cell lines - Nature

Leukemia (1997) 11, 1554–1564  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

JURL-MK1 (c-kithigh/CD30−/CD40−) and JURL-MK2 (c-kitlow/CD30+/CD40+) cell lines: ‘two-sided’ model for investigating leukemic megakaryocytopoiesis R Di Noto1, L Luciano2, C Lo Pardo3, F Ferrara4, F Frigeri5, O Mercuro1, ML Lombardi1, F Pane5, C Vacca3, C Manzo1, F Salvatore5, B Rotoli2 and L Del Vecchio3 Divisione di Oncologia Sperimentale C, Istituto Nazionale dei Tumori; 2Divisione di Ematologia Clinica, Universita` Federico II; 3Servizio di Immunoematologia and 4Divisione di Ematologia, Ospedale A Cardarelli; 5CEINGE-Biotecnologie Avanzate, Dipartimento di Biochimica e Biotecnologie Mediche, Universita` Federico II, Naples, Italy

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Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient’s fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gpIb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURLMK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2Ra, while a marked expression of CD116/GM-CSF-R and CDw123/IL3Ra is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURLMK1 phenotype, causing an increased expression of plateletassociated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURLMK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a ‘two-sided’ model for investigating new aspects of megakaryocytopoiesis. Keywords: megakaryoblastic cell lines; megakaryocytopoiesis; CD30; c-kit; CD117

Introduction Chronic myelogenous leukemia (CML) is characterized by progressive accumulation of differentiated blood cells derived from a single pluripotent progenitor cell carrying the so-called Philadelphia (Ph) chromosome. Virtually all CML patients enter the blastic crisis (BC) stage, in which the malignant cells may show characteristics of various cell types, including those of the megakaryocytic lineage.1 Cell lines derived from patients with CML-BC may be useful for investigating hematopoietic cell growth and differentiation, providing information on the action of biological agents in CML cells, as well as on the cascade of molecular events associated with this myeloproliferative disorder.2 Due to the scarceness of megakaryocytes (MK) in normal bone marrow, the establishment of MK cell lines can provide models in order to perform studies on megakaryocytopoi-

Correspondence: L Del Vecchio, Via Manzoni 24, 80123 Naples, Italy Received 20 January 1997; accepted 12 May 1997

esis.3,4 We report here the establishment of two novel Ph+ MKoriented cell lines, JURL-MK1 and JURL-MK2, obtained from a single patient affected by CML-BC. We show that JURL-MK1 and JURL-MK2 are characterized by divergent phenotypes, karyotypes and responses to cell growth/differentiation promoters. Materials and methods

Case history In December 1983, a 63-year-old male patient was admitted to the Hematology Department for leukocytosis and spleen enlargement. He was diagnosed as having Ph+ CML and was treated with busulfan until July 1987, when he was switched to hydroxyurea. In November 1993, he complained of general malaise and showed progressive unresponsive splenomegaly. Peripheral blood counts were: Hb 8.7 g/dl, platelets 22 000/ml and WBC 140 000/ml with 65% blast cells. Bone marrow examination revealed more than 90% blast cells with myeloid appearance, which was confirmed by a cell surface marker study (CD33+, CD36+, CD45RO+, CD43+, CD117+, CD25±, CD34−, HLA-DR−, CD11a−). Chromosome analysis confirmed the Ph+ karyotype; no additional abnormalities were found. A diagnosis of myeloid BC was made. The patient died of complications (heart failure) a few days later.

Establishment of cell lines Mononuclear cells from a peripheral blood sample drawn during the blastic phase (November 1993) were separated by Ficoll–Hypaque (d = 1.077) density gradient centrifugation at 850 g for 20 min and seeded at 2 × 106/ml in 35-mm Petri dishes, containing 2 ml Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l dextrose (ICN, Oxford, UK), supplemented with 20% fetal bovine serum (FBS; Gibco, Life Technologies, Paisley, UK), 2 mM L-glutamine (Gibco), 10−5 M 2-mercaptoethanol and antibiotics. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere and fresh DMEM was added at 7-day intervals, replacing half of the medium each time. Neither growth factors nor exogenous feeder layers were used. In January 1994, the cells began to grow slowly, with large round cells proliferating on a substrate of fibroblast-like cells. When the cells showed a doubling time of 7 days (May 1994), they were transferred to tissue culture flasks (Falcon; Becton Dickinson, San Jose, CA, USA), and, within a few weeks (July 1994), a series of 12 subcultures were obtained. In this phase, the fibroblast-like cells disappeared. Four months later (November 1994), two subcultures began to grow vigorously, and two cell lines (JURL-MK1 and JURL-MK2) were judged to

JURL-MK1 and JURL-MK2 megakaryoblastic cell lines R Di Noto et al

be established. The cells grew in single cell suspensions, and the later passages showed doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). The cells grew at an optimal cell density of 1.5 × 106 (minimum: 8 × 105 cells/ml; maximum: 2.5 × 106 cells/ml). JURL-MK1 was split 1:2 every 48 h or 1:3 every 72 h, while JURL-MK2 was split 1:2 every 72 h. Aliquots of both JURL-MK1 and JURL-MK2 cells were cryopreserved in FBS/DMSO (6:1). Although we could maintain both JURL-MK1 and JURL-MK2 cells for 3 years in stable condition, the cryopreserved cells were routinely thawed and cultured for further characterization of the cell lines. A test for mycoplasma contamination (Mycoplasma PCR ELISA; Boehringer Mannheim, Mannheim, Germany) was performed at regular intervals, but no contamination was found in either cell culture. Cell lines were established at the Divisione di Oncologia Sperimentale C, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione G Pascale, Naples, Italy.

