Human Megakaryoblastic Cell Line Research Articles

A sensitive human cell line based bioassay for megakaryocyte growth and development factor or thrombopoietin

We have developed a simple, rapid, sensitive bioassay for megakaryocyte growth and development factor (MGDF) or thrombopoietin (TPO) based on its ability to stimulate the proliferation of the human megakaryoblastic cell line MO7e. The bioassay takes only 24 h, is reproducible and sensitive to less than 10 pg/ml of MGDF. It can be made specific for MGDF by including neutralizing antibodies specific for MGDF, and is suitable for detection of MGDF in both plasma and serum samples.

TPO/c-mpl Ligand Induces Tyrosine Phosphorylation of Multiple Cellular Proteins Including Proto-oncogene Products, Vav and c-Cbl, and Ras Signaling Molecules

Thrombopoietin (TPO)/c-mpl ligand is a hematopoietic stimulates proliferation and maturation of megakaryocytes. To analyze the downstream of the c-mpl product, we used a human megakaryoblastic cell line, Mo7e, that has been proved to be responsive to TPO in terms of DNA synthesis. In this study, we found that TPO treatment resulted in tyrosine phosphorylation of Jak-2 kinase. Moreover, it was revealed that several functional molecules involved in the Ras signaling pathway, Shc and Sos, were phosphorylated by treatment with TPO. Finally, tyrosine phosphorylation of the proto-oncogene products, Vav and c-Cbl, has been proved to be induced by TPO. These results suggest that TPO could activate several signaling pathways including the Jak/Stat pathway, the Ras pathway and possibly another pathway involving the c-Cbl proto-oncogene product.

Selective Inhibition of Thrombin Receptor-mediated Ca2+ Entry by Protein Kinase C β

Thrombin initiates many physiological processes in platelets and other megakaryocyte-lineage cells by interacting with surface receptors and generating rises in cytoplasmic Ca2+; these rises result from both Ca2+ release from intracellular stores and receptor-mediated Ca2+ entry. Regulators that limit Ca2+ entry after its initiation by thrombin have not been identified. In this study, prevention of expression of a single protein kinase C isoenzyme (PKC beta) by antisense cDNA overexpressed in HEL cells, a human megakaryoblastic cell line that expresses thrombin receptors, promotes thrombin receptor-mediated Ca2+ entry without altering thrombin-induced intracellular release of Ca2+. The cytoplasmic Ca2+ rise initiated by endoperoxide analogs was not affected by inhibiting PKC beta. Overexpression of a cDNA encoding wild-type PKC beta mutated to prevent recognition by the antisense cDNA abolished the enhancement of Ca2+ influx following thrombin. Thus, PKC beta appears to be a specific negative regulator of thrombin receptor-mediated Ca2+ entry.

Identification of heterotrimeric GTP-binding proteins in human megakaryoblastic leukemia cell line, MEG-01, and their alteration during cellular differentiation

Various heterotrimeric GTP-binding proteins (G proteins) are possible to have important functions in hematopoietic cells. However, there has been no information regarding their expression in magakaryoblasts and/or megakaryocytes. In the present study, protein contents of seven G protein α subunits (Gs α, Gi2 α, Gi3 α, Gz α, G11 α, Gq α and G12 α) and β subunit in a human megakaryoblastic leukemia cell line, MEG-01, were analyzed by immunoblotting. Immature MEG-01 cells expressed the α subunits of Gs, Gi2, Gi3, Gz, G11 and G12 at protein molecule level. During the 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-induced differentiation process, the contents of Gi2 α and Gi3 α increased, whereas the protein levels of Gz α, Gs α, Gil a and G12 α were observed to hardly change, β Subunit was also observed to be present in immature MEG-01 cells and to increase continuously throughout the differentiation process. For the expression of Gi2 α and β subunits, chronic TPA-treatment was required although Rac2, a low Mr GTP-binding protein, was expressed abundantly by only 30 min-TPA-treatment followed by 3 day-culture.

