Establishment of a Human Megakaryoblastic Cell Line (T-33) from Chronic Myelogenous Leukemia in Megakaryoblastic Crisis1

[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 37Â°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 37Â°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 isothiocyanate; 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 4Â°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. Maturation Induction. T-33 cells were cultured for 5 days at a con centration of 10 cells/ml in fresh medium containing 10 mol/liter TPA. Measurement of Ionized Calcium. The whole procedure was processed in accord with the method already described (16). This measurement was performed in order to examine the responsiveness of T-33 to thrombin. Human thrombin was purchased from Midorijuji (Osaka, Japan).

RESULTS Cell Culture. The cultured cells did not decrease in number after the beginning of the culture and began to grow rapidly in 2 weeks, and have been growing well in single cell suspension with a doubling time of 24-36 h. A low percentage of giant round cells was observed. Morphology of T-33 Line Cells. The cells are usually round in shape, measuring 20-30 urn in diameter, intermingled with a few multilobulated giant round cells measuring up to 60 /Â¿m in diameter (Fig. 1, A-C). Cytoplasms were basophilic with vacuoles and a ruffled cell surface. Chromosomal Analysis. 49 of 50 cells examined exhibited a female hyperdiploid karyotype, 51,xx,t(9;22)(q34qll), lp+,+4,+9,+ 19,+Ph"1 and one cell showed another karyotype 49,xx,t(9;22),+6,+9,+ 10,Ph'. The Ph' chromosome, t(9;22), was positive in all karyotypes analyzed. Cytochemical Studies. Most cells were strongly positive for periodic acid-Schiff reactions in both the smears and deparaf- fmated sections of pelleted cells. Cells were strongly positive for NAE and became weakly positive after NaF treatment. They were weakly positive for NBE and almost negative after NaF treatment. Vacuoles were positive for Sudan black. Myeloper- Fig. 1. Cell morphology of the T-33 cell-line cells: A, an inverted phase 4 The print of karyotype was shown in a previous report (13). contrast microscopic picture of the T-33 cells cultured for 1 year (X 160; bar, 20 Â¿im).B,smear of T-33 cells stained with MGG, showing granules in the cytoplasm (x 1050; bar, 20 urn). C, a large cell with a polylobulated nucleus and large granular dots (MGG; x 1050; bar, 20 Â¿im). 6138

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. A HUMAN MEGAKARYOBLASTIC CELL LINE oxidase and AS-D chloroacetate esterase were not demon- 341 383r strated before and after TPA treatment. Similar results were obtained constantly three times. The data were shown in Table M HLA-DR MY-4 1. ni Cell Surface Markers and Intracytoplasmic Antigens. As shown in Table 2 and Fig. 2, among the cell surface markers of unfixed cells analyzed by fluorescence-activated cell sorting, 0 HLA-DR, MY-7, MY-9, and Yukb were positive, while other 100 200 markers were not detected on the cell surface. As also shown 383 in Table 2, when intracytoplasmic antigens of T-33, acetone- fixed, were examined with the indirect immunoperoxidase TP-82 (PAP) staining method using monoclonal antibodies (TP-80, 192 TP-82, TM-83, and 5F1) and a polyvalent antiserum (anti- Yukb), over 80% of cells were positive for platelet GP Hb-IIIa

XI (Fig. 3) and Ilia, and 77% was positive for GP Ib. 99% of cells E were positive for Factor VIII. Erythroid markers such as gly- D 100 200 256 100 200 256 cophorin A and hemoglobin were not revealed. O) Ultrastructures. T-33 cells exhibited a moderate number of Ã³O 325 383r