Morphology, cytochemistry and cell marker analysis Cytospin preparations of JURL-MK1 and JURL-MK2 cells were stained with May–Gru¨nwald–Giemsa and morphology was evaluated under a light microscope. Cytochemical stainings were performed for myeloperoxidase (MPO), alkaline phosphatase (AP), a-naphtyl acetate esterase (ANAE) and periodic acid-Schiff (PAS). Cell surface antigens were tested by flow cytometry (Cytoron Absolute; Ortho Diagnostic Systems, Raritan, NJ, USA). Cell viability was routinely assessed by analysis of light scatter properties. Living cells were also scored daily under a phase contrast microscope. The membrane expression of differentiation antigens was investigated with a panel of monoclonal antibodies (MoAb) directly coupled with fluorescein (FITC) or phycoerythrin (PE), including OKT11-FITC (CD2), Leu9-FITC (CD7), OKB19-FITC (CD19), OKB2-FITC (CD24), LeuM9-PE (CD33), LeuM7-PE (CD13), OKM5-FITC (CD36), Leu15-PE (CD11b), LeuM5-PE (CD11c), 17F11-PE (CD117), anti-GPA-FITC (glycophorin-A), anti-HLA-DR-PE (HLA-DR), IOM34-PE (CD34), HPCA-2-PE (CD34), anti-IIIa-FITC (CD61), IOP41a-FITC (CD41a), IOP42b-FITC (CD42b), IOP62-FITC (CD62P), IOP63-FITC (CD63), FMC8-FITC (CD9), Leu8-PE (CD62L), Leu44-FITC (CD44), IOT16-FITC (CD11a), Leu19-PE (CD56), IOL54-FITC (CD54), 2H4-PE (CD45RA), UCHL1-PE (CD45RO), Ki-1-FITC (CD30), IOT14-PE (CD25), Leu23-FITC (CD69) and IL2RbChFITC (CD122). Antibodies within the ‘OK’ series were obtained from Ortho Diagnostic Systems. ‘IO’ monoclonals, along with 17F11 and anti-GPA were obtained from Immunotech (Marseille, France). ‘Leu’ panel, HPCA-2 and anti-HLADR were purchased from Becton Dickinson. Anti-IIIa, UCHL1 and Ki-1 were obtained from DAKO (Glostrup, Denmark). FMC8 was obtained from Seralab (Crawley Down, UK), IL2RbCh from CLB (Amsterdam, The Netherlands) and 2H4 from Coulter Electronics (Hialeah, FL, USA). CD43 was studied by indirect immunofluorescence, using Leu22 (Becton Dickinson). CD40, CD126 (IL6-Ra) and CD130 (IL6Rb, gp130) were also studied by indirect immunofluorescence using, respectively, the MoAbs G28-5, MT-18 and AM64, obtained at the V International Workshop on Leukocyte Differentiation Antigens. Other cytokine receptors were studied by indirect immunofluorescence using MoAbs obtained at the VI International Workshop on Leukocyte Differentiation Antigens. In particular, the MoAbs used were SCO4 (CD116, GM-

CSF-R), hIL-1R-M1 (CD121a, IL1R type 1), hIL-1R2-M22 (CD121b, IL1R type 2), 9F5 (CDw123, IL3Ra), S456C9 (CD124, IL4R), R34.34 (CD127, IL7R), 3B5 (CD132, IL2R t chain), 4G8 (CD135, Flt3). As negative controls, IgG1, IgG2a and IgG2b immunoglobulin, purchased from Immunotech and Ortho Diagnostic Systems, were utilized in direct and indirect immunofluorescence experiments. As second step reagent for indirect immunofluorescence, a FITC-conjugated goat anti-mouse antiserum (Ortho Diagnostic Systems) was employed. In all immunofluorescence experiments, non-specific bindings due to Fc receptors were avoided by pre-incubating the cells at 4°C with rabbit immunoglobulin. Intracellular hemoglobin was detected by intracytoplasmic indirect immunofluorescence after cell permeabilization (Becton Dickinson permeabilizing solution), using a rabbit polyclonal antiserum (Dako) and a FITC-conjugated swine anti-rabbit antiserum (Dako) as second step reagent. The findings obtained for individual cell lines were expressed as a percentage of positive cells with the background subtracted. The mean channel of fluorescence intensity of histograms characterized by unimodal distribution was directly obtained from the cytometer and recorded.

Karyotype and bcr rearrangement analysis Chromosome analysis was performed as follows: the cultured cells were treated with 75 mM hypotonic KCl solution for 30 min at 37°C and then fixed with methanol/acetate (3:1) solution. The chromosomes were banded by tripsin-Giemsa staining.5 The rearrangements of M-bcr were tested in DNA samples obtained from the patient’s bone marrow cells and from the cell lines by Southern analysis, using a standard technique.6 Briefly, 15 mg of genomic DNA was digested with a three-fold excess of BglII, HindIII, EcoRI or BamHI restriction enzymes (New England Biolabs, Beverley, MA, USA), electrophoresed on an 0.8% agarose gel, blotted on positively charged nylon membranes (Hybond N-plus, Amersham, Buckingham, UK), and hybridized to 50 ng of a32P labelled 0.7 kb HindIIIBamHI genomic probe encompassing exons 2 and 3 of the M-bcr region of the bcr gene.7 A method based on reverse-transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of hybrid bcr/abl transcripts in the patient’s blood and in the cell line samples.8

