Functional and morphological differentiation induction of a human megakaryoblastic leukemia cell line (MEG-01s) by phorbol diesters

The effect of differentiation induction by a tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on a clonal human megakaryoblastic cell line, MEG-O1s, was investigated, and a prominent response was demonstrated. The cells became weakly adherent, developed conspicuous cytoplasmic blebs, and displayed mature megakaryocytic characteristics by light microscopy such as the development of azurophilic cytoplasmic granules and a mosaic pattern of oxyphilic patches, multiplication of nuclei, and enhancement of the PAS reaction and alpha-naphthyl acetate esterase staining. Ultrastructural studies demonstrated the development of prominent cytoplasmic blebs, budding of blebs, and multiplication of nuclei. Numerous granules with central nucleoids that are similar to alpha granules had developed as well as granules with high electron density and clearly demarcated zones. Surface marker analysis revealed a moderate increase in IgG Fc receptor levels and a profound decrease in C3 receptor sites. By an immunofluorescent technique using monoclonal and polyclonal antibodies, a dramatic change in the expression of several megakaryocyte-platelet-specific proteins was demonstrated. All the proteins that had been expressed before induction such as platelet glycoprotein (GP) IIb/IIIa, fibrinogen, von Willebrand factor, factor XIIIA, beta-thromboglobulin (beta-TG), and HLA class 1 antigen were profoundly enhanced after induction by TPA. Induction by TPA led to the expression of fibronectin and factor V, which were not detected on nontreated cells. An ultrastructural immunoperoxidase study demonstrated platelet GPIb and GPIIb/IIIa in both plasma membranes and protein synthesis areas such as perinuclear cisternae and endoplasmic reticulum after TPA induction. beta-TG was also observed in some cytoplasmic granules of TPA-treated cells. TPA remarkably increased the secretion of beta-TG into the culture medium of MEG-01s. Ploidy was also increased from 2C to 4C to 4C to 8C. Similar maturation of MEG-01s was induced by other phorbol diester analogues such as phorbol-12,13-dibutyrate, but not by phorbol itself. These results indicate that phorbol diester, TPA, can bring about differentiation and maturation of a human megakaryoblastic cell line (MEG-01s) and that MEG-01s cells will provide a useful model for studying megakaryocytic differentiation and numerous megakaryocyte-platelet-specific proteins.     

K-252a-induced polyploidization and differentiation of a human megakaryocytic cell line, Meg-J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg-J. In the presence of K-252a (an indolocarbasole derivative), Meg-J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIIa and GPIb. Thrombopoietin (TPO) promoted the K-252a-induced polyploidization and megakaryocytic differentiation. In the process of K-252a-induced polyploidization, levels of expression of both cdc2 and cyclin B₁ were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg-J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B₁ complex, a critical kinase involved in G2/M cell cycle transition.     

Uncoupling in the expression of platelet GP IIb/IIIa in human endothelial cells and K562 cells: absence of immunologic crossreactivity between platelet GP IIb and the vitronectin receptor alpha chain [see comments]

Platelet glycoproteins (GP) IIb and IIIa exist as noncovalently associated Ca++-dependent heterodimer complexes within the platelet membrane and express the major platelet alloantigens Leka (Baka) and PIA1 (Zwa), which are genetic markers of GP IIb and GP IIIa, respectively. Since heterodimers immunologically related to platelet GP IIb/IIIa have been identified in a number of nucleated cell types, we tested anti-Leka and anti-PIA1 antiserum, polyclonal anti-platelet GP IIb/IIIa IgG, as well as a panel of 28 monoclonal anti-GP IIb, GP IIIa, or complex dependent anti-GP IIb/IIIa antibodies on endothelial cells, peripheral blood mononuclear cells, and the erythroleukemic cells HEL and K562 in order to determine whether nucleated cell GP IIb/IIIa related proteins and platelet GP IIb/IIIa are immunologically related. Using immunofluorescence, immunoblotting, and immunoprecipitation experiments, evidence is presented that (1) the alloantigen Leka is not expressed in endothelial cells of an individual whose platelets are of the Leka/PIA1 phenotype, whereas the PIA1 alloantigen is readily detectable in these cells, (2) that in contrast to HEL cells, which express platelet GP IIb/IIIa and are of the Leka/PIA1 phenotype, platelet GP IIb is immunologically undetectable in 12-O-tetradecanoyl- phorbol-13-acetate (TPA)-treated K562 cells despite the presence of platelet GP IIIa, and (3) that peripheral blood mononuclear cells do not express platelet GP IIb or GP IIIa on their cell surface.     

