Leukemia (2000) 14, 142–152  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Establishment and characterization of a new human megakaryoblastic cell line (SET-2) that spontaneously matures to and produces -like particles K Uozumi1, M Otsuka1, N Ohno1, T Moriyama1, S Suzuki1, S Shimotakahara1, I Matsumura2, S Hanada1 and T Arima1

1Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima; and 2Department of Hematology and Oncology, Osaka University Medical School, Osaka, Japan

A new factor-independent megakaryoblastic cell line, desig- described. To date, only three cell lines (Meg-01,2,16 M-07e4 nated SET-2, was established from the peripheral of a and NS-Meg9) that spontaneously developed giant cells with patient with leukemic transformation of essential thrombocy- themia (ET). SET-2 expressed CD 4, 7, 13, 33, 34, 36, 38, 41, multilobed nuclei and produced platelet-like particles in 61, 71, 117, 126, 130 and c-mpl. In addition, it spontaneously culture supernatant have been reported. produced numerous platelet-like particles in liquid culture. We established a new human factor-independent mega- These particles were shown to be the same size as normal karyoblastic cell line, designated SET-2, from the peripheral , and to express CD 36, 38, 41, 61 and 71. Proliferation blood of a patient with leukemic transformation of essential of SET-2 was not influenced by thrombopoietin (TPO) and other thrombocythemia (ET). This cell line was shown to be unique, hemopoietic cytokines. SET-2 was found to express the plate- let-specific proteins such as platelet factor 4 and ␤-thrombo- in that it spontaneously matured to multinucleated megakary- globulin. The levels of expression were not altered by TPO. ocytes and produced platelet-like particles in culture super- SET-2 also secreted interleukin-6 into the supernatants, as well natants without addition of TPO and/or IL-6. As far as we as normal megakaryocytes. These results suggest that SET-2 know, SET-2 is the only human megakaryoblastic cell line spontaneously matures to megakaryocytes and produces plat- derived from ET. This cell line may be useful in the study of elet-like particles. These findings indicate that SET-2 may be the proliferation and differentiation of cells in ET and useful for investigating the proliferation and differentiation mechanisms of leukemia cells and the role of c-mpl on mega- in vitro megakaryocytopoiesis and thrombopoiesis. karyoblasts, megakaryocytes, and platelets in ET. Leukemia (2000) 14, 142–152. Keywords: megakaryoblastic cell line; SET-2; ET; platelet-like particles Materials and methods

Introduction Patient Several human megakaryoblastic and megakaryocytic cell lines have already been established and reported.1–10 Prolifer- A 71-year-old female was admitted to Kagoshima University ation in some cytokine-dependent megakaryocytic leukemia Hospital because of progressive malaise and fever on 25 May cell lines is stimulated by TPO.11–13 Expression of c-mpl is also 1995. She had a 3-year history of ET treated with platelet- observed in some megakaryocytic cell lines (eg M-07e and lowering chemotherapy with 50–100 mg of ranimus- CMK).14 In CMK, it has been observed that treatment with the tine (methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-␣- phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) D-glucopyranoside; MCNU) intermittently. At diagnosis of ET, induces dissociation of cytoplasm into numerous platelet-like the patient had no symptoms. Her peripheral blood counts segments. This alteration has not been observed in untreated were as follows: (RBC) 4.2 × 1012/l, hemoglobin cells.3 Similarly, the human leukemia cell line with mega- 13.1 g/dl, platelet 1.4 × 1012/l, and (WBC) karyocytic features, UT-7, contained multinucleated cells 14.2 × 109/l. Differential count of WBC revealed neutrophilia infrequently, then increased in size and developed multilobed and no immature and no . A bone mar- nuclei following treatment with phorbol 12-myristate 13-acet- row aspiration showed marked hyperplasia of the megakary- ate (PMA).5 Other megakaryoblastic cell lines, MKPL-16 and ocytes and the granulocytes. The patient had stainable iron in MEG-A2,8 also persistently developed giant cells carrying bone marrow and was not iron deficient. There was no fibrosis multilobed nuclei and occasionally multinucleated cells, of bone marrow. The leukocyte alkaline phosphatase activity respectively, without treatment with PMA. However, these was in the normal range. The bone marrow cytogenetic study two cell lines do not produce platelet-like particles in culture revealed a normal karyotype with 46, XX. The Philadelphia supernatants. Recently, a TPO-dependent human megakaryo- chromosome was absent. Therefore, the patient was diag- cytic cell line, UT-7/TPO, has been reported.15 This unique nosed as ET according to the criteria established by the Poly- cell line was heterogenous in size and contained about 5% cythemia Vera Study Group.17 At the current admission, her large multinucleated cells. A large multinucleated cell had peripheral blood counts were as follows: RBC 5.1 × 1012/l, processed pseudopods similar to a normal and hemoglobin 12.5 g/dl, platelet 0.06 × 1012/l with many bizarre small particles on the surface membrane. However, pro- giant platelets, WBC 94.4 × 109/l with 48.5% blasts. The bone duction of platelet-like particles from UT-7/TPO was not marrow aspiration was dry tap. Although the French–Amer- ican–British cooperative group (FAB) classification18 was undetermined, she was diagnosed with leukemic transform- ation of ET. Cytogenetic study and immunophenotyping of Correspondence: K Uozumi, Second Department of Internal Medi- cine, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, leukemia cells at leukemic transformation were not done. The Kagoshima 890-8520, Japan; Fax: +81-99-264-3504 patient died of hepatic and renal failure due to disseminated Received 11 January 1999; accepted 20 August 1999 intravascular coagulation on 2 June 1995. A new human megakaryoblastic cell line, SET-2 K Uozumi et al 143 Cell culture serum against extracellular domain of c-mpl19 by the Kirin Brewery Co., Ltd (Gunma, Japan). On 26 May 1995, a heparinized peripheral blood sample was obtained with the patient’s informed consent. Mononuclear cells were separated by gradient centrifugation using a cell Chromosome analysis separation tube (LeucoPREP; Becton Dickinson, Lincoln Park, NJ, USA). The cells were cultured in 25 cm2 flasks (Corning, Cultured cells were incubated for 1 h at 37°C in the presence Iwaki Glass, Tokyo, Japan) at a concentration of 1.0 × 106 of Colcemid (GIBCO BRL) at a concentration of 0.05 ␮g/ml. cells/ml in 10 ml of Dulbecco’s modified Eagle’s minimal Then, they were treated with 75 mmol/l KCl hypotonic sol- essential medium (DMEM) (GIBCO BRL; Life Technologies, ution for 30 min at 37°C and fixed by methanol-acetic acid Grand Island, NY, USA) containing 10% heat-inactivated fetal (3:1). Chromosomes were banded by the trypsin-Giemsa bovine serum (FBS) (Sanko Junyaku, Co. Ltd, Tokyo, Japan), methods. Karyotypes were described according to the Inter- − 10 5 M 2-mercaptoethanol (2-ME) (Nacalai Tesque, Kyoto, national System for Human Cytogenetic Nomenclature.20 Japan), 100 U/ml penicillin G (PCG) (Meiji Seika, Co. Ltd, Tokyo, Japan), 100 ␮g/ml streptomycin (SM) (Meiji Seika), and 10 ml MEM non-essential amino acid (NEAA) solution Detection of Epstein–Barr virus and mycoplasma (GIBCO BRL). All cultures were maintained in a humidified ° atmosphere at 5% CO2 and 37 C. The cultured medium was The cell line was determined to be free of mycoplasma con- changed once a week. The cell line was cloned by the limiting tamination by using enzyme-linked immunosorbent assay kit dilution method. Culture supernatants were harvested at (Boehringer Mannheim Biochemica, Mannheim, Germany) medium change and immediately centrifuged at 800 g for according to the manufacturer’s instructions. Testing for ° − ° 10 min at 4 C. Supernatants were stored in aliquots at 80 C Epstein–Barr virus infection was performed by EBV-encoded until analyzed. Cell count and viability were assessed by small RNA 1 (EBER-1) in situ hybridization (ISH) method as trypan blue dye exclusion. described previously.21