In situ hybridization (ISH) for Epstein–Barr virus (EBV) nuclear RNA Detection of the two nuclear RNAs encoded by the EBV (EBER) was performed with an ISH kit (Dako) used in accordance with the manufacturer’s instructions. Cytospins of the cell lines were fixed in 4% formalin, air-dried, rehydrated in pure water and treated with proteinase K diluted in tris-buffered saline (TBS). Following two washings in pure water and immersion in 95% ethanol, the slides were air-dried and incubated at 55°C for 30 min with a FITC-conjugated EBV peptide nucleic acid (PNA) probe complementary to the nuclear EBER. FITC-conjugated PNA probe directed against glyceraldehyde 3-phosphate dehydrogenase cellular RNA and FITC-conjugated random PNA probes were used as positive and negative controls, respectively. Following the hybridization step, the

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slides were immersed in preheated stringent wash solution at 55°C for 25 min with shaking. Then, the slides were incubated with an anti-FITC AP-conjugated antibody for 30 min at room temperature. Following repeated washings in TBS and in pure water, the slides were incubated with two to three drops of substrate (5-bromo-4-chloro-3-indolylphosphate, BCIP) combined with nitroblue tetrazolium (NBT) and an inhibitor of endogenous AP (levamisole), immersed in tap water for 5 min and mounted in glycergel.

Molecular typing of HLA class I and class II HLA class I and class II typing of the patient’s fresh cells and the derived cell lines JURL-MK1 and JURL-MK2 was performed using the PCR-sequence specific primers (SSP) method described by Olerup et al.9,10 Commercial kits (Dynal, Oslo, Norway) for PCR-SSP low-resolution of HLA-A, -B, -C, DRB1 loci and PCR-SSP high-resolution of DRB1*01, DRB1*15/16, DRB5, DQB1 loci were used according to the manufacturer’s instructions. Genomic DNA from the patient’s fresh cells and from the derived cell lines was extracted as previously described.11 Amplification was carried out using Taq-polymerase (Perkin-Elmer Roche, Branchburg, NJ, USA). After PCR, electrophoresis on 2% agarose gel of amplified samples was carried out to detect both the presence of amplified control bands and amplification of allele- or group-specific bands. Since each primer pair gives rise to an amplified band which can be identified by its molecular weight, this was tested by using DNA molecular weight marker VI (Boehringer Mannheim). Alleles were assigned according to the Nomenclature for Factors of the HLA System.12

Ploidy studies JURL-MK1 and JURL-MK2 cells were harvested, washed twice with Ca2+/Mg2+-free PBS and stained with a propidium iodide (PI) ipotonic staining solution (PI 50 mg/ml in 0.1% sodium citrate, plus RNAse, 1 mg/ml) (all chemicals were from Sigma Chemical Co, St Louis, MO, USA). Briefly, one milliliter of PI solution was added to 1 × 106 cells and incubated for 30 min at room temperature in the dark. The DNA content was analyzed by a FACScan flow cytometer (Becton Dickinson). Normal human leukocytes were used as the standard in all experiments. Ploidy was defined by the DNA index (DI), which represents the DNA content of G0/G1 cells in comparison to the diploid leukocyte standard. The DI was calculated by dividing the mode channel value of the cell line G0/G1 peak by the mode channel value of the diploid leukocyte G0/G1 peak.

Induction of cell differentiation Induction of differentiation was studied utilizing the following agents: 12-O-tetradecanoyl-phorbol-13-acetate (TPA; Sigma Chemical Co, 10−7 M), all-trans retinoic acid (ATRA; Sigma Chemical Co, 0.5 mM), dimethyl sulfoxide (DMSO; Sigma Chemical Co, 1.2%, v/v), cytosine-arabinoside (Ara-C; Upjohn, Kalamazoo, MI, USA, 5 × 10−9M) and hemin (Sigma Chemical Co; 0.1 mM). TPA was dissolved in 100% ethanol, ATRA in 100% DMSO, Ara-C and hemin in DMEM with 20% FBS. The inducers were further diluted to a final concentration in culture medium. Before each experiment, the cells were

washed twice with DMEM and cultured for 4 days at a density of 1 × 106/ml under the aforementioned culture conditions. Differentiating agents were added to the cultures during logarithmic growth. Control cultures without inducers were always established simultaneously. Blast cell immunophenotyping was performed using the described panel of MoAbs, after 4 days of liquid culture with and without inducers. The analytical gate included a percentage of cells higher than 90%. The absolute number of viable cells before and after incubation was calculated cytometrically, and only experiments revealing comparable values were included in the analysis.

Proliferation assay The proliferative response of the cell lines to various hematopoietic effector molecules was examined by standard 3H-thymidine incorporation and b-scintillation counting (Liquid Scintillation Systems; Beckman Instruments, Fullerton, CA, USA). The cells were washed extensively prior to the proliferation experiments and the number of viable cells was quantitated by flow cytometry, according to their light scatter properties. The cells were seeded in triplicate in 100 ml medium in flat-bottomed 96-well plates and incubated in the absence or presence of cytokines, at a concentration of 2 × 105 cells/ml. For the last 6 h of the 48 h incubation period, 1 mCi methyl3 H-thymidine (Amersham-Buchler, Braunschweig, Germany) was added to each well. The panel of recombinant human cytokines was composed as follows (in parentheses, standard abbreviation, supplier and concentrations at which the cytokines were applied): granulocyte colony-stimulating factor (GCSF; Chugai-Rhone Poulenc, Vitry-sur-Seine, France, 10 ng/ml); granulocyte–macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Innishannon, Eire, 2.5 ng/ml), interleukin-2 (IL-2; Proleukin, EuroCetus, Amsterdam, The Netherlands, 50 U/ml), interleukin-3 (IL-3; Sandoz Pharma, Basel, Switzerland, 100 U/ml), interleukin-6 (IL-6; Biosource International, Camarillo, CA, USA, 100 U/ml), interleukin-11 (IL-11; Biosource International, 100 ng/ml) stem cell factor (SCF/kit ligand; Biosource International, 50 ng/ml). SCF was also used in combination with IL-3 and GM-CSF, at the concentrations mentioned. These assays were performed three times on different occasions, using the entire panel of factors. Moreover, dose–response curves (one cytokine per experimental session) were determined with: G-CSF 0.1 ng/ml to 100 ng/ml; GM-CSF 0.25 ng/ml to 250 ng/ml; IL-2 0.5 U/ml to 500 U/ml; IL-3, IL-6 and IL-11 1 U/ml to 1000 U/ml; SCF 0.5 ng/ml to 500 ng/ml.