Soluble c‐kit molecule in serum from healthy individuals and patients with haemopoietic disorders

The proto-oncogene, c-kit, encodes a transmembrane tyrosine kinase receptor (KIT) and plays an important role in haemopoiesis. We have identified a 95 kD soluble form of KIT (S-KIT) in culture supernatant of human megakaryoblastic cell line, CMK. To study the physiological significance of S-KIT, we have established a sensitive sandwich ELISA system. Serum samples from healthy individuals contained detectable amounts of S-KIT. Next, we determined a total of 220 samples from 134 patients with haemopoietic disorders. A considerable number of patients with acute myeloid leukaemia (AML), especially those with more immature phenotypes (M0, M1 or M2) had elevated levels of serum S-KIT. Those levels decreased to the normal range after effective chemotherapy. In chronic myeloid leukaemia, patients with myeloid blastic crisis showed markedly elevated levels of serum S-KIT. In contrast, S-KIT levels decreased in cases with either acute or chronic lymphoid leukaemia. There was a tendency for patients with severe aplastic anaemia to show decreased levels, but it was not significant. In myelodysplastic syndrome, S-KIT levels appeared to vary by subsets, with higher concentration in more advanced forms of the disease. Although the functional role of S-KIT is not yet elucidated, these results suggest that the serum S-KIT levels may reflect the pathological states of various haematological disorders.

Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e).

The role of the lyn product (p53/p56lyn), a membrane-associated protein tyrosine kinase in the signaling pathway used by granulocyte macrophage-CSFR (GM-CSFR) was investigated by using the GM-CSF-dependent human megakaryoblastic leukemia cell line M-07e. M-07e cells express GM-CSFR and are dependent on GM-CSF for survival and proliferation in vitro. Treatment with anti-lyn Abs coimmunoprecipitated, along with lyn product, the beta subunit of GM-CSFR and a phosphoprotein with a molecular mass of 120 kDa (p120) in the lysates of M-07e cells but not in the lysates of human monocyte-derived macrophages (HMDM) or human lymphoid leukemia cells. That the 120-kDa phosphoprotein coimmunoprecipitated by anti-lyn Abs is the beta subunit of GM-CSFR was confirmed in the immunoprecipitates (IP) of M-07e cells with the use of an agarose-conjugated anti-p-tyr mAb. The formation of GM-CSF/GM-CSFR/lyn signaling complexes was verified in an autoradiographic study with anti-lyn IP of M-07e cells that had been bound with 125I-labeled recombinant human (rh)GM-CSF. The p120 protein (beta subunit) was not detected in the IP of M-07e cells with anti-fyn or anti-PI3 Abs. A direct association of Lyn kinase with the beta subunit of GM-CSFR was illustrated with a reversed approach showing the recovery of Lyn protein in anti-beta (CRS1) but not anti-alpha IP of M-07e cells that had been starved for a prolonged period. Finally, the interaction of Lyn kinase with the GM-CSFR complexes was further corroborated using anti-GM-CSF (G133) mAb, which coimmunoprecipitated both the p120 beta subunit and lyn product in the lysates of M-07e cells that had been bound with rhGM-CSF before cell lysis. Removal of rhGM-CSF from culture medium for 10 to 12 h resulted in a marked decrease in lyn-associated kinase activity but not the beta subunit/lyn kinase complex formation. Taken together, our results showed that, in M-07e cells, Lyn protein tyrosine kinase (p53/p56lyn) is stably associated with a constitutively phosphorylated beta subunit of the GM-CSFR in a manner that seems to be independent of lyn kinase activity.

Suppression of the Delayed Rectifier Type of Voltage Gated K+ Outward Current in Megakaryocytes From Patients With Myelogenous Leukemias

In normal human megakaryocytes, we identified a delayed rectifier type of voltage-gated outward K+ current (DRK). In two human megakaryoblastic tumor cell lines (DAMI, CHRF-288-11) and the human erythroleukemia cell line (HEL) the DRK current was not detected. To determine if the absence of the DRK current in the tumor cells is the result of the underlying malignant state, we examined megakaryocytes from myelogenous leukemia patients. In 24 of 29 megakaryocytes from the myelogenous leukemia patients, the DRK current was greatly suppressed, whereas in the remaining 5 megakaryocytes a normal large amplitude DRK current was present. We had the opportunity to reexamine megakaryocytes from a patient with acute promyelocytic leukemia (M3), after chemotherapy. Whereas the DRK current was suppressed before treatment, the current reappeared after chemotherapy. Exposure to the adenylate cyclase activator, forskolin, caused the appearance of a voltage-gated outward current in the megakaryocytes of patients with acute myelogenous leukemia. This finding suggests either that the channels underlying the DRK current are present but somehow suppressed in megakaryocytes from these patients or that forskolin induces a different voltage-gated outward current. We suggest that the megakaryocytes from the myelogenous leukemia patients with suppressed DRK current are abnormal, whereas the others may be normal megakaryocytes. The suppression of the DRK current may be a contributory factor to the dysregulation of thrombopoiesis (Zittoun et al: Semin Hop Paris 44:183, 1968 and Rabellino et al: Blood 63:615, 1984) in myelogenous leukemias.

