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- MATERIALS AND METHODS
The platelet-sized particle formation in the human megakaryoblastic leukaemia cell line MEG-01 and its subline MEG-01s was examined. MEG-01 and MEG-01s cells spontaneously released platelet-sized particles into the culture medium, in which the cells occasionally extended cytoplasmic processes similar to those of megakaryocyte proplatelets. Scanning electron microscopic images showed cytoplasmic processes elongated from blebs on the MEG-01 and MEG-01s cell surface and were constricted between segments of platelet size. Immunofluorescence staining with anti-tubulin antibody showed that the cytoplasmic processes contained microtubules that were organized into a ring, which is a characteristic of circulating platelets. Some platelet-sized particles, probably released by ruptures at the sites of the process constriction, were metabolically active in an MTT assay (about 50%). Some particles also expressed the platelet-specific glycoproteins GPIIb, IIIa and GMP-140. Rarely, in response to thrombin, particles underwent a shape change from spherical to a shape with irregular membrane protrusions and fine filopodia, and aggregating with one another. The particles also had increased GMP-140 (P-selectin) expression with the addition of thrombin. These results show the usefulness of the MEG-01 and MEG-01s cell lines for the study of thrombopoiesis.
Megakaryocyte precursors originate from pluripotential stem cells and undergo a complex maturation process including the formation of a polyploid nucleus ( Hoffman, 1989). Platelets are shed from mature megakaryocytes by a series of events which are still incompletely understood; the main reason for this is the difficulties in obtaining sufficient numbers of megakaryocytes for studies of megakaryocytopoiesis and thrombopoiesis.
These studies have been advanced through use of in vitro culture systems with haemopoietic stem cells ( Bridell et al, 1991 ; Inoue et al, 1993 ; Debili et al, 1995 ; Choi et al, 1995 ; Guerriero et al, 1995 ; Cramer et al, 1997 ) and with cell lines of megakaryocytic lineage ( Gewirtz et al, 1982 ; Tabilio et al, 1984 ; Ogura et al, 1985 ; Sledge et al, 1986 ; Tayrien et al, 1987 ; Seigneurin et al, 1987 ; Greenberg et al, 1988 ; Tange et al, 1988a ; Sato et al, 1988 ; Komatsu et al, 1991 ). However, little has been reported regarding the platelet formation and platelet shedding which are the terminal differentiation process of thrombopoiesis. Recently, Choi et al (1995 ) and Cramer et al (1997 ) demonstrated that CD34+ cells from normal human peripheral blood or human bone marrow differentiate to mature megakaryocytes capable of producing functional platelets under in vitro culture conditions. Although these two studies made important progress in the morphological characterization of platelet formation, some problems remain for biochemical analysis including the purity of CD34+ cells in the starting cell population and the cell quantities for analysis.
Using several human cell lines exhibiting some morphological and biochemical characteristics of megakaryocytes, it has been revealed by fluorescence and electron microscopy that these cells release platelet-like particles ( Sledge et al, 1986 ; Greenberg et al, 1988 ; Tange et al, 1988b ; Takeuchi et al, 1991 ; Nagano et al, 1992 ). In a few cell lines, however, these particles have not yet been isolated or characterized. The clonal human megakaryoblastic leukaemia cell line MEG-01 and its subline MEG-01s, established by Ogura et al (1985 , 1988), display phenotypic properties that closely resemble those of megakaryoblasts but not other blood cell lineages. These properties demonstrate increased expression after treatment of the cells with phorboldiester, TPA ( Murate et al, 1991 ). The cell lines also release particles identified by a characteristic marginal microtubule and by the localization of platelet-specific glycoprotein (GPIIb/IIIa) in the plasma membrane ( Takeuchi et al, 1991 , 1995). Inhibitors of DNA synthesis, such as aphidicolin, enhance the particle release ( Takeuchi et al, 1995 ). On the basis of these studies, we have examined the mechanism(s) by which particles are formed from MEG-01 and MEG-01s cells and now report that these cells formed long beaded processes which resembled those observed in normal megakaryocytes. Their rupture may yield particles with sizes and functions similar to individual platelets.
