Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1–163 residues of hTPO (hTPO163) including the N-terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332). In order to clarify the structural and physicochemical properties of hTPO163, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and −16.7% for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154–332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.

Rabbit anti-serum against a myeloid-cell-specific peroxidase (Mpo) of Xenopus laevis was generated to identify myeloid cells in adult and larval animals. Smears of blood samples from adult hematopoietic organs were co-stained with Mpo and with XL-2, a mouse monoclonal antibody against a leukocyte common antigen. Lymphocytes found in the thymus and spleen were XL-2(+)Mpo(-) and granulocytes found in peripheral blood cells and the spleen were XL-2(+)Mpo(+), indicating that double-staining with these two antibodies allowed classification of the leukocyte lineages. Immunohistochemical analysis of larval organs showed that XL-2(+)Mpo(-) cells were scattered throughout the liver, whereas XL-2(+)Mpo(+) cells were present mainly in the cortex region. Interestingly, a cluster of XL-2(+)Mpo(+) cells was found in the region of the larval mesonephric rudiment. The ratio of XL-2(+)Mpo(+) cells to XL-2(+) cells in the mesonephric region was approximately 80%, which was much higher than that found in other hematopoietic organs. In order to elucidate the embryonic origin of the myeloid cells in the tadpole mesonephros, grafting experiments between X. laevis and X. borealis embryos were performed to trace the X. borealis cells as donor cells. Among the embryonic tissues examined, the tailbud tissue at the early neurula stage contributed greatly to the myeloid cluster in the mesonephric region at stage 48. Therefore, at least four independent origins of the myeloid cell population can be traced in the Xenopus embryo.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 angstrom using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (Cp) measured by ITC (-1.52 +/- 0.05 kJmol(-1)K(-1)) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 approximate to 0.25 kJmol(-1)K(-1)) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 angstrom using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (Cp) measured by ITC (-1.52 +/- 0.05 kJmol(-1)K(-1)) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 approximate to 0.25 kJmol(-1)K(-1)) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. (C) 2015 Elsevier Inc. All rights reserved.

Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre-(NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. beta 2-globin mRNA expression peaked at day 7 after the start of culture. Larval beta 2-globin was highly expressed in NF56-derived cells, while adult beta 2-globin was detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of epor and gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

<p>The acquisition of fundamental information by the use of recent technologies, including omics-based molecular analyses and total RNA sequencing, has opened the door to further advances in physiological studies on new animal models. Currently, we are endeavoring to develop a comparative hematology protocol in order to build a discovery platform. All vertebrates, with the exception of a few species, have universally peripheral erythrocytes and hemoglobin, suggesting erythropoiesis to be an evolutionary index.</p>

We investigated the effect of the solution flow on crystalline morphology in off-axis 4H-SiC solution growth. In particular, we focused on the relationship between the Si solution flow and step flow directions. In step parallel flow in which the solution drifted transversely to the step flow direction of the off-axis substrate, it was possible to attain a better surface morphology than when the solution drifted in the other direction. Furthermore, the surface morphology was found to be improved as the flow velocity increased. These improvements in morphological stability are presumed to be caused by the aligning of the solute concentration fluctuation along the steps.

Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre-(NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. beta 2-globin mRNA expression peaked at day 7 after the start of culture. Larval beta 2-globin was highly expressed in NF56-derived cells, while adult beta 2-globin was detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of epor and gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated thrombocytes instead of platelets, the features of nucleated thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO (xlTPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xlTPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X. laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xlTPO produced mature, spindle-shaped thrombocytes that were activated by thrombin, thereby altering their morphology. XlTPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xlTPO enhanced viability of peripheral thrombocytes, indicating the xlTPO-Mpl pathway stimulates anti-apoptotic in peripheral thrombocytes. The development of thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200).

Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1-3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12+ thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12+ thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.

Prostate-specific membrane antigen (PSMA) is an attractive target for treatment of prostate cancer. Using the PSMA-recognizing mouse monoclonal antibody 2C9 obtained in our previous study, the biological activities of PSMA antibody were evaluated. Mouse-human chimeric IgG1 of 2C9 (KM2777) showed antibody-dependent cellular cytotoxicity activity against PSMA-expressing prostate cancer cells in the presence of human peripheral blood mononuclear cells (PBMCs). To increase lymphocyte-mediated cytotoxicity of KM2777, C-terminus interleukin-2 (IL-2)-fused KM2777 (KM2812) was constructed. KM2812 retained binding activity to PSMA and exhibited growth-stimulating activity equivalent to IL-2 on the IL-2-dependent T-cell line CTLL-2. Moreover, KM2812 exhibited enhanced cytotoxic activity against PSMA-expressing prostate cancer cells in the presence of PBMCs compared with KM2777. In a xenograft tumor model using PSMA-expressing prostate cancer cells, KM2812 exhibited marked antitumor activity, accompanied by complete regression of tumor in some of the KM2812-treated mice. These results suggest that KM2812 has a therapeutic potential for prostate cancer by stimulating lymphocyte-mediated antitumor cytotoxicity.

Tyrosine kinase inhibitors (TKIs) are highly effective in the treatment of chronic myelogenous leukemia (CML), but there have been a few adverse event reports describing gastrointestinal bleeding. We clinically analyzed two patients who developed intestinal bleeding during the administration of TKIs for CML. Platelet counts of both patients were normal. The patients showed endoscopic findings characterized by mildly hemorrhagic mucosa. The imatinib patient was diagnosed by capsule endoscopy of the small intestine, and required frequent blood transfusions. The dasatinib patient showed occult bleeding due to CD8-positive colitis. We should adequately recognize that gastrointestinal bleeding may occur during the administration of TKIs.

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.

Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 μg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models. © 2013 Yamawaki et al.

The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

Endothelial cells (ECs) lining the blood vessels serve a variety of functions and play a central role in the homeostasis of the circulatory system. Since the ductus arteriosus (DA) has different arterial characteristics from its connecting vessels, we hypothesized that ECs of the DA exhibited a unique gene profile involved in the regulation of DA-specific morphology and function. Using a fluorescence-activated cell sorter, we isolated ECs from pooled tissues from the DA or the descending aorta of Wistar rat fetuses at full-term of gestation (F group) or neonates 30 minutes after birth (N group). Using anti-CD31 and anti-CD45 antibodies as cell surface markers for ECs and hematopoietic derived cells, respectively, cDNAs from the CD31-positive and CD45-negative cells were hybridized to the Affymetrix GeneChip® Rat Gene 1.0 ST Array. Among 26,469 gene-level probe sets, 82 genes in the F group and 81 genes in the N group were expressed at higher levels in DA ECs than in aortic ECs (p<0.05, fold change>2.0). In addition to well-known endothelium-enriched genes such as Tgfb2 and Vegfa, novel DA endothelium-dominant genes including Slc38a1, Capn6, and Lrat were discovered. Enrichment analysis using GeneGo MetaCore software showed that DA endothelium-related biological processes were involved in morphogenesis and development. We identified many overlapping genes in each process including neural crest-related genes (Hoxa1, Hoxa4, and Hand2, etc) and the second heart field-related genes (Tbx1, Isl1, and Fgf10, etc). Moreover, we found that regulation of epithelial-to-mesenchymal transition, cell adhesion, and retinol metabolism are the active pathways involved in the network via potential interactions with many of the identified genes to form DA-specific endothelia. In conclusion, the present study uncovered several significant differences of the transcriptional profile between the DA and aortic ECs. Newly identified DA endothelium-dominant genes may play an important role in DA-specific functional and morphologic characteristics. © 2013 Liu et al.

Background:We previously described a primitive cell population derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMCs), which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s) derived from circulating CD14- cells. The present study was conducted to identify factors that induce MOMC differentiation.Methods:We cultured CD14+ monocytes on fibronectin in the presence or absence of platelets, CD14- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages.Results:The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF)-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1′s critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14+CXCR4high cell population.Conclusion:The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs. © 2013 Seta et al.

Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2′-deoxyuridine (BrdU). We detected 0.12×108 xlEPOR+ BrdU+ cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR+/ BrdU+ cells (approximately 1.30×108 cells) in the liver. The xlEPOR+ cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia. © 2013 Springer-Verlag Berlin Heidelberg.

Hematopoietic responses to environmental factors are not fully characterized. Polycythemia has been reported during exposure to low temperatures in ectothermic animals. The relationship between the causes of polycythemia and erythropoiesis during low temperature exposure is not fully understood. In this study, we exposed C57BL/6 mice to 5°C and monitored the blood cell counts and erythropoiesis. The hematocrit level increased from 45.6 to 52.2% after 14 days. Likewise, the hemoglobin concentration, initially 15.1 g dl-1, rose to 16.0 g dl-1. The reticulocyte production index significantly increased from 4 to 8% after 7 days. We examined the anatomy and cell composition of the spleens of the mice. On day5, the spleens were ̃6 mg g-1 of body mass, which was twofold greater than the spleens on day0. Flow cytometry showed fourfold more proerythroblasts on day5, compared with day0. Additionally, the number of late-stage mature erythroblasts increased on day14. Erythropoietin mRNA levels increased in the kidneys, and hypoxia-inducible genes were enhanced in the kidney. Our findings indicated that low ambient temperature is a novel erythropoietic stress, which induces polycythemia by enhanced erythropoiesis. © 2013. Published by The Company of Biologists Ltd.

Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type-associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs. © 2013 Yusuke Yoshioka et al.

Iron is fundamental for sustaining life for living organisms, and the iron metabolism is finely regulated at different levels. In cancer cells, deregulation of the iron metabolism induces oxidative stress and drives tumor progression and metastasis; however, the molecular mechanisms of iron homeostasis are not fully understood. Here we found that iron deficiency as well as hypoxia promoted microRNA-210 (miR-210) expression. A central mediator of miR-210 transcriptional activation is the hypoxia-inducible factor (HIF)-1α, and the hypoxia-response element in the miR-210 promoter is confirmed experimentally. This is in agreement with the data from in vivo studies that have demonstrated the presence of miR-210-expressing cells at the chronic hypoxic regions of xenografted tumors. Furthermore we found two essential molecules for iron homeostasis, iron-sulfur cluster scaffold protein (ISCU) and transferrin receptor 1 (TfR), are a direct target of miR-210. Transfection ofmiR-210decreases the uptake of transferrin by inhibiting the expression of TfR. In addition, inhibition of miR-210 by anti-miR-210 up-regulates ISCU expression. These findings suggest that miR-210 works as an iron sensor and is involved in the maintenance of iron homeostasis by sustaining the TfR expression level to stimulate cell proliferation and promote cell survival in the hypoxic region within tumors. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Diminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in odianisidine-positive erythrocytes in the hepatic sinusoid 5 days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis. © 2012. Published by The Company of Biologists Ltd.

C7α-substituted estradiols bind to estrogen receptors in cell nuclei, yet these derivatives remain little used in bioimaging. Here, we describe a fluorescent derivative of estradiol (E2) with a boron-dipyrromethene (BODIPY) moiety attached to C7α, synthesized by olefin metathesis reaction of 7α-allylestradiol and 9-decenyl-BODIPY. In ovariectomized rats and non-ovariectomized mice, E2-BODIPY promoted the growth of uterine tissue similar to the effect of estradiol. Twenty-four hours after subcutaneous injection of E2-BODIPY in non-ovariectomized mice, we observed fluorescence of E2-BODIPY in the nuclei of uterine epithelial cells. Our findings suggest that fluorescence microscopy can localize this derivative in E2-responsive cells during normal development and tumorigenesis in vivo. © 2012 Elsevier Inc. All rights reserved.

We previously reported monocyte-derived multipotential cells (MOMCs), which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14+ monocytes requires their binding to extracellular matrix (ECM) protein and exposure to soluble factor(s) derived from circulating CD14 - cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α5 integrin antibody or a synthetic peptide that competed with the RGD domain for the β1-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α5β1 integrin. © 2012 Noriyuki Seta et al.

Background: Acquisition of drug-resistance in cancer has led to treatment failure, however, their mechanisms have not been clarified yet. Recent observations indicated that aberrant expressed microRNA (miRNA) caused by chromosomal alterations play a critical role in the initiation and progression of cancer. Here, we performed an integrated genomic analysis combined with array-based comparative hybridization, miRNA, and gene expression microarray to elucidate the mechanism of drug-resistance.Results: Through genomic approaches in MCF7-ADR; a drug-resistant breast cancer cell line, our results reflect the unique features of drug-resistance, including MDR1 overexpression via genomic amplification and miRNA-mediated TP53INP1 down-regulation. Using a gain of function study with 12 miRNAs whose expressions were down-regulated and genome regions were deleted, we show that miR-505 is a novel tumor suppressive miRNA and inhibits cell proliferation by inducing apoptosis. We also find that Akt3, correlate inversely with miR-505, modulates drug sensitivity in MCF7-ADR.Conclusion: These findings indicate that various genes and miRNAs orchestrate to temper the drug-resistance in cancer cells, and thus acquisition of drug-resistance is intricately controlled by genomic status, gene and miRNA expression changes. © 2011 Yamamoto et al; licensee BioMed Central Ltd.

