EXPERIMENTAL
CELL
RESEARCH
193,223-226
(1991)
SHORT NOTE Production of Platelet-like Particles by a Human Megakaryoblastic Leukemia Cell Line (MEG-01) KIKUKO
TAKEUCHI,*-’
MICHINORI
OGURA,~ HIDEHIKO
SAITO,~
MOTONOBU
SATOH,~ AND MASAO TAKEUCHI~
*Ehime College of Health Science, Takooda, Tobe, Iyo-gun, Ehime 791-21; tAichi Cancer Hospital, Kanokoden, Chigusa-ku, Nagoya 464; *Nagoya University School of Medicine, Chigusa-ku, Nagoya 464; and Slnstitute for Fermentation, Osaka, Yodogawa-ku, Osaka 532, Japan
MATERIALS The distribution of microtubules and platelet-specific glycoproteins (GPIIb/IIIa) in particles was probed by an immunofluorescence method using anti-tubulin and anti-GPIIb/IIIa antibodies to identify whether particles released from a human megakaryoblastic cell line (MEG-01) are platelets. The fluorescence image showing anti-tubulin staining of the particles revealed a characteristic ring structure observed in platelets. Anti-platelet GPIIb/IIIa antibody staining showed an image in which small patches or spots were seen throughout the particle with brighter staining at the periphery. No significant difference was observed between these particles and human blood platelets under immunofluorescent staining. These results show that 6 1x11 MEG-01 cells released platelet-like particles. Academic
Press,
Inc.
INTRODUCTION
One of the barriers to the characterization of human megakaryocytopoiesis and to the clarification of the mechanism of platelet production has been the difficulty in obtaining sufficient numbers of megakaryocytes for study. Thus, a great deal of effort has been directed to establishing cell lines that express megakaryocytic properties. Several human megakaryocytic cell lines from patients with leukemia which exhibit some morphological and biochemical characteristics of megakaryocytes have been established [l-lo]. However, no evidence that platelets were formed by these cell lines has been provided. The clonal line, MEG-01, which was established by Ogura et al. [4] does not express properties of other blood cell lineages, but has phenotypic properties that more closely resemble a megakaryoblast [4, 51. The present report documents our observation that MEG-01 cells produced platelet-like particles. i To whom dressed.
correspondence
and reprint
requests
should
be ad-
AND
METHODS
Materials. Cells: A human megakaryoblastic leukemia cell line, MEG-01, IF0 50151, was established in May of 1983. The cells were cultivated in RPM1 1640 medium supplemented with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY) at 37°C in a humidified atmosphere of 5% CO,. On Day 5 after MEG-01 cells were seeded at 1 X lo5 ml-’ in tissue culture wells with glass coverslips filled with 1 ml medium, the cells and platelet-like particles on the coverslips were washed in phosphate-buffered saline and fixed in 3.7% paraformaldehyde at room temperature and treated with acetone at -20°C. Human platelets: Blood was drawn from healthy human volunteers by venipuncture into a solution of citrate-citric acidglucose (final concentrations 14.1 mM sodium citrate, 10.8 mM citric acid, 22.6 mM glucose) and then subjected to centrifugation at 500g for 10 min. Platelets in the supernatant were fixed in 3.7% paraformaldehyde, put on a coverslip by cytospin, and then permeabilized with acetone at -20°C. Indirect immunofluorescence. MEG-01 cells on the coverslips were incubated in monoclonal antibody against n-tubulin (Amersham) at 37°C for 60 min and then in FITC-conjugated anti-mouse Ig at 37°C for 60 min. For double-staining against n-tubulin and actin, the samples were stained first with anti-tubulin and FITC-conjugated anti-mouse Ig and then with rhodamine-phalloidin (Molecular Probes, Inc.). For double-staining against a-tubulin and GpIIb/IIIa, the samples were stained first with anti-tubulin and FITC-conjugated anti-mouse Ig and then with monoclonal antibody to GPIIb/IIIa (clone HPL 1, Kyowa Medex Co., Ltd.) and Texas red-conjugated anti-mouse Ig sheep antibody (Amersham).