Morphological and cytochemical analysis Southern blot analysis of BCR gene rearrangement

Morphological observation of live cultured cells was made DNA extraction of SET-2 cells and Southern blot analysis were using an inverted microscope. Light microscopic examin- performed using standard methods. Approximately 10 ␮gof ations were performed on May–Gru¨nwald-Giemsa-stained extracted DNA was enzymatically digested with Bg/II and cytospin preparations. Myeloperoxidase (MPO), periodic acid- BamHI (TaKaRa Biomedicals, Tokyo, Japan). Digested DNA ␣ Schiff (PAS), -naphthylbutyrate esterase (ANBE), and naph- was hybridized to the 5Ј-bcr probe. thol ASD chloroacetate esterase (NASDCAE) were stained by standard methods. DNA fingerprinting

Electron microscopy DNAs were extracted from the primary leukemia cells, the resulting cell line and TAMA (control) cell line. Fingerprinting SET-2 cells were fixed with 2.0% glutaraldehyde for 120 min was carried out using a multilocus probe, designated 33.6 and at 4°C. The specimens were then washed with sodium caco- 33.15 (Non-Isotopic Chemiluminescent Enhanced (NICE) dylate and post-fixed with 1% osmium tetroxide, and embed- Probes; Cellmark Diagnostics, Abingdon, UK),22 as described ded in epoxy resin. They were examined with Hitachi H-7000 in detail previously.23,24 Briefly, 20 ␮g of DNA were digested electron microscope (Hitachi Ltd, Tokyo, Japan). to completion with HinfI and separated by electrophoresis on a 0.7% agarose gel. DNA were transferred by blotting to a Hybond-N membrane (Amersham, Buckinghamshire, UK), Flow cytometry and hybridized with the alkaline phosphatase-labelled probes 33.6 and 33.15. Chemiluminescent detection was performed Surface antigens of SET-2 were examined with the direct using CDP-Star (Tropix, Bedford, MA, USA). immunofluorescent method. Monoclonal anti-CD 36, anti-CD 62p, anti-CD 117, and CD 126 were obtained from Immuno- tech (Marseille, France). Monoclonal anti-CD 34 and anti-CD Measurement of IL-6, TPO, ␤-thromboglobulin (␤-TG), 130 were obtained from Becton Dickinson (San Jose, CA, and platelet factor 4 (PF4) by enzyme immunoassay USA) and BioSource International (Camarillo, CA, USA), (EIA) respectively. All other monoclonal antibodies were purchased from DAKO (Glostrup, Denmark). Cells were incubated with SET-2 cells were cultured at a concentration of 1.0 × 105 each monoclonal antibody at 4°C for 30 min, then washed cells/ml. Concentrations of IL-6 in culture supernatants at day twice by cold phosphate-buffered saline (PBS) with 2% bovine 7 were assayed with an EIA kit (Immunotech). The detection serum albumin (BSA). The cells were analyzed using a flow limit of IL-6 is 0.1 pg/ml. EIA procedures for TPO were cytometer (EPICS Elite) (Coulter Electronics, Hialeah, FL, described in detail previously.25 The detection limit of TPO USA). Expression of c-mpl on SET-2 cells was also examined was 0.06 fmol/ml. ␤-TG and PF4 in culture supernatants were with the indirect immunofluorescent method using rabbit anti- measured using EIA kits (Asserachrom ␤-TG and Asserachrom c-mpl lgG. Rabbit anti-c-mpl lgG was purified using a protein PF4; Diagnostica Stago, Asnieres, France). The detailed A Sepharose (Pharmacia Biotech, Uppsala, Sweden) from anti- procedures were described previously.5

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 144 Hematopoietic growth factors and reagents stimulation index (SI) Ͼ1.5 (SI; mean OD with various cytokines/mean OD with control). Recombinant human TPO26 was generously provided by the Kirin Brewery Co., Ltd (Gunma, Japan). Recombinant human erythropoietin (EPO) was a kind donation from the Chugai Reverse transcriptase-polymerase chain reaction (RT- Pharmaceutical Co., Ltd (Tokyo, Japan). Recombinant human PCR) analysis of TPO mRNA interleukin-3 (IL-3), recombinant human IL-6, recombinant human – colony-stimulating factor Isolation of total cellular RNA and RT-PCR analysis were per- (GM-CSF) and recombinant human stem cell factor (SCF) were formed as previously described.26 To amplify human TPO all purchased from GIBCO BRL Life Technologies. Recombi- cDNA, we used a sense primer (5Ј-GGCCAGAATGGA- nant human interleukin-11 (IL-11) was obtained from Immu- GCTGACTGAATTG-3Ј, nucleotides −7 to 18) in combination genex Corp (Los Angeles, CA, USA). Recombinant human leu- with a reverse primer (5Ј-TCCTACAAGCATCAGG- kemia inhibitory factor (LIF) and recombinant human tumor AAACGCACC-3Ј, nucleotides 476 to 501). For internal con- necrosis factor-␣ (TNF-␣) were obtained from Boehringer trol, ␤-actin specific primers were used. Size marker was Mannheim Biochemica. PMA, Calphostin C, and BSA (bovine ␸X174/HaeIII (Toyobo, Tokyo, Japan). The sequence of the albumin, fraction V) were purchased from Sigma (St Louis, PCR fragment was analyzed using the dideoxynucleotide ter- MO, USA). minal method30 to confirm the amplification of the correct sequence of TPO cDNA.

