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A Novel Human Myeloid Leukemia Cell Line, NKM-1, Coexpressing Granulocyte Colony-Stimulating Factor Receptors and Macrophage Colony-Stimulating Factor Receptors'

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A Novel Human Myeloid Leukemia Cell Line, NKM-1, Coexpressing Granulocyte Colony-Stimulating Factor Receptors and Macrophage Colony-Stimulating Factor Receptors' (CANCER RESEARCH 50. 7703-7709. December 1. I990| A Novel Human Myeloid Leukemia Cell Line, NKM-1, Coexpressing Granulocyte Colony-stimulating Factor Receptors and Macrophage Colony-stimulating Factor Receptors' Takae Kataoka,2 Yoshihisa Morishita, Michinori Ogura, Yasuo Morishima, Masayuki Towatari, Yukio Kalo, Hideo Inoue, and Hidehiko Saito The First Department of Internal Medicine, Nagoya University School of Medicine fT. K., Y. M., Y. M., M. T., H. SJ. Showa-ku, Nagoya 466; Department ofllemalology and Chemotherapy, Aichi Cancer Center Hospital [M. O.], Chikusa-ku. Nagoya 464; Department of Internal Medicine, Showa Hospital [Y. A'./. Konan 483; and Kirin Brewery Co. [H. I./, Shihuya-ku, Tokyo ¡50,Japan ABSTRACT respond to a CSF and the absolute number of receptors ex pressed for that factor (12, 13). This lack of correlation may be A novel human myeloid leukemia cell line, NKM-1, was established due to the heterogeneity of the leukemic cell population in a from a patient with acute myeloid leukemia (FAB classification \12). patient or to the fact that receptor levels are measured on whole The cells were positive for myeloperoxidase staining and cluster of leukemic cell populations, which may not accurately reflect differentiation 15 cell surface antigen. Radiolabeled recombinant human granulocyte (G) colony-stimulating factor (CSF) was used, and 60 specific receptor levels for each CSF on relatively rare clonogenic cells. binding sites/cell with a Kd 100 pmol/liter were demonstrated on the cell Therefore, it would be advantageous to use a homogeneous cell surface. '"I-G-CSF binding was not inhibited by interleukin-3, granulo- population when we study how CSFs regulate proliferation of cyte-macrophage CSF, or macrophage (M) CSF. NKM-1 cells also myeloid leukemic cells. expressed M-CSF" receptors detected by c-fms mRNA expression. In Permanent cell lines of myeloid leukemia cells are considered concordance with the receptor expression, NKM-1 cells proliferated in to be the clonal expansion of hematopoietic stem cells at some response to exogenous G-CSF or M-CSF in a dose-dependent manner stage of maturation. Among established myeloid cell lines, (0.1-100 ng/ml), while interleukin-3 or granulocyte-macrophage CSF however, proliferative responses to exogenous CSFs were rarely had no effect. Colony-forming capacity of NKM-1 cells in semisolid agar observed. This may be accounted for by the rarity of the was also enhanced with the addition of 10 ng/ml of G-CSF or M-CSF expression of CSF receptors on cell lines (13) or by the diverse but decreased at higher concentrations. During CSF stimulation, no signal transductions through the binding to its receptors. Here remarkable changes were observed morphologically and phenotypically. The stimulatory effect of G-CSF" and M-CSF on the cell growth was we present a novel human myeloid leukemia cell line, NKM-1, additive. Neither G-CSF-binding capacity nor c-fms mRNA expression which expresses both G-CSF receptors and M-CSF receptors. was altered by pretreatment with M-CSF or G-CSF, respectively. This The cells proliferate remarkably in response to exogenous G- cell line may provide a useful in vitro model for the study of CSF roles CSF and M-CSF. in myeloid leukemia cell proliferation. MATERIALS AND METHODS INTRODUCTION Case History. The cell line described in this report was derived from AML' is characterized by excessive proliferation of myeloid a peripheral blood sample of a 33-year-old male with AML. On October precursors and maturation arrest resulting in accumulation of 19, 1981, he was admitted to Daido Hospital (Nagoya, Japan) because immature hematopoietic cells. In the presence of appropriate of gingival bleeding and epigastralgia. Physical examination revealed growth factors, leukemic cells proliferate in suspension culture mild splenomegaly and numerous ecchymotic spots. Peripheral blood or form colonies in semisolid media. Recent studies describing cell counts at the time of admission were as follows: RBCs, 3.65 x lO'V'iter; platelets, 17 x lO'/lUer; and WBCs, 108 x 10'/liter with the effects of recombinant human hematopoietic growth factors 74% leukemic blasts. The number of iliac bone marrow nucleated cells on AML blasts have revealed that cells from most patients was 450 x 10g/liter with 88.4% blasts. Both prothrombin time and proliferate in response to IL-3 (1-4), GM-CSF (1-9), G-CSF (1-5, 9, 10), or M-CSF (4, 11). The ability of a growth factor partial thromboplastin time were markedly prolonged, the plasma fibrinogen level was 95 mg/dl, and the serum fibrinogen degeneration to exert its biological effect is linked to the expression of products value was 80 pg/ml. AML [FAB classification M2 (14)] with receptors