RESULTS
AND
DISCUSSION
Platelets have a characteristic cytoskeleton composed of the microtubules that exist in a circumferential band just inside the plasma membranes. The discoid shape of platelet has been thought to be supported by the microtubule band [ 111. MEG-01 cells produced spontaneously many particles. To identify whether the particles released from MEG-01 cells are platelets, we used microtuble bands as a marker of platelets. This structure is easily detected as a closed brightly fluorescent ring when probed by an immunofluorescence method with anti-tubulin [12]. We observed many brightly stained rings around and in the diffusely stained remnants of MEG-01 cells (Fig. 1). By changing the focus many rings could also be seen inside the remnants of
223 All
Copyright 0 1991 rights of reproduction
0014.4827/91 $3.00 by Academic Press, Inc. in any form reserved.
224
SHORT
NOTE
FIG. 1. Fluorescent image of microtubule rings around and in remnants of the MEG-01 cells. MEG-01 cells on a coverslip were fixed and stained with anti-tuhulin. Note that many microtuhule rings with diameters from 2 to 4 pm (arrows) were near and in the cytoplasm of diffusely stained remnants of the MEG-O; cells. Scale bar, i0 pm.
MEG-01 cells. Although anti-tubulin staining of the particles usually showed either a dot pattern or a thin diffuse image (Fig. 2a), some particles have a clear ringlike structure (Fig. 2b). On the other hand, rhodaminephalloidin staining of the same particles showed an interesting fluorescence pattern which consisted of a meshwork and a peripheral circle beneath the surface membrane (Figs. 2c and 2d). We have previously suggested that similar microfilament organization plays an important role in the maintenance of the discoid shape of resting bovine platelets [13]. To ascertain that the particles derived from MEG-01 cells are platelets, the particles were double-stained with anti-tubulin and anti-platelet glycoproteins, GPIIb/IIIa [ 141, antibodies. The fluorescence image showing anti-tubulin staining of the particle revealed a typical ring structure (Fig. 3b), while anti-platelet GPIIb/IIIa staining showed the image in which small patches or spots were seen throughout the cytoplasm against the faint diffuse background staining (Fig. 3d). Brighter staining at the periphery showed that the glycoproteins exist on the surface of particles (Fig. 3d). No significant difference was observed between these particles and human blood platelets in immunofluorescent staining images (Figs.
3a and 3~). Consequently, MEG-01 cells produce platelet-like particles. Radley and Hartshorn [ 151 have recently shown that a marginal coil of microtubules does not form until after platelet liberation from megakaryocytes. Therefore, a dot pattern observed in the particle (Fig. 2a) may be an immature microtubule ring shortly after liberation from MEG-01 cells. Figure 2b shows that the microtubule rings seem slack, probably representing a more mature microtubule ring than that in Fig. 2a. The mechanism of platelet production from megakaryocytes remains controversial [ 161. The appearance of many particles in Fig. 1 favors the possibility that the particles, the marginal microtubule ring of which was formed in the MEG-01 cytoplasm, are liberated all at once from MEG-01 as cytoplasmic segregation in normal megakaryocyte. On the other hand, the particles observed in Fig. 2 were seen attached on the MEG-01 cell surface (Figs. 2a and 2c) or connected to processes of MEG-01 cytoplasm (Figs. 2b and 2d). The surface appearance supports the possibility that an additional mechanism for particle release from MEG-01 cell is by fragmentation of peripheral cytoplasm, including process. From these results, we propose two possible mechanisms for platelet-like
SHORT
NOTE
225
FIG. 2. Particles on the MEG-01 cell surface. MEG-01 cells and particles were double-stained with anti-tubulin (a, b) and rhodaminephalloidin (c, d). (a) is the same field as (c); (b) is the same field as (d). Anti-tubulin staining showed the pattern in dots (a, arrows) and slack rings (b, arrows), while rhodamine staining showed an interesting pattern which consisted of a meshwork and a peripheral circle beneath the
FIG. 3. Localization of the microtubule and platelet-specific surface glycoprotein (GPIIb/IIIa) in particles liberated from MEG-01. A human platelet (a, c) and a MEG-01 particle (b, d) were doublestained with anti-tubulin (a, b) and anti-GPIIb/IIIa (c, d). Anti-tubulin staining of MEG-01 particle revealed a typical ring structure (b), while anti-GPIIb/IIIa staining showed small patches or spots throughout the cytoplasm. Scale bar, 2 pm.
