American Journal of Hematology 34:154-156 (1990)

Varied Expressions of Receptors on the Human Megakaryoblastic Cell Line MEG-01 by Monoclonal Antibodies Nobutaka Imamura, Deodatus M. Mtasiwa, Tominari Inada, Atsushi Kuramoto, Michinori Ogura, and Hidehiko Saito Department of Internal Medicine, Research institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima (N.I., D.M.M., T.I., A.K.), Department of Hematology and Chemotherapeutics, Aichi Cancer Center Institute, (M.O.), and The First Department of Internal Medicine, Nagoya University School of Medicine (H.S.), Nagoya, Japan

We herein describe the regulation of specific receptors on the megakaryoblastic cell line MEG-01 by means of fluorescence-activated cell sorting (FACS IV) with a panel of monoclonal antibodies (MAbs). MEG-01 cells expressed Gpllb/llla (CD41a) and OKM5 (CD36) antigens on their cell surface, whereas they showed only a little expression of GPlb (CD42b), suggesting that these are megakaryoblastic cells. The up regulation of von Willebrand factor receptor (GPlb) and thrombospondin receptor (GPIV) and the down regulation of fibrinogen receptor (GPllbillla) and C3bi receptor (CDllb) were found by incubation with MAbs AN51 (CD42b), OKM5 (CD36), J15 (CD4la), and OKMl (CDllb), respectively. This phenomenon was enhanced in the Ca2+ containing medium, except for the experiment with OKM5 (CD36) MAb. We thus suggest that several kinds of receptors on the surface of MEG-01 cells are dexterously regulated through stimulation from outside the cell. Key words: platelet membrane receptors, MEG-01 cell, flow cytometric analysis

INTRODUCTION

Monoclonal Antibodies

Usually platelets adhere to the subendothelium of damaged blood vessels where they become stimulated by agonists and aggregate to arrest bleeding. These processes are mediated by the binding of either plasma protein or platelet-secreted proteins to specific receptor sites on the platelet cell surface. The receptor sites, which are normally present on both the cell-surface of megakaryocytes and on platelets, are necessary for adhesion, which makes them important elements in primary hemostasis [ I ] . We hereby describe the up and down regulation of specific reccptors on the megakaryoblastic cell MEG-O 1 .

Monoclonal antibodies (MAbs) were obtained through the courtesy of Drs. A. J. McMichael (Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, England) [ 3 ] , H. Shiku (Department of Oncology, Nagasaki University, Nagasaki, Japan) [4], and G . Goldstein (Ortho Pharmaceutical Co., Raritan, NJ, USA) [ S ] and Ortho Diagnostic Systems K.K. (Tokyo, Japan), Fujisawa Pharmaceutical Co. (Osaka, Japan), and the Japan Scientific Instrument Company (Tokyo, Japan).

MATERIALS AND METHODS Cell Culture

The MEG-OI cells were established from a male patient in the megakaryoblastic crisis of chronic myelogenous lcukemia in 1984 and have been maintained in continuous suspension culture for more than 400 passages 121. ci-,

1990 Wiley-Liss, Inc.

Received for publication December 1. 1989; accepted December 12, 1989. Address reprint requests to Dr. Nohutaka Imarnura, Department of Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Kasumi 1-2.3. Minami-Ku, Hiroshima, 734, Japan.

Brief Report: Varied Receptor Expressions on MEG-01

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Fluorescence intensity Fig. 1. Varied expressions of the regulation of receptors on the human megakaryoblastic leukemia cell line, MEG-01, by monoclonal antibodies. Up regulation by the MAbs AN5l(a) and OKMS(b) as well as the down regulation by MAbs J15(c) and OKMl(d) are both evident.

Indirect lmmunofluorescence Testing

Cells (1 X lo5) were incubated with 10 pl of MAbs for 1 hr at 37°C in phosphate-buffered saline (PBS) or Hank’s balanced saline solution (HBSS) containing 1.3 mol/L Ca2+. As control, cells were incubated with 10 ~1 of MAbs for 1 hr at 4°C. Cells were washed twice with PBS medium to remove unbound antibodies and then incubated with fluorescein isothiocyanate (F1TC)-conjugated F(ab’), fragment of sheep antimouse immunoglobulin (Ig) reactive with gamma or mu (Tago, Burlingame, CA) for 30 min at 4°C. After incubation, cells were washed twice and analysed by flow cytometry under the same condition. In order to determine the extent of nonspecific binding of the FITC-conjugated F(ab’), fragment of antimouse Ig, the cells were preincubated with either medium or non-reactive monoclonal antibodies. The percentage of positive fluorescing cells was adjusted by substracting the percentage of non-specific fluorescing cells (usually less than 1 .O%) as described previously [6,7]. An indirect cytoplasmic immunofluorescence test

was carried out by means of a previously described method [8]. Flow Cytometric Analysis