MY-9 Table 1 Cytochemistry of T-33 -/+; 1-10%, +; 11-30%, ++; 31-60%, +++; 61-100%. ,63 Periodic acid-Schiffs Â«-Naphthylacetate esterase NAE + NaF a-Naphthyl butyrate esterase + " O 100 200 256 "O 100 NBE + NaF ' -/+ 200 256 Alkaline phosphatase + Fluorescence Intensity i Log Scale) Acid phosphatase + Acid phosphatase + tartrate Fig. 2. Flow cytometric study of the surface phenotypes of T-33 cells, showing (i-Glucuronidase + strong positivity for HLA-DR, Yukb, MY-7, and MY-9, and weak positivity for Myeloperoxidase â€” MY-4 but negativity for TP-82 on the cell surface. Thin lines, negative controls Naphthol AS-D chloroacetate esterase (unstained in HLA-DR, stained with only fluorescein isothiocyanate-conjugated " Strong in intensity. goat anti-mouse immunoglobulins in TP-80, MY-4, MY-7, and MY-9, and with only fluorescein isothiocyanate-conjugated rabbit anti-human immunoglobulins in Yukb). Thick lines, surface phenotypic patterns of T-33 stained with each Table 2 Reactivity of antibodies with T-33 antibody (anti-HLA-DR. TP-82. Yuk", MY-4, MY-7, or MY-9). Direct or indirect immunofluorescence assays for cell surface markers and indirect immunoperoxidase (PAP) staining for cytoplasmic antigens were per formed as described in the text. Reactivity is expressed as the percentage of cells microvilli and ruffles of the cell surface. A small number of that demonstrated fluorescence or 3,3-diaminobenzidine tetrahydrochloride stain. cells possessed long cytoplasmic processes (Fig. 4). % of positive T-33 cells showed a round nucleus with thin marginal chro- cells matin and a few nucleoli and a rather wide cytoplasm with AntibodiesTP80TP82TM83P-2Anti-YukbÂ°5F1 PAPNc moderate number of rough endoplasmic reticulum, mitochon GPIlblllaPlatelet >80+N dria, and open vesicles, and a varying-sized Golgi apparatus GPIlblllaPlatelet >80+N (Fig. 5A). The cytoplasm also contained a few small granules GPIlblllaPlatelet >80+N GPIlblllaPlatelet -"76 (Fig. 5A, insert). After TPA induction, larger cells with a GPIliaPlatelet >80+N polylobulated nucleus and an eccentric cytoplasm increased in (Immunoteck)4F9 GPIbvWFHuman 77+N number, accounting for up to 20% of whole cells examined. (Immunoteck)Anti-Factor99+>80+NNNNNN30938978NNNNNNNNN' VIII*(DAKO)P-12D2-10Anti-Hb Factor VIII re These often showed a membrane complex connecting with an latedantigenThrombospondinGlycophorin open canalicular system or a complex of the DM characteristic of megakaryocytic lineage (Fig. SB). AHuman (DAKO)*Anti-Si hemoglobinSadult A few cells exhibited PPO in the perinuclear space alone. 00*Anti-Leu 100proteinGranulocyte, After TPA treatment, more than 20% of cells showed increased MlAnti-Leu monocyteMonocyteMonocyte M3Anti-MY intensity of PPO in the perinuclear space and rough endo 4Anti-MY (CD14)Granulocyte, plasmic reticulum, but the Golgi apparatus and granules were 7Anti-MY monocyte(CD13)Monocyte still negative for PPO (Fig. 6, A-C). 9Anti-HLA-DRAnti-Leu Responsiveness to Thrombin. In order to test the responsive (CD33)HLA-DRPan-T ness of T-33 cells to thrombin, luminescent response of IO4/ 1Anti-Leu cell(CDS)Ts/c mm3 aequorin (Ca2+-sensitive photoprotein)-loaded T-33 cells 2Anti-Leu cell(CDS)Th/i 3Anti-Leu cell(CD4)Pan-T to 1.0 unit/ml thrombin in 4-(2-hydroxyethyl)-l-piperazineeth- 4Anti-Leu cell(CD3)Natural anesulfonic acid-Tyrode's buffer containing 1 mM Ca2+ was 7Anti-Leu cellHLA-DCBkiller 10Anti-Leu measured as an index of calcium influx which indicates a 12Anti-CALLAVJ41Â° cellALL' response to thrombin. A response to thrombin of T-33 was antigenAdult T -cell leukemia an evident (Fig. 7). tigen (ATLA)FACS Patient'sserum.* Polyclonalantibody.c DISCUSSION N,negative.d -, not performed.SpecificityPlatelet There have been only a few reports of human megakaryo- ALL, acute lymphocytic leukemia. blastic cell lines although a number of cases with acute mega- 6139

Fig. 3. PAP staining ofT-33 cells using an anliplatelct GP llb-IIla monoclonal antibody (TP-80). Positivi!)- is evident. (3.3-diaminobenzidine telrahydrochloride: x 1000: bar. 20