Oxygen is essential for the survival of animals. Red blood cells in the circulation, i.e. peripheral erythrocytes, are responsible for transporting oxygen to tissues. The regulation of erythropoiesis in vertebrates other than mammals is yet to be elucidated. Recently we identified erythropoietin, a primary regulator of erythropoiesis, in Xenopus laevis, which should enable us to identify target cells, including erythroid progenitors, and to investigate the production and development of erythroid cells in amphibians. Here, we established a semi-solid colony-forming assay in Xenopus laevis to clarify the existence of colony-forming unit-erythroid cells, the functional erythroid progenitors identified in vitro. Using this assay, we showed that recombinant xlEPO induces erythroid colony formation in vitro and detected an increased level of erythropoietin activity in blood serum during acute anemic stress. In addition, our study demonstrated the possible presence of multiple, non-x/EPO, factors in anemic serum supportive of erythroid colony formation. These results indicate that erythropoiesis mediated by erythropoietin is present in amphibian species and, furthermore, that the regulatory mechanisms controlling peripheral erythrocyte number may vary among vertebrates. © 2011. Published by The Company of Biologists Ltd.

Objective: Erythropoietin (EPO) and its receptor (EPOR) are key regulators of red blood cell production in mammals and fish. We aimed to investigate the structural and functional conservation of the EPO-EPOR system in amphibian erythropoiesis, using Xenopus laevis as a model. Materials and Methods: X. laevis epo (xlepo) complementary DNA was identified by referring to the Xenopus tropicalis genome database. Biological activity of recombinant xlEPO expressed in COS-1 cells was evaluated using xlEPOR-expressing murine FDC/P2 cells and human EPO-dependent UT-7/EPO cells. Expression of xlepo messenger RNA in adult X. laevis tissues in the normal state and under the condition of phenylhydrazine-induced anemia was evaluated by real-time reverse transcription polymerase chain reaction. Results: In the encoded protein, the positions of four cysteine residues were conserved; however, xlEPO had only 38% identity with human EPO. N-glycosylation sites were absent. Recombinant xlEPO induced proliferation of cell lines expressing xlEPOR and UT-7/EPO, confirming biological activity and cross-species reactivity. Despite little primary amino acid sequence similarity, the evolutionary highly conserved sequence NFLRGK was identified in the EPOR-binding site 1 region as in the human EPO protein. Strong expression of xlepo messenger RNA was detected in the lung and liver, especially in fractionated hepatocytes. No marked increase in xlepo expression was seen in the lung and liver of phenylhydrazine-induced anemic X. laevis. Conclusion: We confirmed that xlEPO is the ligand to the previously reported xlEPOR in X. laevis. xlEPO shares structural and functional similarities and differences with mammalian counterparts, and regulation of xlepo expression and its influence on the erythropoietic system appears to be unique. © 2010 ISEH - Society for Hematology and Stem Cells.

Background and Aim: Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo. Methods: In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum. Results: We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation. Conclusions: Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo. Thus, ASC may be a superior choice for the establishment of a therapy for injured liver. © 2008 The Authors.

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC. © 2009 Informa UK Ltd.

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl4-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor α (IL-1Rα), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BMMSCs and NHDFs. Thus, our findings suggest that ATMSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment. ©AlphaMed Press.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through mRNA degradation or translation inhibition. It has not yet been clearly elucidated whether miRNAs participate in haematopoietic processes such as cell differentiation, apoptosis and maintenance of adequate levels of circulating blood cells. A human miRNA microarray was used to analyze miRNA expression in the erythropoietin-dependent cell line UT-7/EPO compared to other factor-dependent UT-7 cell lines. Among 324 human miRNAs, MIRN188, MIRN362 and MIRN210 levels were significantly elevated in UT-7/EPO cells, and stimulation with EPO in UT-7 cells increased the level of these three miRNAs. Notably, knockdown of MIRN210 in UT-7/EPO cells led to apoptosis. In mouse fetal liver cells, MIRN210 expression was twofold higher in TER-119-positive cells than in TER-119-negative cells. The expression of MIRN210 was elevated during erythroid maturation in vitro. These data suggest MIRN210 to be a member a new class of regulatory miRNAs that might play an important role in erythroid maturation. © 2008 The Authors.

The specific features of the plasticity of adult stem cells are largely unknown. Recently, we demonstrated the hepatic differentiation of human adipose tissue-derived mesenchymal stem cells (AT-MSCs). To identify the genes responsible for hepatic differentiation, we examined the gene expression profiles of AT-MSC-derived hepatocytes (AT-MSC-Hepa) using several microarray methods. The resulting sets of differentially expressed genes (1639 clones) were comprehensively analyzed to identify the pathways expressed in AT-MSC-Hepa. Clustering analysis revealed a striking similarity of gene clusters between AT-MSC-Hepa and the whole liver, indicating that AT-MSC-Hepa were similar to liver with regard to gene expression. Further analysis showed that enriched categories of genes and signaling pathways such as complementary activation and the blood clotting cascade in the AT-MSC-Hepa were relevant to liver-specific functions. Notably, decreases in Twist and Snail expression indicated that mesenchymal-to-epithelial transition occurred in the differentiation of AT-MSCs into hepatocytes. Our data show a similarity between AT-MSC-Hepa and the liver, suggesting that AT-MSCs are modulated by their environmental conditions, and that AT-MSC-Hepa may be useful in basic studies of liver function as well as in the development of stem cell-based therapy. © 2008 The Authors.

Fluorescence labeling of a cytokine at a specific site is required for observing cytokine-receptor interactions in living cells at the single-molecule level. Here, we demonstrated the C-terminus-specific fluorescence labeling of histidine-tagged thrombopoietin (TPO), a ligand for Mpl, with desthiobiotin-tagged fluorescent puromycin. Fluorescent TPO, purified by tandem affinity purification, stimulated the proliferation of Mpl-expressing cells. Within 10 min of its addition, fluorescent TPO was found to be diffusely distributed on the cell membranes of Mpl-expressing cells, and gradually accumulated to form fluorescent spots. This method is applicable for studying the spatial and temporal distributions of cytokines in individual living cells. © 2008 The Society for Biotechnology, Japan.

Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males. © 2007 Zoological Society of Japan.

Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val8]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val8]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val8]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val8]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val8]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species. © 2006 Elsevier Inc. All rights reserved.

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in CAPN3, which encodes an intracellular cysteine protease. To elucidate the fundamental molecular changes that may be responsible for the pathological features of LGMD2A, we employed cDNA microarray analysis. We divided LGMD2A muscles into two groups according to specific pathological features: an early-stage group characterized by the presence of active necrosis and a regeneration process and a later-stage group characterized by the presence of lobulated fibers. After comparing the gene expression profiles of the two groups of LGMD2A muscles with control muscles, we identified 29 genes whose mRNA expression profiles were specifically altered in muscles with lobulated fibers. Interestingly, this group included genes that encode actin filament binding and regulatory proteins, such as gelsolin, PDZ and LIM domain 3 (PDLIM3) and troponin I1. Western blot analysis confirmed the upregulation of these proteins. From these results, we propose that abnormal increased expression of actin filament binding proteins may contribute to the changes of the intra-myofiber structures, observed in lobulated fibers in LGMD2A. © 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society.

The FGF-4 (fibroblast growth factor 4, known as HST-1) protein is an important mitogen for a variety of cell types. However, only limited information is available concerning tissue distribution and the biological role of FGF-4 in the brain. In situ hybridization analysis revealed localization of mouse Fgf-4 mRNA in the normal postnatal mouse hippocampus, subventricular zone (SVZ), and the rostral migratory stream where new neurons generate, migrate, and become incorporated into the functional circuitry of the brain. We also investigated whether FGF-4 could promote both proliferation and differentiation of the neural progenitor cells by using an in vitro neurosphere assay. The addition of recombinant FGF-4 generated large proliferative spheres that have a multipotent differentiation ability. Furthermore, recombinant FGF-4 significantly promotes neuronal differentiation in attached clonal neurosphere culture. These findings suggest that FGF-4 has an ability to promote neural stem cell proliferation and neuronal differentiation in the postnatal brain. © FASEB.

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3β/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se. Copyright © 2005 by the American Association for the Study of Liver Diseases.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3′-hydroxykinase p110-α-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-α levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA. © 2005 by The National Academy of Sciences of the USA.

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key mechanism of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. A cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, tentatively named xlEPOR, was cloned from a cDNA library of Xenopus laevis immature erythrocytes. The comparative identities of the deduced entire amino acid sequence to mammalian EPO receptors were quite low, although functional domains indispensable for erythropoietic activities were found in the molecule. Northern analysis revealed that xlEPOR were expressed in peripheral blood cells. In the peripheral blood of phenylhydrazine-treated adult Xenopus, immature erythrocytes expressing xlEPOR were identified by in situ hybridization and immunostaining with polyclonal antibodies to xlEPOR. To confirm the biological functions of this molecule, the extracellular domain of xlEPOR (i.e., soluble xlEPOR) was administered to adult Xenopus by consecutive intracardiac injection. The peripheral erythrocyte counts were decreased gradually; meanwhile, immature erythrocytes appeared in the circulation, demonstrating that xlEPOR plays a significant physiological role in erythropoiesis in Xenopus laevis. © 2005 The Japanese Biochemical Society.

Previous in vitro studies demonstrated that thrombopoietin (TPO) acts on platelets to activate a variety of intracellular signaling pathways and to enhance platelet sensitivity to multiple agonists. Little is known, however, about whether TPO exerts prothrombotic effects in vivo. The aim of this study was to examine the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a pegylated N-terminal domain of human TPO, in a rat model of venous thrombosis. A microthrombus was photochemically induced on the vessel wall of a mesenteric venule, but the vessel was not occluded by it. A single intravenous injection of PEG-rHuMGDF (3 μg kg -1) after the thrombus generation into normal rats enhanced the thrombus size, resulting in transient thrombotic occlusion in the majority of rats. Stimulatory effects on thrombus growth were also observed following administration of glycosylated recombinant human full-length TPO (6 μg kg-1). In rats rendered thrombocytopenic by total body irradiation, however, PEG-rHuMGDF, even at 300 μ kg-1, did not induce a significant increase in thrombus size or thrombotic occlusion. Platelets from thrombocytopenic rats had decreased surface levels of c-Mp1 and decreased sensitivity to PEG-rHuMGDF in an in vitro aggregation response. Thus, decreased prothrombotic effects of PEG-rHuMGDF in thrombocytopenic rats might be the result not only of low platelet counts but also of decreased platelet reactivity to PEG-rHuMGDF. These results indicate that PEG-rHuMGDF has little effect on venous thrombus formation in thrombocytopenic states associated with high endogenous TPO levels. © 2005 International Society on Thrombosis and Haemostasis.

We examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on the development of L-8057, a murine megakaryoblastic leukemia that expresses the thrombopoietin receptor c-Mpl, in mice. PEG-rHuMGDF administration prolonged survival of L-8057 leukemic mice, in which L-8057 cell growth in the spleen was decreased. L-8057 cells harvested from PEG-rHuMGDF-treated leukemic mice had decreased ability to generate leukemic colonies in vitro as well as to induce leukemia in vivo. PEG-rHuMGDF administration also resulted in prolonged survival of mice transplanted with a c-Mpl-expressing erythroleukemia, but had no effect on survival of mice transplanted with a myeloblastic leukemia that does not possess c-Mpl. Thus, PEG-rHuMGDF suppresses the development of c-Mpl-expressing leukemia in vivo in mice. © 2004 Elsevier Ltd. All rights reserved.

Gonadal differentiation in some species of amphibians is sensitive to steroids. The phenotypic sex of XX/XY-type frogs such as Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles, but little is known about the molecular mechanism of this sex reversal. To elucidate the mechanism of the sex differentiation, we examined the role of P450 aromatase (P450arom), an enzyme that converts testosterone to estrogen, during gonadal differentiation of amphibians. In this study, we first cloned a P450arom cDNA homolog of the frog R. rugosa and analyzed by RT-PCR its expression profile in developing and in female-to-male sex-reversed gonads. P450arom expression was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary in which only immature oocytes were observed. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, when oocytes were growing; and it was no longer seen in adult ovaries. By RT-PCR, we also examined the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it declined rapidly after the injection of testosterone. In contrast, no change in the SF-1 (also known as Ad4BP) expression was observed. Moreover, to identify the type(s) of cells expressing P450arom protein, we performed immunostaining with an antibody against frog P450arom protein. Cells giving positive signals were observed around oocytes in the ovary of frogs 1 month after metamorphosis. They were identified as follicle cells by both light and electron microscopy. The results, taken together, indicate that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa. © 2004 Elsevier Inc. All rights reserved.

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.

