mCi) ATP g 32 P (Amersham, France) using 20 U T4 polynucleotide kinase (New England Biolabs, USA) for 1 h at 378C. The labelled probe was then purified on a G25 Sephadex column. The membrane was prehybridized for 5 h at 658C and hybridized overnight at 658C with the labelled probe (5 ? 10 6 cpm / ml) in a solution containing 6X SSC, 5X Denhardt, 0.5% SDS, 100 mg / ml salmon sperm DNA. The membrane was washed at 658C for 30 min in 2X SSC, 30 min in 2X SSC, 0.1% SDS and finally 10 min in 1X SSC, then exposed to Kodak X-Omat AR film with two intensifying screens at 2 808C for 6 days.

2.4. Immunocytochemical procedures Cells were rinsed twice with PBS, centrifuged (32.2g, 5 min) in a Cytospin 2 centrifuge (Shandon, France) and fixed in methanol at 2 208C for 5 min. After drying, the cells were rehydrated with PBS for 10 min and treated for indirect immunofluorescence. They were incubated for 60 min at room temperature with a monoclonal antibody to ET-1 (MA3-005, Affinity Bioreagents, USA), 1:60 dilution of mouse ascitic fluids in PBS for MEG-01, DAMI and GPIIb / IIIa positive bone marrow cells, and 1:30 dilution for HEL. They were then rinsed 3 times for 10 min with PBS, incubated with the second antibody: FITC-conjugated rabbit F(ab9)2 anti-mouse IgG (Dako, France), 1:50 dilution in PBS for 30 min, washed 3 times for 10 min with PBS and mounted with Citifluor (Citifluor Ltd, UK). All these procedures were carried out at room temperature. Controls for non-specific immunostaining had used purified IgG1 from mouse ascitic fluid (Immunotech S.A., France) instead of primary antibody. It was used at 1:10

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dilution in PBS (3 or 6 times less diluted than primary antibody).

2.5. ET extraction from culture medium and radioimmunoassay Cells were washed twice with RPMI, replaced at 2 ? 10 5 cells / ml in a serum-free RPMI medium, then subjected to further incubations for different times at 378C under 95% air–5% CO 2 , in the absence or with 5 U / ml thrombin. After centrifugation, 2 ml of each conditioned medium were acidified with trifluoroacetic acid (TFA) and applied to Sep-Pak C 18 cartridges (Peninsula, USA) previously equilibrated with methanol and rinsed with 0.1% TFA. The columns were washed with 5 ml 0.1% TFA and 5 ml 1% acetonitrile in 0.1% TFA. ETs were eluted with 3 ml 60% acetonitrile in 0.1% TFA. After evaporation under a stream of nitrogen, the dried material was dissolved in 0.5 ml RIA buffer (0.1 M sodium phosphate buffer, pH 7.5, containing 0.1% BSA and 0.1% Triton X-100). The recovery of synthetic ET-1 during this procedure was 85%. The reconstituted samples were subjected to radioimmunoassay, using a commercial RIA kit (Amersham, France). The assay was ET(1-21) specific with no cross reactivity with big-ETs and unrelated peptides.

Fig. 1. Analysis of PCR amplified cDNA from HEL, MEG-01 and DAMI cell lines and normal GPIIb / IIIa bone marrow positive cells. RT–PCR was performed as described in Section 2 and the products were analysed on 1.5% agarose gels. Lanes 1, 4 and 7: molecular weight markers; lane 2: RNA from HEL; lane 3: PCR without cDNA; lane 5: RNA from MEG-01; lane 6: RNA from DAMI; lane 8: RNA from GPIIb / IIIapositive bone marrow cells; lane 9: RT–PCR without RNA. The size of the amplified products (453 bp) is indicated with arrows.

2.6. Effect of ET receptor agonists and antagonist on MEG-01 cell growth 1 ml cell suspension in the exponential phase of growth was plated in 24-well plates at a starting concentration of 2 ? 10 5 cells. The culture conditions were as described with 0, 1, 5 or 10% FCS, without or with ET-1 (1, 10, 100 nM and 1 mM), sarafotoxin 6C (1 mM), PD 142893 (10 mM) and ET-1 antibody (1:1000 and 1:100 dilution). After 48 h, cell counts and cell size profiles were performed using a ZM Coulter counter (Coultronics SA, France).

