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1.
Biotechnol J ; 5(4): 385-92, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20235145

ABSTRACT

The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by beta-catenin protein transduction. Constitutively active beta-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active beta-catenin protein was added to HEK-293 cells, and induction of several Wnt/beta-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active beta-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active beta-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.


Subject(s)
Drug Carriers/chemistry , Kidney/physiology , Polyethyleneimine/chemistry , Signal Transduction/physiology , Transduction, Genetic/methods , Wnt Proteins/metabolism , beta Catenin/metabolism , Cations , Cell Line , Transfection/methods , beta Catenin/genetics
2.
Stem Cells Dev ; 18(7): 1081-92, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19025339

ABSTRACT

Thrombopoietin interactions with its receptor, Mpl, play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation. In this study, we report that the mast cell restricted progenitor cells (MCP) and the mast cell precursors in the bone marrow of wild-type mice express Mpl on their surface. Furthermore, targeted deletion of the Mpl gene in mice decreases the number of MCP while increasing the number of mast cell precursors present in the marrow and spleen. It also increases the number of mast cells present in the dermis, in the peritoneal cavity, and in the gut of the mice. In addition, serosal mast cells from Mpl(null) mice have a distinctive differentiation profile similar to that expressed by wild-type dermal mast cells. These results suggest that not only does ligation of thrombopoietin with the Mpl receptor exert an effect at the mast cell restricted progenitor cell level, but also plays an unexpected yet important role in mast cell maturation.


Subject(s)
Cell Differentiation , Dermis/cytology , Hematopoietic Stem Cells/cytology , Mast Cells/cytology , Receptors, Thrombopoietin , Animals , Bone Marrow/metabolism , Cell Proliferation , Dermis/metabolism , Hematopoietic Stem Cells/metabolism , Intestinal Mucosa/metabolism , Intestines/cytology , Mast Cells/metabolism , Mice , Mice, Mutant Strains , Organ Specificity/genetics , Peritoneal Cavity/cytology , Spleen/cytology , Spleen/metabolism , Thrombopoietin/genetics , Thrombopoietin/metabolism
3.
Biochem Biophys Res Commun ; 377(1): 205-9, 2008 Dec 05.
Article in English | MEDLINE | ID: mdl-18840400

ABSTRACT

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.


Subject(s)
Aorta/metabolism , Gene Expression Regulation, Developmental , Glypicans/genetics , Gonads/metabolism , Hematopoiesis/genetics , Mesonephros/metabolism , Animals , Aorta/cytology , Aorta/embryology , Cell Line , Cell Proliferation , Fetal Blood/cytology , Fetal Blood/metabolism , Gene Expression Profiling , Glypicans/physiology , Gonads/cytology , Gonads/embryology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Mesonephros/cytology , Mice , Stromal Cells/cytology , Stromal Cells/metabolism , Transfection
4.
J Hum Genet ; 53(5): 447-453, 2008.
Article in English | MEDLINE | ID: mdl-18322642

ABSTRACT

Gene therapy using cDNA driven by an exogenous promoter is not suited for genetic disorders that require intrinsic expression of a transgene, such as hyperimmunoglobulin (Ig)M syndrome (HIGM), which is caused by mutations in the CD40L gene. The human artificial chromosome (HAC) vector has the potential to solve this problem, because it can be used to transfer large genomic fragments containing their own regulatory elements. In this study, we examined whether introduction of a genomic fragment of CD40L via the HAC vector permits intrinsic expression of the transgene and has an effect on immunoglobulin secretion. We constructed an HAC vector carrying the mouse CD40L genomic fragment (mCD40L-HAC) in Chinese hamster ovary (CHO) cells and transferred the mCD40L-HAC vector into a human CD4-positive active T-cell line (Jurkat) and a human myeloid cell line (U937) via microcell-mediated chromosome transfer (MMCT). The mCD40L-HAC vector permits mCD40L expression in human active T cells but not in human myeloid cells. The mCD40L-HAC also functions to stimulate mouse B cells derived from CD40L(-/-) mice, inducing secretion of IgG. This study may be an initial step toward the therapeutic application of HAC vectors for intrinsic expression of genes, a potential new direction for genome-based gene therapy.


Subject(s)
CD40 Ligand/genetics , Chromosomes, Artificial, Human/genetics , Gene Transfer Techniques , Genetic Vectors , Genome, Human/genetics , Immunoglobulins/biosynthesis , Animals , CD40 Ligand/physiology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Humans , Hypergammaglobulinemia/genetics , Hypergammaglobulinemia/metabolism , Jurkat Cells , Mice , Mice, Knockout , U937 Cells
5.
Stem Cells ; 26(4): 912-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18276801

ABSTRACT

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow-derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.