Objective. We examined the stimulatory effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production in male (NZW × BXSB) Fl (W/B F1) mice, a murine model of idiopathic thrombocytopenic purpura. Materials and Methods. A cohort of 19- to 25-week-old, severely thrombocytopenic male W/B F1 mice were given PEG-rHuMGDF at different dosing schedules. Before and at various times after therapy, platelet counts, reticulated platelets, platelet lifespan, and levels of platelet-associated immunoglobulin G were measured. Analysis of megakaryocytic cells was performed. Results. Treatment of male W/B F1 mice with PEG-rHuMGDF (30 μg/kg/day) three times per week for several weeks resulted in sustained thrombocytosis, accompanied by increased megakaryocytopoiesis in both the bone marrow and spleen. The degree of the platelet response to PEG-rHuMGDF varied between individual mice, likely reflecting the heterogeneity of the disease. Production of new platelets in response to PEG-rHuMGDF was manifested by an increase in reticulated platelets. Levels of platelet-associated immunoglobulin G decreased inversely during periods of thrombocytosis. PEG-rHuMGDF therapy also improved thrombocytopenia in male W/B F1 mice refractory to splenectomy. Platelet lifespan was not affected by PEG-rHuMGDF. Male W/B F1 mice treated with pegylated murine MGDF, a homologue of PEG-rHuMGDF, had persistent thrombocytosis for at least 7 months, suggesting that antiplatelet antibody production was not enhanced. Conclusion. PEG-rHuMGDF therapy potently stimulated platelet production, effectively ameliorating thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Thrombocytopenia is one of the major complications of liver cirrhosis. Except for hypersplenism associated with portal hypertension, it is not known which abnormalities of thrombopoiesis cause thrombocytopenia. To evaluate thrombopoiesis in liver cirrhosis, we analyzed thrombopoietin (TPO) and its receptor c-Mp1 levels in cirrhotic patients. Expression of c-Mp1 on platelets and the serum level of TPO were investigated from 38 patients with various stages of liver cirrhosis by flow-cytometric analysis and enzyme-immuno assay. Samples obtained from 22 individuals without evidence of liver disease were used as controls. Neither platelet counts nor TPO levels correlated with disease progression defined by the Child-Pugh classification. c-Mp1 was constitutively expressed on the platelets of cirrhotic patients, and its expression level was reduced with disease progression defined by the Child-Pugh classification. In this study, serum TPO did not fluctuate according to the grade of cirrhosis. However, its receptor c-Mp1, which is expressed on platelets, was decreased significantly in severely cirrhotic patients with thrombocytopenia. Thus, a correlation between reduced c-Mp1 expression and the progression of liver cirrhosis was demonstrated. We conclude that, in addition to hypersplenism, the reduced expression of c-Mp1 may play a significant role in the thrombocytopenia observed in severe liver cirrhosis. © 2002 Elsevier Science B.V. All rights reserved.

To clarify the mechanisms underlying thrombocytosis secondary to infections, we longitudinally studied serum levels of thrombopoietin (TPO) and interleukin (IL)-6 in 15 infants and young children with prominent thrombocytosis (platelets >700 × 109/l) following acute infections and 116 age-matched controls using an enzyme-linked immunosorbent assay. The subjects included nine patients with bacterial infections, three with viral infections and three with non-determined pathogens. TPO values in the controls were 2.24 ± 0.87 fmol/ml (mean ± SD) with a 95% reference interval of 0.85-4.47 fmol/ml. In the first week of infection, platelet counts were normal, but TPO values increased (∼10.73 fmol/ml). TPO levels peaked on day 4 ± 2 at 6.44 ± 2.37 fmol/ml and then fell gradually. When platelet counts peaked in the second and third weeks, TPO levels were similar to the controls. IL-6 levels in the first week rose and dropped more rapidly than TPO. Serum TPO values were significantly correlated with C-reactive protein levels (r = 0.688, P < 0.001) and IL-6 levels (r = 0.481, P = 0.027). These results suggest that TPO contributes to thrombocytosis following infections in conjunction with IL-6, arguing for additional regulatory mechanisms of blood TPO levels.

Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS. © 2002 Wiley-Liss, Inc.

We studied the effect of thrombopoietin (TPO) on interleukin-3 (IL-3)-dependent bone marrow cell colony formation of mice to clarify the role of protein kinase C (PKC) in the signal transduction of TPO for the proliferation of primitive hematopoietic progenitors. TPO alone hardly yielded colonies. However, TPO in combination with IL-3 increased colony numbers synergistically from 2- to 4-fold, compared with those supported by IL-3 alone. Serial observation of colony development showed that TPO may hasten the appearance of colonies by shortening the dormant period (Go) of primitive progenitors. Immunocytochemical studies on PKC isoforms in progenitor cells stimulated with TPO have revealed that the expression pattern of PKC-ε is changed, but not that of PKC-α, -β, -γ, -δ or -ζ. Selective PKC inhibitors, such as calphostin C and GF 109203X, and PKC-ε-specific translocation inhibitor peptide abrogated the enhancing effect of TPO on IL-3-dependent colony formation and the changes in the intracellular expression pattern of PKC-ε. These data taken together suggest that TPO has a direct effect on primitive progenitors and enhances IL-3-dependent colony formation, at least partly through the activation of PKC-ε.

We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

Thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

To verify pathophysiological mechanisms underlying thrombocytosis in low-birth-weight (LBW) preterm babies, we evaluated kinetic changes in platelet counts and thrombopoietic cytokines including thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 in 24 uncomplicated preterm infants. Platelet counts in cord blood (CB) (265 ± 64 × 109/l) were similar to adult levels, increased by d 14 (473 ± 140 × 109/l), and then remained fairly constant. Thrombocytosis (> 500 × 109/l) was observed in 9/24 (38%) subjects. Mean TPO level in CB was 5.11 ± 1.51 fmol/ml, peaked at d 2 (7.64 ± 3.28 fmol/ml), decreased at d 5 (3.93 ± 1.67 fmol/ml), and thereafter kept fairly constant during the remaining neonatal period. Compared with term infants, mean TPO levels of preterm infants in CB and at d 2 were significantly higher (P < 0.01). There was an inverse correlation between platelet counts and TPO levels (r = 0.45, P < 0.001, n = 88). Preterm neonates with thrombocytosis had significantly higher TPO values in CB than those without thrombocytosis (P < 0.05). There was no significant relationship between platelet counts and IL-6. IL-11 was not detectable. These results suggest that an early elevation of serum TPO levels is related to the subsequent thrombocytosis in LBW preterm infants.

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl+/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N. linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to 1/10 that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C- terminal region was found to be important for secretion of TPO. Among six N- glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn- 234, were found to be critical for secretion, and two other locations, Ash- 319 and Ash-327, did not affect the secretion.

At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitosis, which is characterized by DNA replication without cell division. Although a number of cell cycle regulatory molecules have been identified, the precise roles of these molecules in megakaryocytic endomitosis are largely unknown. In a human interleukin-3-dependent cell line transfected with the thrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO or the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation. We found that TPO stimulation or Ha-Ras(G12v) expression led to up-regulation of cyclin D1, cyclin D2, and cyclin D3 expression. In addition, expression levels of cyclin A and cyclin B were reduced during the total course of both TPO- and Ha- Ras(G12V)-induced megakaryocytic differentiation, thereby leading to decreased cdc2 kinase activity. Neither the induced expression of cyclin D1, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce megakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpression of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinase activity may participate in megakaryocytic differentiation.

Background and Aims: The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. The mechanisms by which levels of TPO mRNA are regulated in cirrhosis have not been elucidated. In this study, we investigated some possible mechanisms. Methods: We used three experimental models: bone marrow suppression, acute liver injury and primary cultured hepatocytes. We used northern blots to assess the kinetics of TPO mRNA expression in the livers of irradiated rats (with and without cirrhosis) in acute liver injury and in primary cultured hepatocytes treated with hepatotoxin or cytokines. Results: Although the bone marrow was hypocellular, there was no apparent enhancement of TPO mRNA expression in the irradiated rats with cirrhotic livers compared with the unirradiated rats with cirrhotic livers. There were no conspicuous changes in hepatic TPO mRNA expression between the livers of the control rats and the three models of acute liver injury. There were no conspicuous changes in the levels of TPO mRNA between control hepatocytes and hepatocytes treated with hepatotoxin or cytokines. Conclusions: Our results suggest that bone marrow is not a regulator of hepatic TPO production in cirrhosis. The reduced TPO mRNA expression found in cirrhotic rats may not result merely from serious cellular damage; it may be associated with cirrhosis-specific regulatory mechanisms for the expression of the TPO gene. Further studies are needed to search for other factors that may induce reduced TPO expression. (C) 2000 Blackwell Science Asia Pty Ltd.

Serum thrombopoietin (TPO) levels in 50 essential thrombocythemia (ET) patients were measured using a highly sensitive sandwich ELISA. In nine cases, TPO levels were measured at two points with different platelet counts. ET patients showed significantly higher serum TPO levels (n = 59, 2.70 ± 2.74 fmol/mL, P < 0.0001) than those of normal individuals (n = 29, 0.83 ± 0.36 fmol/mL). Twenty-three previously untreated ET patients also showed significantly higher serum TPO levels (1.33 ± 0.75 fmol/mL, P = 0.0066) than normal individuals. Extremely high serum TPO levels (5.46 ± 3.68 fmol/mL) were observed in ET patients with normal platelet counts. Furthermore, a strong inverse correlation was found between serum TPO levels and platelet counts in ET patients (R = -0.729, P < 0.0001). This inverse correlation also held for each of nine cases with two-point TPO measurements. In the clinical course of ET, megakaryocyte mass may parallel the platelet mass before and after chemotherapy. Although it is unknown whether overproduction of TPO exists or not in ET, total platelet and megakaryocyte mass, i.e., the total number of c-Mpl, may play a role to regulate serum TPO levels. (C) 2000 Wiley-Liss, Inc.

Thrombopoietin (TPO), a major cytokine involved in megakaryocytopoiesis/thrombopoiesis, may be effective for treatment of the thrombocytopenia associated with myelodysplastic syndromes (MDS). However, it has been unclear whether TPO stimulates proliferation of MDS blasts, as observed in de novo acute myeloid leukemia. This study examined this concern. When marrow cells from 37 MDS cases were cultured with or without recombinant human PEGylated TPO, TPO increased the blast number (stimulation index ≥1.5) in 9 of 16 high-risk MDS cases (refractory anemia with excess blasts [RAEB] and RAEB in transformation) and 4 of 10 cases with MDS transformed to acute leukemia (MDS-AL), but none of 11 cases with low-risk MDS (RA and RA with ringed sideroblasts). When the cell cycle of cultured cells was determined by three-color flow cytometry, TPO activated the cell cycle of MDS cells (causing a decrease in Go-phase cells) in most of the cases whose blast number increased in response to TPO. Reverse transcriptase-polymerase chain reaction analysis detected TPO receptor messenger RNA in purified blasts from all six cases examined, irrespective of the response of their blasts to TPO in culture. Analysis of the patients' characteristics identified a high-serum lactate dehydrogenase (LDH) value as being associated with blast proliferation in high-risk MDS cases (p = 0.0036). We conclude that TPO stimulates in vitro proliferation of blasts from a fraction of MDS patients. High-risk MDS patients, especially those who have a high-serum LDH value, and MDS-AL patients should be monitored with particular care in clinical trials of TPO for MDS.

We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38-c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.

Thrombocytosis is occasionally seen in patients with carcinomas and has been assumed to be attributable to interleukin-6 or granulocyte-macrophage colony-stimulating factor produced by carcinoma cells. In this study, we clarified whether thrombopoietin (TPO) is involved in carcinoma-associated thrombocytosis. Expression of TPO mRNA was observed in the majority of 27 carcinoma cell lines as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). There were 6 PCR products differing in size; sequence analysis showed the full-length TPO mRNA (TPO-1), 12- and 116-bp deleted variants (TPO-2 and TPO-3, respectively), and 3 novel isoforms (197- and 128- bp deleted forms and a 60-bp insert form of TPO-3; named TPO-4, TPO-5, and TPO-6, respectively). Of 27 lines, 24 expressed TPO-1 mRNA with various other isoforms. Culture supernatants of COS-1 cells transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive cells, whereas Western blot analysis on the cell lysates demonstrated TPO-5 but not TPO-6 protein, suggesting poor extracellular secretion (TPO-5) or poor protein synthesis (TPO-6). TPO protein was detected in 10-fold concentrated culture supernatants of cells of these carcinoma lines, with a median concentration of 0.38 fmol/mL as evaluated by enzyme-linked immunosorbent assay. High blood TPO levels were observed with a median value of 3.46 fmol/mL (range, 0.34 to 8.67 fmol/mL) in patients with advanced carcinomas associated with thrombocytosis. These results indicate that thrombocytosis in patients with carcinomas might be caused, at least in part, by TPO produced by carcinoma cells.