Cell lines Aggregation studies HepG2,27 a human hepatocellular carcinoma cell line, was obtained from the Japanese Cancer Research Bank (Tokyo, Culture-derived platelet-like particles were isolated from Japan). TAMA is a CD 34+ immature myeloid leukemia cell 7-day cultured supernatants as previously described.31 Briefly, line derived from acute myelogenous leukemia (AML) estab- harvested supernatants were centrifuged at 180 g for 15 min lished in our laboratory in 1994. at room temperature. Half of the centrifuged supernatants was taken from each tube and pooled to one tube. The pooled supernatants were re-centrifuged at 250 g for 15 min to pellet Serum-free culture cells carried over from culture. Then, supernatants containing platelet-like particles were finally centrifuged at 2800 g for SET-2 cells were washed twice with serum-free DMEM con- 10 min at room temperature. The pellets containing platelet- − taining 10 5 M 2-ME, 100 U/ml PCG, 100 ␮g/ml SM, and like particles were resuspended in DMEM and rinsed three 10 ml MEM NEAA solution, or twice with the same medium times with DMEM without FBS and incubated with or without ° supplemented with 5% BSA. The cells were then cultured in 0.8 U/ml of thrombin (Sigma) at 37 C for 10 min. Platelet-like the serum-free medium or the medium supplemented with 5% particles were then cytocentrifuged on to glass slides at 250 g ° for 5 min. The slides were air-dried and stained by the May– BSA at 37 Cin5%CO2. Gru¨nwald-Giemsa method.

Cell proliferation assay Platelet-like particle viability To quantitate the proliferation of SET-2, MTT [3-(4, 5-dime- thylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (Sigma) To examine whether the platelet-like particles released from rapid colorimetric assay was used as previously reported.28 In SET-2 were viable, we used the MTT rapid colorimetric assay. brief, triplicate aliquots of cells (1.0 × 104 cells resuspended Various numbers of platelet-like particles were incubated in ° in 100 ␮l DMEM supplemented with 10% FBS in the absence 0.1 ml DMEM with MTT at 37 C for 4 h. or presence of various concentrations of cytokines and reagents) were cultured in 96-well flat-bottom tissue culture plate (Falcon 3072; Becton Dickinson Labware, Lincoln Park, Results NJ, USA) for 72 h at 37°C. The concentrations of cytokines and reagents were as follows: EPO (10 U/ml), GM-CSF Establishment of the cell line (5 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml), IL-11 (100 ng/ml), LIF (10 ng/ml), SCF (50 ng/ml), TNF-␣ (20 ng/ml), TPO Approximately 4 weeks after the beginning of the culture, cells − (10 ng/ml), and PMA (10 7 M). Additive and synergistic effects consistently proliferated as a single cell suspension. From the of TPO and other cytokines and reagents on the proliferation suspension culture, a new cell line, designated SET-2, was of SET-2 cells were analyzed under the same concentrations cloned by the limiting dilution method. At present, the cells of cytokines and reagents in the presence of TPO (10 ng/ml). have continued to grow in liquid culture for more than 3 years MTT (10 ␮l of 5 mg/ml solution in PBS) was added to each using the same culture conditions. Doubling time was about well for the final 4 h of culture. Then, 100 ␮l of acid isopro- 36 h. The growth of SET-2 cells was retarded in serum-free panol (0.04 N HCL in isopropanol) was added to all wells and medium without 5% BSA more than with 5% BSA. Sequential mixed. The optical density (OD) was then measured on the observations found that most SET-2 cells died within 10 days microplate reader (MPR A4) (Toyo Soda, Tokyo, Japan) with in serum-free medium without 5% BSA (data not shown). a test wavelength of 570 nm and a reference wavelength of Technical aspects of cell culturing are listed in Table 1. DNA 620 nm. PKC inhibitor (Calphostin C)29 was added at a con- fingerprinting proved unequivocally that SET-2 was indeed centration of 250 nM. Positive stimulation was defined as a derived from the patient described (Figure 1). The cultures are

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 145 Table 1 Synopsis of data on SET-2

Parameter SET-2

Cell phenotype megakaryoblast Original disease essential thrombocythemia Patient 71-year-old female Status of disease at leukemic transformation Source peripheral blood Year of establishment 1995 − Culture medium DMEM with 10% FBS and 10 5 M 2-ME Cell storage condition cells could be frozen in 90% FBS + 10% DMSO stored in liquid nitrogen, and successfully recovered with a viability of approximately 80% Cell propagation cells were split every 7 days at 1:10 to 2:5 Maximum cell density c. 1.0 to 2.0 × 106 cells/ml Minimum cell density c. 1.0 to 2.0 × 104 cells/ml Doubling time c. 36 h In situ morphology single, round cells in suspension, partly in clumps Cytogenetics 47–48, X, add (X) (p22) [5], del (X) (p21) [5], add (1) (p36) [2], add (4) (p1?2) [2], −5 [10], add (7) (q32) [10], −9 [10], del (10) (q24) [2], add (12) (p13) [5], add (13) (q3?2) [2], −14 [10], add (16) (q22) [10], add (17) (q25) [4], add (19) (p13) [10], −21 [10], −22 [10], +1ෂ3r, +5ෂ7 mar [cp10]

Cytokine proliferative effect none

free from Epstein–Barr virus and mycoplasma (data not tern, and most of the giant multinucleated cells showed diffuse shown). positive for PAS (data not shown).