3. Results

Fig. 2. Southern-blot analysis of PCR products from MEG-01, HEL and DAMI cell lines. After denaturation then transfer to Hybond N membrane, they were probed with a 32 P labelLed oligonucleotide specific for an ET-1 sequence. Lanes 1 to 3 represent blots from MEG-01, HEL and DAMI, respectively.

3.1. Cellular expression of ET-1 mRNA 3.2. Immunocytochemistry Fig. 1 shows that reverse transcription reactions followed by PCR amplified a cDNA fragment of the expected size (453 bp) in HEL, MEG-01 and DAMI cell lines. This fragment was not amplified when cDNA was omitted in the reaction mixture. The RT–PCR products were shown to be specific to ET-1 mRNA by Southern blot hybridization with a specific labelled ET-1 oligonucleotide probe (Fig. 2). The RT–PCR procedure applied to total RNA purified from GPIIb / IIIa positive cells sorted from human bone marrow also amplified a 453 bp fragment, which showed ET-1 mRNA expression in these cells (Fig. 1).

Immunoreactive ET-1 was detected by indirect immunofluorescence in the three human megakaryoblastic cell lines MEG-01, HEL and DAMI as well as in GPIIb / IIIapositive human bone marrow cells (Fig. 3). When the monoclonal primary antibody to ET-1 was replaced by IgG1 from mouse ascitic fluid, no fluorescent signal could be observed (not shown).

3.3. Immunoreactive ET in the cell culture medium ET (1-21) was detected in the culture medium of the

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Fig. 3. Immunofluorescence staining of ET-1 immunoreactivity on cytocentrifuged preparations of MEG-01, HEL and DAMI megakaryoblastic cell lines and for GPIIb / IIIa-positive bone marrow cells. The first antibody was a monoclonal antibody directed against ET-1, and the second was a FITC-conjugated rabbit F(ab9)2 anti-mouse IgG. a: MEG01, b: HEL, c: DAMI, d: GPIIb / IIIa-positive bone marrow cells. First antibody diluted 1:60 for MEG-01, DAMI and bone marrow cells, 1:30 for HEL, magnification ( 3 55).

three cell lines and in that of GPIIb / IIIa-positive bone marrow cells. A serial dilution curve of extracts obtained from MEG-01 cells paralleled that of standard ET-1 in the RIA (data not shown). The amounts of ET (pg / 10 6 cells) found in the spent media of HEL, DAMI, MEG-01 and bone marrow cells after 24 h incubation were 1.560.6, 1.160.5, 4.360.8 and 2.860.7, respectively (m6SE, n:5). We also showed that ET was released as a function of time from the three cell lines and that thrombin treatment caused a significant increase in release from these cells, especially from MEG-01 (Fig. 4).

3.4. Action of ET agonists and antagonist on MEG-01 growth ET-1, sarafotoxin 6C or PD 142893 did not modify the proliferation of MEG-01 cells, whereas FCS, as expected, increased it in a concentration-dependent fashion. Neither ET-1 nor PD 142893 nor sarafotoxin 6C affected the FCS-induced increase in MEG-01 cell proliferation (Fig. 5). Lower concentrations of ET-1, as well as a neutralizing

Fig. 4. Time-course for the release of immunoreactive ET from MEG-01, HEL, and DAMI (upper to lower level) megakaryoblastic cell lines, in the absence (s) or presence (m) of 5 U / ml thrombin. Cells were incubated for 0–8 h in a serum-free medium. The spent medium was collected at the indicated times and assayed for the ET concentration as described in Section 2. Values represent mean6SE of 5 individual experiments. * p , 0.05 by Student’s t-test.

ET-1 antibody also had no effect on cell number (not shown). None of the agents used above were able to modify the size distribution of the MEG-01 cell population (cell diameter: 13–23 mm, with a mean of 17 mm), not shown.

4. Discussion The present study shows for the first time that human

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Fig. 5. Effect of ET-1, of the selective ETB receptor agonist sarafotoxin 6C (SRT 3 6C) and of the non-selective ET receptor antagonist PD 142893 on MEG-01 cell proliferation. Cells were plated at the initial density of 0.2 3 10 6 cells / ml and treated with drug solvent (Controls), ET-1, SRT 3 6C or PD 142893 at different FCS concentrations. Cells were counted after 48 h drug exposure. Columns are means and vertical bars SE of 4–6 individual experiments. *p , 0.05 by Student’s t test.