Thrombopoietin (TPO) is important as the physiologic regulator of platelet production. High-altitude hypoxia is a well-known cause of polycythemia and thrombocytopenia in animals. Fifty-two Wistar rats were housed for 0.5 to 21 days in a mechanical chamber in an environment equivalent to that found at 5500 m to determine (a) the cellular localization of TPO and (b) whether the decreased platelet and megakaryocyte counts in rats exposed to a hypobaric hypoxic environment (HHE) are associated with an altered TPO mRNA expression. In normal rats, there were high levels of TPO mRNA in the liver and kidney, intermediate levels in the brain and large intestine, and low levels in the skeletal muscle and small intestine. TPO mRNA and protein were expressed in Purkinje cells and neuronal cells in the brain, in proximal tubular cells and the mesangial cells of the glomeruli in the kidney, in hepatocytes and biliary duct epithelial cells, in absorptive epithelial cells in the large intestine, in the epidermis, and in the lung. The platelet count in the blood and megakaryocyte counts in the bone marrow and spleen were all decreased significantly after 5 or more days of exposure to HHE. In major producers such as the liver and kidney and in minor producers such as the brain, TPO mRNA levels, which tended to be decreased after 0.5 to 3 days of exposure to HHE, had returned to normal by about Day 5 or 7. Thus, during the HHE period with a decreased platelet count, no changes in TPO mRNA levels were detected in these three organs. In conclusion, we have demonstrated that TPO production occurs in various types of cells. In HHE, however, factors other than TPO may be involved in hypobaric hypoxia- induced thrombocytopenia in rats.

The effects of thrombopoietin (TPO) and/or stem cell factor (SCF) on the development of human mast cells from CD34+ bone marrow (BM) cells were investigated using a serum-deprived liquid culture system. Mast cells were identified by measurement of intracellular histamine content, immunocytochemical staining, and flow cytometric analysis. Whereas SCF alone generated only a small number of tryptase+ cells, the addition of TPO to the culture containing SCF resulted in an apparent production of mast cells from 3 weeks until at least 15 weeks. Some of the cells reacted with an antichymase monoclonal antibody as well. Based on the effects of growth factor(s) on a later phase of the mast cell growth, TPO may stimulate an early stage of mast cell development in combination with SCF, whereas subsequent growth seems to be supported by SCF alone. Single-cell culture studies indicated that the CD34+CD38-c-kit+ cells and CD34+CD38+c-kit+ cells were responsible for the SCF + TPO-dependent mast cell production. Two- step culture assays clearly showed that mast cells originated from multilineage colony-forming cells that had potential to differentiate into neutrophil/mast cell lineages, neutrophil/macrophage/mast cell lineages, or neutrophil/macrophage/mast cell/erythroid lineages. These results suggest that TPO plays an important role in the development of human mast cells from CD34+ BM cells in concert with SCF, and provide direct evidence of the differentiation into the mast cell lineage of human multipotential BM- derived progenitors.

In the present study, we investigated the effects of stem cell factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34+ cells using a serum-deprived liquid culture system. Although SCF alone supported a modest production of neutrophilic cells and a remarkable generation of mast cells, the addition of TPO to the culture containing SCF caused an apparent generation of neutrophilic cells, identified by immunocytochemical staining and flow cytometric analysis. The significant production of neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF). The addition of neutralizing antibody against G-CSF or GM-CSF did not influence the SCF + TPO-dependent neutrophilic cell production. A single-cell culture study showed that not only CD34+CD38+c-kit+ cells but also CD34+CD38-c-kit+ cells were responsible for the neutrophilic cell generation. In clonal cell cultures, GM progenitors as well as erythroid progenitors and multipotential progenitors expanded in the cultures supplemented with SCF and TPO. The neutrophilic cells grown by SCF + TPO were at myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the CD34-c-kit(-/low) CD15+ cells grown by SCF + TPO into a culture containing SCF + G-CSF permitted both the terminal maturation into segmented cells and the appearance of ALP and CD35. These results indicate the existence of a G-CSF/GM-CSF-independent system of neutrophilic cell production.

Thrombopoietin (TPO) isolated from thrombocytopenic plasma of various animal species has previously been shown to comprise only truncated forms of the molecule, presumably generated by proteolysis. Native TPO has now been partially purified from normal human plasma by immunoaffinity chromatography and was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) consisting of 332 amino acid residues, and a minor peak corresponding to a smaller molecular size. Immunoblot analysis also revealed that most plasma-derived TPO migrated at the same position as fully gly- cosylated rhTPO, corresponding to molecular size of ~ 80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from healthy individuals. These results indicate that the truncation of circulating TPO is not related to disease pathophysiology, and that the predominant form of TPO in blood is a biologically active ~ 80- to 100-kDa species. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin, indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-mediated cleavage.

Thrombopoietin (TPO) is a ligand for c-mpl, which regulates the differentiation and maturation of megakaryocytes. Essential thrombocythaemia (ET) is a clonal myeloproliferative disorder. It has been reported that the platelet count declines during pregnancy in ET patients. We examined serial changes in the serum TPO level during the course of pregnancy in a patient with ET. The serum TPO level showed significant negative correlation with the platelet count. Although it mimicked the normal feed back system, the TPO levels were consistently higher than the normal upper limit. Accumulation of these data will be helpful in revealing the pathogenesis of ET and the decline in the platelet count during pregnancy.

Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all-trans retinoic acid (RA) treatment. During differentiation-inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10-20d of all-trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation-inducing therapy, and it would appear that TPO levels are directly regulated by all-trans RA during the first 10-20d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all-trans RA treatment, suggesting a close relationship between TPO and EPO signalling.

We report a case of an 11-year-old boy who underwent successful bone marrow transplantation for X-linked hyper-IgM syndrome (XHIM). The donor was an HLA-matched brother. The patient was conditioned with busulfan, cyclophosphamide and anti-thymocyte globulin. He received 4.7 x 108 marrow cells per kg from the donor. Prophylaxis against graft-versus-host disease consisted of cyclosporine and short-term methotrexate. The clinical course after the bone marrow transplantation was uneventful, and 12 months after transplantation the patient was doing well with no need for therapy. We examined expression of the CD40 ligand (CD40L) on the patient's activated T lymphocytes and in vitro production of immunoglobulins by his lymphocytes. Although expression of CD40L was totally absent before the bone marrow transplant, subnormal expression appeared after the transplantation. In vitro production of IgG and IgA also was improved by the transplant. Based on our experience bone marrow transplantation appears to be a reasonable therapeutic option for patients with XHIM if HLA-matched family donors are available.

To clarify the relationship between the carboplatin AUC and the extent of damage to thrombopoiesis, we monitored both endogenous serum TPO kinetics and carboplatin pharmacokinetics after single-agent carboplatin administration. Previously untreated 12 patients with stage IV non-small-cell lung cancer were enrolled. The actual carboplatin AUC showed a significant positive correlation with the maximum increase ratio of TPO (TPO(max)/TPO(day1) (r = 0.74, p = 0.014). Furthermore, the increase ratio of TPO at one week after administration of carboplatin (TPO(day8)/TPO(day1) showed a significant negative correlation with the following platelet nadir around day 19 (r = -0.84, p = 0.005). By monitoring the changes in endogenous TPO concentration, we could estimate the degree of thrombocytopenia and determine the indication, and thus the optimal timing of prophylactic administration of TPO before platelets are markedly reduced.

We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease-free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73-5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r=0.27) or in children alone (r=0.12). In children >1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = -0.257 + 4.039 x plasma TPO values (r=0.951, P<0.001, n=22). This study is the first to report age-dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.

Three patients with liver cirrhosis (LC) and a bleeding tendency due to marked thrombocytopenia of less than 20 × 109/1 were admitted to our hospital for further examination. Bone marrow examination revealed megakaryocytic hypoplasia in all three patients. All patients exhibited amegakaryocytic thrombocytopenic purpura, myelodysplastic syndrome, or bone marrow hypoplasia. 111In-labeled platelet kinetic studies revealed decreased platelet production in all patients. Although serum thrombopoietin (sTPO) levels are usually within the normal level in patients with LC, the sTPO levels of our patients were about 10 times higher than the levels of normal subjects (1.22 ± 0.37 fmol/ml): 13.34, 16.79, and 10.46 fmol/ml, respectively. These sTPO data supported our findings of decreased megakaryopoiesis. Our findings suggest that examination of sTPO lev els is useful in determining the etiology of marked thrombocytopenia in LC patients. © 1999 The Japanese Society of Hematology.

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.

Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c-Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30-0.93 vs. 3.70, 1.38-8.33, P < 0.001), although platelet counts were similar (mean 321 x 109/l, range 42- 1,040 vs. 253 x 109/l, 124-381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = -0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c-Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production.

The effects of thrombopoietin (TPO; c-mpl ligand), FLT3/FLK-2 ligand (FL), and interleukin-6 (IL-6) on the survival of murine hematopoietic long- term reconstituting cells (LTRC) were studied by using lineage-negative, Sca- 1-positive, c-kit-positive (Lin-Sca-1+c-kit+) marrow cells from 5- fluorouracil-treated mice. We tested the ability of these cytokines to maintain the viability of LTRC by transplanting the cultured cells to lethally irradiated Ly-5 congenic mice together with compromised marrow cells. As a single agent, only TPO could maintain the LTRC. Neither IL-6 nor FL was effective by itself, but they acted synergistically to maintain the LTRC. We examined whether the maintenance of LTRC by these cytokines was due to the survival of stem cells or was the result of active cell divisions and self-renewal. To monitor cell division, we used membrane dye PKH26. Enriched cells were stained with PKH26 on day 0 and incubated in suspension culture with TPO or with IL-6 and FL for 7 days. On day 7, PKH26(low) and PKH26(high) cells were prepared by sorting and their in vivo reconstituting abilities were tested by transplantation into lethally irradiated Ly-5 congenic mice together with compromised marrow cells. PKH26(high) populations cultured with both TPO alone and the combination of IL-6 and FL showed greater reconstitution activity than that of PKH26(low) populations. These data indicate that TPO alone and the combination of IL-6 and FL can support the survival of stem cells without stimulating their active cell proliferation.

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although, TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPOinduced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPOinduced proliferation and differentiation, c-mpl was introduced into F- 36P, a human interleukin-3 (IL-3)dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate an differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F- 36P-mpl cells, and their effects on TPQ-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ~30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-ras(G12C)) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-ras(G12v)-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells-with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Thrombopoietin (TPO), or c-MPL ligand, is the primary regulator of megakaryocyte and platelet production. TPO receptors expressed on human megakaryocytes derived from peripheral blood (PB) and cord blood (CB) progenitors cultured in the presence of TPO have now been analyzed quantitatively. Like those on human PB platelets, TPO receptors on the cultured megakaryocytes exhibited a molecular mass of approximately 80 kDa. Various characteristics of PB- and CB-derived megakaryocytes indicated that the former were more mature than the latter. Both PB- and CB-derived megakaryocytes expressed a single class of high-affinity TPO receptors, with 1933 ± 772 (n = 3) and 184 ± 48 (n = 4) sites per cell, respectively. These data indicate that the number of TPO receptors on human megakaryocytes increases with cell maturation.

Thrombopoietin (TPO) is the recently isolated lineage-dominant hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In vivo studies have shown that daily multiple injections of pegylated human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule related to human TPO, modified with polyethylene glycol, greatly improve thrombocytopenia and in most cases anemia and neutropenia in myelosuppressed animal models. In this study, we further examined various administration protocols of PEG-rHuMGDF on thrombocytopenia in mice treated with a combination of irradiation and carboplatin. After the myelosuppressive treatment on Day 0, mice received the same amount of PEG-rHuMGDF beginning on Day 1 by a single, 3 times (on alternate days), or 7 day daily administration. A single injection of PEG- rHuMGDF significantly reduced the severity and duration of thrombocytopenia and anemia with a concomitant accelerated recovery of megakaryocytic and erythroid progenitors in the bone marrow, similar to the 2 other administration protocols. As the start of a single injection of PEG-rHuMGDF was delayed, its therapeutic effects were attenuated. These results indicate that an administration of PEG-rHuMGDF at an earlier time after the myelosuppressive treatment is necessary to improve thrombocytopenia and anemia.

Marked thrombocytosis (over 50 x 104/μl) is frequently seen in patients with hepatoblastoma. Thrombopoietin (TPO), c-mpl ligand, has recently been purified as the major physiological regulator of the thrombopoiesis and is mainly produced in the liver. Since it is possible that TPO participates in thrombocytosis and the tumor growth of this particular hepatic tumor, serum TPO levels in addition to interleukin 1β (IL-1β) and IL-6 levels were assessed in seven untreated patients by using a sandwich enzyme-linked immunosorbent assay. High serum TPO levels were observed in all of the examined patients. The level ranged from 3.15 to 11.02 (mean ± standard deviation; 6.08 ± 1.25) fmol/ml. IL-6 levels were also somewhat higher than normal. Platelet counts, however, appeared to correlate more with serum TPO levels (p = 0.1) than with IL-1β (p = 0.5) and IL-6 (p = 0.2) levels. Furthermore, using the reverse transcriptase polymerase chain reaction method, the expression of c-mpl mRNA was found in five of eight hepatoblastoma tissues as well as TPO mRNA in all eight tissues. These observations suggest that thrombocytosis in hepatoblastoma patients results from the production of cytokine members, including TPO, within tumor tissues. Additionally, it is possible that TPO might act as a type of autocrine and/or paracrine system for cellular growth in this tumor.