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- MATERIALS AND METHODS
Attempts to develop an in vitro system using cell lines to clarify the mechanisms of platelet production have been made by many investigators, and the release of platelet-like particles has already been reported in several human cell lines of megakaryotic lineage ( Sledge et al, 1986 ; Greenberg et al, 1988 ; Tange et al, 1988b ; Takeuchi et al, 1991 ; Nagano et al, 1992 ). However, little is known of how the release actually occurs. We have previously shown that MEG-01 and MEG-01s cells release platelet-like particles ( Takeuchi et al, 1991 , 1995) and the results of the present study suggest that the release occurs via a mechanism such as the flow model ( Radley & Scurfield, 1980; Stenberg & Levin, 1989). This contention is based on the following evidence. First, the MEG-01 and MEG-01s cells extended processes with constrictions ( Figs 12–3), which closely resemble those found in in vivo megakaryocytes ( Radley & Scurfield, 1980; Scurfield & Radley, 1981) and those supported in culture ( Choi et al, 1995 ; Cramer et al, 1997 ). The process formation first observed in megakaryocytes in bone marrow has been demonstrated in cultures of megakaryocytes of many kinds of animals ( Scurfield & Radley, 1981; Leven & Yee, 1987; Topp et al, 1990 ; Inoue et al, 1993 ; Debili et al, 1995 ; Choi et al, 1995 ; Guerrieróet al, 1995 ). Cytoplasmic processes often develop constrictions between segments of platelet size that give a beaded appearance. A rupture at the site of constriction is thought to release the platelets. Second, the particle fractions prepared from MEG-01 and MEG-01s culture media contained short fragments with tapered ends, often at opposite ends (Fig 3A), and long fragments with several constrictions ( Figs 3B and 3C), but mainly particles without a tail. The fragments with a tail were thought to be shapes detected immediately after rupture of the cytoplasmic processes attached to the MEG-01 cell body. The fragments are comparable to the proplatelet process observed in megakaryocytes. Third, the immunofluorescence imaging using anti-tubulin antibody showed that the cytoplasmic processes were microtubule-containing structures ( Figs 1E and 1F). As shown in our previous reports ( Takeuchi et al, 1991 , 1995), and by the present results, microtubule rings were observed not only in the cytoplasmic processes of MEG-01 cells in culture medium ( Figs 1E and 1F), but also in some platelet-sized particles fractionated from MEG-01 culture medium ( Takeuchi et al, 1995 ). Approximately 20–30% of the fractionated particles had microtubule rings, which were not localized as a mem-brane cytoskeleton in the subplasmalemmal region of the particles, but were located in a coil only in the equatorial region ( Takeuchi et al, 1995 ). Thus, we have used the distinctive morphology of microtubule organization as the first indication that the particles were similar to platelets. The presence of microtubule rings in both cytoplasmic processes and particles from MEG-01 cells indicated that particle formation in MEG-01 cells is similar to platelet formation from megakaryocyte processes. Radley & Haller (1982) observed that spontaneous cytoplasmic process formation was reversed by vincristine. Similarly, Leven & Yee (1987) indicated that both colchicine and vincristine inhibited the process formation stimulated by thrombocytoplasmic plasma. Thus, the formation of microtubule-based cytoplasmic processes may be an important step in platelet formation, although it is still unclear what role the microtubules play in cytoplasmic fragmentation. The flow model suggests that the demarcation membrane is involved in the formation of attenuated processes by megakaryocytes ( Stenberg & Levin, 1989). In our present study we could not examine these relationships because of the difficulty due to the low frequency of the formations of both demarcation membrane and cytoplasmic processes; MEG-01 and MEG-01s cells are megakaryoblasts at a relative early stage in which those structures are seldom found and divide predominantly into daughter cells. Only a few cells differentiate to develop the cytoplasmic processes which break, releasing small fragments, thus forming platelets.