Suppression of the delayed rectifier type of voltage gated K+ outward current in megakaryocytes from patients with myelogenous leukemias

In normal human megakaryocytes, we identified a delayed rectifier type of voltage-gated outward K+ current (DRK). In two human megakaryoblastic tumor cell lines (DAMI, CHRF-288–11) and the human erythroleukemia cell line (HEL) the DRK current was not detected. To determine if the absence of the DRK current in the tumor cells is the result of the underlying malignant state, we examined megakaryocytes from myelogenous leukemia patients. In 24 of 29 megakaryocytes from the myelogenous leukemia patients, the DRK current was greatly suppressed, whereas in the remaining 5 megakaryocytes a normal large amplitude DRK current was present. We had the opportunity to reexamine megakaryocytes from a patient with acute promyelocytic leukemia (M3), after chemotherapy. Whereas the DRK current was suppressed before treatment, the current reappeared after chemotherapy. Exposure to the adenylate cyclase activator, forskolin, caused the appearance of a voltage-gated outward current in the megakaryocytes of patients with acute myelogenous leukemia. This finding suggests either that the channels underlying the DRK current are present but somehow suppressed in megakaryocytes from these patients or that forskolin induces a different voltage-gated outward current. We suggest that the megakaryocytes from the myelogenous leukemia patients with suppressed DRK current are abnormal, whereas the others may be normal megakaryocytes. The suppression of the DRK current may be a contributory factor to the dysregulation of thrombopoiesis (Zittoun et al: Semin Hop Paris 44:183, 1968 and Rabellino et al: Blood 63:615, 1984) in myelogenous leukemias.

Herbimycin A Inhibits Phorbol Ester-Induced Morphologic Changes, Adhesion, and Megakaryocytic Differentiation of the Leukemia Cell Line, MEG-01

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces rapid changes in the morphology of the human megakaryoblastic leukemia cell line, MEG-01, as well as changes in adhesion and megakaryocytic differentiation. To investigate the signal transduction pathway of these three phenomena, we studied the effect of herbimycin A, an inhibitor of tyrosine kinase (TK) and the effects of calphostin C, a specific inhibitor protein kinase C (PKC) on TPA treated MEG-01 cells. Both herbimycin A and calphostin C inhibited all three TPA-induced phenomena, suggesting that both pathways are required for these phenomena. Herbimycin A but not calphostin C blocked the tyrosine phosphorylation of cellular proteins. Immunohistochemical staining of PKC using an anti-PKC monoclonal antibody showed that herbimycin A did not interfere with the translocation and subsequent down regulation of PKC induced by TPA, suggesting that the TPA-induced effect on PKC (translocation and probably its activation) is not dependent on TK. Induction of c-fos and c-jun expression by TPA was inhibited by both herbimycin A and calphostin C, suggesting that both PKC and TK pathways are necessary for the induction of the TPA-induced transcription factor AP1, which is a known TPA-inducible early immediate gene product. Taken together, our results show that the tyrosine kinase signal transduction system as well as the PKC pathway is indispensable for the TPA-induced phenomena of morphologic change, cell attachment, early immediate gene expression, and lineage-specific phenotypic expression in the MEG-01 cell line.

Cyclic‐AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain incompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPIb expression. Moreover, although these agents used alone did not affect GPIIb/IIIa expression, they markedly reversed phorbol ester-induced GPIIb/IIIa expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPIb and GPIIb/IIIa expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis.

Relation between membrane fluidity and signal transduction in the human megakaryoblastic cell line MEG-01

The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC 14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-O1, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol /phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC,4 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 · 10 −9 cm 2/s at a low C/P decreasing to 2.55 · 10 −9 cm 2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22°C There were only little differences in phosphatidylinositol metabolism, Ca 2+ influx or mobilization and prostaglandin I 2-induced formation of cyclic AMP. At 37°C cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca 2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20–0.31 the influence on signal processing is minor.

Expression of Endothelin and ETB Receptors in the Megakaryoblastic MEG-01 Cell Line

The presence of endothelin (ET) receptors and the nature of the subtype and expression of ET were investigated in the human megakaryoblastic cell line MEG-01. By the RT-PCR procedure, we have shown that both ETA and ETB receptor subtype mRNAs are expressed in the cells. However, binding experiments have shown that the selective ETB receptor antagonist BQ788, but not the selective ETA receptor antagonist BQ123, competes with the specific binding of [125I]ET-1. Using immunocytochemistry, RIA, and RT-PCR Southern blot, we have shown that MEG-01 cells express ET-1. In addition, ET (1-21)-like immunoreactivity was released from the cells into the culture medium, and this release was modulated by thrombin. These data suggest that an ET-1-mediated autocrine loop could occur in the human megakaryoblastic MEG-01 cell line.