megakaryoblastic leukemia cell lines express ET-1 mRNA, produce and release the mature peptide. Although these cell lines, in particular MEG-01 and DAMI, are generally considered to be good cell models of the megakaryocyte lineage, they nonetheless exhibit some phenotypic properties of other hematopoietic lineages. This raises the question as to whether or not ET-1 expression is specifically related to the megakaryoblastic-like phenotype or to that of an additional other hematopoietic lineage. The fact that the three cell lines used here have some properties of the megakaryoblastic lineage in common, along with different additional properties highlights a relationship between the megakaryocyte lineage phenotype and the ET-1 expression. Indeed, HEL expresses erythroid properties [11], MEG-01 cells were shown to exhibit a B-cell antigen, but were devoid of erythroid phenotype [12], whereas the DAMI cell line does not express the lymphoid, monocyte, granulocyte or macrophage specific antigens, but bears glycophorin A [13]. The further observation that GPIIb / IIIa-positive cells purified from human bone marrow also express ET-1 is clear evidence of a strong link between ET-1 expression and the megakaryocyte lineage. The three leukemia megakaryoblastic cell lines and bone marrow GPIIb / IIIa-positive cells were able to release ET-1 in their culture medium in a time-dependent manner. However, the amounts released were less than those secreted by human umbilical vein endothelial cells [19], bovine endothelial cells or the human endothelial cell line EA.hy.926 [20]. As the three cell lines used were shown to be thrombin sensitive [14], and as thrombin has been found to be one of the factors which enhance prepro-ET mRNA expression in several cell types and / or release of mature peptides from

these cells [21–25], we investigated whether thrombin was able to release ET-1 into the conditioned medium. Our data showed that thrombin caused a marked increase in ET-1 release from MEG-01 cells, and to a lower extent from HEL and DAMI cell lines. Given that thrombin was able to enhance ET-1 as early as 2 h after the onset of treatment, it is likely that ET-1 production was mainly post-transcriptionally regulated in response to thrombin stimulation of megakaryoblastic cell lines, as it was in astrocytes [24]. Previous results from our laboratory have shown that the MEG-01 megakaryoblastic cell line, in contrast to HEL and DAMI, which were insensitive to ETs [14], responded to ET isoforms by increasing intracellular ionized calcium and inositol phosphates [26], and predominantly expressed ETB receptor subtypes [27]. As MEG-01 cells also express ET-1 mRNA, contain ET-1 and release ET-1 into their culture medium, we examined the effects of ET-1, of the selective ETB agonist sarafotoxin 6C, of the non-selective ET receptor antagonist PD 142893 and of ET-1 neutralizing antibody on MEG-01 cell proliferation, in order to determine whether this cell line could represent a novel model of autocriny for ETs. Our data showed that none of these agents were able to alter cell growth in basic or FCS stimulated medium. These negative results suggest that ET-1 released from the cells did not appear to participate, either alone or in association with FCS growth factors, in the proliferation of MEG-01 cells. In the MEG-01 cell line, an autocrine / paracrine action on growth mediated by ET-1 was not thus observed, as it was in some other cell types (see [2] and [3] for review). The observation that MEG-01 cells only bear the ETB receptor subtype [27], which mediates nitric oxide release, could explain the lack of effect of ET agonists or antagonist on cell growth. On the

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other hand, the ET-1-releasing effect of thrombin on MEG-01 cells could not be related to the previous observation that thrombin is antiproliferative on this cell line [28]. Indeed, ET-1 is neither proliferative nor antiproliferative on MEG-01 cells, as demonstrated above, and did not modify cAMP levels in these cells [26], whereas thrombin was shown to exert its antiproliferative action via a cAMPdependent mechanism [28]. When considering differentiation, one of the main steps of megakaryoblast maturation to megakaryocyte is increased size, a property also exhibited by phorbol ester stimulated MEG-01 cells [28]. However, the ET analogs tested were unable to modify the MEG-01 size distribution, which showed a lack of effect of ETs on this differentiation parameter. The present findings that the human malignant cells HEL, DAMI and MEG-01, which present megakaryoblastic cell characteristics, express ET-1 mRNA, contain ET-1 and produce ET-1 raised the question as to whether these characteristics could be related to hematological malignancies which specifically affect the megakaryoblast–platelet lineage or to the megakaryoblastic phenotype. The subsequent finding that human normal marrow cells of the megakaryocyte–platelet lineage also expressed and released ET-1 indicates, most likely, the presence of an intrinsic ET system in bone marrow, like the postulated local renin–angiotensin [29], but its role on hematopoiesis remains to be determined.

Acknowledgments The authors thank Mrs C. Bellegarde for her editorial assistance. This study was supported by INSERM (France).

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