Thrombopoietin (TPO), the c-Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c-Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full-length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.

It is widely accepted that thrombocytopenia associated with liver cirrhosis is caused by increased platelet destruction in the enlarged spleen, but this issue has not yet been analysed sufficiently in terms of platelet production. Thrombopoietin is produced mainly in the liver and strongly promotes platelet production. We studied serum thrombopoietin and the levels of its mRNA in liver tissue of cirrhotic patients and also in a rat model of liver cirrhosis. Furthermore, to clarify the influence of the spleen, we investigated thrombopoietin mRNA in splenectomized rats. The serum thrombopoietin level in humans with liver cirrhosis was not significantly reduced instead of thrombocytopenia. The expression of thrombopoietin mRNA in liver tissue decreased with the progression of liver cirrhosis in both patients and the rat model and no compensatory expression was observed in other organs or nonparenchymal cells. The level of thrombopoietin mRNA did not differ significantly in splenectomized cirrhotic rats before or after administration of dimethylnitrosamine, but was lower than that in splenectomized rats without cirrhosis. We conclude that thrombocytopenia in liver cirrhosis is caused not only by platelet destruction but also by decreased platelet production, perhaps due to reduction of thrombopoietin mRNA in the liver.

To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 ± 2.02%: mean ± SD) relative to normal controls (2.17 ± 0.90%), but serum TPO concentrations (1.91 ± 1.27 fmol/l) did not differ significantly from the normal range (1.43 ± 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 ± 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 ± 1.11 x 109/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 ± 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.

We have isolated and characterized a thrombopoietin (TPO)-dependent BF-TE22 cell line endogenously expressing murine Mp1, which is a subclone of murine pro-B Ba/F3 cells. TPO stimulated the proliferation of BF-TE22 cells in a dose-dependent manner, and also induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens. The results indicate that the murine Mp1 on BF-TE22 cells can transmit both proliferation and megakaryocyte lineage-specific differentiation signals to cells. Furthermore, it was shown that IL-3 inhibits the TPO-induced differentiation signals of BF-TE22 cells. These results suggest that the signals mediated by IL-3 predominate over thor of TPO in BF-TE22 cells. Thus, BF-TE22 cells will be useful for the biological and biochemical studies of the TPO-Mp1 signal transduction mechanism.

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG- rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 μg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit-spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG- rHuMGDF (80 μg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG- rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG- rHuMGDF, a single injection of recombinant human granulocyte colony- stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 μg/kg) treatment even when rhG-CSF doses were escalated to 1,000 μg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

We investigated in detail the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization in male BDF1 mice. Treatment with PEG- rHuMGDF for 5 d stimulated a striking expansion of the circulating levels of multiple types of colony-forming units in culture (CFU-c), including CFU- granulocyte-macrophage, CFU-megakaryocyte, burst-forming units-erythroid, and multipotent CFU-c, and primitive day-12 CFU-spleen. All of these progenitors were mobilized into the peripheral blood (PB) with similar kinetics; their numbers peaked after the cessation of treatment and then declined earlier than platelet numbers peaked. The maximal increase in any of the four CFU-c in the PB was attained with at least 300 μg/kg/d of PEG-rHuMGDF, whereas peripheral platelet counts plateaued at 30 μg/kg/d. Adoptive transfer with PB from PEG- rHuMGDF-treated donor mice resulted in greater survival of lethally irradiated recipients. The majority of the recipients that survived at 187 d after transplantation with PEG-rHuMGDF-mobilized PB showed significant donor engraftment at the progenitor cell level. The combined administration of appropriate doses of PEG-rHuMGDF and recombinant human granulocyte colony-stimulating factor induced a synergistic increase in the circulating levels of the four CFU-c compared to either factor alone. These results indicate that PEG-rHUMGDF as a single agent can mobilize a full spectrum of PBPCs in mice.

The serum thrombopoietin (TPO) levels in 61 idiopathic thrombocytopenic purpura (ITP) patients were found to be slightly increased compared with those of 29 normal subjects. The TPO levels of the 15 ITP patients who had a poor response to steroid therapy (i.e. an unchanged platelet count) were higher than those of the 22 ITP patients who had a good response to steroid therapy (i.e. an increased platelet count) and the normal subjects. The TPO levels in the 15 ITP patients whose platelet count was higher than 10 x 104/μl after the discontinuation of steroid therapy significantly higher than those of the normal subjects. The platelet-associated immunoglobulin G (PAIgG) levels in the ITP patients who had a poor response to steroid therapy were slightly increased compared with the normal subjects and the ITP patients who had a good response to the steroid therapy and the nine ITP patients who did not undergo the steroid therapy. The serum TPO level was negatively correlated only with the megakaryocyte count in the ITP patients, and the megakaryocyte count in the ITP patients who had good responses to the steroid therapy was higher than that in those who had poor responses. These data suggest that serum TPO levels might be important for the prediction of the outcome of ITP patients who receive steroid therapy.

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, c-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

We evaluated the effects of human thrombopoietin (TPO) alone, or in combination with other several hematopoietic factors, on megakaryocyte colony growth from human umbilical cord blood CD34+ cells in serum-depleted agar cultures. The addition of TPO alone had a concentration-dependent effect on the number of pure megakaryocyte colonies and of megakaryocytes per colony (colony size). The maximally stimulating concentration of TPO generated a greater number of megakaryocyte colonies and larger megakaryocyte colony size compared with the stimulation observed with an optimal concentration of human interleukin-3 (IL-3) or stem cell factor (SCF). At the high concentration of TPO that yielded the maximum colony numbers, a substantial proportion of megakaryocyte colonies contained 100 or more cells per colony. The combination of TPO plus SCF resulted in a synergistic enhancement of both the number and size of megakaryocyte colonies. Among the combinations of TPO plus other cytokines tested, IL-3 plus TPO had a modest effect on megakaryocyte colony numbers. The generation of megakaryocyte colonies from subpopulations of CD34+ cells was further examined. The addition of TPO alone induced a greater number of megakaryocyte colonies from CD34+CD41+ cells compared with CD34+CD41- cells, and TPO plus IL-3 exerted a synergistic effect on the number of megakaryocyte colonies only from CD34+CD41- cells. In contrast to the effects on colony numbers, TPO induced larger megakaryocyte colony sizes from CD34+CD41- cells, compared with CD34+CD41+ cells. In the case of HLA-DR expression, TPO and IL-3, administered singly or in combination, induced similar megakaryocyte colony numbers and sizes from CD34+DR+ and CD34+DR- subpopulations. Ploidy analysis revealed that the majority of megakaryocytes generated from cord blood CD34+ cells in serum-free liquid cultures containing TPO displayed 2N and 4N values, suggesting that they were immature. These results indicate that, compared with IL-3 and SCF, TPO has more potent proliferative effect on human cord blood megakaryocyte progenitors, leading to greater numbers of megakaryocyte progenitors triggered for both growth and cell division, and synergizes with SCF to enhance megakaryocyte colony growth.

To clarify the role of thrombopoietin (c-Mp1 ligand, TPO) in 'hypersplenic' thrombocytopenia, we used an enzyme-linked immunosorbent assay to examine changes in serum TPO levels accompanied with splenectomy in 6 patients with liver cirrhosis, 4 patients with gastric cancer, and 2 patients with lymphoid malignancies. We also measured serum levels of other thrombopoietic cytokines such as interleukin-6 (IL-6) and erythropoietin. Platelet counts reached a maximum at day 14 after splenectomy in all subjects. In patients with liver cirrhosis, a lower elevation of platelet counts was observed compared with that in patients with gastric cancer. Serum TPO levels gradually elevated after splenectomy and reached a maximum 3.5 days after splenectomy in noncirrhotic patients, whereas peak serum TPO levels were delayed until day 7 in the cirrhosis group. IL-6 and erythropoietin showed similar kinetics between cirrhotic and noncirrhotic patients. These findings suggest that transient thrombocytosis after splenectomy may be associated with an alteration in the site of TPO catabolism by platelets from spleen to the blood and that deterioration of TPO production may play a role in thrombocytopenia in liver cirrhosis.

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO-R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I-TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects (P<0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB- t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB-t. The number of TPO-R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB-t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB-t. Thus, in addition to thrombocytopenia, a reduced platelet TPO-R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO-R and marrow megakaryocytes, such as blasts expressing TPO-R, may operate in RAEB/RAEB-t.

We have tested the effects of steel factor (SF) the ligand for flt3/flk2 (FL) and thrombopoietin (TPO, Mpl ligand), on the proliferation of primitive human bone marrow progenitors in serum-deprived culture. Varying combinations of SF, FL and TPO supported formation of only few colonies from CD34+/c- Kit(low)/CD38(neg/low) cells. However, the addition of interleukin 3 (IL-3) to the three cytokines significantly increased the number of colonies. When this population of cells was tested in suspension culture for one week for production of colony-forming cells there was synergism among SF, FL and TPO. Addition of IL-3 to the three cytokines further increased the number of erythroid colony-forming cells. The effects of these four factors on CD34+/c- Kit(low)/CD38(high) cells were merely additive. Studies of individual CD34+/c-Kit(low)/CD38(neg/low) cells demonstrated the direct effects of SF, FL and TPO. In the presence of SF, FL and TPO, approximately half of the individual CD34+/c-Kit(low)/CD38(neg/low) cells proliferated in seven day suspension culture. Addition of IL-3 to the combination of SF, FL and TPO did not increase the frequencies of proliferating clones, but increased the size of individual clones. These observations suggest that SF, FL and TPO play important roles in survival and proliferation of primitive human hematopoietic progenitors.

In a case control study, serum levels of thrombopoietin (TPO) were determined by a sandwich ELISA in 20 patients (median age, 7 years; range, 2- 56 years) with various malignancies who received high-dose chemotherapy and a stem cell rescue operation. The patients received two different transplant modalities: (a) 12 patients received purified autologous peripheral blood CD34+ cells; and (b) 8 patients received cells in the CD34(-) fraction, which still contains many CD34+ cells. No significant differences were observed between the two groups with regard to the duration required to achieve an absolute granulocyte count of >0.5 x 109/liter, the duration of dependence on platelet transfusion, or the number of platelet transfusions. In both groups, the serum TPO levels were inversely correlated with the circulating platelet count. Multivariate analysis demonstrated that significant determinants of the serum TPO level included the circulating platelet count (standardized regression coefficient = -0.5179), transplantation with cells in the CD34(-) fraction (0.2414), solid tumor (0.1420), and the age of the patient (-0.1236; r2 = 0.3021; P < 0.0001). These results suggest that the mode of stem cell support (i.e., the presence of accessory cells in the inoculum), age, or the type of preceding chemotherapy affects serum TPO levels after transplantation.

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl proto-oncogene). In an effort to determine the pathophysiological role of TPO-c-Mpl system in essential thrombocythemia (ET), we have examined the levels of serum TPO and the expression and function of platelet c-Mpl in 17 patients with ET. In spite of extreme thrombocytosis, serum TPO levels were slightly elevated or within normal range in most, if not all, patients with ET (mean ± SD, 1.31 ± 1.64 fmol mL), as compared with normal subjects (0.76 ± 0.21 fmol/mL). Flow cytometric and Western blot analyses revealed that the expression of platelet c-Mpl was strikingly reduced in all patients with ET. Furthermore, the expression of platelet c-mpl mRNA was found to be significantly decreased in the ET patients tested. In contrast, almost identical levels of GPIIb/IIIa protein and mRNA were expressed in platelets from ET patients and normal controls. In addition to expression level, activation state of platelet c-Mpl was investigated in ET patients. Immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TP-induced protein-tyrosine phosphorylation, including c-Mpl- tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl-mediated signaling pathway was not constitutively activated in platelets of ET patients. These results suggested that the TPO- c-Mpl system may not be directly linked to pathogenesis of ET, and that gene(s) mutated in ET may be important in regulating the levels of c-mpl gene expression in addition to the growth and differentiation of multipotential hematopoietic stem cells.

Thrombopoietin (TPO), produced mainly in the liver, is a major regulator of platelet production. Serum TPO levels are generally increased in thrombocytopenia. We report a case of a 12-year-old boy with chronic severe thrombocytopenia, achondroplasia and nephritis. Severe chronic thrombocytopenia was found at 9 months of age. It was resistant to any treatment. Studies on megakaryocytic colonies in vitro revealed that the marrow cells responded well to TPO and no plasma inhibitor was found. Although hepatic function test results were normal, serum TPO levels in the patient (0.94 fmol/ml) were consistent with those in age-matched children (0.49 1.75 fmol/ml). Chronic thrombocytopenia requires individual evaluation before clinical trials with TPO.