Expression of Endothelin and ETB Receptors in the Megakaryoblastic MEG-01 Cell Line

Summary: The presence of endothelin (ET) receptors and the nature of the subtype and expression of ET were investigated in the human megakaryoblastic cell line MEG-01. By the RT-PCR procedure, we have shown that both ETA and ETB receptor subtype mRNAs are expressed in the cells. However, binding experiments have shown that the selective ETB receptor antagonist BQ788, but not the selective ETA receptor antagonist BQ123, competes with the specific binding of [125I]ET-1. Using immunocytochemistry, RIA, and RT-PCR Southern blot, we have shown that MEG-01 cells express ET-1. In addition, ET (1–21)-like immunoreactivity was released from the cells into the culture medium, and this release was modulated by thrombin. These data suggest that an ET-1-mediated autocrine loop could occur in the human megakaryoblastic MEG-01 cell line.

Interleukins and colony stimulating factors in human myeloid leukemia cell lines.

The present review has summarized the expression, production and effects of the human interleukins (IL) 1-11 and myelopoietic colony stimulating factors (CSF) in the established myeloid leukemia cell lines and in cells from patients with acute myeloid leukemia as well as the oncogene expression reported in these myeloid leukemia cell lines. The genetic dissection of leukemic myelopoiesis may provide new perspectives for the control of myeloid leukemias. Based on their expression of phenotypic markers (e.g., surface antigens, cytochemical staining, etc.), myeloid cell lines can be further subdivided into myelogenous, monocytic, erythroid and megakaryoblastic leukemia cell lines. Due to the close relationship of erythroid and megakaryoblastic progenitor cells and to the existence of a probably common precursor cell giving rise to these two different cell lineages, many megakaryoblastic cell lines express erythroid markers (e.g., expression of hemoglobin or glycophorin A) and conversely cell lines with a predominant erythroid profile might display megakaryoblastic features (e.g., platelets peroxidase or glycoproteins CD41, CD42b or CD61). The recent cloning of the specific cytokine: thrombopoietin (TPO) and its receptor generated a strong interest in these particular myeloid cell lines that are discussed in more detail in the present review. Both normal and leukemic megakaryocytopoiesis are stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, GM-CSF/IL-3 fusion protein, IL-6, IL-11 and TPO but inhibited by IL-4, interferon-alpha (IFN-alpha) and IFN-gamma. Human megakaryoblastic leukemia cell lines have common biological features: high expression of the megakaryocytic specific antigen (CD41); high expression of early myeloid antigens (CD34, CD33 and CD13); constitutive expression of IL-6 and platelet-derived growth factor; a complex karyotype picture; expression of c-kit (the stem cell factor receptor); growth-dependency or -stimulation by IL-3 and/or GM-CSF; and in vivo tumorigenicity in mice associated with marked fibrosis. Whereas numerous chemical and biologic agents induce granulocytic and/or monocytic differentiation of myeloid leukemia cell lines, only a few agents including phorbol myristate acetate, vitamin D3, IFN-alpha, IL-6 and thrombin have been reported to induce megakaryocytic differentiation in the megakaryoblastic leukemia cells.

Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.

The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53 +/- 0.06) x 10(-9) cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99 +/- 0.31) x 10(-9) cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D-Phenylalanyl-L-prolyl-L-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.

Growth and Differentiation of the Human Megakaryoblastic Cell Line (ELF-153): A Model for Early Stages of Megakaryocytopoiesis

Abstract ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte- macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Growth and differentiation of the human megakaryoblastic cell line (ELF- 153): a model for early stages of megakaryocytopoiesis

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Interleukin-3 and granulocyte-macrophage colony-stimulating factor induce activation of the MAPKAP kinase 2 resulting in in vitro serine phosphorylation of the small heat shock protein (Hsp 27)

