[CANCER RESEARCH 48. 6137-6144, November 1. 1988] Establishment of a Human Megakaryoblastic Cell Line (T-33) from Chronic Myelogenous Leukemia in Megakaryoblastic Crisis1 Tsuyoshi Tange,2 Kazuhiko Nakahara, Kinuko Mitani, leharu Yamasaki, Hideyuki Yasuda, Fumihiko Tanaka, Masaru Mizuguchi, Hideaki Oda, Yutaka Yatomi, Riichiro Takanashi, Seitoku Fujioka, Kazuyoshi Yamaguchi, and Yoshinori Urano Department of Pathology and the First Department of Internal Medicine, University of Tokyo, and Mitsui Memorial Hospital, Tokyo, Japan ABSTRACT peripheral blood of a female with megakaryoblastic crisis of CML (13) and here report this cell line termed as T-33 as a A megakaryoblastic cell line, termed T-33, was established from the human megakaryoblastic cell line capable of differentiating into peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis. T-33 cells megakaryocyte. have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for MATERIALS AND METHODS over 2 years. Giemsa-banded karyotypes were female hyperdiploid with Case History. A 55-year-old female was diagnosed as Ph'-positive a modal chromosomal number of 51, all cells including Philadelphia chromosome. The cells showed strong positivity for periodic acid-Schiff CML and treated with busulfan at Mitsui Memorial Hospital (Tokyo) and a-naphthyl acetate esterase, and weak for a-naphthyl butyrate in 1978. In 1983, blastic crisis of CML was suspected because of the esterase, but were negative for myeloperoxidase. Flow cytometric analysis appearance of hip joint pain but the bone marrow was still in the of cell surface markers showed the existence of HLA-DR, MY-7, MY- chronic phase with myelofibrosis. Thereafter, vincristine, prednisolone, 9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes. and 6-mercaptopurine were given when the leucocyte count increased. Most of the cells fixed with acetone were positive for Factor VIII, platelet On the last admission in August, 1985, the leucocyte count was 13,200 glycoprotein Hb-IIIa, Ilia (Yukb), and Ib, but negative for glycophorin A (blasts, 2.5%) and the bone marrow was dry tap, the biopsy revealing osteomyelofibrosis. The patient became refractory' to any drugs and the and hemoglobin. Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% leucocyte count increased up to 26,700 (blasts, 15.5%). Then, ultra- after the treatment with 12-O-tetradecanoylphorbol-13-acetate. The cells structural study of the peripheral blood blasts revealed the positivity contained small dense granules negative for platelet peroxidase, their for platelet peroxidase. leading to a diagnosis of megakaryoblastic crisis number increasing threefold after 12-0-tetradecanoylphorbol-13-acetate of CML. The patient died of pulmonary edema on October 30, 1985. Cell Culture. On October 23, 1985, 10 ml of heparinized peripheral treatment. Such treated cells frequently showed a complex of the demar blood was obtained, when the white blood cell count was 5000/mm3 cation membrane in the cytoplasm. T-33 responded thrombin to exhibit calcium influx. This cell line may be useful for the study of the early (blasts, 50%). The sample was diluted in 0.1 mol PBS. pH 7.3, and the mononuclear cells were separated by Ficoll-Conray gradient centrifu- stage of megakaryocytic differentiation in human megakaryopoiesis. gation. After washing three times in PBS, cells were cultured at a concentration of 106/ml in Falcon plastic tissue culture bottles contain INTRODUCTION ing 7 ml of RPMI 1640 medium (GIBCO, Grand Island, NY) and 10% PCS (Flow Laboratories, North Ryde, NSW, Australia) with penicillin Since it became possible to identify a megakaryoblast by ( 100 U/ml) and streptomycin (50 Mg/ml) at 37°C,5%CO2. The cultured demonstrating ultrastructural PRO,3 platelet GP Ib and Hb- cells were fed once or twice weekly by half medium change. Illa, and coagulant factors such as factor VIII or IV (1-4), an Morphological Observation. Cultured cells were observed by the increased number of cases with megakaryoblastic leukemia or Nikon inverted-phase contrast microscope (Nikon, Tokyo). The cells megakaryoblastic crisis of CML have been reported (2, 5-7). were smeared on the slide glass and stained with May-Gruenwald- Giemsa or Wright's staining. Recently, a few megakaryoblastic cell lines such as MEG-01 have been established (8, 9) but MEG-01 did not morphologi Chromosomal Analysis. In absence of Colcemid, the cultured cells were treated with 75 mmol/liter KC1 hypotonie solution for 30 min at cally show any evident megakaryocytic differentiation (8). Cell 37°C,andfixed with methanol-acetic acid solution. Chromosomes were lines established by Morgan et al. did not exhibit any granules banded by the trypsin-Giemsa method. in the cytoplasm in spite of the development of a megakaryocy Cytochemistry. The cultured cells suspended in 10% bovine serum tic membrane