Morphological and cytochemical analysis Electron microscopy Leukemic blasts and bizarre giant platelet in the patient’s fresh peripheral blood are shown in Figure 2a and b. Blast cells were mostly round or ovoid with a modal diameter of about Ultrastructural analysis of untreated SET-2 cells showed the 15 to 20 ␮m, and had large round to ovoid nuclei with a few presence of dense electron granules resembling platelet-spe- prominent nucleoli and basophilic cytoplasms. No Auer rods cific ␣-granules and numerous mitochondria in the cytoplasm. were seen. Demarcation membrane-like vesicles and tubules were not Figure 3 illustrates the SET-2 cells observed after May– identified. Irregular cytoplasmic projections and budding were Gru¨nwald-Giemsa staining. Among the small round cells, observed (Figure 5a). Platelet-like particles also contained multinucleated giant cells whose diameters were approxi- several granules (Figure 5b). mately three to eight times larger than single suspended cells were observed occasionally (about 5%). So many platelet-like particles were also seen, but proplatelet formation was not seen in the suspension culture using phase-contrast Surface markers analyzed by flow cytometry microscopy (Figure 4a). Platelet-like particles appear to be released by the dissociation of cytoplasms. Like the original blasts, SET-2 cells were mostly round to ovoid with a similar SET-2 cells were the population in the gate (a). On a log scale modal diameter, and had large round to ovoid nuclei with a for forward and side scatter, platelet-like particles (the popu- few nucleoli and basophilic cytoplasms with several cytoplas- lation in the gate (b)) showed the same size as normal plate- mic protrusions (Figure 3a). Giant multinucleated cells had lets. As summarized in Table 2, SET-2 cells distinctly basophilic cytoplasms with a few vacuoles, several cytoplas- expressed platelet-megakaryocytic lineage antigens (except mic protrusions, and platelet-like particles on the cell surface. CD 42b and CD 62p), myeloid-lineage antigens, and multi- These cells showed morphologic characteristics of mature potent-hematopoietic progenitor antigens. Immature lymph- megakaryocytes with multilobular nuclei (Figure 3b). Platelet- oid-lineage antigens (CD 4 and CD 7) were also expressed, like particles were mostly round with a modal diameter of 2 but mature T- and B-lineage antigens were not expressed. SET- to 4 ␮m. Microscopically, platelet-like particles were dis- 2 cells were negative for anti-erythroid-lineage antibody and tinguished from cellular debris (Figure 4b). Platelet-like par- anti-Fas antibody. Platelet-like particles (gate b) also expressed ticles had scant basophilic cytoplasms and azurophilic gran- platelet-lineage antigens, but had relatively low levels of CD ules (Figure 4c). The cells stained negative for MPO and 34. CD 2, 3, 10, and 19 were all negative on SET-2 cells. As NASDCAE but positive for ANBE. A low percentage of small illustrated in Figure 6, SET-2 showed c-mpl and CD 41 round cells showed positive for PAS staining in a dotted pat- expression on the cell surface.

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 146

a

b

Figure 2 May-Gru¨nwald-Giemsa-staining of fresh leukemic blasts. (a) Blast cells were round or ovoid with a modal diameter of about 15 to 20 ␮m, and had a few nucleoli and basophilic cytoplasms (original Figure 1 DNA fingerprinting of the primary leukemia cells and cell magnification ×600). (b) Bizarre giant platelet with a diameter of about lines. DNA digested with Hinfl was size-separated on an agarose gel 15 ␮m (original magnification × 600). by electrophoresis, Southern blotted, hybridized with the alkaline phosphatase-labelled multilocus probe 33.6. Data with probe 33.15 was not shown. Lane 1, TAMA, myeloid leukemia cell line established in our laboratory. Lane 2, primary leukemia cells. Lane 3, SET-2. Lane 4, DNA size marker. the MTT assay. As shown in Figure 8, SET-2 did not respond to TPO, EPO, TNF-␣ and Calphostin C. SET-2 responded to IL-3, IL-6, GM-CSF, and to a lesser degree, IL-11, SCF and LIF. Cytogenetic analysis However, stimulation index of all the cytokines were lesser than 1.5. Additive and synergistic effects were not evident Chromosome analysis of SET-2 cells revealed a hyperdiploid between TPO and other cytokines and reagents in the growth karyotype, with a modal chromosome number of 47 (range, activity of SET-2 (data not shown). To investigate the action 45 to 48). The composite karyotype was 47–48, X, add (X) mechanisms of PMA for SET-2, Calphostin C was added to the (p22) [5], del (X) (p21) [5], add (1) (p36) [2], add (4) (p1?2) culture. Calphostin C did not influence the proliferation of [2], −5 [10], add (7) (q32) [10], −9 [10], del (10) (q24) [2], SET-2 in the presence of PMA (data not shown). add (12) (p13) [5], add (13) (q3?2) [2], −14 [10], add (16) (q22) [10], add (17) (q25) [4], add (19) (p13) [10], −21 [10], −22 [10], +1 ෂ3r, +5 ෂ7mar [cp10]. The Philadelphia chromosome Quantitative analysis of ␤-TG and PF4 in culture was negative in all karyotypes analyzed. 5Ј-BCR rearrange- supernatants ment was not detected by Southern blot analysis using 5Ј-bcr probe (data not shown). SET-2 produced ␤-TG but not PF4 in the absence of TPO and PMA (Figure 9). PMA increased the production of ␤-TG and PF4. TPO did not influence the production of ␤-TG and PF4 Proliferative responses to cytokines by SET-2 cells.

The growth curves stimulated by TPO and PMA are shown in Figure 7. TPO did not stimulate proliferation of SET-2 cells. Measurement of IL-6 and TPO in culture supernatants PMA-treated SET-2 cells lost their proliferative capacity but increased platelet-like particles in liquid culture (data not To clarify the possibility of autocrine growth of SET-2 cells, shown). We also investigated proliferative responses of SET-2 both IL-6 and TPO in culture supernatants were analyzed at cells to other hematopoietic growth factors and reagents using day 7. TPO was not detected in culture supernatants. Small

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 147

a a

b b

Figure 3 May–Gru¨nwald-Giemsa-staining of SET-2 cells. (a) Baso- philic cytoplasm and many cytoplasmic protrusions are demonstrated (original magnification × 200). (b) Multinucleated with a few cytoplasmic vacuoles, cytoplasmic protrusions, and small platelet-like particles on the surface of the cell (original magnification × 400).

amounts of IL-6 (5.4 ± 0.9 pg/ml) released into the culture medium were detected.

RT-PCR analysis of TPO mRNA

RT-PCR analysis of TPO mRNA revealed that the 508 bp of amplified fragments of human TPO cDNA was detected in c HepG2, but not in SET-2 (data not shown). For internal con- trol, human ␤-actin was amplified in both HepG2 and SET-2. Figure 4 Light microscopical appearance of platelet-like particles. (a) Phase-contrast photomicrograph of SET-2 cells in liquid suspension culture. Numerous platelet-like particles are seen among round or ovoid SET-2 cells. (original magnification × 80). (b) May–Gru¨nwald- Aggregation of platelet-like particles Giemsa staining of platelet-like particles. Arrow head indicates cellu- lar debris (original magnification × 200). (c) Various-sized platelet-like As a function of culture-derived platelet-like particles, aggre- particles with scant basophilic cytoplasms and azulophilic granules gation in response to thrombin was investigated. Culture- (original magnification × 800). derived platelet-like particles incubated in the presence of thrombin did not aggregate (data not shown). Discussion