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have previously been reported to induce rapid phosphorylation of the mitogen-activated protein (MAP) kinase. However, little is known about signaling events initiated by both hematopoietins that occur downstream of the MAP kinase. MAP kinase has been shown to phosphorylate the AP-1 transcription factor and also to activate two kinases designated insulin-stimulated protein kinase-1 and MAP kinase- activated protein (MAP-KAP) kinase 2. We show here that IL-3 and GM-CSF induce MAPKAP kinase 2 activity in the human megakaryoblastic leukemia cell line MO7 and phosphorylate the human small heat shock protein Hsp 27 on serine residues in vitro. GM-CSF also induced Hsp 27 phosphorylation in neutrophils in a range similar to that observed in MO7 cells, suggesting that MAPKAP kinase 2-mediated Hsp 27 activation occurs independently of proliferation. Hsp 27 phosphorylation was dose- dependent, occurred as early as 5 minutes after factor exposure, and was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A. Furthermore, the protein phosphatase A2 abolished IL-3- and GM-CSF-induced serine phosphorylation of Hsp 27. Taken together, our findings indicate that tyrosine phosphorylation of MAP kinase is a prerequisite for serine phosphorylation of Hsp 27, which is mediated by MAPKAP kinase 2. Hsp 27 has shown activation-dependent translocation from the cytosolic to the nuclear region and has been linked to the cellular stress response. However, its precise function is largely unknown. Our data identify Hsp 27 as a target of the IL-3/GM-CSF stimulation pathway that involves MAP kinase and MAPKAP kinase 2. In addition, our results indicate that Hsp 27 may be target of phosphorylation events not only in the stress response but also in unstressed cells responding to cytokine stimulation.

Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor Induce Activation of the MAPKAP Kinase 2 Resulting in In Vitro Serine Phosphorylation of the Small Heat Shock Protein (Hsp 27)

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have previously been reported to induce rapid phosphorylation of the mitogen-activated protein (MAP) kinase. However, little is known about signaling events initiated by both hematopoietins that occur downstream of the MAP kinase. MAP kinase has been shown to phosphorylate the AP-1 transcription factor and also to activate two kinases designated insulin-stimulated protein kinase-1 and MAP kinase-activated protein (MAP-KAP) kinase 2. We show here that IL-3 and GM-CSF induce MAPKAP kinase 2 activity in the human megakaryoblastic leukemia cell line MO7 and phosphorylate the human small heat shock protein Hsp 27 on serine residues in vitro. GM-CSF also induced Hsp 27 phosphorylation in neutrophils in a range similar to that observed in MO7 cells, suggesting that MAPKAP kinase 2-mediated Hsp 27 activation occurs independently of proliferation. Hsp 27 phosphorylation was dose-dependent, occurred as early as 5 minutes after factor exposure, and was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A. Furthermore, the protein phosphatase A2 abolished IL-3- and GM-CSF-induced serine phosphorylation of Hsp 27. Taken together, our findings indicate that tyrosine phosphorylation of MAP kinase is a prerequisite for serine phosphorylation of Hsp 27, which is mediated by MAPKAP kinase 2. Hsp 27 has shown activation-dependent translocation from the cytosolic to the nuclear region and has been linked to the cellular stress response. However, its precise function is largely unknown. Our data identify Hsp 27 as a target of the IL-3/GM-CSF stimulation pathway that involves MAP kinase and MAPKAP kinase 2. In addition, our results indicate that Hsp 27 may be target of phosphorylation events not only in the stress response but also in unstressed cells responding to cytokine stimulation.

Detection of Plasminogen Activator Inhibitor-1 (PAI-1) mRNA in Human Megakaryocytes by In Situ Hybridization

Platelets have been described to contain a large proportion of the circulating plasminogen activator inhibitor type 1 (PAI-1) which is released on platelet activation. This protein could be taken up by platelets from the plasma or synthesized by megakaryocytes (MKs). Recently, PAI-1 mRNA has been detected in a human megakaryoblastic leukemia cell line (MEG-01) by the polymerase chain reaction (PCR). However, a direct-demonstration of its presence in normal human MKs is lacking. In order to prove directly the megakaryocytic origin of platelet PAI-1, the MEG-01 cell line, human bone marrow enriched in MKs, and bone marrow smears from allogeneic bone marrow transplantation donors were investigated for the presence of PAI-1 mRNA using in situ hybridization (ISH). Specimens of bone marrow were first stained with May-Grünwald Giemsa (MGG) for cell identification according to their morphology. Subsequently, the same slides were used for ISH. PAI-1 mRNA was clearly demonstrated in the MEG-01 cell line and in MKs, and its presence correlated with the detection of PAI-1 antigen by immunocytochemistry. PAI-1 mRNA was also detected in morphologically characterized mature granulocytes of marrow samples.