Expression of high affinity binding sites for erythropoietin on L8057 cells, a mouse megakaryoblastic cell line, associated with cell differentiation.

In this study, specific binding sites were examined for erythropoietin (EPO) on the mouse leukemic cell line, L8057. This cell line is megakaryoblastic in origin as evidenced by an enlargement of cell size, multinuclearity, intense activity of acetylcholinesterase, more expression of glycoprotein IIb and IIIa antigen, and higher ploidy distribution after the treatment with 12-o-tetradecanoylphorbor-13-acetate (TPA). The original undifferentiated cells possessed a single class of low affinity binding sites for recombinant human (rh) EPO with a Kd of 3.5 nM. Following the treatment with TPA, high affinity binding sites (Kd; 440 pM) were expressed in addition to the low affinity sites. EPO stimulated the incorporation of 3H-leucine into TPA-treated L8057 cells, and the maximal effect of EPO was observed at the same order as the Kd value of high affinity sites. The present data demonstrates that the expression of high affinity binding sites for EPO is associated with the differentiation of L8057 cells which have megakaryocytic characteristics. Furthermore, protein synthesis stimulated by EPO may be mediated through the high affinity sites.     

Megakaryocytic Maturation is Regulated by Maintaining a Balance Against Cytokine Induced-cell Proliferation: Steel Factor Retards Thrombopoietin-induced Megakaryocytic Differentiation While Synergistically Stimulating Mitogenesis

Using a factor-dependent cell line M07ER, which contains a stably transduced human erythropoietin (EPO) receptor gene in human megakaryoblastic cell line MO7e and which resulted in concomitant expression of EPO receptor, c-Mpl and c-Kit, we investigated the biological effects of these cytokines in terms of cell growth and differentiation. Thrombopoietin (TPO), EPO and Steel factor (SLF) all stimulated MO7ER cell proliferation in a dose-dependent manner. Combined stimulation of cells with SLF plus either TPO or EPO resulted in striking synergistic enhancement of MO7ER cell growth as compared with each cytokine alone, whereas combination of TPO plus EPO showed only an additive effect on cell proliferation. With regards to cell differentiation, either TPO or EPO treatment induced enhancement of platelet glycoprotein (GP) IIb/IIIa and GPIb expression. SLF induced GPIIb/IIIa and GPIb expression, but the effect was much weaker than that of EPO or TPO. However, addition of SLF to either TPO- or EPO- containing cultures (which induced potent mitogenesis in MO7ER cells) resulted in suppression of these megakaryocyte specific antigens. Addition of low-dose cytosine arabinoside (Ara-C)(1 to 10 ng/ml) enhanced TPO- or EPO- induced megakaryocytic differentiation in MO7ER cells while mildly suppressing cell growth. Treatment the cells with low-dose Ara-C plus TPO plus SLF overrode the proliferative enhancing effects of SLF and induced GPIIb/IIIa and GPIb expression as efficient as TPO alone. Retardation of TPO-induced megakaryocytic maturation was also observed in normal murine bone marrow cells by combined stimulation with TPO and SLF as assessed by the numbers of acetylcholinesterase staining-positive cells and megakaryocyte nuclear polyploidy. These results suggest that megakaryocytic maturation is, at least in part, regulated by countering cytokine-induced cell proliferation.     