Fluorescent-positive cells were analysed in a fluorescence-activated cell sorter (FACSIV, Becton Dickinson, Mountain View, CA) with a focused 488 nm Argon laser (Spectra-Physics, Mountain View, CA). Fluorescent signals above 520 nm were identified for the analysis of 20.000 cells. RESULTS AND DISCUSSION

Immuno-phenotypically, the differentiation stage for the MEG-01 cell line was considered to belong to the megakaryoblast, as demonstrated by both the presence of megakaryocyte-platelet-related cell-surface antigens, GPIIbAIIa (J15, P2, and HPL2:CD41a), Plt-1, OKM6, OKM5 (CD36), and Ia (HLA-DR) and the lack of GP Ib (AN51:CD42b) [9,10].

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Brief Report: lmamura et al.

Figure 1 shows the up (a;GPIb (von Willebrand factor receptor), b; Thrombospondin receptor) and down (c;GPIIbiIIIa (fibrinogen receptor), d;C3bi receptor) regulation of the corresponding receptors by incubation with MAbs, AN51(CD42b), OKM5(CD36), J15(CD41a), and OKM I(CD1 lb), respectively. This phenomenon was enhanced in the Ca’+-containing medium (HBSS) except for the experiment with OKM5 MAb, which suggests that the mechanism for the former phenomenon was induced by a CA’+-dependent system, whereas the latter was caused by a Ca’ +-independent one. On the basis of these findings, we conclude that several kinds of receptors on the cell-surface of the megakaryoblastic cell line (MEG-01 ) are dexterously regulated through stimulation from outside the cell. Furthermore, MAbs J15 and OKMl may be useful tools for the treatment of chemotherapy-resistant megakaryoblastic leukemias by the means of serotherapy utilizing them coupled with various kinds of toxic substances. REFERENCES Kroll MH, Schafer AI: Biochemical mechanisms of platelet activation. Blood 74:l 1x1, 1989. Ogura M. Morishima Y, Okumura M. Hotta T , Takamoto S, Ohno R, Hirabayaahi N, Nagura H. Saito H: Functional and morphological diflerentiation induction 01‘ a human megakaryoblastic leukemia cell line (MEG-01s) by phorbol dieaters. Blood 7 2 4 9 . 1988.

3 . Vainchenker W. Descharnps JF, Bastin JM. Guichard J , Titeux M, Breton-Gorius J, McMichael AJ: Two monoclonal anti-platelet antibodies as markers of human megakaryocyte maturation; immunofluorescent staining and platelet peroxide detection in megakaryocyte colonies and in vivo cells from normal and leukemic patients. Blood 9 . 5 1 4 , 1982. 4. Hayashi K, Furukawa K, Takamoto S, Shiku H , Yamada K: Analysis of cell hurface molecules on human platelet with monoclonal antibodies. I . Identification of four platelet-specific surface molecules. Acta Haematol (Basel) 7.5: 141, 1986. 5 . Talk MA, Rao PE, Westberg E, Allergar N , Makowski M , Mittler RS, Goldstein G: Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies. Cell lmmunol 78:83, 1983. 6 . Imamura N. Tanaka R, Kajihara H, Kuramoto A: Analysis of peroxidase negative acute unclassifiable leukemias by monoclonal antibodies. I . Acute niyelogenous leukemia and acute myelomonocytic leukemia. Eur J Haematol 41:420, 1988. 7 Imamura N , Kuramoto A: Analyak of peroxidase negative acute leukemias by monoclonal antibodies. 111. Acute lymphoblastic leukemia. J Clin Lab Analysis 3 : 8 8 . 1989. 8 lmarnura N. Yanagihara K , Kusunoki Y: Tumor-associated antigen of spontaneous mammary tumor in rats. Oncology 41:25.5, 198.5. 9 Imamura N, Kajihara H, Kuramoto A: Flow cytometric analysis or peroxidase negative acute leukemias by monoclonal antibodies. 11. Acute megakaryoblastic and acute promegakaryocytic leukemia. Leuk Kes 12:279. 1988. I 0 lmainura N , lnada T , Mtasiwa DM, Kuramoto A: Demonstration of TSP receptor both on the cell surface and in the cytoplasm of the megakaryoblastic leukemia cell line (MEG-01). Am J Hematol 32:78, 1989.

Varied expressions of receptors on the human megakaryoblastic cell line MEG-01 by monoclonal antibodies.

We herein describe the regulation of specific receptors on the megakaryoblastic cell line MEG-01 by means of fluorescence-activated cell sorting (FACS...
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