MEG-01 and MEG-01s particles are similar to platelets in their distinctive morphology of microtubule organization and because of the presence of several proteins important for platelet function (GPIb, GPIIb/IIIa ( Takeuchi et al, 1991 , 1995) and P-selectin). In addition, this study showed the functional similarities between MEG-01 and MEG-01s particles and platelets with respect to thrombin-induced shape change (Fig 5B), aggregation (Fig 5C) and expression of GPIIb/IIIa and P-selectin (Fig 7), although the number of shape-changed particles and the amount of aggregated particles were very small. If many dead particles and cell fragments were contained, these results are reasonable. The MTT assay results also support such a possibility (Fig 4). The amount of formazan generated in this assay is directly proportional to the activity of mitochondria. The particle fraction had only about 50% of the metabolic activity of platelet fractions. It seems likely that half of the particles were inactive or dead, but the possibility that all of the particles had low enzyme activity could not be excluded. As MEG-01 and MEG-01s cells are metabolically active cells, such a contamination in the particle fraction may give incorrect results. The microscopic images, in which the formazans produced by MEG-01 and MEG-01s cells were large and thick, and those by MEG-1 and MEG-01s particles and human platelets were very thin (data not shown), clearly showed that the particle fraction did not contain MEG-01 and MEG-01s cells.
Platelet activation requires various membrane glycoproteins specific to platelets, such as GPIIb/IIIa in the surface membrane and P-selectin in the α-granular membrane. The present MEG-01 particles certainly displayed P-selectin expression, increased by thrombin. The low level of the expression probably indicates the presence of particles with inactive P-selectin and/or without P-selectin. The platelets generated from CD34+ cells in vitro ( Choi et al, 1995 ; Cramer et al, 1997 ), mostly lose their discoid-shape and extend spiky and bulky pseudopods as if they were activated. They showed no marginal microtubule bands characteristic of the discoid-shaped platelets. In contrast, shape change and aggregation were so markedly impaired in many MEG-01 particles that they could not be detected by aggregometer. Their ring-like structure of marginal microtubule bands was therefore retained ( Figs 1E and 1F). These differences in features between MEG-01 particles and culture-derived platelets may be related to the levels of expression of glycoproteins, GPIIb/IIIa and P-selectin, which respond to the stimulus and recruit adjacent platelets to form aggregates accompanying the shape change. The culture-derived platelets expressed higher levels of the glycoproteins than MEG-01 particles, and thus were able to induce their aggregation. Although other components in the signal transduction pathway are also required for platelet aggregation, their expression in MEG-01 particles was not examined in this study, except for mito-chondria dehydrogenase activity analysed by MTT assay.
Unfortunately we could not separate the beaded processes connected with the MEG-01 and MEG-01s cell bodies which probably may be functional. The centrifuged culture supernatants contained mainly particles/fragments detached from the cell body. The connected processes were in the pellet together with the MEG-01 and MEG-01s cells. In addition, we could not include any steps to enrich the metabolically active particles in our preparation, as they may decrease sensitivity to stimuli during preparation.
The morphological features observed here in MEG-01 and MEG-01s cells closely approximate those in megakaryocytes, thus allowing the cell lines to be a useful model for the study of megakaryocyte maturation as evidenced by the process formation and cytoplasmic fragmentation into platelets. To obtain a large yield of functional particles, many problems remain to be solved, including candidate factors for maturation, the fragmentation of cytoplasmic processes and particle preparation.
We believe that studies with the MEG-01 and MEG-01s cell lines will contribute to development of new forms of treatment of megakaryoblastic leukaemia based on the use of various components including cytokines to induce differentiation in malignant cells and to clarify the nature of the clinical abnormalities in megakaryopoiesis.