Subject(s)
Cell Differentiation/physiology , Mast Cells/cytology , Mast Cells/physiology , Stem Cells/cytology , Stem Cells/physiology , Thrombopoietin/physiology , Animals , Cell Differentiation/drug effects , Cells, Cultured , Male , Mast Cells/drug effects , Mice , Mice, Knockout , Stem Cells/drug effects
6.
Cancer Sci ; 98(10): 1597-603, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17645774

ABSTRACT

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.


Subject(s)
Cytokine Receptor gp130/pharmacology , Dendritic Cells/metabolism , Erythroid Precursor Cells/metabolism , Membrane Proteins/pharmacology , Myeloid Progenitor Cells/metabolism , Receptors, Interleukin-6/metabolism , Receptors, Notch/metabolism , Cells, Cultured , Flow Cytometry , Granulocytes/metabolism , Humans , Interleukin-3/metabolism , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins , Ligands , Monocytes/metabolism
7.
Biochem Biophys Res Commun ; 353(4): 992-8, 2007 Feb 23.
Article in English | MEDLINE | ID: mdl-17214966

ABSTRACT

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.


Subject(s)
Gene Silencing , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Chromatin Immunoprecipitation , CpG Islands/genetics , DNA Methylation , Gene Expression , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/genetics , Mice , Mice, Knockout , NIH 3T3 Cells , Nuclear Proteins/genetics , Polycomb Repressive Complex 1 , Polycomb-Group Proteins , Protein Binding , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Two-Hybrid System Techniques
8.
Stem Cells ; 24(11): 2456-65, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16857897

ABSTRACT

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice, which showed myeloid, B, T, and natural killer cells as well as CD133(+)CD34(+) cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.


Subject(s)
Cell Proliferation , Hematopoietic Stem Cells , Membrane Proteins/metabolism , Receptors, Fc/metabolism , AC133 Antigen , ADP-ribosyl Cyclase 1/analysis , Animals , Antigens, CD/analysis , Antigens, CD34/analysis , Cell Differentiation/drug effects , Cell Lineage , Cell Proliferation/drug effects , Cells, Cultured , Cytokine Receptor gp130/metabolism , Fetal Blood/cytology , Fetal Blood/immunology , Fetal Blood/metabolism , Glycoproteins/analysis , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Immunomagnetic Separation , Interleukin-3/metabolism , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Peptides/analysis , Receptors, Fc/genetics , Receptors, Interleukin/metabolism , Receptors, Interleukin-6 , Receptors, Notch/metabolism , Recombinant Fusion Proteins/metabolism
9.
Proc Natl Acad Sci U S A ; 103(7): 2202-7, 2006 Feb 14.
Article in English | MEDLINE | ID: mdl-16461905

ABSTRACT

The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a Gata2 promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following Gata2-directed GFP fluorescence, and that they can also be monitored in vivo. Each individual GFP-positive cell lay in a G0/G1 cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered.


Subject(s)
Bone Marrow Cells/cytology , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Hematopoietic Stem Cells/cytology , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Ataxin-1 , Ataxins , Bone Marrow Cells/physiology , Cell Cycle/genetics , Cell Differentiation/genetics , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/metabolism , Mice , Mice, Mutant Strains , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Osteoblasts/cytology , Osteoblasts/metabolism , Promoter Regions, Genetic
10.
J Biol Chem ; 281(7): 4495-506, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16354670

ABSTRACT

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to alpha-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.


Subject(s)
Endosomes/physiology , Lysosomes/physiology , Q-SNARE Proteins/physiology , Amino Acid Sequence , Animals , Apoptosis , Lipofuscin/biosynthesis , Mice , Molecular Sequence Data , NIH 3T3 Cells , Q-SNARE Proteins/chemistry , R-SNARE Proteins/physiology , RNA, Small Interfering/pharmacology
11.
J Hum Genet ; 51(2): 147-150, 2006.
Article in English | MEDLINE | ID: mdl-16333524

ABSTRACT

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.


