A microtubule associated protein (hNUDC) binds to the extracellular domain of thrombopoietin receptor (Mpl)

Human NUDC (hNUDC) was initially characterized as a nuclear migration protein based on the similarity of its C-terminus to that of fungal NUDC from Aspergillus nidulans. However, hNUDC is a 331 amino acid protein whereas fungal NUDC is 198 amino acids in length. The extra N-terminal portion of hNUDC has no known function or homology to other proteins. In this study, we report the binding of hNUDC to the extracellular domain of the thrombopoietin receptor (Mpl) as detected by the yeast two-hybrid system, GST pull-down, and co-immunoprecipitation. Our deletion analysis demonstrated that amino acids between positions 100 and 238 as the critical domain mediating the hNUDC and Mpl interactions as detected by the two-hybrid system and GST pull-down assay. Immunofluorescence staining of human megakaryocyte cells indicated that hNUDC and Mpl colocalized at all stages of megakaryocyte development. Substantial colocalization of hNUDC with microtubules was also detected around nuclei and elongated microtubular structures, especially in proplatelet extensions.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.     

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102–251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu²²⁸ and Leu²³⁰, as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp²³⁵ and Leu²³⁹ had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.     

The effect of cytokines on the ploidy of megakaryocytes

The effects of recombinant cytokines on the ploidy of human megakaryocytes derived from megakaryocyte progenitors were studied using serum-free agar cultures. Nonadherent and T cell-depleted marrow cells were cultured for 14 days. Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet IIb/IIIa. The ploidy of individual megakaryocytes in colonies was determined by microfluorometry with DAPI (4',6-diamidino-2-phenylindole) staining. Recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner. However, both rhIL-3 and rhGM-CSF had no definite ability to increase the ploidy values. Recombinant human erythropoietin (rhEpo) or recombinant human macrophage colony-stimulating factor (rhM-CSF) by itself did not stimulate the growth of megakaryocyte progenitors. rhEpo or rhM-CSF, however, stimulated increases in the number, size and ploidy values of megakaryocyte colonies in the presence of rhIL-3 or rhGM-CSF. Recombinant human interleukin 6 (rhIL-6) showed no capacity to generate or enhance megakaryocyte colony formation when added to the culture alone or in combination with rhIL-3. rhIL-6, however, increased the ploidy values in colonies when added with rhIL-3. These results show that rhEpo, rhM-CSF and rhIL-6 affect endomitosis and that two factors are required for megakaryocyte development.     

Immature megakaryocytes in the mouse: In vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes

An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr^-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEHl-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division.     

Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro

The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both.     

Purification and properties of a megakaryocyte stimulatory factor present both in the serum-free conditioned medium of human embryonic kidney cells and in thrombocytopenic plasma

Megakaryocyte stimulatory factor (MSF) has been purified to homogeneity (7.5 X 10(5)-fold) from serum-free conditioned medium obtained from cultured human embryonic kidney cells and to near homogeneity (1.44 X 10(7)-fold) from thrombocytopenic rabbit plasma. MSF activity from either source was assayed by its ability to enhance the rate of synthesis of platelet factor 4-like proteins in a rat promegakaryoblast cell line. The 125I-labeled factor prepared from human embryonic kidney cell conditioned medium is homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing in the presence of 9.2 M urea. MSF obtained from the above source is an acidic protein (pI = 5.1) with an Mr = 15,000 which stimulates platelet factor 4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF was also purified from thrombocytopenic rabbit plasma by a nearly identical isolation procedure, and 125I-labeled factor prepared from this source also possessed an Mr = 15,000. MSF exhibited no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.     

Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators

We have previously reported that heparin is capable of stimulating in vitro and in vivo megakaryocytopoiesis in mice and has a thrombopoietic effect when given in chronic immune thrombocytopenic purpura and that heparin and several other glycosaminoglycans (GAGs) promote the growth of human megakaryoblastic cell lines in the presence of serum. We show here that GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), dermantan sulfate (DS), and hyaluronic acid (HA), also stimulate in vitro growth of murine megakaryocyte progenitors and augment the diameter of individual megakaryocytes in the presence of serum. However, in a serum-free agar system, the GAGs alone had no effect on megakaryocyte colony formation, suggesting that GAGs cooperate with some serum factor(s) to exert their activity. We also show that heparin significantly potentiates the megakaryocytopoietic activity of C-Mpl ligand and interleukin (IL)-6 but not IL3, GM-CSF, SCF, and Epo. In addition, the GAGs significantly neutralize the inhibitory action of platelet factor 4 (PF4) and transforming growth factor β1 (TGFβ1) on megakaryocyte colony growth. These results demonstrate a stimulating activity of GAGs on megakaryocytopoiesis by modifying the activity of several growth-regulating factors. © 1996 Wiley-Liss, Inc.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).     