Platelet-like particle viability A novel human megakaryoblastic cell line, SET-2, that belongs to an immature megakaryocytic lineage was established. Until In MTT assay, the absorbances were approximately pro- now, all established megakaryoblastic and megakaryocytic portional to the number of platelet-like particles (data not cell lines have been derived from the megakaryoblastic crisis shown). The microscopic images of formazans produced by of patients with chronic myelogenous leukemia (CML) or with platelet-like particles were very thin (data not shown). acute megakaryoblastic leukemia (FAB-M732).9 Recently, a

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 148 Table 2 Surface phenotypes of SET-2 cells

CD Positive cells (%)

(Gate) ab

4 58.5 NT 5 negativea NT 7 99.3 NT 9 negative NT 13 98.6 NT 14 negative NT 20 negative NT 25 negative NT 33 99.2 NT 34 96.4 33.0 36 92.0 44.9 38 93.6 42.6 41 99.8 48.2 42b negative NT 61 88.4 64.7 62p negative NT 71 98.2 89.3 95 negative negative 117 36.1 NT 126 84.2 NT 130 32.5 NT HLA-DR 99.8 NT Gly-A negative NT

aNegative, Ͻ10% of the cells were positive under the condition in which the negative control showed 5% of positive background. The population in the gated area (a) in linear-scaled FS (forward scatter) and SS (side scatter) are round to ovoid-shaped SET-2 cells. The population in gated area (b) in logarithmic-scaled FS and SS are normal platelet-sized particles. NT, not tested.

Figure 5 Electron microscopic appearance of SET-2 cells and plat- elet-like particles. (a) Transmission electron micrograph of SET-2 cells showing a nucleus with prominent nucleolus, and cytoplasm contain- ing numerous mitochondria, Golgi apparatus, and electron dense granules resembling ␣-granules (white arrowhead). Although irregular cytoplasmic protrusions and cytoplasmic budding (black arrowheads) are observed, no definite demarcation membranes are noted (bar 1 ␮m; original magnification × 5000). (b) Ultrastructure of platelet-like particles. Several electron dense granules are seen (bar 2 ␮m; Figure 6 Flow cytometric analysis of c-mpl and CD41 expression original magnification ×6000). on SET-2 cells. The cells were labeled with rabbit anti-c-mpl IgG and anti-CD41 monoclonal antibody (gray profile) or with a nonspecific isotype control IgG (open).

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 149

Figure 9 Time-course of increase in production of ␤-thromboglob- ␤ × 5 Figure 7 Growth curves of SET-2 cells in suspension culture in the ulin ( -TG) and platelet factor 4 (PF4). SET-2 cells (1 10 /ml) were − −7 absence or presence of TPO (10 ng/ml) and PMA (10 7 M). Doubling cultured in the absence or presence of PMA (10 M) and TPO time was calculated as being about 36 h. Medium, DMEM containing (10 ng/ml), and the culture medium was harvested. The values rep- 10% FBS as described in Materials and methods. resent the mean from two independent cultures. Medium, DMEM con- taining 10% FBS as described in Materials and methods.

SET-2 cells were identified as a megakaryoblastic leukemia cell line34 from their appearance as small round blastic cells mixed with a few multinucleated giant cells; from their expression of antigens of the platelet-megakaryocytic lineage, such as CD 33, 34, 36, 38, 41, 61 and 71; and from their synthesis of proteins associated with megakaryocyte matu- ration, such as PF4 and ␤-TG. SET-2 cells spontaneously pro- duce numerous platelet-like particles in supernatants. How- ever, typical proplatelet formation was not seen. Platelet-like particles appear to be released by the dissociation of cyto- plasms. Appearance of multinucleated giant cells in suspen- sion culture has occasionally been detected in several cell lines,2,4–6,8,9,15 and CMK cells treated with PMA have been found to display morphologic characteristics of mature megakaryocytes with multilobular nuclei.35 Among them, only three cell lines (MEG-01, M-07e and NS-Meg) have been shown to spontaneously produce platelet-like particles, sug- gesting megakaryocytic terminal differentiation. CMK also shows the release of numerous segments only upon treatment with TPA.3 CMK 11–5,36 a subclone of CMK with more mature characteristics than CMK itself, as well as M-07e,4 have been Figure 8 The response of SET-2 cells to several growth factors and observed to release platelet-like particles in the absence of reagents. Proliferative responses of SET-2 cells were analyzed by the 16 4 MTT assay (see Materials and methods). MTT reduction was measured TPA. Platelet-like particles from MEG-01 and M-07e have after 3 days of culture. The values represent the mean ± s.d. from not been compared in detail with normal platelets. Prolifer- triplicate cultures. Horizontal broken line indicates SI = 1.5. ation of M-07e was enhanced by TPO but not IL-6 and IL- 11.37 Those from CMK11–5 possessed many of the same properties as normal platelets except that they were unable to new megakaryoblast cell line10 has been established from a aggregate.36 NS-Meg,9 a megakaryoblastic/erythroid cell line patient with AML (FAB-M1). To date, however, no human derived from a patient with CML in blast crisis, also spon- megakaryoblastic cell lines have been established from a taneously produces platelet-like particles. However, this cell patient with ET. One myeloid cell line, Marimo,33 established line also did not exhibit a proliferative response to IL-6 or IL- from a patient with ET has only been reported previously. 11. Platelet-like particles released by SET-2 were the same size However, Marimo did not have megakaryoblastic and platelet as normal platelets in peripheral blood. Ultrastructurally, these markers, and was derived from a therapy-related AML clone particles containing electron dense granules appeared to be but not an ET clone. Thus, this is the first report of a megakar- normal platelets. In addition, to ascertain whether these plate- yoblastic cell line derived from a patient with ET. Unfortu- let-like particles released from SET-2 were not cellular debris, nately, it is unclear whether SET-2 cells were really derived we examined the viability of platelet-like particles using MTT from an ET clone or therapy-related AML clone, because bone assay. The amount of formazan generated in this assay is marrow cells of our patient had a normal karyotype at chronic directly proportional to the activity of mitochondria in the pla- phase of ET. telet-like particles. Very thin formazans showed that the plate-

