Dami Cell Line Research Articles

Differential quantitative zinc-induced expression of human metallothionein isogenes in haematopoietic precursor cell lines

The expression pattern of functional members of the metallothionein (MT) gene family was studied in the haematopoietic precursor cell lines, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 with 10% (v/v) foetal calf serum, with or without different zinc supplements. Expression of MT isogenes was analysed by quantitative real-time PCR (RT-PCR) using mRNA extracted from cultured cells. The more mature K562, DAMI, and MEG-01 cell lines exhibited transcription of all MT isogenes, except MT-3 and MT-4. Relative quantitative expression of MT isogenes in the mature cell lines such as K562, DAMI, and MEG-01 was higher than in the immature ELF-153 cell line. Immunohistochemical staining (IHC) reveals an increased MT protein biosynthesis in more mature cell lines such as K562, DAMI and MEG-01 greater than in the immature ELF-153 cell line. Real-time PCR and immunohistochemical staining for investigating the effect of phorbol ester and hemin (haematopoietic differentiation stimuli) on expression of MT isogenes in K562 cells reveals that phorbol ester induces increased MT transcription and biosynthesis. Therefore, to our knowledge, the role of MT in differentiation in human haematopoietic precursor cell lines is here reported for the first time.

Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.

Probing platelet factor 4 α‐granule targeting

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets

AbstractThrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme that after activation down-regulates fibrinolysis. Platelets are known to contain antifibrinolytic factors that are secreted during platelet activation. Therefore, the presence of TAFI in platelets was analyzed. TAFI was identified in platelets in a concentration of about 50 ng/1 × 109 platelets and was secreted on platelet activation. Thrombin-mediated activation of platelet-derived TAFI resembled that of plasma-derived TAFI with respect to stimulation by thrombomodulin and spontaneous loss of activity at 37°C. The different glycosylation of platelet-derived TAFI compared with plasma-derived TAFI suggests that platelet-derived TAFI is synthesized in the megakaryocyte. This suggestion was substantiated by the detection of mRNA in the megakaryocytic cell lines DAMI and CHRF, representing the intermediate and late stages of megakaryocyte development. These results establish the presence of TAFI in platelets and suggest a role for platelet-derived TAFI in the protection of the clot against fibrinolysis.

Expression of transient receptor potential mRNA isoforms and Ca 2+ influx in differentiating human stem cells and platelets

Store-regulated Ca 2+ entry (SOCE) is an important mechanism of elevating cytosolic [Ca 2+] i in platelets, though the Ca 2+ influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34 +) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61 +/CD42b low) and mature (CD61 +/CD42b high) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14 +/CD61 −/CD42b −) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca 2+ after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III.

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Megakaryocyte maturation is associated with expression of the cxc chemokine connective tissue activating peptide ctap III

Abstract Connective tissue activating peptide III (CTAP III) is a CXC chemokine, derived from platelet basic protein (PBP), which has been identified in platelets, activated macrophages, neutrophils and in T lymphocytes. CTAP III can support stem cell derived hematopoiesis yet clearly inhibits the proliferation of committed megakaryocytes (MK) progenitors. This investigation was aimed at characterizing CTAP III in human MK and determining it association with proliferation and differentiation. We report high expression of CTAP III in mature MK from normal human bone marrow (BM) and from BM of patients with immune thrombocytopenic purpura (ITP) or chronic myeloid leukemia (CML) and in ex-vivo expanded MK. Immunostaining with anti-CTAPIII antibodies demonstrated its prominent presence in platelet producing like zones within the cytoplasm and intense staining around the periphery of the large mature MK. In logarithmically growing cell lines DAMI, CHRF-288 or MEG01 megakaryoblasts only 15% of the cells, which were primarily large cells (>30 μM), expressed CTAPIII. The addition of phorbol ester PMA, which causes proliferation arrest and differentiation induction, stimulated high levels of CTAPIII expression after 24 hours in more than 75% of the cells, being expressed in both the small cells and the large cells. RT-PCR reveled upregulation of CTAPIII mRNA after one hour of exposure to PMA which was sustained for 24 hours. While the biological function of CTAPIII in MK is not known, these studies demonstrate that molecular upregulation of CTAP III in MK is associated with maturation and like other chemokines may be involved in proliferation arrest and cellular interactions with extracellular matrix.

Induction and enhancement of normal human megakaryocyte polyploidization are concomitant with perturbation in the actin metabolism.