system (9). A few megakaryocytic cell lines such albumin were smeared on the slide glass, air dried, and fixed by the use as EST-IU and CHRF-288 have also been reported (10-12). of published methods for cytochemical stainings. EST-IU seems to lack the DM, while CHRF-288 is a mega Antibodies. Most of the monoclonal antibodies used were purchased karyocytic tumor cell line forming a s.c. tumor in nude mice, from Becton-Dickinson Co. (Sunnyvale, CA) except MY-4 (CD 14), but nonleukemic. We have established a human cell line releas MY-7 (CD 13), and MY-9 (CD 33) which were from Coulter Co. ing a virus-like particle into the culture supernatant from the (Hialeah, FL). TP-80 (14), TP-82 (15). and TM-83, murine IgG mono clonal antibodies specific for human platelet GP Ilb-IIIa were kindly Received 12/28/87; revised 6/25/88; accepted 7/26/88. provided, TP-80 and TP-82 were by Dr. Hiroo Maeda (University of The costs of publication of this article were defrayed in part by the payment Tokyo Hospital), and TM-83 was by Dr. Kenjiro Tanoue (Tokyo of page charges. This article must therefore be hereby marked advertisement in Metropolitan Institute of Medical Science, Tokyo). Anti-Yukh. kindly accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported in part by Grant-in-Aids for Scientific Research (61304036) from provided by Dr. Yoichi Shibata (Toranomon Hospital, Tokyo), is a the Ministry of Education and for Cancer Research (61-11) from the Ministry of maternal polyvalent antibody, human IgG, reacted to fetal platelets. Health and Welfare in Japan. Immunofluorescence Technique. For direct staining, 5 x IO5of T-33 2To whom requests for reprints should be addressed, at the Department of were suspended in 20 ^1of each FITC-conjugated monoclonal antibody Pathology, Faculty of Medicine, the University of Tokyo, 7-3-1, Hongo. Bunkyo- (anti-Leu-1, -2, -3, -4, -7, -10, -11, -12, -Ml, and -M3, anti-HLA-DR, ku. Tokyo 113. Japan. 1The abbreviations used are: PPO, platelet peroxidase; GP. glycoprotein; anti-CALLA, or anti-IL-2) and incubated on ice for 30 min. The cells CML, chronic myelogenous leukemia: Ph', Philadelphia chromosome: NAE, a- were then washed three times with PBS containing 2% fetal calf serum naphthyl acetate esterase; NBE, a-butyrate esterase; DM, demarcation membrane; (PBS-FCS) and resuspended in 1 ml of PBS-FCS. PBS, phosphate buffered saline; PCS, fetal calf serum; FITC, fluorescein isothi- ocyanate; PAP, peroxidase-anti-peroxidase;TPA. 12-O-tetradecanoylphorbol-13- For indirect staining, the same number of T-33 were suspended in acetate. 20 M' of a nonlabeled antibody (MY-4, MY-7, MY-9, TP-80, TP-82, 6137 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. or Yukb) and incubated on ice for 30 min. After washing three times with PBS-FCS. the cells were resuspended in 20 ^1 of appropriately diluted FITC-conjugated goat anti-mouse immunoglobulins (Cappel; Copper Biochemical, Inc., Malvenu PA) or FITC-conjugated rabbit anti-human immunoglobulins (kindly provided by Dr. Ko Okumura, Juntendo University School of Medicine, Tokyo), followed by incuba tion for 30 min on ice. Then, the cells were washed three times and suspended in 1 ml of PBS-FCS. Cytofluorographic analysis was per formed by a fluorescence-activated cell sorter, FACS-420 (Becton Dick inson, Sunnyvale, CA). Immunostaining of Intracytoplasmic Antigens. After the cells were washed three times, suspended in 10% bovine serum albumin solution, smeared on slides, and fixed with acetone, cytoplasmic antigens such as platelet GP (Ilb-IIIa, Illa, Ib), Factor VIII, hemoglobin A, and glycophorin A were stained with the indirect PAP immunostaining method using monoclonal or polyvalent antibodies. Scanning Electron Microscopy. Cells in the culture bottle were fixed with 1% glutaraldehyde for 30 min and postfixed with 0.5% osmium tetroxide for 20 min. Fixed cells were dehydrated in a graded series of ethanol and coated with gold. Samples were observed by a scanning electron microscope (Hitachi S450LB). Transmission Electron Microscopy. Cultured cells were centrifuged at 3000-5000 rpm for 10 min by Eppendorfs centrifuge and the pelleted cells were fixed in 2.5% glutaraldehyde for 2 h at 4°Cand postfixed in 1% osmium tetroxide for 1 h. Fixed cells were dehydrated in a graded series of ethanol and embedded in EPON. Ultrathin B sectioning was performed with a diamond cutter and sections were stained with uranyl acetate and lead citrate, and observed by JEM 100C electron microscopy (JEM, Tokyo). Ultrastructural Platelet Peroxidase (PPO). Cells unfixed or fixed with periodate-lysine-paraformaldehyde fixative solution were incu bated in 3,3-diaminobenzidine tetrahydrochloride solution according to the method previously described (2). Then, the procedure was proc essed in the same way as described above for the transmission electron microscopy.