Results

Morphology and cytochemical characteristics Phase contrast microscopy revealed in both lines cell size heterogeneity, with the presence of giant cells (3% in both cell lines) which were twice the size of the majority of cells. May– Gru¨nwald–Giemsa-stained cytospin preparations showed that JURL-MK1 cells were round, with large nuclei containing one to several prominent nucleoli. The cytoplasm was basophilic with many vacuoles. Almost all cells showed cytoplasmic protrusions. JURL-MK1 also occasionally showed cells charac-


terized by a larger size and lobulated nuclei (Figure 1A). JURLMK2 morphology was similar, although a few differences could be noted. In particular, JURL-MK2 exhibited a cytoplasm with frequent azurophilic granules, and cytoplasmic protrusions were observed in a limited fraction of cells. A large proportion of JURL-MK2 cells had the appearance of large cells with multi-lobulated nuclei (Figure 1B). As far as cytochemical reactions are concerned, both JURL-MK1 and JURL-MK2 cells were strongly positive for PAS and weakly positive for ANAE. MPO and AP activity was not observed (data not shown).

Immunophenotype The surface marker analysis of JURL-MK1 and JURL-MK2 cells is reported in Table 1. Both cell lines were clearly positive for CD33, CD63, CD43 and CD45RO. In addition, in both lines a limited but consistent percentage of cells expressed gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gpIb (CD42b), glycophorin A (GPA), hemoglobin and CD34 (tested by two different MoAbs, IOM34 and HPCA-2). Searching for differences between the two lines, we found that CD69 was relatively more often expressed on JURL-MK1 cells, whereas CD13, CD44, CD54, CD30 and CD40 were specific features of JURL-MK2 cells. Furthermore, HLA-DR was expressed by most JURL-MK2 cells and by a minority of JURL-MK1 cells. Among cytokine receptors, CD117 (SCF-R) was strongly displayed by a large proportion of JURL-MK1 cells but hardly

Table 1

Surface marker analysis of the two novel MK cell lines

JURL-MK1

JURL-MK2

1.1 1.2 1.0 2.9

1.3 1.1 5.8 7.5

10.6 90.9

59.1 98.9

1.2 1.8

0.9 3.7

Stem Cell HLA-DR CD34

48.1 2.3

96.7 2.0

Platelet CD9 CD36 CD41a CD42b CD61 CD63

2.8 30.0 40.1 1.0 31.8 77.0

3.7 55.0 28.9 2.5 25.6 98.0

Adhesion CD11a CD11b CD11c CD43 CD44 Cd45RA CD45R0 CD54 CD62L CD62P

1.8 1.3 0.7 99.0 8.9 1.0 84.4 7.7 1.5 1.3

4.5 6.4 1.1 99.1 33.0 5.4 99.0 98.8 3.4 2.5

Activation CD56 CD69

2.9 28.8

13.4 11.1

NGF-R superfamily CD30 CD40

1.1 0.2

95.8 78.8

Cytokine receptors CD25 CD116 CD117 CD121a CD121b CD122 CDw123 CD124 CD126 CD127 CD130 CD132 CD135

90.8 24.3 95.6 1.1 4.2 1.1 0.9 1.8 1.1 1.5 1.7 1.3 2.8

94.7 47.6 18.9 2.5 5.0 1.5 57.6 5.4 9.2 2.9 1.5 2.7 2.9

Lymphoid CD2 CD7 CD19 CD24 Myeloid CD13 CD33 Erythroid GPA Hemoglobin

Data are expressed as a percentage of positive cells. Relevant figures (.15%) are in bold. During the last year, the phenotypic characterization was repeated monthly, with reproducible results. Data are referred to one representative characterization.

Figure 1 Microphotographs of JURL-MK1 (A) and JURL-MK2 (B) cells (May–Gru¨nwald–Giemsa staining, × 1000).

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Figure 2 JURL-MK1 (a) and JURL-MK2 (b) Giemsa-banded karyotype, displaying 39 and 72 chromosomes, respectively. For details see Results section.


detectable on a minority of JURL-MK2 cells. Both cell lines were clearly positive for CD25 (IL2-Ra chain), while CD122 (IL2-Rb chain) and CD132 (IL2-Rt chain) were undetectable. CD116 (GM-CSF-R) was more frequently expressed by JURLMK2 cells as compared to JURL-MK1, while CDw123 (IL3Ra chain) was exclusively expressed by JURL-MK2. The other cytokine receptors tested were unexpressed by both the cell lines (Table 1).