Megakaryocyte polyploidization results from the lack of cytoplasmic separation while the nucleus keeps dividing. To investigate the role of actin in the megakaryocyte polyploidization, three human cell lines with megakaryocytic properties (DAMI, HEL and K562) were incubated in the presence of cytochalasin B, an inhibitor of actin polymerization. These data were then compared with normal megakaryocytes. Compared with control conditions, cells cultured in the presence of cytochalasin B revealed an augmentation of cell size and ploidy and an arrest of cell proliferation. The expression of platelet membrane glycoproteins Ib, IIb/IIIa, IIIa and thrombospondin and transferrin receptors was augmented after treatment with cytochalasin B. Physiologically, the role of actin in inducing polyploidization could be related to an imbalance between G- and F-actins. To test this hypothesis, we measured G-, F- and total actin in cytochalasin B-treated cells. Actin was found to be increased significantly in cytochalasin B-treated DAMI and HEL cell lines. In contrast, the G/F-actin ratio was not affected by cytochalasin B. To confirm these actin changes in physiological megakaryocytopoiesis, G- and F-actin contents were then estimated in normal megakaryocytes. The G- and F-actin contents of megakaryocytes from eight normal patients exponentially decreased from 2 to 128n, whereas the total actin content per cell kept increasing. The G/F ratio was unaffected. Polyploidization of human megakaryocytes results from either a diminution of actin synthesis or an increased actin turnover, which in turn possibly abrogates the formation of the actin cleavage furrow in telophasis.

Augmentation of the number of nucleolar organizer regions in human megakaryocyte cell lines after induction of polyploidization by a microtubule inhibitor.

Megakaryocyte polyploidization is an advantageous and regulated mechanism that leads to an increase in platelet production. In megakaryocytic cell lines, polyploidization can be obtained by using colchicine, an inhibitor of the tubulin spindle. The nucleolar organizer regions (AgNORs) are parts of nucleolar DNA transcribed into ribosomal RNA and are detected by the silver-staining technique. Their number is proportional to protein synthesis. To estimate protein synthesis in polyploid megakaryocytes, AgNORs are measured in three cell lines with megakaryocyte properties (DAMI, HEL and K562) after a 4-day culture in the presence or absence of colchicine. The mean number of AgNORs per cell was 16+/-4 (mean+/-SEM), 24+/-3 and 14+/-3 for DAMI, HEL and K-562 cell lines respectively. The addition of colchicine (10 ng mL[-1]) significantly increased the number of AgNORs per cell (DAMI 556%, HEL 338% and K-562 300% of controls, P < 0.05 using the t-test). Moreover, the number of nucleoles per cell after the addition of colchicine was augmented significantly (DAMI 246%; HEL 237% and K-562 148% of controls, P < 0.05 using the t-test). The total protein content estimated by Bradford's method increased significantly to 226%, 215% and 304% of controls in DAMI, HEL and K562 respectively (P < 005 using the t-test). After treatment with colchicine, the endomitotic index (EI) [mean of (log2 DNA content expressed in N)-1] measured by flow cytometry (and reflecting ploidy) increased to 234%, 255% and 301%, respectively, in DAMI, HEL and K-562 cell lines (P < 0.05 using the t-test). Concomitantly, the number of AgNORs per unit of DNA increased in the DAMI and HEL cell lines (P < 0.05 using the t-test) from 48+/-8 and 39+/-5, respectively, to 79+/-11 and 61+/-10. In contrast, the number of nucleoles and the total protein content per endomitotic index were not affected by colchicine (P > 0.05 using the t-test), but the number of nucleoles per endomitotic index of DAMI cells was affected. The increase in the number of the NORs induced by an agent known to stimulate polyploidization of megakaryocytic cell lines suggests that polyploidization occurs by a proportional increase in protein synthesis per DNA unit.

Early Contamination of the Dami Cell Line by HEL

Early Contamination of the Dami Cell Line by HEL

Antiproliferative effects of imidazo[1,2-β]pyrazine derivatives on the dami cell line

