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THE BIOLOGY of MEGAKARYOCYTES Monday THE BIOLOGY OF MEGAKARYOCYTES Monday 152 ANALYSIS OF PHORBOL ESTER STIMULATED HUMAN MEGAKARYOCYTE DEVELOPMENT. R. Hoffman, B.J. Roth, G. W. Sledge, Jr., J. Straneva, and J. Brandt. Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA. The events that occur during the terminal maturation of human megakaryocyte& are poorly characterized. To examine these events, a recently characterized human megakaryocytic cell line (EST-IU, Cancer Res. 46: 2155-2159, 1986) was exposed to 12-0-tetradecanoyl-phorbol-13-ace ts te (TPA), as well as 2 non- transforming phorbol esters (4 alpha phorbol and 4 bets phorbol 12 alpha, 13 alpha diace ts te) at the identical concentrations. Morphologic changes, including cellular attachment to untreated plastic or glass, occurred within 4 hrs of treatment with TPA. Treatment of EST-IU cells with e1 ther of the 2 non-transforming phorbols (4-alpha phorbol, or 4-be ts phorbol, 12-be ts, 13-alpha diace ts te) failed to change morphology, DNA content, or expression of surface membrane glycoproteins or alpha-granule constituents when compared to control cells. TPA treatment resulted, however, in ..!!fOfound changes in adherence to plastic by the EST-IU cells, with an obvious dose-response relationship. At a 5 X 10 M TPA, cellular attachment was noted as early as 4 hours following treatment, !gd was complete by 16 hours, at which time > 95% of treated cells were attached. Following TPA treatment at 5 X 10 M, a number of morphologic changes occurred, including marked cellular flattening, the appearance of extensive cytoplasmic budding, and the development of numerous filopodia. Cells treated with either of the non-transforming phorbols as assessed by propidium iodide staining and flow cytometric analysis failed to exhibit a crenge in ploidy, although TPA reproducibly altered this parameter of megakaryocyte development. Cells treated with 10- -~ TPA have -'lfproxima tely the same proportion of cells in the 4N and 8N peaks as control cells. Following exposure to 10 M and 10 M TPA, there was an apparent shift of cells out of the 4N peak to 8N and 16N levels, and even the appearance of a small percentage of 32N cells. The DNA content of TPA-tres ted cells was also assessed by Feulgen staining and microdensitometry. Those cells (5%) which failed to adhere following TPA treatment were analyzed separately, and showed a very different ploidy distribution than the adherent cell population. Over 85% of adherent cells have a ploidy > 16N, with some cells attaining the 128N level. Treatment of cells with either of the 2 non-transforming phorbols failed to affect the expression of ~ftctor V, Factor VIII:RAg, beta-thromboglobulin, fibrinogen, or platelet glycoproteins. Cells treated with 5 X 10 M TPA similarly do not significantly increse the expression of Factor V, fibrinogen, or beta-thromoglobulin over that observed in control cells. The expression of both Factor VIII:RAg and platelet glycoproteins however, increase in TPA-trested cells. A similar increase in the expression of platelet glycoprotein IIb/IIIA using the mouse monoclonal C 7 was also observed. Those cells ths t express the highest levels of Factor VIII:RAg and platelet glycoprotelns following phorbol treatment also demonstrated the highest ploidy levels and also are the largest cells as measured by forward angle light scatter during flow cytometry. These studies indicate that TPA treatment of EST-IU cells inltia tes a cascade of events characterized by cellular adherence, increases in cell size and DNA con tent, and enhanced expression of platelet glycopro teins and Factor VIII:RAg. These events appear to occur in concert and closely resemble information that is available concerning maturation of normal rodent and human megakaryocytes. Although it is important to emphasize that EST-IU cells are leukemic and thus intrinsically different from normal human megakaryocytes, their availability and dynamic responses to TPA will provide an appropriate cellular model with which to study megakaryocyte ma tura tlon. 153 PRODUCTION AND LOCALISATION OF ALPHA-GRANULE PROTEINS IN MATURING MEGAKARYOCYTES: AN OVERVIEW ON ULTRA­ STRUCTURAL ASPECTS OF MEGAKARYOCYTE MATURATION. Elisabeth M. Cramer, John F. Martin, William Vainchenker, Janine Breton-Gorius, INSERM U91, Hopital Henri Mondor, Creteil, FRANCE, and Wellcome Research Laboratories, Beckenham, U.K. In order to study the production of a-granule proteins in maturing megakaryocytes, we used immunocytochemical techniques performed on cultured and enriched bone marrow megakaryocytes. Cultures were prepared from bone marrow CFU-MK with the methylcellulose and plasma clot techniques. Preparation of bone marrow megakaryocytes was carried out from human or pig rib marrow separated on percoll gradient and counterflow centrifugation. Megakaryocyte preparations were 90% pure and represented 85% of those in the whole marrow. Activation was prevented with prostacyclin and prefixation with low concentration glutaraldehyde. A panel of monoclonal and polyclonal antibodies, against different platelet membrane glycoproteins and against cytoplasmic antigens (such as von Willebrand Factor (vWF), fibrinogen (Fg) and thrombospondin (TSP)) was used and observed by immunofluorescence or by immunogold in electron microscopy. The first megakaryocytic precursors, promegakaryoblasts (PMKB) identifiable by these antibodies were found at day 5 of culture. They had the size of lymphocytes, were labelled for GP lib, Ilia, and IIb-IIIa complex but not for GPib which appeared later. Platelet peroxidase was also present, otherwise these cells were devoid of a­ granules and only a few of them exhibited a diffuse pattern for vWF immunolabelling. One day later membrane GPib and diffuse cytoplasmic labelling for vWF were detected in the majority of PMKB. At day 9 of culture, this This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. pattern of labelling for vWF became more intense and granular. The same pattern was observed for TSP and platelet factor 4. Immunoelectron microscopy showed that in immature megakaryocytes isolated from human bone marrow, labelling for vWF and TSP was observed in vesicles located in the Golgi region; in addition numerous small granules less than 0.1~m in diameter, round or elongated in shape, were labelled for these antigens. In mature human megakaryocytes, the labelling for these cytoplasmic antigens was restricted to the platelet a-granules in a distribution pattern similar to that of platelet a-granules. However, the labelling for Fg was consistently less intense in the granules of immature and mature megakaryocytes than in platelets. Because in platelets a-granule immunolabelling for vWF is associated with tubular structures which are specially prominent in porcine species, we studied vWF and tubular structures in pig megakaryocytes. Standard and immunoelectron microscopy revealed the simultaneous appearance of both in the small vesicles located in the Golgi area in the small immature a-granules and later in the mature a-granules. In mature megakaryocytes, labelling for vWF was intense and restricted to the a-granules. It was distributed eccentrically as in porcine blood platelets. Gold particles were often eccentrically located at one pole of the a-granule either labelling only its periphery or outlining one side of an elongated granule. Standard electron microscopy showed that tubular structures were very numerous in the mature a-granules, regularly spaced; arranged in parallel and usually located at one side of the granule. On the other hand platelets from pigs with homozygous von Willebrand disease were found to be completely devoid of both tubular structures and immunolabelling for vWF suggesting that the tubules represent the vWF itself. In acute megakaryoblastic leukemia, several phenotypes of PMKB were found in different patients, which corresponded to the stages of maturation of cultured megakaryocytes from CFU-MK. In conclusion, immunolabelling methods combined with megakaryocyte enrichment techniques are useful tools to study the origin of megakaryocyte (and platelet) granular proteins. 43 .
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