Cytogenetic analysis JURL-MK1 has a hypodiploid karyotype (33–43 chromosomes). The predominant karyotype was 39XY,−4,−5,−9, der(9)t(9;22)(q34q11),−11,−12,i(17),−18,−19, −21,der(22)t(9;22)(q34q11),+mar1(six metaphases) (Figure 2a). JURL-MK2 was near-triploid (66–80 chromosomes). One paradigmatic karyotype is presented in Figure 2b (72XXY, +1,+3,+3,+4,+5,+5,+6,+7,+8,+8, der(9)t(9;22)(q34q11), +der(9) t(9;22) (q34q11),+10,+10,+12,+12,+13,+14,+15,+15, +16,i(17),+18,+21,+21, der(22)t(9;22)(q34q11),+22, +der(22) t(9;22)(q34q11). Among the 24 cells analyzed, three metaphases displayed a pattern identical to that described, whereas 14 metaphases had similar findings but with additional random gains. The karyotypes have been tested on different occasions, with identical results.

no staining, indicating that the cells were not infected with the Epstein–Barr virus (data not shown).

Molecular typing HLA class I and class II genotype of the patient’s fresh cells and of the derived cell lines was: A*33,68; B*13,14; Cw* 06,08; DRB1*0102, 1501; DRB5*0101; DQB1*0501,0602. As a representative experiment, PCR-SSP high resolution of DRB1*15/16 is presented in Figure 3. Results showed positive DNA amplification of internal control (429 bp) in all gel lanes, whereas specific DNA amplification, found in gel lanes 1, 3, 5, 10, 12, identified the presence of the DRB1*1501.

Cellular ploidy analysis Flow cytometry analysis demonstrated that the G0/G1 peak of JURL-MK1 was comparable to the diploid peak of normal leukocytes (DI = 1.03), while the G0/G1 peak of JURL-MK2 indicated hyperdiploidy (DI = 1.9) (data not shown).

In situ hybridization (ISH) for Epstein–Barr virus (EBV) nuclear RNA Immunocytochemistry testing with FITC-conjugated EBV PNA probe complementary to the two nuclear EBER RNAs revealed

Figure 3 PCR-SSP high resolution of DRB1*15/16. A total of 12 reactions were performed per DNA sample. Each reaction contained a primer pair for the amplification of a specific allele and another one for the amplification of the internal control (429 bp). (a) DNA from the patient’s fresh cells; (b) DNA from JURL-MK1; (c) DNA from JURL-MK2. m: standard molecular weight markers.

Figure 4 Southern analysis of the M-bcr region. Lane 1: DNA samples from the patient at diagnosis; lane 2: DNA samples from JURL-MK1 cell line; lane 3: DNA samples from JURL-MK2 cell line. SM: molecular size markers.

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Detection of BCR rearrangements By Southern analysis of the M-bcr region following BglII digestion, DNA samples extracted from fresh bone marrow cells and from both cell lines shared the same rearranged band of 9.1 kb (Figure 4). No rearranged band was detected after digestions with the restriction enzymes (HindIII (Figure 4) and BamHI (not shown). These data were consistent with a hybrid bcr/abl gene with the junction b3/a2. Indeed, RT-PCR analysis revealed in all samples a p210 type of bcr/abl transcript with the b3/a2 junction. b2a3 transcript was not detectable by RTPCR (data not shown).

Induction of cell differentiation Exposure of the two cell lines over 4 days to Ara-C, ATRA and hemin did not show significant effects on the cell surface marker pattern when compared to the immunophenotype of non-induced cells. By converse, exposure of JURL-MK1 to TPA and of JURL-MK2 to TPA or DMSO produced relevant surface marker pattern modifications. Marked differences were observed when analyzing the expression of CD117 and CD30, along with a marker of MK differentiation, CD41a (Figure 5). In particular, in JURL-MK1 we observed a significant TPA-driven increase of CD41a expression (Figure 5a), while DMSO had no effect (Figure 5b). By contrast, in JURLMK2 we observed comparable results by both TPA (Figure 5c)

and DMSO (Figure 5d): a clear-cut increase of CD41a was paralleled by a significant augmentation of CD117 expression and a dramatic decrease of CD30 display. Dual fluorescence experiments were performed by simultaneously analyzing CD30 and CD117 expression. A clear-cut reciprocal modulation of CD30 and CD117 was observed in JURL-MK2 cells treated with either TPA (Figure 6) or DMSO. Along with these main results documented in Figures 5 and 6, TPA stimulation exerted on JURL-MK1 a number of additional effects, ie a marked increase in the percent expression of CD44, CD54 and CD69 (from 8.9 to 76.1%, from 7.7 to 47.2% and from 28.8 to 95.4%, respectively). On the same cells, the only effect produced by DMSO was a decrease in HLA-DR+ cells, from 48.1 to 19.6% (data not shown). In JURL-MK2, TPA stimulation increased CD44 and CD69 expression (from 30.0 to 94.5% and from 11.1 to 81.8%, respectively), while reducing CD54 and CD40 expression, from 98.8 to 74.4% and from 78.8 to 30.6%, respectively. Similar results were obtained with DMSO. In addition, in the JURL-MK2 cell line, the mean channel of fluorescence intensity of HLA-DR positive cells after exposure to DMSO was significantly lower as compared to control cultures (data not shown).

Effect of growth factors on JURL-MK1 and JURL-MK2 proliferation The JURL-MK1 and JURL-MK2 cell lines grow in the absence of any added growth factor. However, the effects of selected

Figure 5 Surface marker analysis of the two lines before (T0) and after (96 h) treatment with TPA or DMSO. (a) JURL-MK1 treated with TPA; (b) JURL-MK1 treated with DMSO; (c) JURL-MK2 treated with TPA; (d) JURL-MK2 treated with DMSO. Data are expressed as a percentage of positive cells and are referred to one representative experiment. Similar results were obtained in three independent experiments.