The effect of recombinant erythropoietin on murine megakaryocyte colony formation

We investigated the effect of a recombinant human erythropoietin preparation (recombinant Epo) on murine megakaryocyte (MK) colony formation in serum-free and serum-containing culture systems, in order to study the relationship between Epo and megakaryopoiesis. Pokeweed mitogen spleen-conditioned medium (PWM-SCM), a standard source of MK colony stimulator, dose-dependently stimulated MK colony formation in the two culture systems. The plating efficiency of serum-free cultures was almost equal to that of cultures containing serum. Recombinant Epo also dose dependently stimulated MK colony formation in serum-containing cultures. However, in serum-free cultures recombinant Epo alone did not stimulate the growth of MK colonies; with the addition of fetal calf serum (FCS) to the serum-free cultures, recombinant Epo induced the growth of MK colonies. Furthermore, recombinant Epo enhanced MK colony formation through the stimulation of PWM-SCM or murine interleukin 3 (IL-3) in serum-free cultures. Our data show that Epo can act as a stimulator of megakaryopoiesis in collaboration with a factor in serum, or with an MK colony stimulator such as IL-3.     

Recombinant gibbon interleukin-3 stimulates megakaryocyte colony growth in vitro from human peripheral blood progenitor cells

Gibbon interleukin-3 (rIL-3) has recently been cloned and found to have a high degree of homology with the human IL-3 molecule. In this investigation, we evaluated the effects of gibbon rIL-3 on normal human peripheral blood megakaryocyte progenitor cell growth in vitro. Gibbon rIL-3 exhibited substantial megakaryocyte colony stimulatory activity (Meg-CSA), supporting peak colony numbers at a concentration of 1 U/ml. Megakaryocyte colony growth induced by rIL-3 reached 58% of the maximum achieved with the active, Meg-CSA-containing protein fraction of aplastic canine serum. Increasing gibbon rIL-3 concentrations also stimulated a 4-5-fold increase in megakaryocyte colony size and resulted in a decrease in geometric mean megakaryocyte ploidy. Ploidy values fell from 8.5N +/- 1.4 (+/- SEM) at an rIL-3 concentration of 0.1 U/ml to a minimum of 2.9N +/- 0.3 at 10 U/ml. In the presence of rIL-3 at 1.0 U/ml, megakaryocyte colony growth was linear with cell plating density and the regression line passed approximately through the origin. The effects of rIL-3 on megakaryocyte colony growth were independent of the presence of T-lymphocytes in the cultures. Cross-species evaluation of murine and gibbon IL-3 indicated that its bioactivity is species restricted. Murine IL-3 did not support colony growth from human megakaryocyte progenitors and gibbon rIL-3 showed no activity in stimulating acetylcholinesterase production by murine bone marrow cells. Gibbon rIL-3 is a potent stimulator of the early events of human megakaryocyte progenitor cell development promoting predominantly mitosis and early megakaryocytic differentiation.     

Human dermal fibroblasts express multiple bFGF and aFGF proteins

Summary We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

Essential thrombocythemia: impaired regulation of megakaryocyte progenitors

In this paper, the in vitro growth of bone marrow early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) progenitors was evaluated in 18 essential thrombocythemia (ET) patients and 22 normal control subjects. BFU-meg clonality was demonstrated both in normal and ET bone marrows, cultivating these primitive progenitors at limiting dilutions in plasma clot assay: 1 to 7 BFU-meg/2.5 x 10(4) mononuclear non-adherent cells were observed, with a strong correlation in ET [r = 0.955 stimulated by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) plus recombinant human interleukin (rhIL) 3], as well as in normal controls (r = 0.969). In order to clearly elucidate the in vitro response of ET megakaryocyte (meg) progenitors to recombinant growth factors, the interference of accessory cells (i.e., monocytes, T lymphocytes, and natural killer cells) and human serum were avoided by performing experiments on CD34+ cells in a serum-free fibrin clot assay. The number of both early and late meg progenitors in ET was significantly increased in response to rhIL-3, rhIL-3 plus rhIL-6, and rhIL-3 plus rhGM-CSF, but not in response to rhGM-CSF alone. Furthermore, both meg progenitors were investigated for their response to rh transfer growth factor (TGF)-beta 1, tested at concentrations from 0.01 to 10 ng/ml. rhTGF-beta 1 was able to inhibit CFU-meg and BFU-meg in a dose-response manner normal, whereas ET CFU-meg appeared less sensitive to the lower doses investigated (p less than 0.05) and ET BFU-meg were slightly reduced in number only at the higher concentrations of rhTGF-beta 1 (p less than 0.01). Our data suggest that the increased thrombopoiesis in ET may depend on an increased sensitivity of meg progenitors to some of the physiological growth factors and to a disrupted sensitivity to at least one negative regulator of megakaryocytopoiesis. Since these abnormalities involve both meg progenitors, this can be considered a demonstration that the neoplastic event hits the most primitive hemopoietic progenitors.