Since cyclic 3',5'-adenosine monophosphate (cAMP) is involved in cell proliferation and as previous data showed that imidazo[1,2-α]pyrazine derivatives (PAB12, PAB30, PAB40, SCA40, SCA41, and SCA44) inhibited cAMP breakdown by a phosphodiesterase (PDE)-inhibitory effect, the aim of the present study was to investigate the effects of these derivatives on proliferation of the Dami cell line in relation with their actions on cAMP content and on PDE isoenzymes isolated from Dami cells. SCA41 and SCA44 inhibited cell growth in a dose-dependent manner, while SCA40 and PAB40 induced a weak inhibition. Growth inhibitions were 40%, 91%, and 60% for SCA41, SCA44 (at 100 μM), and IBMX (at 1000 μM), respectively, and could not be related to their effects on cAMP levels. In addition, although all compounds potentiated cAMP formation by prostaglandin E1 (PGE1), no potentiations were observed when the antiproliferative effects of SCA41 and SCA44 were considered. Investigation of derivatives on PDE isoenzymes III, IV, and V indicated non-selective PDE inhibitory effects for SCA41 and SCA44, while SCA40 elicited preferences for type III, and PAB30 and PAB40 preferences for type IV isoenzymes. These effects could not totally explain the antiproliferative activity of the derivatives. The activation of P2 purinoceptors by imidazo[l,2-α]pyrazine did not lead to their antiproliferative effects. Thus, the mechanism of the antiproliferative effects of the compounds remains to be determined. It does, however, depend on the chemical substitutions of the imidazo[l,2-α]pyrazine skeleton and in particular on the 2-carbonitrile presence and the length of the 8-aminoaliphatic group.

Modulation of the Megakaryoblastic Dami Cell Line Differentiation by Phosphodiesterase Inhibitors and Imidazo[1,2‐a]pyrazine Derivatives

Phosphodiesterase inhibitors have been shown to modulate cell differentiation. We have previously shown that a series of imidazo[1,2-a]pyrazine derivatives displayed inhibitory effects on phosphodiesterase isoenzymes types III. IV and V isolated from Dami cells and on Dami cell growth. In the present study we have investigated the effect of these derivatives on the expression of two differentiation markers, glycoproteins Ib and IIb/IIIa of the human megakaryoblastic leukaemic Dami cell line in comparison to those elicited by 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors of types 1 (8-methoxymetyl-1-methyl-3-(2-methylpropyl) xanthine), III (Milrinone), IV (RO-201724) and V (Zaprinast). Imidazo[1,2-a]pyrazine derivatives, 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors, except 8-methoxymethyl-1-methyl-3-(2-methylpropyl) xanthine, decreased glycoprotein Ib expression. SCA40, SCA41, SCA44 and 3-isobutyl-1-methylxanthine-but not the other compounds affected the expression of glycoprotein IIb/IIIa in a positive manner. The effects of imidazo[1,2-a]pyrazine derivatives on glycoprotein expression appeared to be related to their phosphodiesterase inhibitory potency.

Dissociation between phosphodiesterase inhibition and antiproliferative effects of phosphodiesterase inhibitors on the dami cell line

Phosphodiesterase (PDE) inhibitors were shown to inhibit proliferation of various cell types. The present investigation was designed to study the activity of selective PDE inhibitors (8-MeoMIX, milrinone, trequinsin, rolipram, RO-201724, zaprinast, and MY-5445) on the proliferation of the Dami cell line in relation to their effects on cAMP levels and PDE isoenzymes isolated from Dami cells. All compounds, except 8-MeoMIX, elicited antiproliferative effects. Trequinsin, RO-201724, and MY-5445 (100 μM) were found to inhibit cell growth up to 60%, 83%, and 85%, respectively; milrinone, rolipram and zaprinast elicited only weak effects (19–21% at 100 μM). Their growth-inhibitory effects could not be related to their effects on cAMP levels. In addition, although PDE type III and IV inhibitors potentiated cAMP formation due to adenylycyclase activation, no potentiation could be observed when considering their antiproliferative effect. Separation and characterization of PDE of Dami cells revealed the existence of types III, IV, and V isoenzymes. The PDE inhibition found for the PDE inhibitors could not explain their antiproliferative effects. The lack of correlation with cAMP concentrations or PDE inhibition and the high concentrations needed to elicit antiproliferative effects suggest the implication of other parameters, such as cytotoxicity or lipophilicity, or other targets in addition to PDE for the PDE inhibitors tested. Lipophilicity did not seem to be of importance in antiproliferative effects. In contrast, cytotoxic effects, in particular those of trequinsin and MY-5445, could partially explain their negative action on cell growth.

Apoptotic effects of imidazo[1,2- a]pyrazine derivatives in the human Dami cell line

cAMP-elevating agents like phosphodiesterase inhibitors and purines have been shown to induce apoptosis. In the present work we have studied the effects of imidazo[1,2- a]pyrazine derivatives with a purine-like structure: PAB13 (6-bromo-8-(methylamino)imidazo[1,2- a]pyrazine), PAB15 (6-bromo-8-(ethylamino)imidazo[1,2- a]pyrazine), PAB23 (3-bromo-8-(methylamino)imidazo[1,2- a]pyrazine) on the growth of the Dami cell line in comparison to that of adenosine. The growth effect of PAB13, PAB15 and PAB23 was investigated in relation to their phosphodiesterase-inhibitory action and their activity on purinoceptors. Inhibition in cell growth was up to 71.0%, 76.3% and 89.7% for PAB23, PAB13 and PAB15, respectively and 100% for adenosine. Cell viability was affected in a concentration-dependent manner by PAB13, PAB15 and adenosine, with a correlation between growth inhibition and cytotoxicity. These effects of imidazo[1,2- a]pyrazine derivatives were found to be unrelated to an action on purinoceptors, but rather appear quantitatively linked to their ability in inducing apoptosis through their cAMP-increasing and phosphodiesterase-inhibitory potency.