Megakaryocytic Maturation is Regulated by Maintaining a Balance Against Cytokine Induced-cell Proliferation: Steel Factor Retards Thrombopoietin-induced Megakaryocytic Differentiation While Synergistically Stimulating Mitogenesis; Hematopoiesis

Using a factor-dependent cell line MO7ER, which contains a stably transduced human erythropoietin (EPO) receptor gene in human megakaryoblastic cell line MO7e and which resulted in concomitant expression of EPO receptor, c-Mpl and c-Kit, we investigated the biological effects of these cytokines in terms of cell growth and differentiation. Thrombopoietin (TPO), EPO and Steel factor (SLF) all stimulated MO7ER cell proliferation in a dose-dependent manner. Combined stimulation of cells with SLF plus either TPO or EPO resulted in striking synergistic enhancement of MO7ER cell growth as compared with each cytokine alone, whereas combination of TPO plus EPO showed only an additive effect on cell proliferation. With regards to cell differentiation, either TPO or EPO treatment induced enhancement of platelet glycoprotein (GP) IIb/IIIa and GPIb expression. SLF induced GPIIb/IIIa and GPIb expression, but the effect was much weaker than that of EPO or TPO. However, addition of SLF to either TPO- or EPO- containing cultures (which induced potent mitogenesis in MO7ER cells) resulted in suppression of these megakaryocyte specific antigens. Addition of low-dose cytosine arabinoside (Ara-C)(1 to 10 ng/ml) enhanced TPO- or EPO- induced megakaryocytic differentiation in MO7ER cells while mildly suppressing cell growth. Treatment the cells with low-dose Ara-C plus TPO plus SLF overrode the proliferative enhancing effects of SLF and induced GPIIb/IIIa and GPIb expression as efficient as TPO alone. Retardation of TPO-induced megakaryocytic maturation was also observed in normal murine bone marrow cells by combined stimulation with TPO and SLF as assessed by the numbers of acetylcholinesterase staining-positive cells and megakaryocyte nuclear polyploidy. These results suggest that megakaryocytic maturation is, at least in part, regulated by countering cytokine-induced cell proliferation.     

Platelet factor 4 gene expression in a human megakaryocytic leukemia cell line (CMK) and its differentiated subclone (CMK11-5)

The human cell line CMK spontaneously expresses megakaryocytic characteristics and can be induced to differentiate into mature megakaryocytes after exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In comparison with CMK, we have examined the characteristics of a subclone, designated as CMK11-5, that is morphologically larger than the parent clone CMK and contains multinucleated giant cells. All CMK11-5 cells were positive for platelet peroxidase (PPO) activity, and some contained abundant alpha-granules and well-developed demarcation membranes. CMK cells had few demarcation membranes and alpha-granules, and 10% of these cells were found not to possess PPO activity. Phenotypic analysis revealed the percentage of CMK11-5 cells for platelet glycoprotein (GP) IIb/IIIa (CD41a), and GPIIIa (CD61) was greater than that for CMK cells. On the basis of these findings, CMK11-5 cells were considered to be more differentiated than CMK cells. We further examined the expression of GPIIb and platelet factor 4 (PF4) mRNA by Northern blot hybridization using 32P-labeled cDNA probes for GPIIb and PF4 in CMK and CMK11-5 cells. CMK cells exhibited mRNA for GPIIb, and its expression was augmented by TPA addition, but not PF4. In contrast, CMK11-5 cells were found to contain mRNA for GPIIb and PF4, and their mRNA levels were increased by the addition of TPA. The immunoreactive PF4 antigen was not detected in the TPA-treated CMK11-5 cells or in the culture medium of these cells. These results indicate that expression of mRNA for PF4 is a useful marker for the identification of mature megakaryocytes. Detection of mRNA for PF4 is a more sensitive method for characterization of megakaryocytic cells than that for the PF4 antigen.     