Although thrombopoietin (TPO) is known to play a fundamental role in both megakaryopoiesis and thrombopoiesis, the molecular mechanism of TPO- induced megakaryocytic differentiation is not known. In a human megakaryoblastic leukemia cell line, CMK, that showed some degree of megakaryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cip1), but not p27(Kip1), p16(INK4A), p15(INK4B), or p18(INK4C), was found to be upregulated in an immediately early response to TPO. The expression of p21 was found to be sustained over a period of 5 days by treatment with TPO in large polyploid cells that developed in response to TPO, but not in small undifferentiated cells, indicating a close correlation between the ligand-induced differentiation and p21 induction in CMK cells. To examine potential roles of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gene, β-galactosidase, and were cultured with medium alone for 5 days. The ectopic expression of p21 or p27 but not of p16 led to induction of megakaryocytic differentiation of CMK cells. Overexpression of the N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient to induce megakaryocytic differentiation, whereas that of the C- terminal domain (aa 76 to 164) had little or no effect on morphological features. Furthermore, we found that although TPO induced tyrosine phosphorylation of both STAT3 and STAT5 in CMK cells, only STATS showed binding activities to potential STAT-binding sites that locate in the promoter region of p21 gene (p21-SIE sites), thereby leading to transactivation of p21. These results suggested that p21 induction, possibly mediated through activated STATS, could play an important role in TPO- induced megakaryocytic differentiation.

A heterogeneity in the molecular weight (M(r)) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low M(r) peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.

Thrombopoietin (TPO) is the major regulator of the proliferation and differentiation of megakaryocyte precursors through interaction with its receptor encoded by the c-mpl protooncogene. We established the human TPO-dependent leukemia cell line, UT-7/TPO. In these cells, TPO activated protein kinase C (PKC) in a time dependent manner. Subsequently, the c-myc gene was transiently induced to a maximal level 60-90 minutes after TPO exposure. In addition, we found that stimulating UT-7/TPO cells with TPO rapidly induces the significant accumulation of inositol 1,4,5-trisphosphate (Ins-P3), leading to the mobilization of calcium from intracellular stores. Taken together, the activation of PKC and subsequent c-myc gene induction are involved in the TPO-induced cellular response(s), presumably through the activation of PLC.

The serum levels of thrombopoietin (TPO) were measured in 16 patients with thrombotic thrombocytopenic purpura (TTP), 12 with hemolytic uremic syndrome (HUS), 10 with aplastic anemia (AA), 10 with disseminated intravascular coagulation (DIC), and 71 with idiopathic thrombocytopenic purpura (ITP). The serum TPO levels were measured with a sensitive sandwich enzyme-linked immunosorbent assay. The serum TPO level in the ITP group (1.68 ± 0.85 fmol/ml) were not significantly increased compared with those of the normal subjects. The TPO levels in the TTP (2.77 ± 1.38 fmol/ml) and HUS groups (5.77 ± 4.41 fmol/ml) were higher than those of the normal subjects. The patients with AA (12.7 ± 8.0 fmol/ml) and those with DIC (13.3 ± 5.7 mol/ml) had significantly higher serum TPO levels than did the normal subjects and ITP patients. The TPO levels were well correlated with the platelet counts in the TIP patients, and were negatively correlated with the platelet counts in the ITP patients. These results suggest that the serum TPO levels in some thrombocytopenic diseases are regulated not only by the platelet count and the megakaryocyte mass, but also by other factors.

Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed. © 1997, Informa UK Ltd All rights reserved: reproduction in whole or part not permitted. All rights reserved.

We measured and the endogenous plasma concentration of thrombopoietin (TPO) in 76 patients with acquired aplastic anaemia (AA) by a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). The plasma TPO concentrations were significantly higher in AA patients when compared to healthy control subjects (P<0.0001) and there was a significant negative correlation between plasma TPO concentrations and platelet counts in 54 AA patients who had not received any platelet transfusions prior to sampling. On the other hand, there was no statistically significant correlation between the TPO concentrations and platelet counts in 22 AA patients who had previously received platelet transfusions. We studied serial changes of plasma TPO concentration in 24 patients showed an increase in their platelet counts following bone marrow transplantation or immunosuppressive (IS) therapy. Although a decrease in plasma TPO concentration as observed, levels remained above the range of normal healthy controls even in the patients who attained normal platelet counts following therapy. A decrease in TPO concentrations was observed in only half of the responders to IS therapy. Whether exogenous TPO will result in increased platelet counts in AA patients with high TPO levels remain to be determined.

In a serum-free liquid culture, thrombopoietin (TPD) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 x 105) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1β and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1α was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription-polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

Formation of proplatelets from megakaryocytes is believed to be the first step of platelet production in vitro. In this study, we evaluated the effects of recombinant human thrombopoietin (hTPO) on the development of proplatelets from a GpIIb/IIIa+ population of rat bone marrow cells highly enriched for late megakaryocyte progenitors (GpIIb/IIIa+ CFU-MK) that we recently found to be a primary target population of TPO. Quantitative measurement of hTPO-induced proplatelet formation was performed in liquid cultures. Proplatelet formation from megakaryocytes derived from GpIIb/IIIa+ CFU-MK in the presence of hTPO began on day 4 of culture and peaked the following day. On day 5 of culture, lower concentrations of hTPO expanded the number of megakaryocytes, increased the number of proplatelets and the percentage of proplatelet-developing megakaryocytes. Increasing hTPO concentrations resulted in a modest decrease in proplatelet development. We next used hTPO to derive immature or mature megakaryocytes from GpIIb/IIIa+ CFU-MK. These populations of cultured megakaryocytes spontaneously formed proplatelets when recultured in the absence of exogenous hTPO. The addition of hTPO at higher concentrations modestly augmented proplatelet production from immature megakaryocytes derived from 2-day liquid cultures. However, either murine interleukin-6 (IL-6) or human IL-11, but not rat IL-3, was more potent than hTPO in augmenting proplatelet formation from immature megakaryocytes. Each of these four cytokines had an inhibitory effect on proplatelet formation from more differentiated megakaryocytes derived from 3-day liquid cultures. These results indicate that TPO enhances proplatelet production primarily by stimulating CFU-MK to increase the number of proplatelet-forming megakaryocytes and that its action is clearly different from those of other cytokines that also stimulate megakaryocytopoiesis.

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma- containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU- Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst- promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.

To elucidate the physiologic role of thrombopoietin (TPO) for hematologic reconstitution following allogeneic bone marrow transplantation (BMT), serum TPO levels as well as interleukin-3 (IL-3), IL-6 and IL-11 were serially measured in 55 samples from 3 patients who underwent allogeneic BMT using an enzyme-linked immunosorbent assay (ELISA). The TPO level was higher in the serum taken during marrow aplasia than in the pretransplant serum. The serum TPO levels and platelet counts showed a strong inverse relationship in all patients examined. We also sequentially measured endogenous serum TPO levels before and within 36 h after platelet transfusions. Endogenous serum TPO levels were inversely correlated with platelet mass following platelet transfusions. Serum levels of IL-3 had no apparent correlation with platelet counts and serum levels of IL-11 remained below the detection levels (31.3 pg/ml) in all samples. Serum levels of IL-6 were high during myeloaplasia and more upregulated in the febrile period. These findings support the view that TPO is the central regulator for megakaryopoiesis in vivo and the rationale for its clinical use after allogeneic BMT.

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony-stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.

We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is capable of improving thrombocytopenia and promoting thrombopoietic reconstitution following lethal irradiation and bone marrow transplantation (BMT) in mice. Immediately after receiving 10 Gy whole body irradiation (day 0), male C3H/HeN mice were inoculated with 106 bone marrow cells obtained from syngeneic mice. Circulating platelet counts decreased to below 4% of the normal counts with a nadir on day 10, and then returned to the normal level on day 28 in the control mice undergoing BMT. Subcutaneous consecutive treatment with PEG-rHuMGDF at doses from 10 to 300 μg/kg/day from day 1 for 13 days significantly improved the platelet nadir and promoted platelet recovery. The white blood cell counts and hemoglobin concentration following BMT were not influenced by the PEG-rHuMGDF. PEG-rHuMGDF-injection starting from day 5 did not improve the platelet nadir following BMT. Furthermore, administration with PEG-rHuMGDF on alternate days at 55.7 μg/kg/day for 7 days or at an interval of 3 days at 78 μg/kg/day for 4 days (twice a week for 2 weeks) had a significant efficacy, but these administration regimens had less efficacy than consecutive administration at 30 μg/kg/day for 13 days. The numbers of megakaryocytes and megakaryocyte progenitor cells decreased to 5 and 0.2% of normal level, respectively, in the control mice. Consecutive administration of PEG-rHuMGDF enhanced the recovery of the mean number of these cells compared to those in vehicle-treated mice, although such effects were not statistically significant except for the number of megakaryocyte progenitors on day 12. These results suggest that consecutive treatment with PEG-rHuMGDF beginning from the day after BMT may be effective in improving thrombocytopenia following BMT.

Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal rule in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy- induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia: it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG- CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

The in vivo effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human thrombopoietin modified with polyethylene glycol, were investigated in normal Balb/c mice. PEG-rHuMGDF was more potent in producing platelets and the dose-response curve was sleeper compared with the case of the nonpegylated form of this molecule. Five consecutive injections with PEG- rHuMGDF caused a dose-dependent increase in peripheral platelet counts with a peak on day 8. There was a dose-dependent rise in platelet counts on day 8 at daily doses from 0.333 to 30 μg/kg. Intermediate doses of PEG-rHuMGDF (1.111 to 10 μg/kg/day) caused a significant decrease in mean platelet volume, and conversely, higher doses of PEG-rHuMGDF (30 to 270 μg/kg/day) induced a dose-dependent increase in mean platelet volume. There was a dose-dependent decrease in hemoglobin concentration with a minimum on day 8 but no significant reduction in reticulocyte counts following PEG-rHuMGDF administration. White blood cell counts were unchanged by PEG-rHuMGDF treatment. Marrow megakaryocyte size enlarged in 1.5-fold and the number of marrow megakaryocytes increased to sixfold by consecutive administration of PEG-rHuMGDF at 30 μg/kg/day. A twofold increase in the number of marrow megakaryocytic progenitor cells (colony-forming units-megakaryocyte) was also observed. Marrow erythroid progenitor (colony-forming units-erythroid) counts decreased but splenic colony-forming units-erythroid, marrow and splenic erythro/myeloid progenitor cell counts, and splenic granulocyte/macrophage progenitor cell counts increased with PEG-rHuMGDF treatment. Marrow and splenic erythroid burst-forming cells were unchanged. These results indicate that PEG-rHuMGDF, a truncated molecule of thrombopoietin, is a potent stimulator for megakaryopoiesis and thrombopoiesis, and also affects the development of other hematopoietic cells in normal mice.

We assayed serum thrombopoietin (TPO) levels in amegakaryocytic thrombocytopenia (AMT) and immune thrombocytopenic purpura (ITP) patients by using a newly established enzyme-linked immunosorbent assay (ELISA). TPO levels in AMT patients were quite high (mean ± SD = 13.7 ± 11.2 fmoles/ml, n = 4), whereas those in ITP patients were only slightly higher (1.25 ± 0.39, n = 12) than those of the healthy donors (0.55 ± 0.2, n = 20). Furthermore, in ITP patients no correlation was observed between platelet counts and serum TPO levels (correlation coefficient = 0.14). We further assayed serum TPO levels sequentially during steroid treatment in patients with AMT and ITP. In one AMT patient serum TPO levels started to decrease in accordance with the increase of megakaryocyte counts, which preceded the increase in platelet counts. However, in ITP patients serum TPO levels did not change significantly throughout the course of the treatment despite the recovery of platelet counts. Based on these findings, we conclude that serum TPO levels may be regulated at least in part by megakaryocyte counts.

To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c-mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL3-dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc-associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte-specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N-terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C-terminal region may also play important roles in transducing the differentiation signals.

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic levels in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

To clarify the role of c-Mpl ligand (thrombopoietin: TPO) in liver cirrhosis (LC), we examined serum TPO levels (sTPO) in patients with LC (N = 44), chronic hepatitis (CH; N = 13) and healthy controls (N = 41) by an enzyme-linked immunosorbent assay. Although platelet counts of all LC patients (89 ± 59 X 109/l; mean ± SD) were lower than those of controls and CH patients, sTPO levels in LC patients (1.23 ± 0.51 fmol/ml) were the same as those in controls (1.22 ± 0.37) and CH patients (1.18 ± 0.36). Platelet counts were significantly higher in splenectomized patients than in unsplenectomized patients, but the sTPO level did not differ between these two groups. In LC patients, the sTPO level was not correlated with the platelet count, but was correlated with prothrombin time, activated partial thromboplastin time, and total bilirubin, indicating that production of TPO in the liver decreases slightly with the development of liver dysfunction. Our findings suggest that production of TPO is maintained in LC patients and their thrombocytopenia is not due to a defect in platelet production.