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Figure 6 Four-dimensional flow cytometry analysis of JURL-MK1 and JURL-MK2 cells prior to and following induction of differentiation with TPA (4-day exposure). Viable cells were identified in a dual parameter cytogram of orthogonal and 180° light scatter, then cells were analyzed in two color cytograms for their expression of a combination of CD30 (x axis) and CD117 (y axis). Vertical and horizontal cursors were set on the basis of the reaction with isotype-matched controls. (a) JURL-MK1 without TPA; (b) JURL-MK1 with TPA; (c) JURL-MK2 without TPA; (d) JURL-MK2 with TPA. In JURL-MK2, TPA induced a measurable up-modulation of CD117-c-kit, accompanied by a significant reduction of CD30 expression. DMSO produced similar results.

cytokines on the proliferation of these cells were assessed by measuring the 3H-thymidine incorporation into DNA. The values were calculated in comparison to the incorporation level without growth factor (51 000 ± 5200 c.p.m.) and are averages of triplicates. SCF stimulated the 3H-thymidine incorporation (1.5-fold basal level) of JURL-MK1 cells, while it was ineffective on JURL-MK2 (Figure 7). No synergistic effect with IL-3 or GM-CSF was observed. G-CSF, GM-CSF, IL-2, IL-3, IL6 and IL-11 had no significant effect on 3H-thymidine incorporation in either JURL-MK1 or JURL-MK2. Three different assays gave identical results. Dose–response curves, performed with all factors, confirmed that SCF stimulated JURL-MK1 (but not JURL-MK2) in a concentration-dependent manner with the maximal stimulation at 50 ng/ml (data not shown). Discussion Cell lines of CML origin, representing the early differentiation stages of various hemopoietic lineages are useful models to investigate biological events occurring in this type of leukemia.2 Moreover, among the various CML cell lines, cells arrested at the MK differentiation stage can provide new insights into biological studies on megakaryocytopoiesis, which are often hampered by difficulty in obtaining MK progenitors from normal human bone marrow.3,4 In the present study, we describe two novel MK-oriented cell lines, estab-

lished from the peripheral blood of a patient with Ph+ CMLBC, which are characterized by different cytogenetic, phenotypic and functional features. The possibility of generating more than one cell line from a single patient has been previously described, for example as regards the myelomonocytic cell line ME-1.13 There are two possible explanations for the generation of morphologic, cytochemical, phenotypic and cytogenetic changes in different sublines: the occurrence of an intracellular stochastic event or, alternatively, the establishment of unforeseeable and imperceptible divergent conditions in split subculture microenvironments. In order to obtain unequivocal evidence to demonstrate that our cell lines are derived from the same ancestry, as well as to formally exclude cross-contamination and misidentification, we showed the HLA class I and class II molecular typing of the original cells in direct comparison with the resulting cell lines. As for cytogenetic differences, JURL-MK1 is hypodiploid, whereas JURL-MK2 is near triploid. This finding, obtained by conventional cytogenetic analysis, was confirmed by the results of ploidy analysis by flow cytometry, although the concordance between these two methods cannot be absolute. In order to clarify the differentiation stage of the cell lines JURL-MK1 and JURL-MK2 we evaluated a large series of cell markers, including platelet glycoproteins, the CD34 antigen and erythromyeloid specificities. A ‘pure’ MK cell line should display the following biological characteristics: expression of


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Figure 7 Effect of growth factors on the proliferation of JURL-MK1 (a) and JURL-MK2 (b) cells. The values were calculated in comparison to the incorporation level without growth factor (51 000 ± 5200 c.p.m.) and are averages from triplicates. Three independent experiments gave identical results.

the MK specific antigen CD41; expression of c-kit/CD117; MK differentiation induced by TPA.4 The absence of the CD42b/gpIb molecule does not preclude the attribution of MK origin to JURL-MK1 and JURL-MK2, since even in other previously described MK cell lines its expression is variable.14 Moreover, when typing acute leukemia cells, CD41a+/CD42b− cells are considered as megakaryoblasts,15 while the simultaneous expression of CD41a and CD42b has been referred to as characteristic of (pro)megakaryocytic leukemia. At the same time, the constitutive expression of CD61/gpIIIa along with CD36/thrombospondin receptor15 provides additional

evidence for the MK nature of JURL-MK1 and JURL-MK2 cell lines. JURL-MK1 and JURL-MK2 do not express CD34. It is not clear whether this phenotype reflects a relatively late differentiation stage, as suggested by the existence of CD34−/CD61+ MK precursors16 or, alternatively, if it indicates a malignancyrelated surface membrane abnormality. The lack of CD34 positivity cannot be due to a defective reactivity in the reagents used, since the finding was confirmed using two different MoAbs. Of note, other published MK lines are characterized by low or even absent CD34 expression.4 The pheno-