Human chronic myeloid leukemic cell line with positive Philadelphia chromosome exhibits megakaryocytic and erythroid characteristics

A cell line (LAMA-84) has been established from the blood of a patient with chronic myeloid leukemia in acute phase. LAMA-84 cells retained the patient's chromosome abnormalities, i.e., triplication of all chromosomes except chromosome 18, the presence of Philadelphia (Ph) chromosome in 4-5 copies, and the presence of chromosome markers. LAMA-84 cells have morphological features of undifferentiated blast cells, but analyses have indicated that they belong to the megakaryocytic lineage; platelet peroxidase (PPO) was found in 8.5% of cells; LAMA-84 cells reacted spontaneously with poly- and monoclonal antibodies against the platelet glycoproteins (GP) IIb, IIIa, and the GPIIb/IIIa complex, whose presence was confirmed by crossed immunoelectrophoresis. LAMA-84 cells lack the membrane characteristics of lymphoid and mature granulocytic cells but do, however, react with certain antibodies to immature myeloid cells. Furthermore, they are positive with an antiglycophorin antibody, and contain alpha- and gamma-globin mRNA, thus demonstrating erythroid marker expression. Thus LAMA-84 is a tripotent, megakaryocytic, erythroid, and granulocytic cell line. The megakaryocytic and erythroid markers were enhanced by the addition of DMSO, butyrate, TPA, and hemin. The LAMA-84 cell line represents an interesting tool for the study of megakaryocytic and erythroid differentiation and the mechanisms of neoplastic growth.     

A functional role of mitogen-activated protein kinases, Erk1 and Erk2, in the differentiation of a human leukemia cell line, UT-7/GM: A possible key factor for cell fate determination toward erythroid and megakaryocytic lineages

The mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation and differentiation. In this study, we examined the roles of 2 members of the MAP kinase family, extracellular signal-regulated kinase 1 (Erk1) and Erk2, in erythropoietin (EPO)-induced erythroid differentiation and thrombopoietin (TPO)-induced megakaryocytic differentiation. UT-7/GM was used as a model system because this cell line is an erythroid/megakaryocytic bipotent cell line that can be induced to differentiate into the erythroid and megakaryocytic lineages by EPO and TPO, respectively. The kinetics of activation of Erk1 and Erk2 were examined during erythroid and megakaryocytic differentiation of UT-7/GM cells. EPO induced a transient activation of these kinases, peaking after 1 minute of stimulation and then declining quickly almost to the basal level. In contrast, TPO-induced activation of the kinases peaked at 10 minutes and persisted for up to 60 minutes, similar to the activation by granulocyte-macrophage colony-stimulating factor. The percentage of EPO-induced hemoglobin-positive cells was elevated by the addition of PD98059, a specific inhibitor of MEK1 (MAP kinase/ERK kinase 1). In contrast, PD98059 clearly reduced the amount of glycoprotein IIb/IIIa antigens induced by TPO on UT-7/GM cells. Thus, inactivation of Erk1 and Erk2 kinases promoted EPO-induced erythroid differentiation and suppressed TPO-induced megakaryocytic differentiation of UT-7/GM cells. In conclusion, the activation of Erk1 and Erk2 kinases may be a critical event in the determination of cell fate and the differentiation processes of the erythroid and megakaryocytic lineages.     

Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest

The maturation of megakaryocytes in vivo requires polyploidization or repeated duplication of DNA without cytokinesis. As DNA replication and cytokinesis are tightly regulated in somatic cells by cyclins and cyclin-dependent kinases, we sought to determine the pattern of cyclin gene expression in cells that undergo megakaryocytic differentiation and polyploidization. The Dami megakaryocytic cell line differentiates and increases ploidy in response to phorbol 12-myristate 13-acetate (PMA) stimulation in vitro. We used Northern blotting to analyze mRNA levels of cyclins A, B, C, D1, and E in PMA-induced Dami cells and found that cyclin D1 mRNA levels increased dramatically (18-fold). Similar increases in cyclin D1 mRNA were obtained for other cell lines (HEL and K562) with megakaryocytic properties, but not in HeLa cells. The increase in cyclin D1 was confirmed by Western immunoblotting of PMA-treated Dami cells. This finding suggested that cyclin D1 might participate in megakaryocyte differentiation by promoting endomitosis and/or inhibiting cell division. To address these possibilities, we constructed two stable Zn+2-inducible, cyclin D1-overexpressing Dami cell lines. Cyclin D1 expression alone was not sufficient to induce polyploidy, but in conjunction with PMA-induced differentiation, polyploidization was slightly enhanced. However, unlike other cell systems, cyclin D1 overexpression caused cessation of cell growth. Although the mechanism by which cyclin D1 may affect megakaryocyte differentiation is not clear, these data demonstrate that cyclin D1 is upregulated in differentiating megakaryocytic cells and may contribute to differentiation by arresting cell proliferation.     

Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia.

The blockade of the platelet integrin glycoprotein (GP) IIb/IIIa has proved to be an effective antiplatelet therapy. Profound thrombocytopenia has repeatedly been described as an adverse effect in patients treated with GP IIb/IIIa inhibitors, but its mechanism has not been elucidated yet. With use of flow cytometry, the activation status of platelets was monitored in 26 patients presenting with acute myocardial infarction who were treated with the GP IIb/IIIa inhibitor abciximab alone or in combination with the fibrinolytic agent reteplase. Fibrinogen and PAC-1 (a GP IIb/IIIa activation-specific monoclonal antibody) binding, as well as P-selectin expression on unstimulated platelets were constant in 25 patients throughout a follow-up of 7 days. In 1 patient (D.F.), the percentage of platelet-binding fibrinogen increased from 2.2% to 17.8%, for PAC-1 from 2.8% to 13.2%, and for P-selectin expression from 10.2% to 58.3% 10 minutes after the start of treatment. Furthermore, D.F. had a decrease in single platelet count in ethylenediaminetetraacetic acid-, citrate-, and heparin-anticoagulated and native blood. Blood films revealed platelet aggregates. In vitro testing of D.F.'s blood 2 and 4 weeks after initial admission demonstrated a reinduction of fibrinogen and PAC-1 binding to platelets, an increase of P-selectin expression, and formation of platelet aggregates following exposition of platelets to abciximab in vitro. In summary, this report describes the induction of platelet activation by a GP IIb/IIIa inhibitor in vivo and reinduction in vitro in direct association with thrombocytopenia. Platelet activation by GP IIb/IIIa inhibitors may be one potential mechanism for GP IIb/IIIa inhibitor-induced thrombocytopenia.     

Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01

OBJECTIVE: Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dependent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to study how PGHS-1 expression is regulated in platelets. MATERIALS AND METHODS: Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures. RESULTS: We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population. CONCLUSION: We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.     

Regulation of megakaryocytopoiesis in an in vitro stroma model: preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

OBJECTIVE: Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. MATERIALS AND METHODS: Human CD34(+) cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. RESULTS: After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the beta(1) integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. CONCLUSION: In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.     

Establishment of a novel human megakaryoblastic leukemia cell line, MEG- 01, with positive Philadelphia chromosome