particle production in MEG-01 cells. They must be clarified in further detail. Several human megakaryotic cell lines from patients with leukemia were established [l-lo]. However, the cell lines except MEG-01 [4] and CHRF-288 [9] express characteristics associated not only with megakaryocyte lineages but also with other nonmegakaryocyte lineages. In some of the lines [4, 91, the expression of megakaryotic markers increased under phorbol myristate acetate (TPA) treatment. The treatment of MEG01 with TPA induced morphological and biochemical differentiation in more mature megakaryocytes [ 181. In this report, we showed that MEG-01 is the first cell line that has the ability to produce platelet-like particles, the most important function of the megakaryocyte. Therefore, this cell line will provide a useful model for elucidating the mechanisms of platelet production and regulation of megakaryocytopoiesis [19]. Also the immunofluorescent technique using anti-tubulin antibody to detect platelets will greatly facilitate megakaryocytopoiesis research. We are deeply indebted to Prof. Motoo, Nojima of the Ehime College of Health Science, Dr. Teiji, Iijima, and Dr. Tohru, Hasegawa, of
SHORT the Institute for Fermentation, Osaka, for continuing encouragement throughout this work. This research was supported in part by a grantin-aid for medicinal research from the Fugaku Trust. REFERENCES 1. 2. 3.
4. 5. 6.
7. 8.
Gewirtz, A. M., Burger, D., Radio, T. A., Benz, E. J., Jr., and Hoffman, R. (1982) Blood 60, 785-789. Martin, P. (1982) Science 216, 1233-1235. Sledge, G. W., Jr., Glant, M., Jansen, J., Heetrema, N. A., Roth, B. J., Goheen, M., and Hoffman, R. (1986) Cancer Res. 46,21552159. Ogura, M., Morishima, Y., Ohno, R., Kato, Y., Hirabayashi, N., Nagura, H., and Saito, H. (1985) Blood 66, 138441392. Ogura, M., Tanabe, N., Nishioka, J., Suzuki, K., and Saito, H. (1987) Blood 70,301L306. Avanzi, G. C., Lista, P., Giovinazzo, B., Miniro, R., Saglio, G., Benetton, G., Coda, R., Cattoretti, G., and Pegoraro, L. (1988) Brit. J. Haematol. 69, 359-366. Greenberg, S. M., Rosenthal, D. S., Greeley, T. A., Tantravahi, R., and Handin, R. I. (1988) Blood 72, 1968-1977. Seigneurin, D., Champelovier, P., Mouchiroud, G., Berthieer, R., Leroux, D., Prenant, M., McGregor, J., Starch, J., Morle, F.,
Received September 11, 1990 Revised version received November
6, 1990
NOTE
9. 10.
11. 12. 13. 14. 15.
16. 17. 18.
19.
Micouin, C., Pietrantuono, A., and Kolodie, L. (1987) Exp. Hematol. 15, 822-832. Witte, D. P., Harris, R. E., Jenski, L. J., and Lampkin, B. C. (1986) Cancer 58, 238-244. Komatsu, N., Suda, T., Mori, M., Tokuyama, N., Sakata, Y., Okada, M., Nishida, T., Hirai, Y., Sato, T., Fuse, A., and Miura, Y. (1989) Blood 74,42-48. Behnke, 0. (1970) Reu. Exp. Pathol. 9, l-92. Debus, E., Weber, K., and Osborn, M. (1981) Eur. J. Cell Biol. 24, 455.52. Takeuchi, K., Kuroda, K., Ishigami, M., and Nakamura, T. (1990) Enp. Cell Res. 186, 374-380. Furukawa, K., Hayashi, K., Naoe, T., Takamoto, S., Shiku, H., and Yamada, K. (1987) Acta Haematol. Japan. 50, 914-925. Radley, J. M., and Hartshorn, M. A. (1987) Blood Cells 12,603610. Stenberg, P. E., and Levin, .J. (1987) Blood Cells 15, 23-47. Yamada, E. (1957) Acta Anat. 29, 267-290. Ogura, M., Morishima, Y., Okumura, M., Hotta, T., Takamoto, S., Ohno, R., Hirabayashi, N., Nagura, H., and Saito, H. (1988) Blood 72,49-60. Gewirtz, A. M. (1986) Semin. Hematol. 23, 27-42.