typic pattern CD41a+CD42b−CD34− has been reported on MK cell lines, in the context of a wide spectrum of different antigenic combinations. A data bank search has shown that 28/29 MK cell lines were CD41a+, 15/25 CD42b+ and 18/24 CD34+. The JURL-MK1 and JURL-MK2 cell lines exhibit high levels of the CD45 isoform R0. Recently, we have demonstrated a preferential expression of CD45R0 on CML-BC cells as compared to de novo acute myeloblatic leukemia (AML) blast cells, which very frequently express CD45RA.17 Since CD45R0 has been associated with adhesive cell properties,18,19 JURL-MK1 and JURL-MK2 may be a tool for further analyzing the potential involvement of CD45R0 in MK adhesion processes. In this report we demonstrate that the IL-2 receptor a-chain (CD25) is strongly expressed by both JURL-MK1 and JURLMK2, while b and t chains are cytometrically undetectable. It has been demonstrated that the lack of detection by flow cytometry of the II-2 receptor b chain (p75, CD122) is caused by the low number of molecules per cell and that this chain expression is a common feature of myeloid leukemia cells.20 In non-lymphoid malignancies the expression of CD25 is related to the co-expression of markers of different lineages as well as to the presence of the Philadelphia chromosome. 21 Another relevant finding in this work is the divergent expression of CD117/c-kit, CD116/GM-CSF-R and CDw123/IL3Ra in these two cell lines. The c-kit product, reported on most of the megakaryoblastic cell lines established so far,22,23 was more expressed on JURL-MK1, as compared to JURL-MK2. In accordance with surface phenotype, the growth of the JURL-MK1 cell line could be significantly enhanced by exposure to SCF. By contrast, although CD116 and CDw123 were an almost exclusive feature of JURL-MK2, GM-CSF and IL-3 were unable to stimulate JURL-MK2 cell growth. Moreover, GM-CSF and IL-3 were unable to synergize with SCF on both JURL-MK1 and JURL-MK2 cell lines. These data are divergent from those obtained with another welldescribed MK cell line, ELF-153,3,4 and could enable additional studies concerning the effects of SCF on megakaryocytopoiesis. Further studies on the effects of other hematopoietic cytokines, in particular the recently cloned human recombinant thrombopoietin (TPO) are required. A recent review reported that while none of 30 growth factor independent erythro-MK cell lines respond to TPO with increased proliferation, this growth factor strongly augments the growth of cytokinedependent cell lines (HU-3, M0-7e, TF-1), which can be rendered TPO-dependent and used as bioassays.24 The vast majority of JURL-MK2 cells express CD30, which is a member of the nerve growth factor-receptor (NGF-R) superfamily25,26 and is typically expressed by cells of certain lymphoid neoplasms;27 JURL-MK1 cells lack this antigen. The occasional expression of CD30 in myeloid malignancies has recently been reported, particularly in myelodysplasia-AML. 28 It has also been shown that LAMA-84, an MK-oriented cell line, produces CD30 transcripts.29 The results of published observations indicate that the binding of the CD30 ligand to its receptor confers the capacity for signal transduction to different target cells.29,30 Interestingly, JURL-MK2 (and not JURLMK1) cells express CD40, another member of the NGF-R superfamily. In this respect, it will be of interest to investigate whether CD30 and/or CD40 ligands are able to trigger a biological response in the JURL-MK2 cell line. Treatment of the two novel megakaryoblastic cell lines here reported with differentiation inducers has indicated that JURLMK2 is more inducible as compared to JURL-MK1. In parti-

cular, while JURL-MK1 phenotypic changes are only promoted using a phorbol ester, JURL-MK2 shows a clear-cut response to both TPA and DMSO, as testified by the significant modulation of several surface markers. In addition, TPA and DMSO induced a marked up-regulation of CD117/c-kit expression in JURL-MK2. This finding is in keeping with previous data, indicating that the treatment of MK cell lines with TPA and bryostatin 1 triggers a significant increase in c-kit mRNA levels.22 Our data demonstrate that c-kit expression can be augmented by various differentiation stimuli, not necessarily confined to protein kinase C activators. Differentiation experiments performed on JURL-MK2 indicated a reciprocal modulation of CD30 and CD117 expression, TPAand DMSO-driven CD117 up-modulation being consistently paralleled by decreased CD30 expression. To date, about 20 megakaryoblastic cell lines, including the present two, have been published, generally derived from AML-M7 or CML-BC patients. Some of these cell lines (MEG01, DAMI, CMK, MKPL-1, M-07e, MOLM-1, UT-7) represent nowadays a standard tool for studying MK biology.3,4 In our opinion, our novel cell lines can be employed in studying new aspects of megakaryocytopoiesis, such as the expression and role of NGF-Rs and c-kit, and for further elucidating the biological meaning of the apparently reciprocal regulation of these molecules.

Availability of the cell lines JURL-MK1 and JURL-MK2 cell lines will be made available to outside investigators upon request to the first (RDN) or senior author (LDV).

Acknowledgements R Di Noto was a recipient of an AIRC fellowship throughout the period of establishment of the two cell lines (1993–1995). Ploidy was studied by Dr Stefano Pepe and Dr Angela Ruggiero, Chair of Oncology, Universita` Federico II, Naples.

References 1 Kantarjian HM, Deisseroth A, Kurzrock R, Estrov Z, Talpaz M. Chronic myelogenous leukemia: a concise update. Blood 1993; 82: 691–703. 2 Drexler HG. Leukemia cell lines: in vitro models for the study of chronic myeloid leukemia. Leukemia Res 1994; 18: 919–927. 3 Hassan HT, Freund M. Review of megakaryoblastic cell lines. Characteristic biological features of human megakaryoblastic leukaemia cell lines. Leukemia Res 1995; 19: 589–594. 4 Hassan HT, Drexler HG. Interleukins and colony-stimulating factors in human myeloid leukemia cell lines. Leuk Lymphoma 1995; 20: 1–15. 5 Seabright M. A rapid banding technique for human chromosomes. Lancet 1971; II: 971–972. 6 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press: New York, 1989. 7 Negrini M, Tallarico A, Pazzi I, Castagnosli A, Cuneo A, Castoldi GL. A new chromosomal breakpoint in Ph positive, bcr negative chronic myelogenous leukemia. Cancer Genet Cytogenet 1992; 61: 11–13. 8 Pane F, Frigeri F, Camera A, Sindona M, Brighel F, Martinelli V, Luciano L, Selleri C, Del Vecchio L, Rotoli B, Salvatore F. Complete phenotypic and genotypic lineage switch in a Philadelphia