Activated human platelets express beta2 integrin.

The expression of molecules of the beta2 integrin family (CD11a, CD11b , CD11c, and CD18) was explored on 2 human megakaryocytic cell lines and on platelets from different donors by immunofluorescence and flow cytometry using a large panel of mAb. CD11a, CD11b, CD11c and CD18 were detected on the megakaryocytic cell lines DAMI and HEL. A low and variable expression of CD11a, CD11b and CD18 determinants was also detected on resting platelets; this expression was markedly increased when platelets were activated by thrombin. Expression of CD18 was closely correlated to that of CD11a or CD11b when comparing the fluorescence intensity observed in different experiments. In presence of Ca++, platelets did bind to a RAJI cell line which exhibits a high expression level of CD54. This binding was increased when platelets were activated by thrombin and was decreased by an anti CD11a, CD18 and anti CD54 mAb. This study indicates that human platelets express molecules of the beta2 integrin family, when activated, which allows them to bind to CD54 bearing cells.

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.

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.

Characterization of specific functional receptors for HuIFN‐α on a human megakaryocytic cell line (Dami): expression related to differentiation

Interferon-alpha (IFN-alpha) treatment has been shown to be highly effective in inhibiting human megakaryocytopoiesis and controlling thrombocytosis in patients with myeloproliferative disorders. These observations suggest that IFN-alpha might play some role in the biological feature of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN-alpha on human megakaryocytic cells, i.e. the Dami cell line, and to study the regulation of their expression. Our study demonstrates that [125I]-recombinant human IFN-alpha ([125I]rHu-IFN-alpha) binds to high-affinity specific receptor on these cells. Scatchard analysis of binding data indicates the presence of homogeneous binding sites estimated in the range of 3000-5000, with an apparent equilibrium dissociation constant, Kd, of 1-2 x 10(-9) M. Also, [125I]rHuIFN-alpha binding capacity decreased in Dami cells incubated with unlabelled rHuIFN-alpha. This down-regulation which was dose-dependent appeared to result from a reduction of IFN-alpha cell surface receptors and was observed at doses that elicited antiproliferative effects in Dami cells. Cross-linking of [125I]rHuIFN-alpha to Dami membrane proteins using a bifunctional reagent yielded to a radioactive complex of approximately 150,000 kD on SDS-PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cells as evaluated by surface marker and ploidy analysis, a 3-fold increase of the number of specific membrane receptors for IFN-alpha was observed, without any modification of either the affinity or the M(r) value of the cross-linked complex. Such an increase appeared to be restricted to IFN-alpha receptors; actually it was not observed in [125I]IFN-gamma binding experiments. Transcript analysis indicated that down-regulation and increased expression of the IFN-alpha receptor after PMA treatment are post-transcriptional events.

Expression of the lactotransferrin receptor during the differentiation process of the megakaryocyte Dami cell line

In order to determine whether the human lactotransferrin receptor recently described on platelets was also present on hematopoietic precursors, we investigated its presence and characteristics on the megakaryocytic Dami cell line. The reversible binding of human 5-(([2-(carbo(hydrazino)methyl]thio)acetyl)aminofluorescein-labele d lactotransferrin showed that such a receptor was only present on the subpopulation of the largest cells. The increase in numbers of large cells during culture was paralleled by a concurrent increase in lactotransferrin receptor positive cells. Scatchard analysis of the binding of [125I]-labeled lactotransferrin showed that a single affinity class of binding site was present (Kd = 446 +/- 40 nM) and that there were 52 +/- 3 x 10(5) sites per cell. The mouse monoclonal antibody DP5B3G10, specific for the human lactotransferrin receptor, allowed its characterization as a 105 kDa protein on Western blots. The same monoclonal antibody was used to separate the small and large cell subpopulations of Dami cells by panning. Separate culture of the small cells showed that the receptor appeared prior to and independent from endomitosis. In contrast, GPIb was expressed only by large megakaryocytes. The use of conditioned medium from cultures of whole Dami cell populations indicated that a soluble factor is involved in differentiation, but not in the appearance of the lactotransferrin receptor.