CELL
RESEARCH
193,223-226
(1991)
SHORT NOTE Production of Platelet-like Particles by a Human Megakaryoblastic Leukemia Cell Line (MEG-01) KIKUKO
TAKEUCHI,*-’
MICHINORI
OGURA,~ HIDEHIKO
SAITO,~
MOTONOBU
SATOH,~ AND MASAO TAKEUCHI~
*Ehime College of Health Science, Takooda, Tobe, Iyo-gun, Ehime 791-21; tAichi Cancer Hospital, Kanokoden, Chigusa-ku, Nagoya 464; *Nagoya University School of Medicine, Chigusa-ku, Nagoya 464; and Slnstitute for Fermentation, Osaka, Yodogawa-ku, Osaka 532, Japan
MATERIALS The distribution of microtubules and platelet-specific glycoproteins (GPIIb/IIIa) in particles was probed by an immunofluorescence method using anti-tubulin and anti-GPIIb/IIIa antibodies to identify whether particles released from a human megakaryoblastic cell line (MEG-01) are platelets. The fluorescence image showing anti-tubulin staining of the particles revealed a characteristic ring structure observed in platelets. Anti-platelet GPIIb/IIIa antibody staining showed an image in which small patches or spots were seen throughout the particle with brighter staining at the periphery. No significant difference was observed between these particles and human blood platelets under immunofluorescent staining. These results show that 6 1x11 MEG-01 cells released platelet-like particles. Academic
Press,
Inc.
INTRODUCTION
One of the barriers to the characterization of human megakaryocytopoiesis and to the clarification of the mechanism of platelet production has been the difficulty in obtaining sufficient numbers of megakaryocytes for study. Thus, a great deal of effort has been directed to establishing cell lines that express megakaryocytic properties. Several human megakaryocytic cell lines from patients with leukemia which exhibit some morphological and biochemical characteristics of megakaryocytes have been established [l-lo]. However, no evidence that platelets were formed by these cell lines has been provided. The clonal line, MEG-01, which was established by Ogura et al. [4] does not express properties of other blood cell lineages, but has phenotypic properties that more closely resemble a megakaryoblast [4, 51. The present report documents our observation that MEG-01 cells produced platelet-like particles. i To whom dressed.
correspondence
and reprint
requests
should
be ad-
AND
METHODS
Materials. Cells: A human megakaryoblastic leukemia cell line, MEG-01, IF0 50151, was established in May of 1983. The cells were cultivated in RPM1 1640 medium supplemented with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY) at 37°C in a humidified atmosphere of 5% CO,. On Day 5 after MEG-01 cells were seeded at 1 X lo5 ml-’ in tissue culture wells with glass coverslips filled with 1 ml medium, the cells and platelet-like particles on the coverslips were washed in phosphate-buffered saline and fixed in 3.7% paraformaldehyde at room temperature and treated with acetone at -20°C. Human platelets: Blood was drawn from healthy human volunteers by venipuncture into a solution of citrate-citric acidglucose (final concentrations 14.1 mM sodium citrate, 10.8 mM citric acid, 22.6 mM glucose) and then subjected to centrifugation at 500g for 10 min. Platelets in the supernatant were fixed in 3.7% paraformaldehyde, put on a coverslip by cytospin, and then permeabilized with acetone at -20°C. Indirect immunofluorescence. MEG-01 cells on the coverslips were incubated in monoclonal antibody against n-tubulin (Amersham) at 37°C for 60 min and then in FITC-conjugated anti-mouse Ig at 37°C for 60 min. For double-staining against n-tubulin and actin, the samples were stained first with anti-tubulin and FITC-conjugated anti-mouse Ig and then with rhodamine-phalloidin (Molecular Probes, Inc.). For double-staining against a-tubulin and GpIIb/IIIa, the samples were stained first with anti-tubulin and FITC-conjugated anti-mouse Ig and then with monoclonal antibody to GPIIb/IIIa (clone HPL 1, Kyowa Medex Co., Ltd.) and Texas red-conjugated anti-mouse Ig sheep antibody (Amersham).