karyoblastic leukemia or megakaryoblastic crisis of chronic microscopic study combined with PPO reaction, it has been myelogenous leukemia have been reported (2, 5-7, 17). Re known that PPO-positive megakaryoblasts in a case of acute cently, a few human cell lines of megakaryocytes have also been megakaryoblastic leukemia are also positive for MY-7 and MY- reported (10-12). Since erythroleukemic cell lines such as K562 9 in the granules and on the cell surface.5 Moreover, a new (18, 19). HEL (20), and LAMA-84 (21) also exhibit megakar- human megakaryocytic cell line (CMK) established from acute yocytic phenotypes before or after the induction of maturation, megakaryoblastic leukemia has exhibited high positivity for an exact cellular characterization is necessary to sentence the MY-7 and -9 (28). These indicate that MY-7 and MY-9 are not establishment of a cell line of pure megakaryocytic lineage. specific for granulocytes and monocytes and that their expres T-33 is characterized as a megakaryoblastic cell because of sion as well as HLA-DR cannot exclude the possibility of T-33 the presence of PRO, platelet GP (Ilb-IIIa, Ilia, and Ib), Factor cells being immature megakaryoblasts. T-33 cells reacted with VIII, and ultrastructural characteristics of megakaryocytic lin anti-Yukb serum which recognizes platelet GP Ilia. Although eage such as granules resembling a granules, an open canali- T-33 did not react with most monoclonal antibodies made cular system, and a complex of demarcation membranes. Al against platelets GP Ilb-IIIa (TP-80, TP-82, TM-83, and P-2) though the percentage of PPO-positive cells was very low and and GP Ib (5F1) on the cell surface but reacted to them in the it might be inadequate for a high percentage of platelet GP- cytoplasm when cells were fixed with acetone and stained with positive cells, this discrepancy is supposed to be due to some the indirect immunoperoxidase (PAP) method. We are now problems of cell lines because the similar discrepancy has been investigating the localization of platelet GP in T-33 cells by described in the HEL cells and also no reasonable correlation using immunoelectron microscopy. In the same way, another between the percentage of the positivity for PPO and platelet megakaryoblastic cell line, MEG-01, expressed GP Ib in the GP has been confirmed in previous megakaryocytic cell lines cytoplasm but not on the cell surface although GP Ilb-IIIa was (8-11). As for ultrastructural findings, granules might be iden present both in the cytoplasm and on the cell surface of MEG- tified as Â«granules, being different from lysosomes (22). Cy- 01 (8). According to these facts, it can be thought that the tochemical results also indicate the megakaryoblastic nature platelets GP Ilb-IIIa and GP Ib will move to the cell surface as because cells are strongly positive for periodic acid-Schiff re the cells become mature. Factor VIII definitely exists in T-33 action and NAE, weakly positive for NBE but negative for like MEG-01 (8), CHRF-288 (10), and EST-IU (11). However, myeloperoxidase. A combination of strongly positive NAE and HEL and K562 did not express Factor VIII without induction weakly positive NBE is compatible with megakaryoblastic cells and HEL might slightly express it after induction. Flow cyto and unlikely for monocytic lineage. This combination was pre metric analysis of cellular DNA content of T-33 showed cell viously described in a few cases with acute megakaryocytic ploidys of 2N and 4N, and a small amount of 8N (data not leukemia (23, 24). In flow cytometric studies on the cell surface shown).6 This also indicates that T-33 is an immature mega- markers, T-33 represented nonspecific markers such as HLA- karyoblast. DR, MY-7, and MY-9. It is known that HLA-DR is expressed Although this cell line seems to express many of the pheno- by immature megakaryoblasts (25) and also that MY-9 can be typic characteristics of human megakaryocytic lineage, it does expressed by hemopoietic precursor cells such as CFU-GEMM, CFU-GM, and BFU-E (26). PPO-positive and platelet GP- 5 Dala presented by M. I immuto and Y. Watanabe at the symposium on Megakaryocytic cell lines, Tokyo. October 31. 1987. negative megakaryoblasts may exhibit labeling with anti-MY-9 6 Data presentation was authorized by Dr. Hiroshi Miyazaki. Kirin Brewery- (26, 27). Recently, according to the immuno-gold electron Co.. Ltd.. Maebashi. Japan. 6140

Fig. 4. A scanning electron micrograph of T-33 cell showing long cytoplasmic projections suggestive of megakaryocytic lineage, (x 9900: bar, l urn).

not appear to express any phenotypic properties of other cell megakaryoblastic cells was morphologically observed in vitro in lineage except MY-4, MY-7, and MY-9. T-33 does not include a Day 4 short term culture system derived from the peripheral T- and B-lymphocyte DNA rearrangement (13). Regarding blood of cases with acute megakaryoblastic leukemia or mega erythroid markers, T-33 cells did not exhibit glycophorin A and karyoblastic crisis of CML (30). Human platelet production by hemoglobin A in the stainings with the PAP method using megakaryocytic cell lines has not been described (10-12), al specific antibodies. Western blotting analysis also failed in though in vitro production of platelets in mice was observed in detecting glycophorin A by using a specific polyclonal anti a short term liquid culture (31) and in megakaryocytic colonies body.7 from CFU-Meg growing in a semisolid culture medium (32). Recently, it has been known that erythropoietin induces Presumably, there have been so far no megakaryoblastic cell megakaryocytic maturation (29). Interestingly, however, T-33 lines capable of differentiating into megakaryocytes or yielding seemed to respond to recombinant human erythropoietin (pro platelet particles. Although T-33 has not yet released evident vided by Kirin-Amgen) to proliferate more rapidly when added platelet particles outside, it possesses small dense granules or at the concentration from 0.01 to 2 units/ml according to cell counting by hemocytometer.8 Therefore, it can be expected that Â«-granules,open canalicular systems, and a complex of the DM T-33 might be a useful cell line for the comparative study of surrounding a platelet architecture in the cytoplasm. Therefore, T-33 is a unique human immature megakaryoblastic cell line erythropoietin and thrombopoietin receptors of megakaryocytic cells. Estimation of erythropoietin receptor on T-33 cells is capable of differentiating into the megakaryocytic stage. T-33 cell has been growing well with a doubling time of 24 under way. As for the morphological aspects of megakaryoblastic cells to 36 h in the liquid culture of IBL media (IBL, Fujioka, Japan) cultured in vitro, a certain cytoplasmic maturation of human containing 2% PCS and also has recently been well maintained without serum. Large scaled cell harvesting in the serum-free 7 Data presentation was authorized by Dr. Hiromitsu Okano, Kyushu Cancer Institute, Fukuoka. Japan. condition will lead to a successful purification of specific pro * Unpublished data. teins and cloning of complementary DNA although c-sis gene 6141