specific for that factor on the surface of the cells, disseminated intravascular coagulation was diagnosed. On the second although there is no direct correlation between the ability to hospital day, he suddenly went into a coma and computed tomography scanning showed a massive cerebroventricular hemorrhage. He died the Received 5/29/90; accepted 8/31/90. The costs of publication of this article were defrayed in part by the payment same day. of page charges. This article must therefore be hereby marked advertisement in Cell Culture. On October 19, 1981, a heparinized peripheral blood accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sample was obtained with the patient's consent and brought to the First 1This work was supported in part by a Grant-in-Aid for Scientific Research Department of Internal Medicine. Nagoya University. The mononu- from the Ministry of Education, Science and Culture: by a Grant-in-Aid from the clear cells were separated by Ficoll-Conray gradient centrifugaron and Ministry of Health and Welfare: and by a Grant-in-Aid from the Aichi Blood Disease Research Juridical Foundation in Japan. seeded in Falcon 3013 plastic tissue culture flasks (Falcon, Division of 2To whom requests for reprints should be addressed, at First Department of Becton Dickinson, Oxnard, CA) at 10" cells/ml in RPMI 1640 medium Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho. (GIBCO Laboratories, Grand Island, NY) supplemented with 20% Showa-ku, Nagoya 466. Japan. 3The abbreviations used are: AML, acute myeloid leukemia; IL-3, interleukin- FCS (Flow Laboratories, Stanmore, New South Wales, Australia), 3; CSF, colony-stimulating factor: GM-CSF, granulocyte-macrophage C'SF: G- aqueous penicillin G (100 units/ml), and streptomycin (50 ^g/ml) and CSF, granulocyte CSF; M-CSF. macrophage CSF: FCS, fetal calf serum; MGG, were incubated at 37°Cwith a humidified atmosphere of 5% CO2. May-Grunwald-Giemsa: MPO, myeloperoxidase; CAE, naphlhol AS-Dchloroac- Cultures were fed once weekly by partial replacement of spent medium etate esterase; NBE, a-naphthyl butyrate esterase: CD, cluster of differentialon; NBT. nitroblue tetrazolium; TPA, 12-O-tetradecanoylphorbol-13-acetate; SB, with the fresh medium. No conditioned media or feeder cells were used. sodium butyrate; MTT, 3-(4,5-dimethylthia/ol-2-yl)-2,5-diphenyltetra7.oliuni bro Morphological Studies. The cells were stained with MGG, MPO, mide; Kd. dissociation constant. and dual esterase staining for CAE and NBE according to the standard 7703 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. G-CSF-, M-CSF- RESPONSIVE CELL LINE procedures. Ultrastructural morphology (conventional thin electron above. The recovery of clonogenic cells/2 ml in suspension was calcu microscopy) and MPO were analyzed as described previously (15). lated by multiplying the plating efficiency in agar by the number of Cell Surface Markers. Cell surface antigens were determined by an cells harvested from the suspension culture (25). indirect immunofluorescent method using monoclonal antibodies and Cell Cycle Analysis. DNA histogram analysis with propidium iodide analyzed by flow cytometry (EPICS PROFILE, Coulter Electronics, was performed (26) by staining 1 x IO6leukemic blasts/sample with 50 Inc., Hialeah, FL) as described previously (15). The following monoclo Mg/ml propidium iodide (Sigma Chemical Co.) in 0.1 % sodium citrate nal antibodies were used for the analysis. CD numbers were used from containing 0.2% Nonidet P-40 (Particle Data Inc., Elmhurst, IL) and the designation of the Fourth International Workshop and Conference 250 ^g/ml RNase (Boehringer-Mannheim, Mannheim, West Germany) on Human Leukocyte Differentiation Antigens. 9.6 (CD2), 64.1 (CD3), for 30 min at 0°Candincubated for another 15 min at 37"C. The nuclei 10.2 (CDS) and 7.2 (HLA-DR) were provided by Dr. J. A. Hansen were analyzed by flow cytometry (EPICS PROFILE). The percentage (Fred Hutchinson Cancer Research Center, Seattle, WA). ICIO (CD15) of the cells having S and ( ;.. + M content of DNA was estimated by a was the generous gift of Dr. I. Burnstein, Fred Hutchinson Cancer program developed by Ortho Diagnostics. Research Center. 4A (16) (CD7), NL-1 (17) (CD10), and HPL3 (18) Tritiated Thymidine Incorporation. Cells (6 x 10') were cultured in (CD41) were produced locally. B4 (CD 19), Bl (CD20), MOI (CD1 Ib), 200 »ilofCOSMEDIUM-001 with or without G-CSF in 96-microtiter MY7 (CD13), MY4 (CD14), and MY9 (CD33) were purchased from plates (Nunc). After 20 h of incubation at 37°C,7.4kilobecquerel (kBq) Coulter Co. Glycophorin A (erythrocytes) (Immunotech, Marseille, tritiated thymidine (2.48 x 10* kBq; NEN Research Products, Boston, France), Leu 1Ib (CD16) (Becton Dickinson Co.), anti-CD18 (CD18), MA) was added, and the cells were harvested 4 h later using a Titertek and anti-low affinity Fey receptor (CDw32) (Cosmo Bio Co., Tokyo, harvester 550 (Flow Laboratories) (4). Radioactivity was determined Japan) were also used. with a Beckmann II scintillation counter (Becton Instruments Inc., Chromosome Analysis. The cultured cells were incubated in the Fullerton, CA).
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