RESULTS
AND
DISCUSSION
Platelets have a characteristic cytoskeleton composed of the microtubules that exist in a circumferential band just inside the plasma membranes. The discoid shape of platelet has been thought to be supported by the microtubule band [ 111. MEG-01 cells produced spontaneously many particles. To identify whether the particles released from MEG-01 cells are platelets, we used microtuble bands as a marker of platelets. This structure is easily detected as a closed brightly fluorescent ring when probed by an immunofluorescence method with anti-tubulin [12]. We observed many brightly stained rings around and in the diffusely stained remnants of MEG-01 cells (Fig. 1). By changing the focus many rings could also be seen inside the remnants of
223 All
Copyright 0 1991 rights of reproduction
0014.4827/91 $3.00 by Academic Press, Inc. in any form reserved.
224
SHORT
NOTE
FIG. 1. Fluorescent image of microtubule rings around and in remnants of the MEG-01 cells. MEG-01 cells on a coverslip were fixed and stained with anti-tuhulin. Note that many microtuhule rings with diameters from 2 to 4 pm (arrows) were near and in the cytoplasm of diffusely stained remnants of the MEG-O; cells. Scale bar, i0 pm.
MEG-01 cells. Although anti-tubulin staining of the particles usually showed either a dot pattern or a thin diffuse image (Fig. 2a), some particles have a clear ringlike structure (Fig. 2b). On the other hand, rhodaminephalloidin staining of the same particles showed an interesting fluorescence pattern which consisted of a meshwork and a peripheral circle beneath the surface membrane (Figs. 2c and 2d). We have previously suggested that similar microfilament organization plays an important role in the maintenance of the discoid shape of resting bovine platelets [13]. To ascertain that the particles derived from MEG-01 cells are platelets, the particles were double-stained with anti-tubulin and anti-platelet glycoproteins, GPIIb/IIIa [ 141, antibodies. The fluorescence image showing anti-tubulin staining of the particle revealed a typical ring structure (Fig. 3b), while anti-platelet GPIIb/IIIa staining showed the image in which small patches or spots were seen throughout the cytoplasm against the faint diffuse background staining (Fig. 3d). Brighter staining at the periphery showed that the glycoproteins exist on the surface of particles (Fig. 3d). No significant difference was observed between these particles and human blood platelets in immunofluorescent staining images (Figs.
3a and 3~). Consequently, MEG-01 cells produce platelet-like particles. Radley and Hartshorn [ 151 have recently shown that a marginal coil of microtubules does not form until after platelet liberation from megakaryocytes. Therefore, a dot pattern observed in the particle (Fig. 2a) may be an immature microtubule ring shortly after liberation from MEG-01 cells. Figure 2b shows that the microtubule rings seem slack, probably representing a more mature microtubule ring than that in Fig. 2a. The mechanism of platelet production from megakaryocytes remains controversial [ 161. The appearance of many particles in Fig. 1 favors the possibility that the particles, the marginal microtubule ring of which was formed in the MEG-01 cytoplasm, are liberated all at once from MEG-01 as cytoplasmic segregation in normal megakaryocyte. On the other hand, the particles observed in Fig. 2 were seen attached on the MEG-01 cell surface (Figs. 2a and 2c) or connected to processes of MEG-01 cytoplasm (Figs. 2b and 2d). The surface appearance supports the possibility that an additional mechanism for particle release from MEG-01 cell is by fragmentation of peripheral cytoplasm, including process. From these results, we propose two possible mechanisms for platelet-like
SHORT
NOTE
225
FIG. 2. Particles on the MEG-01 cell surface. MEG-01 cells and particles were double-stained with anti-tubulin (a, b) and rhodaminephalloidin (c, d). (a) is the same field as (c); (b) is the same field as (d). Anti-tubulin staining showed the pattern in dots (a, arrows) and slack rings (b, arrows), while rhodamine staining showed an interesting pattern which consisted of a meshwork and a peripheral circle beneath the
FIG. 3. Localization of the microtubule and platelet-specific surface glycoprotein (GPIIb/IIIa) in particles liberated from MEG-01. A human platelet (a, c) and a MEG-01 particle (b, d) were doublestained with anti-tubulin (a, b) and anti-GPIIb/IIIa (c, d). Anti-tubulin staining of MEG-01 particle revealed a typical ring structure (b), while anti-GPIIb/IIIa staining showed small patches or spots throughout the cytoplasm. Scale bar, 2 pm.