To investigate the pathophysiological role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 84 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzyme immunoassay procedure. The TPO level was 0.84 ± 0.40 f mol/ml in normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission (postchemotherapy group: n = 18; 18.46 ± 9.70) f mol/ml). When the data of normal individuals and the postchemotherapy group were combined, TPO levels were inversely correlated with the platelet count in this combined group. We compared these data of normal individuals and the postchemotherapy group with various hematological disease states. In aplastic anemia (n = 13; 16.03 ± 9.44 f mol/ml, acute lymphoblastic leukemia (n = 5; 10.36 ± 5.57 f mol/ml), malignant lymphoma (n = 6; 2.79 ± 2.27 f mol/ml), multiple myeloma (n = 3; 3.34 ± 0.20 f mol/ml and chronic lymphocytic leukemia (n = 21 1.71 ± 3.91 f mol/ml), the relationship of serum TPO levels and platelet counts was almost the same as in the combined group with normal individuals and the postchemotherapy group. However, the TPO levels were slightly higher in myeloproliferative disorders (n = 12; 1.99 ± 1.47 f mol/ml) and lower in acute myelogenous leukemia (n = 8; 2.27 ± 1.25 f mol/ml), hypoplastic leukemia (n = 3; 2.76 ± 2.23 f mol/ml, myelodysplastic syndrome (n = 2; 0.42 ± 0.60 f mol/ml), liver cirrhosis (n = 2; 1.50 ± 0.92 f mol/ml) and idiopathic thrombocytopenicpurpura (n = 12; 2.08 ± 1.41 f mol/ml), when compared to the regression line for the combined group with normal individuals and postchemotherapy group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.

We have studied the effects of recombinant human thrombopoietin (TPO; mpl ligand) on the proliferation of human primitive hematopoietic progenitors in vitro. CD34+ cells were enriched for cell-cycle-dormant primitive progenitors by separation on the basis of expression of c-kit and CD38. In the presence of varying combinations of TPO, Steel factor (SF), and interleukin-3 (IL-3), CD34+/c-kit(low)/CD38(neg/low) cells produced fewer colonies than CD34+/c-kit(low)/CD38(high) cells. However, when cultured in suspension for 7 days and replated in methylcellulose culture for measurement of colony-forming cells, the former population generated more colony-forming cells than the latter. In suspension culture of CD34+/c- kit(low)/CD38(neg/low) cells, TPO acted synergistically with SF and/or IL-3 in support of the production of colony-forming cells for granulocyte/macrophage colonies, erythroid colonies, and mixed colonies. Culture studies of individual CD34+/c-kit(low)/CD38(neg/low) cells provided the evidence for the direct nature of the effects of TPO. When combined with SF, TPO showed stronger stimulation of production of progenitors in suspension culture than other early-acting factors, such as IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF). TPO may be an important cytokine for in vitro manipulation of human hematopoietic stem cells.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT- 7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT- 7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c- mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NFE2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein, These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 μmol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not effected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 μmol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 μmol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 μg/mL), thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin- induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL- 3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

Thrombopoietin (TPO) is a recently identified hematopoietic growth factor that is essential for the growth and development of megakaryocytes. We have previously shown that TPO induces proliferation of acute myeloblastic leukemia (AML) cells in vitro. In this study, we have examined the expression of TPO and its receptor c-mpl in a series of AML cases and human leukemia cell lines. The mRNA transcripts of TPO were detectable in 18 of 50 AML cases and in some myeloid leukemia cell lines (HEL, M07E and CMK) by means of reverse transcriptase polymerase chain reaction (RT-PCR). In addition, TPO transcripts were coexpressed with c-mpl transcripts in 10 of 50 AML cases and in HEL, M07E and CMK cells. With regard to the French-American-British (FAB) classification, coexpression of TPO and c-mpl was observed with high frequency in AML cases of M7-type. Despite the TPO expression in a substantial fraction of leukemia cells, biological activity of TPO was not found in the conditioned medium that was obtained from cultivation of TPO mRNA-positive leukemia cells. These results suggest that TPO may not commonly participate in the abnormal growth of AML cells as an extracellular autocrine growth factor.

The prohormone convertases PC1/PC3 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. To determine the cellular and subcellular distribution of PC1/PC3 and PC2 in the rat pancreas, we generated their polyclonal antisera in rabbits, using as immunogens two synthetic peptide antigens corresponding to amino acids 442- 459 (ST-28) of PC1/PC3 and 613-629 (ST-29) of PC2 and two bacterially expressed antigens covering amino acids 145-414 (KN-1) of PC1/PC3 and 385- 637 (KN-2) of PC2. Western blot analysis revealed the presence of PC1/PC3 (87 and 68 kDa) and PC2 (75 and 70 kDa) in rat pancreatic islets, indicating that the antisera are specific for the corresponding antigens. Immunocytochemical staining of serial sections demonstrated that the antibody against PC1/PC3 immunostained only insulin-producing cells, whereas the PC2 antibody stained insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells. Double-immunolabeling of the prohormone convertases and pancreatic hormones with gold particles of different sizes revealed that insulin-positive secretory granules were also immunolabeled with PC1/PC3 and PC2 antibodies, whereas glucagon-, somatostatin-, or pancreatic polypeptide-positive granules were labeled only with the PC2 antibody. This differential localization of PC1/PC3 and PC2 provides a further problem on the substrate-specificity of these enzymes in the processing of pancreatic prohormones.

To clarify the regulatory mechanism of thrombopoietin (TPO, c-Mpl ligand) in chronic thrombocytopenic conditions, we determined TPO levels in the sera of patients with aplastic anaemia (AA; n = 26) and idiopathic thrombocytopenic purpura (ITP; n = 32) by an enzyme-linked immunosorbent assay. Despite a similarity in platelet counts, serum TPO levels in the AA group were markedly higher than those in the ITP group: 20.41 ± 9.71 f mol/ml (mean ± SD) and 1.66 ± 0.55 f mol/ml, respectively, both of which were significantly elevated compared to normal subjects (n = 41; 1.22 ± 0.37). In both groups, serum TPO level showed an inverse correlation with the platelet count. We determined the megakaryocyte volume using bone marrow clot section and found that it was markedly small in the AA group; while in the ITP group it was augmented with a correlation to serum TPO level. Our findings suggest that TPO levels may be regulated not only by platelets but also megakaryocytes in AA and ITP.

Using enzyme-linked immunosorbent assays, we measured the concentrations of serum thrombopoietin (TPO) and plasma glycocalicin, a proteolytic fragment of platelet glycoprotein Ibα, in 13 patients with myelodysplastic syndrome (MDS), aplastic anaemia (AA) or idiopathic thrombocytopenic purpura (ITP). In the patients with AA or MDS, the TPO concentrations were remarkably increased, and their glycocalicin concentrations were decreased compared with the normal control individuals. In the patients with ITP; however, the TPO and glycocalicin levels were not changed as much as in the AA/MDS patients in spite of the same degree of thrombocytopenia. During immunosuppressive treatment of ITP patients, there was an inverse relationship between the level of TPO and the platelet count. Thus, measurements of TPO and glycocalicin levels are useful for the diagnosis of thrombocytopenia, and our results from ITP patients did not support the model which suggested the simple feedback regulation of TPO in thrombocytopenia.

We examined the role of the ligand for c-mpl, thrombopoietin (TPO), in murine early haemopoiesis, using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

The serial change of various cytokines in the serum from a patient with cyclic thrombocytopenia is described, Interleukin 7, stem cell factor, and transforming growth factor β1 synchronized with the platelet count, and there was a significant positive correlation between the three cytokines and the platelet count. Levels of macrophage colony-stimulating factor, thrombopoietin, platelet-associated IgG and erythropoietin changed reciprocally with the platelet count, and there was a significant negative correlation between the platelet count and these cytokines except erythropoietin. No cyclic change was observed in IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor, or leukaemia inhibitory factor. These observations suggest that this disease involves two cyclic changes: megakaryocytopoiesis and platelet destruction, in both of which the cytokines play an important role.

Thrombopoietin (TPO) was purified from irradiated thrombocytopenic rat plasma. In the process of purification, some biochemical and biological characteristics were investigated. Rat plasma TPO was extremely hydrophobic and exhibited multiple peaks of activity on gel filtration. Both the low and high molecular weight fractions were separately subjected to further purification. Consequently, a rat TPO cDNA was cloned based on the amino acid sequences of purified rat plasma TPO. It revealed that each final purified rat plasma TPO was not a full-length form. In addition, rat hepatocytes and three rat hepatoma cell lines were found to produce rat TPO. Each native TPO derived from cultured cells was also partially purified, and hepatocyte-derived TPOs were shown to be heterogeneous in molecular weight. To study the structure of TPO, various recombinant TPO molecules were generated. Two disulfide bonds (Cys7-Cys151 and Cys29-Cys85) located in the N-terminal domain of TPO have an important effect on its biological activity. The human TPO muteins, sequentially deleted from the C-terminal, were expressed in COS-1 cells. TPO (1-151) was active, but TPO (1-150), which lacks Cys151, did not exhibit TPO activity. These findings indicate that the region essential for TPO activity is the N-terminal domain, which contains two disulfide bonds. Although the role(s) of the C-terminal domain is not clear at present, the potential N-glycosylation in the C-terminal domain is not directly required for exhibiting TPO activity. ©AlphaMed Press.

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra- and interassay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross-react with a variety of blood components and cytokines to produce false-positive results. The serum TPO concentrations from 29 normal males and 21 females were 0.79 ± 0.35 and 0.70 ± 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukaemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET (n = 6, 2.80 ± 1.55 fmol/ml) were higher than the normal level. The patients with AA (n = 7, 18.53 ± 12.37 fmol/ml) and ALL (n = 5, 10.36 ± 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system, The TPO level (mean ± SD) in ITP patients was mildly elevated (186 ± 1.17 fmol/ml) compared to that in normal subjects (0.76 ± 0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35 ± 642 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.

We examined the effects of recombinant human thrombopoietin (rhTPO) on myelosuppressive chemotherapy-induced thrombocytopenia in cynomolgus monkeys. After treatment with nimustine (ACNU) on day 0, the monkeys intravenously received rhTPO at a dose of 0.04, 0.2 or 1 μg/kg/d or monkey's serum once each day from day 1 to day 2.8. Administration of rhTPO reduced the severity of thrombocytopenia and accelerated the rate of platelet recovery in a dose-dependent fashion. Treatment with the highest rhTPO dose completely prevented thrombocytopenia and stimulated a marked increase in platelet counts over the normal values. Animals treated with ACNU also became neutropenic and slightly anaemic. Administration of rhTPO following ACNU treatment significantly improved neutropenia with increasing doses of rhTPO, but had no effect on anaemia. Compared to the control animals, rhTP0-treated animals exhibited no significant-changes in several serum parameters, C-reactive protein concentration and some blood coagulation profiles within the study period. These results suggest a therapeutic efficacy of rhTPO in improving chemotherapy-induced thrombocytopenia.

The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIlb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa+ population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/III- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIla+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

Thrombopoietin (TPO) stimulates proliferation and maturation of megakaryocytes in vivo and in vitro. In an attempt to better define the regulation and clinical significance of TPO concentrations, we measured endogenous TPO concentrations in 75 patients with acquired aplastic anemia (AA) and 12 normal controls by a sensitive ELJSA. The mean plasma TPO concentration is 1.3 ±0.5 fmoles/ml in normal controls. The plasma TPO concentrations were significantly higher in patients with AA than in normal controls (p<0.05). (VSAA: 26.5 ±10.5, SAA: 21.3 ±9.9, NSAA: 16.9 ±7.6 fmoles/ml, respectively). TPO levels were serially measured in 23 patients undergoing BMT and in 20 patients receiving immunosuppressive (IS) therapy. Although a decrease in TPO concentrations was observed in patients who achieved self-sustaining hematopoiesis following BMT, we did not find decreases of TPO levels in responders to IS therapy. Whether exogenous TPO will result in increased platelet counts in AA patients with high TPO levels remains to be determined.

To investigate the pathophysioJogical role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 87 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzymeimmunoassay procedure. The TPO level was negatively correlated with the platelet count in all 104 subjects investigated. The TPO levels was 0.84 ±0.04 f mol/mL in the normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission. When the data of normal individuals and patients with myelosuppression were combined as a control, TPO levels showed a significant negative correlation with the platelet count in this group (r= -0.768, p<0.0001). In aplastic anemia, acute lymphoblasuc leukemia, malignant lympnoma, multiple myeloma and chronic lymphocytic leukemia, the relationship between serum TPO levels and platelet counts was almost the same as in the control group. However, the TPO levels were slightly higher in myelpproliferative disorders, and lower in acute myelogenous leukemia, hypoplastic leukemia, myelodysplastic syndrome, liver cirrhosis and idiopaUiic thrombocytopenic purpura, when compared with the regression line for the control group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.