1563


1564 9

10 11

12

13

14

15

16

17

18

19

chromosome-positive acute lymphoblastic leukemia. Leukemia 1996; 10: 741–749. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor– recipient matching in cadaveric transplantation. Tiss Antigens 1992; 39: 225–235. Olerup O, Aldener A, Fogdell A. HLA-DQB1 and DQA1 typing by PCR amplification with sequence specific primers (PCR-SSP) in 2 hours. Tiss Antigens 1993; 41: 119–134. Lombardi ML, Mercuro O, Tecame G, Fusco C, Ruocco V, Salerno A, Pirozzi G, Manzo C. Molecular analysis of HLA DRB1 and DQB1 in Italian patients with Pemphigus vulgaris. Tiss Antigens 1996; 47: 228–230. Bodmer JG, Marsh SGE, Albert ED, Bodmer WF, Bontrop RE, Charron D, Dupont B, Erlich HA, Mach B, Mayr WR, Parham P, Sasazuki T, Schreuder GMT, Strominger JL, Svejgaard A, Terasaki PI. Nomenclature for factors of the HLA system, 1995. Tiss Antigens 1995; 46: 1–18. Yanagisawa K, Sato M, Horiuchi T, Hasegawa H, Fujita S. Difference in response to colony-stimulating factors and involvement of protein kinase C signal transduction system in three subclones from the ME-1 cell line and two sublines. Blood 1994; 84: 84–93. Takeuchi S, Sugito S, Uemura Y, Miyagi T, Kubonishi I, Taguchi H, Enzan H, Ohtsuki Y, Miyoshi I. Acute megakaryoblastic leukemia: establishment of a new cell line (MKPL-1) in vitro and in vivo. Leukemia 1992; 6: 588–594. Imamura N, Ota H, Abe K, Kuramoto A. Expression of the thrombospondin receptor (CD36) on the cell surface in megakaryoblastic and promegakaryocytic leukemias: increment of the receptor by megakaryocyte differentiation in vitro. Am J Hematol 1994; 45: 181–184. Debili N, Issaad C, Masse´ JM, Guichard J, Katz A, Breton-Gorius J, Vainchenker W. Expression of CD34 and platelet glycoprotein during human megakaryocytic differentiation. Blood 1992; 80: 3022–3035. Schiavone EM, Lo Pardo C, Di Noto R, Manzo C, Ferrara F, Vacca C, Del Vecchio L. Expression of the leukocyte common antigen (LCA, CD45) isoforms RA and R0 in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis. Br J Haematol 1995; 91: 899–906. Stamenkovic I, Sgroi D, Aruffo A, Sy MS, Anderson T. The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45R0 on T cells and alpha 2-6 sialyltransferase, CD75, on B cells. Cell 1991; 66: 1133–1144. Coombe DR, Watt SM, Parish CR. Mac-1 (CD11b/CD18) and CD45 mediate the adhesion of hematopoietic progenitor cells to

20

21

22 23

24 25

26 27

28

29

30

stromal cell elements via recognition of stromal heparan sulfate. Blood 1994; 84: 739–752. Pizzolo G, Rigo A, Zanotti R, Vinante F, Vincenzi C, Cassatella M, Carra G, Castaman G, Chilosi M, Semenzato G, Zambello R, Trentin L, Libonati M, Perona G. a (p55) and b (p75) chains of the interleukin-2 receptor are expressed by AML blasts. Leukemia 1993; 7: 418–425. Nakase K, Kita K, Otsuji A, Anazawa H, Hoshino K, Sekine T, Shirakawa S, Tanaka I, Nasu K, Tsutani H, Dohy H, Tsudo M. Diagnostic and clinical importance of interleukin-2 receptor alpha chain expression on non-T cell acute leukaemia cells. Br J Haematol 1992; 80: 317–326. Hu ZB, Ma W, Uphoff CC, Quentmeier H, Drexler HG. c-kit expression in human megakaryoblastic leukemia cell lines. Blood 1994; 8: 2133–2144. Morita S, Tsuchiya S, Fujie H, Itano M, Ohashi Y, Minegishi M, Imaizumi M, Endo M, Takano N, Konno T. Cell surface c-kit receptors in human leukemia cell lines and pediatric leukemia: selective preservation of c-kit expression on megakaryoblastic cell lines during adaptation to in vitro culture. Leukemia 1996; 10: 102–105. Drexler HG, Quentmeier H. Thrombopoietin: expression of its receptor MPL and proliferative effects on leukemic cells. Leukemia 1996; 10: 1405–1421. Durkop H, Latza U, Hummel M, Eitelbach F, Seed B, Stein H. Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell 1992; 68: 421–427. Goodwin RG, Armitage RJ. CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF. Cell 1993; 73: 1349–1360. Smith CA, Gruss HJ, Davis T, Anderson D, Farrah T, Baker E, Sutherland GR, Brannan C, Copeland NG, Jenkins NA, Grabstein KH, Gliniak B, McAlister IB, Fanslow W, Alderson M, Falk B, Gimpel S, Gillis S, Din WS, Goodwin RG, Armitage RJ. CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF. Cell 1993; 73: 1349–1360. Masuya M, Kita K, Shimizu N, Ohishi K, Katayama N, Sekine T, Otsuji A, Miwa H, Shirakawa S. Biologic characteristics of acute leukemia after myelodysplastic syndrome. Blood 1993; 81: 3388–3394. Gruss HG, DaSilva N, Zhen-Bo H, Uphoff CC, Goodwin RG, Drexler HG. Expression and regulation of CD30 ligand and CD30 in human leukemia–lymphoma cell lines. Leukemia 1994; 8: 2083–2094. Gruss HJ, Boiani N, Williams DE, Armitage RJ, Smith CA, Goodwin RG. Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines. Blood 1994; 83: 2045–2056.