A megakaryoblastic cell line, designated MEG-01, was established from the bone marrow of a patient with blast crisis of Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia. MEG-01 cells grew in single-cell suspension with a doubling time of 36 to 48 hours. Under the usual culture conditions, approximately half of the cells adhered to the culture flask with extention of pseudopods. MEG-01 cells were positive for the periodic acid-Schiff reaction, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for myeloperoxidase, alpha- naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and alkaline phosphatase. Ultrastructural platelet peroxidase was positive in MEG-01 cells. Cytoplasmic factor VIII (FVIII)-related antigen was weakly positive in larger MEG-01 cells by both an indirect immunofluorescent technique with monoclonal antibodies and a direct immunoperoxidase technique using horseradish peroxidase-conjugated conventional rabbit anti-human FVIII antibody. Platelet glycoprotein (GP) IIb/IIIa antigen was uniformly demonstrated on the surface of MEG- 01 cells by both indirect immunofluorescent and immunoperoxidase techniques using antiplatelet GP IIb/IIIa monoclonal antibodies; platelet GP lb antigen was demonstrated only in the cytoplasm of larger MEG-01 cells. MEG-01 cells possessed no markers for B or T lymphocytes or for myeloid cells. Chromosome analysis of this cell line revealed a human male hyperdiploid karyotype with a modal chromosome number of 56 to 58. The Ph1 chromosome was observed in all karyotypes analyzed. This novel human megakaryoblastic cell line may provide a useful model for the study of human megakaryopoiesis and of the biosynthetic mechanisms of proteins unique to megakaryocytic lineage.     

Stem-cell factor regulates the expression of cyclin A and retinoblastoma gene product in the growth and differentiation pathway of human megakaryocytic cells

The biological effects of c-kit ligand (stem-cell factor: SCF) on an immortalized human megakaryocytic cell line (CMK) was evaluated using methods including the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, surface marker analysis, DNA cell-cycle analysis and immunoblotting. SCF stimulated the growth of CMK cells. Incubation with SCF resulted in increased expression of IIb/IIIa platelet-related glycoprotein (gpIIb/IIIa), indicating enhanced differentiation of CMK cells. Treatment of CMK cells with SCF resulted in a decrease in the subpopulation in the G1 phase, with a reciprocal increase in those in the S phase and the G2+M phase. Moreover, SCF significantly increased cellular expression of cyclin A, a regulatory subunit of cyclin-dependent protein kinase (CDK), and the ratio of phosphorylated/dephosphorylated retinoblastoma gene product (RB protein). These results suggest that SCF stimulates the growth and differentiation of megakaryocytic cells possibly through mechanisms related to the activation of cell-cycle-dependent serine/threonine kinase and inactivation of the nuclear tumor-suppressor gene product.Abbreviations SCF stem-cell factor - G-CSF granulocyte-colony stimulating factor - MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - gpIIb/IIIa IIb/IIIa platelet-related glycoprotein - RB retinoblastoma gene product - ppRB phosphorylated RB - pRB dephosphorylated RB - IL Interleukin - TNF tumor necrosis factor This work was supported in part by a Grant-in-Aid for Scientific Research (06670615) from the Ministry of Education, Science, and Culture, Japan     

Human megakaryocytes. VII. Analysis of megakaryocytes for nuclear DNA content distribution in whole marrow cell suspensions by flow cytometry

These studies were designed to quantitate and determine the DNA content distribution of human marrow megakaryocytes using whole bone marrow. Cellular DNA content within megakaryocytic cells was established by measuring propidium iodide staining in marrow cells expressing platelet glycoprotein IIb/IIIa (Gp IIb/IIIa). These studies were based on the development of a method for rapid analysis of whole marrow cell preparations by a dual fluorescent system. DNA values ranging from 2 to 64 C were observed in all samples tested, with 16 C corresponding to the modal ploidy class representing almost one-half of the cells containing Gp IIb/IIIa. The second most frequent ploidy class corresponded to 32 C, followed by 8 C, with 20% and 15%, respectively. Virtually all high-ploidy megakaryocytes (greater than or equal to 8 C) were of low density (less than 1.050 g/cm3), whereas 2 and 4 C megakaryocytes were evenly distributed between less than or equal to 1.050 and greater than 1.050 g/cm3 marrow cells. These studies conclusively establish DNA content distribution of normal human marrow megakaryocytes and provide a basis for the study of states of altered megakaryocytopoiesis.