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 150 let-like particles fraction did not contain SET-2 cells.38 Taken let c-mpl receptor were markedly reduced and c-mpl- together, platelet-like particles released from SET-2 seem to mediated signaling pathway was not constitutively activated be viable but not dead cellular debris. However, they failed in platelets from ET patients.48 In addition, no mutation of c- to aggregate after being incubated with thrombin. Generally, mpl has been detected in the clonal platelet population platelets derived from patients with ET have been shown to derived from ET patients.49 However, in comparison with nor- exhibit hypoaggregation in response to epinephrine, ADP, col- mal controls’ fluorescence intensity in the flow cytometric lagen and thrombin.39 Since SET-2 was derived from a patient analysis,48 surface expression levels (Figure 6) of c-mpl on with transformation into acute leukemia of ET, it is likely that SET-2 cells were not reduced. Therefore, whether the signal platelet-like particles released by SET-2 would show the same transduction of c-mpl receptor, independent of its ligand, aggregation properties as the platelets from patients with ET. TPO, resulting from an activating mutation of the c-mpl proto- In addition, SET-2 spontaneously released these platelet-like oncogene causes the spontaneous proliferation and differen- particles into the culture supernatants in the absence of PMA, tiation of SET-2 should be studied. SET-2 might be an suggesting this cell line may be useful for studying the mech- important tool to investigate the role of c-mpl on the mega- anisms of platelet formation by megakaryocytes in the karyoblasts, megakaryocytes and platelets in ET. terminal stage of thrombopoiesis. The low levels of IL-6 detected in SET-2 culture super- Similar to MEG-A28 and NS-Meg,9 SET-2 cells expressed the natants at day 7 were the same as those previously observed in megakaryocytic lineage markers, CD4, 7, and HLA-DR anti- culture supernatants of normal megakaryocytes.50 This finding gens. Antigens detected in early hematopoietic progenitors, suggests that SET-2 has the same capacity as that of normal such as CD33 and 34 were also expressed in SET-2. Whereas megakaryocytes51,52 and that of the megakaryoblastic leuke- the erythroid marker, glycophorin A, which is expressed in mia cell line, CMK,53 to secrete IL-6 into the culture medium. other megakaryocytic cell lines, including CMK,3 UT-75 and In conclusion, this new cell line has many interesting NS-Meg,9 was not detected in SET-2. The CD95 (Fas) antigen characteristics, and may be useful for investigating the pro- which, along with its mRNA, is expressed on terminally differ- liferation and differentiation mechanisms of leukemia cells entiated myeloid cells40 but not on CD34+ immature progeni- and the role of c-mpl on megakaryoblasts, megakaryocytes tor cells in the bone marrow41 was not detected in SET-2. and platelets in ET. While further studies are required, SET-2 CD 117 (c-kit) is known to be expressed on stem cells and may prove to be a valuable model for the study of mega- most of the megakaryoblastic cell lines.42,43 Therefore, weak karyocytic differentiation and maturation from megakaryo- expression of CD117 and no expression of CD95 on SET-2 blasts, as well as the regulation of platelet generation by cells also suggested that SET-2 has characteristics of an imma- megakaryocytes in the terminal stage of thrombopoiesis. ture megakaryocytic lineage progenitor. Terminal differentiation of megakaryocytes is characterized by the expression of platelet-specific proteins, including PF4 Acknowledgements and ␤-TG, and the expression of these proteins was observed in SET-2 cells. Although ␤-TG was spontaneously produced We thank Dr K Ueno (Division of Persistent and Oncogenic without stimulation. PF4 was not detected in the absence of Viruses, Center for Chronic Viral Diseases, Faculty of Medi- PMA. After stimulation by PMA, however, the expression of cine, Kagoshima University, Kagoshima, Japan) for his techni- these proteins in culture supernatants was dramatically cal help in the detection of EB virus infection using EBER-1 increased. In contrast, although c-mpl is expressed on the sur- ISH method, and Dr T Tanaka (Teijin Bio Laboratories, Tokyo, face membranes of SET-2 cells, TPO did not stimulate the Japan) for the help provided in performing the DNA finger- expression of PF4 and ␤-TG. These findings suggested that printing. TPO had no effect on the megakaryocytic terminal differen- tiation and maturation of SET-2. Although, like SET-2, the CMK, CMK11–5 and NS-Meg cell lines contain multi- Note nucleated giant cells and produce platelet-like particles, these cell lines differ from SET-2 in that they do not express PF4 44,45 The cell line, SET-2, can be provided to outside investigators mRNA or immunoreactive PF4 in culture supernatants. by written request to K Uozumi. Spontaneous production of ␤-TG means that SET-2 have the same ability to terminally differentiate and mature as normal megakaryoblasts. References Since Calphostin C did not influence the proliferation of SET-2 in the presence of PMA, it suggests that the pathway of 1 Fugman DA, Witte DP, Jones CLA, Aronow BJ, Lieberman MA. In differentiation and proliferation of SET-2 is not associated with vitro establishment and characterization of a human megakaryo- protein kinase C. SET-2 also did not respond to TPO and EPO, blastic cell line. Blood 1990; 75: 1252–1261. which stimulate the megakaryocytic lineage progenitors. SET- 2 Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura 2 did not release TPO into the culture medium and did not H, Saito H. Establishment of a novel human megakaryoblastic leu- express mRNA of TPO, which excludes the possibility of an kemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66: 1384–1392. autocrine growth mechanism of TPO. Point mutations within 3 Sato T, Fuse A, Eguchi M, Hayashi Y, Ryo R, Adachi M, Kishimoto a dimer interface homology domain of c-mpl have been Y, Teramura M, Mizoguchi H, Shima Y, Komori I, Sunami S, Oki- shown to induce constitutive receptor activity and tumorigen- moto Y, Nakajima H. Establishment of a human leukemic cell line icity,46 whereas point mutations in the transmembrane region (CMK) with megakaryocytic characteristics from a Down’s syn- of c-mpl also induce abrogation of the factor dependency of drome patient with acute megakaryoblastic leukaemia. Br J a cell line, as well as tumorigenicity.47 Therefore, we will Haematol 1989; 72: 184–190. 4 Avanzi GC, Brizzi MF, Giannotti J, Ciarletta A, Yang Y-C, Pegoraro examine whether SET-2 has point mutations within interface L, Clark SC. M-07e human leukemic factor-dependent cell line homology domain or transmembrane region of c-mpl. provides a rapid and sensitive bioassay for the human cytokines Recently, it has been reported that surface expression of plate- GM-CSF and IL-3. J Cell Physiol 1990; 145: 458–464.