particle production in MEG-01 cells. They must be clarified in further detail. Several human megakaryotic cell lines from patients with leukemia were established [l-lo]. However, the cell lines except MEG-01 [4] and CHRF-288 [9] express characteristics associated not only with megakaryocyte lineages but also with other nonmegakaryocyte lineages. In some of the lines [4, 91, the expression of megakaryotic markers increased under phorbol myristate acetate (TPA) treatment. The treatment of MEG01 with TPA induced morphological and biochemical differentiation in more mature megakaryocytes [ 181. In this report, we showed that MEG-01 is the first cell line that has the ability to produce platelet-like particles, the most important function of the megakaryocyte. Therefore, this cell line will provide a useful model for elucidating the mechanisms of platelet production and regulation of megakaryocytopoiesis [19]. Also the immunofluorescent technique using anti-tubulin antibody to detect platelets will greatly facilitate megakaryocytopoiesis research. We are deeply indebted to Prof. Motoo, Nojima of the Ehime College of Health Science, Dr. Teiji, Iijima, and Dr. Tohru, Hasegawa, of
SHORT the Institute for Fermentation, Osaka, for continuing encouragement throughout this work. This research was supported in part by a grantin-aid for medicinal research from the Fugaku Trust. REFERENCES 1. 2. 3.
4. 5. 6.
7. 8.
Gewirtz, A. M., Burger, D., Radio, T. A., Benz, E. J., Jr., and Hoffman, R. (1982) Blood 60, 785-789. Martin, P. (1982) Science 216, 1233-1235. Sledge, G. W., Jr., Glant, M., Jansen, J., Heetrema, N. A., Roth, B. J., Goheen, M., and Hoffman, R. (1986) Cancer Res. 46,21552159. Ogura, M., Morishima, Y., Ohno, R., Kato, Y., Hirabayashi, N., Nagura, H., and Saito, H. (1985) Blood 66, 138441392. Ogura, M., Tanabe, N., Nishioka, J., Suzuki, K., and Saito, H. (1987) Blood 70,301L306. Avanzi, G. C., Lista, P., Giovinazzo, B., Miniro, R., Saglio, G., Benetton, G., Coda, R., Cattoretti, G., and Pegoraro, L. (1988) Brit. J. Haematol. 69, 359-366. Greenberg, S. M., Rosenthal, D. S., Greeley, T. A., Tantravahi, R., and Handin, R. I. (1988) Blood 72, 1968-1977. Seigneurin, D., Champelovier, P., Mouchiroud, G., Berthieer, R., Leroux, D., Prenant, M., McGregor, J., Starch, J., Morle, F.,
Received September 11, 1990 Revised version received November
6, 1990
NOTE
9. 10.
11. 12. 13. 14. 15.
16. 17. 18.
19.
Micouin, C., Pietrantuono, A., and Kolodie, L. (1987) Exp. Hematol. 15, 822-832. Witte, D. P., Harris, R. E., Jenski, L. J., and Lampkin, B. C. (1986) Cancer 58, 238-244. Komatsu, N., Suda, T., Mori, M., Tokuyama, N., Sakata, Y., Okada, M., Nishida, T., Hirai, Y., Sato, T., Fuse, A., and Miura, Y. (1989) Blood 74,42-48. Behnke, 0. (1970) Reu. Exp. Pathol. 9, l-92. Debus, E., Weber, K., and Osborn, M. (1981) Eur. J. Cell Biol. 24, 455.52. Takeuchi, K., Kuroda, K., Ishigami, M., and Nakamura, T. (1990) Enp. Cell Res. 186, 374-380. Furukawa, K., Hayashi, K., Naoe, T., Takamoto, S., Shiku, H., and Yamada, K. (1987) Acta Haematol. Japan. 50, 914-925. Radley, J. M., and Hartshorn, M. A. (1987) Blood Cells 12,603610. Stenberg, P. E., and Levin, .J. (1987) Blood Cells 15, 23-47. Yamada, E. (1957) Acta Anat. 29, 267-290. Ogura, M., Morishima, Y., Okumura, M., Hotta, T., Takamoto, S., Ohno, R., Hirabayashi, N., Nagura, H., and Saito, H. (1988) Blood 72,49-60. Gewirtz, A. M. (1986) Semin. Hematol. 23, 27-42.