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Fig. 5. Transmission electron micrographs of T-33 cells: A, a representative cell having an evident nucleolus and a wide cytoplasm with a granule (arrow, inserted) and endoplasmic reticulum (x 7300: bar. 1 (Â¿m).B.a complex of DM in the intermediate zone of the cytoplasm, connecting open canalicular system (x 35,000; bar, 1 /jm).

6142

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'N Fig. 6. Ultrastructural PPO: A, a large cell with a polylobulated nucleus showing positivity for PPO in the perinuclear space, vesicles, and endoplasmic reticulum appearing after TPA induction (x 4000; bar, 1 ^m). B, a Golgi area (G) negative for PPO and endoplasmic reticulum-positive for PPO. N, nucleus: C. cen- triole (x 33,000; bar, l Â¿im).C,dense granules (arrow) negative for PPO. Vesicles, endo plasmic reticulum. and perinuclear space are positive. N, nucleus (x 19,000; bar, l Â¿im).

R. Constitutive and inducible secretion of platelet derived growth factor analogs by human leukemic cell lines coexpressing erythroid and megakary ocytic markers. J. Clin. Invest., 79: 859-866, 1987. 13. Tange, T., Yamasaki, I., Nakahara, K., Nakauchi, H., Hayami, M., Tanaka, F., Milani, K., Fujioka, S., Takanashi, R., Yoshino, T., Ohtsuki, Y., Yama- guchi, K., and Urano, Y. A human cell line of chronic myelogenous leukemia releasing a novel virus like particle. Acta Haematol. Jpn., 51: 76-82, 1988. 14. Sato, T., Shichishima, T., Kimura, H., Uchida, T., Kariyone, S., Ohio, H., and Maeda, H. Immunocytochemical detection of normal and abnormal megakaryocytes using monoclonal antibody to glycoprotein Ilb-IIIa (TP80): the quantitative assay in normal and in leukemic patients. Scand. J. Hae 1min matol., 36: 415-423, 1986. Thrombin, ID/ml 15. Higashihara, M., Maeda, H., Shibata, Y., Kume, S., and Ohashi, T. A monoclonal anti-human platelet antibody: a new platelet aggregating sub stance. Blood, 65: 382-391, 1985. 16. Johnson, P. C., Ware, J. A., Cliveden, P. B., Smith, M., Dvorak, A. M., and Time Salzman, E. W. Measurement of ionized calcium in blood platelets with the Fig. 7. Luminescent response of 1 x IO4aequorin-loaded T-33 cells to 1 unit/ photoprotein Aequorin. J. Biol. Chem., 260: 2069-2074, 1985. ml thrombin in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid-Tyrode's 17. Travis, W. D., Li, C. Y., Banks, P. M., and Nichols, W. L. Megakaryoblastic buffer containing ionized calcium. transformation of chronic granulocytic leukemia. Cancer (Phila.), 60: 193- 200, 1987. 18. Lozzio, C. B., and Lozzio, B. B. Human chronic myelogenous cell line with expression and the subsequent secretion of platelet-derived positive Philadelphia chromosome. Blood, 45: 321-334, 1975. growth factor have not been examined. 19. Totteroo, P. A. T., Massaro, F., Mulder, A., Schreuder-van Gleder, R., and Von Dem Borne, A. E. G. Kr. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. Establishment of a Human Megakaryoblastic Cell Line (T-33) from Chronic Myelogenous Leukemia in Megakaryoblastic Crisis

Tsuyoshi Tange, Kazuhiko Nakahara, Kinuko Mitani, et al.

Cancer Res 1988;48:6137-6144.

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