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 151 5 Komatsu N, Nakauchi H, Miwa A, Ishihara T, Eguchi M, Moroi DNA fingerprint probes for individualization and establishment of M, Okada M, Sato Y, Wada H, Yawata Y, Suda T, Miura Y. Estab- family relationships, determined from extensive casework. Am J lishment and characterization of a human leukemic cell line with Hum Genet 1991; 48: 824–840. megakaryocytic features: dependency on granulocyte–macro- 23 Ha¨ne B, Tu¨mmler M, Ja¨ger K, Schleithoff L, Janssen JWG, Drexler phage colony-stimulating factor, interleukin 3, or erythropoietin HG. Differences in DNA fingerprints of continuous leukemia– for growth and survival. Cancer Res 1991; 51: 341–348. lymphoma cell lines from different sources. Leukemia 1992; 6: 6 Takeuchi S, Sugito S, Uemura Y, Miyagi T, Kubonishi I, Taguchi H, 1129–1133. Enzan H, Ohtsuki Y, Miyoshi I. Acute megakaryoblastic leukemia: 24 MacLeod RAF, Dirks WG, Reid YA, Hay RJ, Drexler HG. Identity establishment of a new cell line (MKPL-1) in vitro and in vivo. of original and late passage Dami megakaryocytes with HEL Leukemia 1992; 6: 588–594. erythroleukemia cells shown by combined cytogenetics and DNA 7 Mouthon MA, Freund M, Titeux M, Katz A, Guichard J, Breton- fingerprinting. Leukemia 1997; 11: 2032–2038. Gorius J, Vainchenker W. Growth and differentiation of the human 25 Tahara T, Usuki K, Sato H, Ohashi H, Morita H, Tsumura H, Mats- megakaryoblastic cell line (ELF-153): a model for early stages of umoto A, Miyazaki H, Urabe A, Kato T. A sensitive sandwich megakaryocytopoiesis. Blood 1994; 84: 1085–1097. ELISA for measuring thrombopoietin in human serum: serum 8 Abe A, Emi N, Kato H, Adachi K, Murate T, Saga S, Ogura M, thrombopoietin levels in healthy volunteers and in patients with Kojima T, Tanimoto M, Morishita N, Kawashima K, Saito H. Estab- haemopoietic disorders. Br J Haematol 1996; 93: 783–788. lishment and characterization of an immature human megakar- 26 Kato T, Ogami K, Shimada Y, Iwamatsu A, Sohma Y, Akahori H, yoblastic cell line, MEG-A2. Leukemia 1995; 9: 341–349. Horie K, Kokubo A, Kudo Y, Maeda E, Kobayashi K, Ohashi H, 9 Tsuyuoka R, Takahashi T, Suzuki A, Sasaki Y, Nakamura K, Fuku- Ozawa T, Inoue H, Kawamura K, Miyazaki H. Purification and moto M, Ohmori K, Ohno Y, Nakao K. A newly established characterization of thrombopoietin. J Biochem 1995; 118: 229– megakaryoblastic/erythroid cell line that differentiates to red cells 236. in the presence of erythropoietin and produces platelet-like 27 Knowles BB, Howe CC, Aden DP. Human hepatocellular carci- particles. Stem Cells 1995; 13: 54–64. noma cell lines secrete the major plasma proteins and hepatitis B 10 Allen RJ, Smith SD, Moldwin RL, Lu MM, Giordano L, Vignon C, surface antigen. Science 1980; 209: 497–499. Suto Y, Harden A, Tomek R, Veldman T, Ried T, Larson RA, Le 28 Mosmann T. Rapid colorimetric assay for cellular growth and Beau MM, Rowley JD, Zeleznik-Le N. Establishment and charac- survival. Application to proliferation and cytotoxicity assays. terization of a megakaryoblast cell line with amplification of MLL. J Immunol Meth 1983; 65: 55–63. Leukemia 1998; 12: 1119–1127. 29 Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C 11 Matsumura I, Kanakura Y, Kato T, Ikeda H, Ishikawa J, Horikawa (UCN-1028C), a novel microbial compound, is a highly potent Y, Hashimoto K, Moriyama Y, Tsujimura T, Nishiura T, Miyazaki and specific inhibitor of protein kinase C. Biochem Biophys Res H, Matsuzawa Y. Growth response of acute myeloblastic leukemia Commun 1989; 159: 548–553. cells to recombinant human thrombopoietin. Blood 1995; 86: 30 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain 703–709. terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463– 12 Drexler HG, Quentmeier H. Thrombopoietin: expression of its 5467. receptor MPL and proliferative effects on leukemic cells. Leukemia 31 Choi ES, Nichol JL, Hokom MM, Hornkohl AC, Hunt P. Platelets 1996; 10: 1405–1421. generated in vitro from proplatelet-displaying human megakaryo- 13 Drexler HG, Zaborski M, Quentmeier H. Thrombopoietin sup- cytes are functional. Blood 1995; 85: 402–413. ports the continuous growth of cytokine-dependent human leuke- 32 Bennett JM, Catovsky D, Daniel M-T, Flandrin G, Galton DAG, mia cell lines. Leukemia 1996; 11: 541–551. Gralnick HR, Sultan C. Criteria for the diagnosis of acute leukemia 14 Matsumura I, Kanakura Y, Ikeda H, Ishikawa J, Yoshida H, Hori- of megakaryocytic lineage (M7). A report of the French–Amer- kawa Y, Nishiura T, Tahara T, Kato T, Miyazaki H, Matsuzawa Y. ican–British Cooperative Group. Ann Intern Med 1985; 103: Coexpression of thrombopoietin and c-mpl genes in human acute 460–462. myeloblastic leukemia cells. Leukemia 1996; 10: 91–94. 33 Yoshida H, Kondo M, Ichihashi T, Hashimoto N, Inazawa J, Ohno 15 Komatsu N, Kunitama M, Yamada M, Hagiwara T, Kato T, Miya- R, Naoe T. A novel myeloid cell line, Marimo, derived from ther- zaki H, Eguchi M, Yamamoto M, Yasusada M. Establishment and apy-related acute myeloid leukemia during treatment of essential characterization of the thrombopoietin-dependent megakaryo- thrombocythemia: consistent chromosomal abnormalities and cytic cell line, UT-7/TPO. Blood 1996; 87: 4552–4560. temporary C-MYC gene amplification. Cancer Genet Cytogenet 16 Takeuchi K, Ogura M, Saito H, Satoh M, Takeuchi M. Production 1998; 100: 21–24. of platelet-like particles by a human megakaryoblastic leukemia 34 Hassan HT, Freund M. Review of megakaryoblastic cell lines. cell line (MEG-01). Exp Cell Res 1991; 193: 223–226. Characteristic biological features of human megakaryoblastic leu- 17 Murphy S, IIand H, Rosenthal D, Laszol J. Essential thrombo- kemia cell lines. Leukemia Res 1995; 19: 589–594. cythemia: an interim report from the Polycythemia Vera Study 35 Komatsu N, Suda T, Moroi M, Tokuyama N, Sakata Y, Okada M, Group. Semin Hematol 1986; 23: 177–182. Nishida T, Hirai Y, Sato T, Fuse A, Miura Y. Growth and differen- 18 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gral- tiation of a human megakaryoblastic cell line, CMK. Blood 1989; nick HR, Sultan C. Proposed revised criteria for the classification 74: 42–48. of acute myeloid leukemia. A report of the French–American– 36 Nagano T, Ohga S, Kishimoto Y, Kimura T, Yasunaga K, Adachi British Cooperative Group. Ann Intern Med 1985; 103: 620–625. M, Ryo R, Sato T. Ultrastructural analysis of platelet-like particles 19 Bartley TD, Bogenberger J, Hunt P, Li YS, Lu HS, Martin F, Chang from a human megakaryocytic leukemia cell line (CMK 11–5). Int MS, Samel B, Nichol JL, Swift S, Johonson MJ, Hsu RY, Parker VP, J Hematol 1992; 56: 67–78. Suggs S, Skrine JD, Merewether LA, Clogston C, Hsu E, Hokom 37 Quentmeier H, Zaborski M, Graf G, Ludwig WD, Drexler HG. MM, Choi E, Pangelinan M, Sun Y, Mar V, McNinch MJ, Simonet Expression of the receptor MPL and proliferative effects of its L, Jacobsen F, Xie C, Shutter J, Chute H, Basu R, Selander L, Trol- ligand thrombopoietin on human leukemia cells. Leukemia 1996; linger D, Sleu L, Padilla D, Trail G, Elliott G, Izumi R, Covey T, 10: 297–310. Crouse J, Garcia A, Xu W, Castillo JD, Biron J, Cole S, Hu MCT, 38 Takeuchi K, Satoh M, Kuno H, Yoshida T, Kondo H, Takeuchi Pacifici R, Ponting I, Saris C, Wen D, Yung YP, Lin H, Bosselman M. Platelet-like particle formation in the human megakaryoblastic RA. Identification and cloning of a megakaryocyte growth and leukaemia cell lines, MEG-01 and MEG-01s. Br J Haematol 1998; development factor that is a ligand for the cytokine receptor mpl. 100: 436–444. Cell 1994; 77: 1117–1124. 39 Rao AK, Carvalho ACA. Acquired qualitative platelet defects. In: 20 ISCN (1995). Mitelman F (ed). An international system for human Colman RW, Hirsh J, Marder VJ, Salzman EW (eds). cytogenetic nomenclature. S Karger: Basel, 1995. and Thrombosis: Basic Principles and Clinical Practice 3rd edn. 21 Chang KL, Chen YY, Shibata D, Weiss LM. Description of an in- JB Lippincott Co: Philadelphia, 1994, pp 685–704. situ hybridization methodology for detection of Epstein–Barr virus 40 Iwai K, Miyawaki T, Takazaki T, Konno A, Ohta K, Yachie A, Seki RNA in paraffin-embedded tissues, with a survey of normal and H, Taniguchi N. Differential expression of bcl-2 and susceptibility neoplastic tissue. Diagn Mol Pathol 1992; 1: 246–255. to anti-Fas-mediated cell death in peripheral blood lymphocytes, 22 Jeffreys AJ, Turner M, Debenham P. The efficiency of multilocus , and . Blood 1994; 84: 1201–1208.