We developed a sandwich enzyme-linked imrnuiiosorbent assay (ELISA) to quantify serum levels of human thrombopoietin (TPO) by using monoclonal and polyclonal antibodies. The. ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intraand inter-assay variation were from 3.0 10 4.9 % and from 5.9 to 6.1 %. respectively, This quantitative limit of the ELISA was 0.09 f moVml m scrum, which represents 3.0 pg of a fulllength TPO peptide in I ml scrum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with hematopoieüc disorders were parallel to the standard curves. The ELISA did no! cross-react with a variety of Wood components and seven other cytokines including rhIL-3. rhlL-6. rhlL-7. rhG-CSF. rhGMCSF, rhEPO and rhSCF. The addition of extracellular domain of (he human c-MpI that may coexist in the blood samples had only a minimal effect on the ELISA. The estimated TPO levels m normal donors and in patients with hematopoietic disorders using the ELISA were as follows. The serum TPO concentrations from 29 normal males aged 24-38 (mean, 31 years) and 21 females aged 20-35 (mean, 26 years) were 0.79±0.35 <mean±SD; range 0.33 10 1.72) and 0.70±0.26 fmol/ml (range 0.33 to 1.33), respectively. No statistical difference in the serum TPO levels between males and females was noted (P-0.710). m addition, there was no statistically significant correlation between the serum TPO levels and platelet counts in the normal individuals. The serum TPO levels in the patients with idiopathic thromboeylopenic purpura (n-5, î.70±0.51 fmol/ml) were higher than the normal level. The patients with plastic anemia (n-7, 18.53± 12.37 fmol/ml) and acute lymphocytic leukemia (n-5, I0.36±5.57 fmoi/ml) had significantly higher serum TPO levels than the normal. Interestingly, in patients with essential thrombocythemia whose platelet counts were increased, the TPO lvcls(n-6, 2.80+L55 fmol/ml) were not decreased but elevated above normal. The established ELISA specific to TPO should prove useful to measure the TPO concentration in serum samples.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin (TPO). To establish a model system for analyzing functional domains of c-Mpl, we searched for a cell line not expressing endogenous c-Mpl. Previously we reported that c-mpl mRNA could not be detected in a human EPO-responsive cell line, UT7/EPO (Blood 82:456,1993; Blood in press). This was confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Based on these observations, we prepared an expression vector containingfulllengthc-mp/ cDNA and transfected it into the UT-7/EPO cells. We isolated 3 clones and examined whether these clones exogenously expressed c-Mpl on the surface of the cells by FACS analysis with an anti-c-Mpl antibody. All these transfectants expressed c-Mpl and grew in response to TPO. Moreover, after incubation with TPO for one week, some of the cells became large and multinucleated, and expressed platelet factor-4 mRNA, suggesting that the TPO signalling pathway mediated through c-Mpl is involved in not only the proliferation but also megakaryocytic differentiation of the transfectants. Therefore, this model system may be useful for analyzing the functional domains of human c-Mpl. We are now preparing several kinds of deletion mutants.

The administration of thrombopoietin (TPO) to mice after chemotherapy and total body irradiation not only promotes recovery from chemotherapy-induced thrombocytopenia, but also relieves anemia. In addition, TPO has burst promoting activity (BPA) in vitro. These observations suggest the influence of TPO on erythropoiesis. To elucidate whether TPO has a direct effect on erythroid differentiation, we established a subclone of a human cytokine-dependent cell line UT7, UT-7/GM Hl. UT-7/GM HI is maintained by GM-CSF and can grow in response to erythropoietin (EPO) or TPO. After a 1 week culture in IMDM medium containing 10% FCS in the presence of I U/ml of EPO or 10 ng/ml of TPO, more than 90 % and 45 % of UT-7/GM HI cells, respectively, were dianisidine-stained. Even after treatment of UT7/GM HI cells with up to 100 ng/ml of TPO, the EPO receptor was not tyrosine phosphorylated. These findings suggest that TPO directly induced erythroid differentiation and that the effect was not achieved via an erythropoietin receptor.

We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst- promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony- stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO.

Thrombopoietin (TPO) is e newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto- oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c- mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1, 100 rats by measuring the production of megakaryo-cytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49%108, respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl. © 1995 by the Journal of Biochemistry.

A cDNA for the thrombopoietin has been cloned by several groups. The recombinant thrombopoietin has been reported to stimulate the megakaryocytopoiesis and thrombopoiesis. Little is known regarding the molecular basis of its effects. To elucidate the molecular mechanism involved in signal transduction, we have investigated the effects of thrombopoietin on platelet tyrosine phosphorylation. We report here that thrombopoietin induced time- and dose-dependent tyrosine phosphorylation of several proteins including Janus kinase 2 (Jak2) and a 52-kD protein, Shc, in human blood platelets. Both Jak2 and Shc were tyrosine phosphorylated within 15 seconds after stimulation. The tyrosine phosphorylation of Jak2 was accompanied by increased kinase activity, whereas Shc tyrosine phosphorylation induced its association with a 25-kD protein, Grb2. Thus, our data suggest that Jak2, Shc, and Grb2 may be involved in signal transduction after ligand binding to c-mpl in human platelets.

Overlapping cDNA clones encoding rat thrombopoietin (TPO) were isolated from liver-derived cell line cDNA libraries and the nucleotide sequences were determined. The deduced 326-amino-acid rat TPO showed significant homology to the known TPO of other species, especially in the N-terminal sequence. © 1995.

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5x104 cells/ml (104 cells/well) with test samples in 96-well tissue culture plates for 4 days at 37°C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples-including a wide variety of cytokines and unknown growth factors, alone or in combinations-and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

Platelet formation, occurring from bone marrow or lung megakaryocytes, has been difficult to study mechanistically. Recombinant human megakaryocyte growth and development factor (rHuMGDF), a recently described cytokine, has now been used to establish an in vitro system in which this important and little understood process occurs. CD34+ cells cultured with rHuMGDF develop into megakaryocytes which form long cytoplasmic extensions (proplatelets) that fragment into platelet‐sized particles (in vitro platelets). Morphologically, in vitro and human plasma‐derived platelets (control platelets) are virtually identical with respect to size, dense granule distribution and ultrastructural features. Functionally, in vitro and control platelets have similar aggregation and activation responses, and similarly incorporate mepacrine into dense granules. These findings suggest that rHuMGDF is sufficient to generate platelet‐synthesizing megakaryocytes from CD34+ cells and provide an experimental setting in which the study of human platelet formation can be adequately performed. Copyright © 1995 AlphaMed Press

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

Thrombopoietin (TPO) is a cytokine which can support the proliferation and differentiation of megakaryocyte progenitor cells, and the maturation of megakaryocytes. We show here that mitogen-activated protein (MAP) kinases, Erk1 and Erk2, are involved in TPO signal transduction in the human TPO-dependent megakaryocytic cell line, UT-7/TPO. TPO induced tyrosine phosphorylation of Erk1 and Erk2 proteins in a dose and time-dependent manner. Moreover, the activation of MAP kinases was actually induced by TPO. These results suggest that MAP kinase activation is involved in the signalling pathway of TPO, as it is for other cytokines, one of which is erythropoietin. © 1995 by Academic Press, Inc.

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

The complete gene for human thrombopoietin (TPO) has been cloned by screening a human genomic library using human TPO cDNA as a probe. This gene is 6.2 kb in length and contains six exons and five introns. It is shown that the human genome contains a single copy of the human TPO gene according to Southern blotting analysis. The transcription initiation site was determined by S1 nuclease mapping. The human TPO gene expressed TPO activity when transfected into COS-1 cells. The human TPO gene has been mapped to chromosome 3q27 by in situ hybridization using a biotin-labeled probe. © 1994.

Cross-linking of the high affinity receptor for IgE (FcεRI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the FcεRI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on FcεRI cross- linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast cells, despite their responsiveness to these cytokines. SPY75 was not physically associated with the receptor or other known signaling molecules. This protein, the mouse homologue of the human HS1 gene product, has putative repetitive helix-turn-helix motifs found in many DNA-binding proteins and a putative nuclear transport signal. It also has a Src homology 3 domain, which is found in many signaling molecules and cytoskeletal proteins. These structural features and the rapid tyrosine phosphorylation on FcεRI cross-linking suggest that the signal generated by FcεRI cross-linking is transmitted through tyrosine phosphorylation of SPY75.

Two novel tyrosine kinase cDNAs were obtained from murine mast cells. These kinases, Emb and Emt, constitute a novel tyrosine kinase subfamily which may also include Tec, a kinase preferentially expressed in liver, and Dsrc28, a fruit fly kinase. Both lack hydrophobic stretches characteristic of the transmembrane domains found in growth factor receptor tyrosine kinases and carboxyl-terminal, negative regulatory tyrosine residue found in Src family kinases. In addition to the Src homology region 2 (SH2) and SH3 domains characteristic of the Src family kinases and other signaling molecules, Emb and Emt share a similar amino-terminal domain comprised mainly of two repeat segments. The emb 2.7-kb transcript was expressed in mast cells, myeloid cells and B lymphocytes while the emt 4.6-kb mRNA in mast cells, myeloid cells and T lymphocytes. The evidence for in vitro tyrosine kinase activity of Emb and Emt proteins is also provided. © 1993 Academic Press.

Several murine monoclonal antibodies (MAbs) specific for human erythropoietin (HuEpo) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag.8-653, with the splenocytes of mice immunized with recombinant human Epo (rHuEpo). Based on epitope analysis by a competitive binding assay, these MAbs could be classified into at least three groups: (1) 1E10, (2) 1H7, (3) 2D6, 3D6 and 3D8. In a sandwich enzyme-linked immunosorbent assay (ELISA), using these MAbs as the solid-phase antibodies, MAb-bound HuEpo was detected with rabbit anti-HuEpo sera. Some combinations of two different classes of MAbs, such as 1H7 and 3D8, were found to capture much more HuEpo than each MAb used individually. Urinary HuEpo (U-HuEpo) was highly purified from the urine of patients with severe aplastic anemia with about 50% final recovery using an immunoaffinity column on which a mixture of 1H7 and 3D8 was immobilized. The purified U-HuEpo had a specific activity of 77 340 U/mg in a radioimmunoassay (RIA) and of 76 673 U/mg using an in vivo bioassay. © 1988.

The asparagine-linked sugar chains of human erythropoietin produced by recombinant Chinese hamster ovary cells and naturally occurring human urinary erythropoietin were liberated by hydrazinolysis and fractionated by paper electrophoresis, lectin affinity chromatography, and Bio-Gel P-4 column chromatography. Both erythropoietins had three asparagine-linked sugar chains in one molecule, all of which were acidic complex type. Structural analysis of them revealed that the sugar chains from both erythropoietins are quite similar except for sialyl linkage. All sugar chains of erythropoietin produced by recombinant Chinese hamster ovary cells contain only the NeuAcα2→3Gal linkage, while those of human urinary erythropoietin contain the NeuAcα2→6Gal linkage together with the NeuAcα2→3Gal linkage. The major sugar chains were of fucosylated tetraantennary complex type with and without N-acetyllactosamine repeating units in their outer chain moieties in common, and small amounts of 2,4- and 2,6-branched triantennary and biantennary sugar chains were detected. This paper proved, for the first time, that recombinant technique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar chains of the native one.

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU‐E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt‐genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt‐genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU‐E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt‐genes may be induced after the stimulation of erythropoietin. Copyright © 1986, Wiley Blackwell. All rights reserved

Two types of human cancers, a renal cell carcinoma and a hepatocellular carcinoma, were investigated in vitro; both produced a marked erythrocytosis in each patient. These tumors, when transplanted into athymic nude mice, produced a remarkable erythrocytosis in the host mice. To analyze this phenomenon, the primary cultures from these xenotransplanted tumors were performed. To obtain pure tumor cell cultures, cells derived from host nude mice were eliminated by the treatment with the antiserum raised against nude mouse cells. Epithelial cells derived from each tumor attached and grew in the cultures. The conditioned media from both tumor cells revealed high erythropoietic stimulatory activities. We have characterized these erythropoietin‐like activities by size‐exclusion high‐performance liquid chromatography. Three peaks of erythropoietin‐like activities were noted after bovine serum albumin region. The molecular weights were estimated at about 55,000, 40,000, and 33,000, respectively. The results suggested that the human renal cell and hepatocellular carcinomas produced erythropoietin‐like activities in vitro in culture and that erythrocytosis found in patients with cancer and in nude mice transplanted with the tumors was attributable to production of the erythropoietin‐like activities by the tumor cells themselves. Copyright © 1985 American Cancer Society