Leukemia A new human megakaryoblastic cell line, SET-2 K Uozumi et al 152 41 Nagafuji K, Shibuya T, Harada M, Mizuno S, Takenaka K, Miya- 48 Horikawa Y, Matsumura I, Hashimoto K, Shiraga M, Kosugi S, moto T, Okamura T, Gondo H, Niho Y. Functional expression of Tadokoro S, Kato T, Miyazaki H, Tomiyama Y, Kurata Y, Matsuz- Fas antigen (CD95) on hematopoietic progenitor cells. Blood awa Y, Kanakura Y. Markedly reduced expression of platelet c- 1995; 86: 883–889. mpl receptor in essential thrombocythemia. Blood 1997; 90: 42 Hu ZB, Ma W, Uphoff CC, Quentmeier H, Drexler HG. c-kit 4031–4038. expression in human megakaryoblastic leukemia cell lines. Blood 49 Kiladjian JJ, Elkassar N, Hetet G, Briere J, Grandchamp B, Gardin 1994; 83: 2133–2144. C. Study of the thrombopoietin receptor in essential thrombocy- 43 Morita S, Tsuchiya S, Fujie H, Itano M, Ohashi Y, Minegishi M, themia. Leukemia 1997; 11: 1821–1826. Imaizumi M, Endo M, Takano N, Konno T. Cell surface c-kit 50 Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro E, receptors in human leukemia cell lines and pediatric leukemia: Macioce G, Pace A, Ziegler B, Hassan HJ, Peschle C. Unilineage selective preservation of c-kit expression on megakaryoblastic cell megakaryocytic proliferation and differentiation of purified hema- lines during adaptation to in vitro culture. Leukemia 1996; 10: topoietic progenitors in serum-free liquid culture. Blood 1995; 86: 102–105. 3725–3736. 44 Adachi M, Ryo R, Sato T, Yamaguchi N. Platelet factor 4 gene 51 Navarro S, Debili N, Le Couedic JP, Klein B, Breton-Gorius J, Doly expression in a human megakaryocytic leukemia cell line (CMK) J, Vainchenker W. Interleukin-6 and its receptor are expressed by and its differentiated subclone (CMK 11–5). Exp Hematol 1991; human megakaryocytes: in vitro effects on proliferation and endo- 19: 923–927. replication. Blood 1991; 77: 461–471. 45 Sasaki Y, Takahashi T, Tanaka I, Nakamura K, Okuno Y, Naka- gawa O, Narumiya S, Nakao K. Expression of prostacyclin recep- 52 Wickenhauser C, Lorenzen J, Thiele J, Hillienhof A, Jungheim K, Schmitz B, Hansmann ML, Fisher R. Secretion of cytokines tor in human megakaryocytes. Blood 1997; 90: 1039–1046. ␣ − − 46 Alexander WS, Metcalf D, Dunn AR. Point mutations within a (interleukin-1 , 3, and 6 and granulocyte–macrophage colony- dimer interface homology domain of c-Mpl induce constitutive stimulating factor) by normal human bone marrow megakary- receptor activity and tumorigenicity. EMBO J 1995; 22: 5569– ocytes. Blood 1995; 85: 685–691. 5578. 53 Fuse A, Kakuda H, Shima Y, Van Damme J, Billiau A, Sato T. 47 Onishi M, Mui AL-F, Morikawa Y, Cho L, Kinoshita S, Nolan GP, Interleukin 6, a possible autocrine growth and differentiation fac- Gorman DM, Miyajima A, Kitamura T. Identification of an onco- tor for the human megakaryocytic cell line, CMK. Br J Haematol genic form of the thrombopoietin receptor MPL using retrovirus- 1991; 77: 32–36. mediated gene transfer. Blood 1996; 88: 1399–1406.

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