Subject(s)
Chromosomes, Artificial, Human/genetics , Chromosomes, Human, Pair 21/genetics , Cord Blood Stem Cell Transplantation/methods , Gene Expression , Genetic Therapy/methods , Hematopoietic Stem Cells/metabolism , Animals , DNA Primers , Flow Cytometry , Genetic Vectors/genetics , Gentamicins , Green Fluorescent Proteins/metabolism , Humans , Mice
12.
Cell Immunol ; 234(2): 77-80, 2005 Apr.
Article in English | MEDLINE | ID: mdl-16095582

ABSTRACT

Notch signaling is required for the early steps in T cell development. However, distribution of Notch ligands in the thymus anlages is not clear. We investigated the expressions of Delta like (Dll) 1 and Dll4 in the mouse thymus anlages. In the normal thymus anlages on embryonic day 13, Dll4 is strongly expressed throughout the epithelial region, but Dll1 is expressed only in scattered cells. In contrast, epithelial cells of the nude thymus anlages express neither Dll1 nor Dll4. These results indicate that expressions of Dll1 and Dll4 in thymic epithelial cells are regulated by Foxn1 transcriptional factor.


Subject(s)
Embryonic Structures , Membrane Proteins/metabolism , Thymus Gland , Animals , Cells, Cultured , DNA-Binding Proteins , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Forkhead Transcription Factors , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , In Situ Hybridization , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/genetics , Mice , Mice, Nude , Receptors, Notch , Signal Transduction/physiology , Thymus Gland/cytology , Thymus Gland/embryology , Transcription Factors
13.
J Biochem ; 137(6): 693-701, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16002991

ABSTRACT

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.


Subject(s)
Biotechnology/methods , Polyethyleneimine/metabolism , Protein Transport , Animals , Avidin , BALB 3T3 Cells , Biotinylation , Carrier Proteins/metabolism , Cations/chemistry , Cations/metabolism , Cell Death , Fibroblasts , Humans , Mice , Microscopy, Confocal , Nerve Tissue Proteins , Polyethyleneimine/chemistry , Ribonuclease, Pancreatic , Streptavidin
14.
Blood ; 105(9): 3493-501, 2005 May 01.
Article in English | MEDLINE | ID: mdl-15665119

ABSTRACT

Idiopathic myelofibrosis (IM) is a disease characterized by marrow fibrosis, abnormal stem/progenitor cell trafficking, and extramedullary hematopoiesis frequently associated with alterations in megakaryocytes (Mks). Mice harboring genetic alterations in either the extrinsic (ectopic thrombopoietin expression, TPO(high) mice) or intrinsic (hypomorphic GATA-1 mutation, GATA-1(low) mice) control of Mk differentiation develop myelofibrosis, a syndrome similar to IM. The relationship, if any, between the pathobiologic mechanism leading to the development of myelofibrosis in the 2 animal models is not understood. Here we show that plasma from GATA-1(low) mice contained normal levels of TPO. On the other hand, Mks from TPO-treated wild-type animals (TPO(high) mice), as those from GATA-1(low) animals, had similar morphologic abnormalities and contained low GATA-1. In both animal models, development of myelofibrosis was associated with high transforming growth factor beta1 (TGF-beta1) content in extracellular fluids of marrow and spleen. Surprisingly, TPO treatment of GATA-1(low) mice restored the GATA-1 content in Mks and halted both defective thrombocytopoiesis and fibrosis. These data indicate that the TPO(high) and GATA-1(low) alterations are linked in an upstream-downstream relationship along a pathobiologic pathway leading to development of myelofibrosis in mice and, possibly, of IM in humans.


Subject(s)
DNA-Binding Proteins/metabolism , Primary Myelofibrosis/etiology , Signal Transduction , Thrombopoietin/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Animals , Bone Marrow/pathology , Cell Differentiation , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Megakaryocytes/pathology , Mice , Mice, Inbred Strains , Models, Animal , Mutation , Primary Myelofibrosis/genetics , Primary Myelofibrosis/pathology , Spleen/pathology , Thrombopoietin/analysis , Thrombopoietin/genetics , Transcription Factors/analysis , Transcription Factors/genetics , Transforming Growth Factor beta/analysis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1
15.
Carcinogenesis ; 25(11): 2239-46, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15297369

ABSTRACT

Inbred mice differ dramatically in their sensitivity to the colon carcinogen, azoxymethane (AOM). Identifying genes associated with this differential susceptibility in mice may ultimately reveal molecular mechanisms responsible for colon carcinogenesis. A cDNA array approach was taken to study gene expression changes induced by AOM in the colons of sensitive (A/J) and resistant (AKR) mice. Among the genes represented on the array, pre-adipocyte factor 1 (Pref-1), associated previously with suppression of adipocyte differentiation, was induced specifically by AOM in the distal colons of sensitive A/J mice (5.4-fold). Reverse transcription-PCR followed by sequence analysis revealed the presence of four alternative splice variants of Pref-1 mRNA in the colon. The potential significance of Pref-1 in colon tumorigenesis was explored in colon cancer cells infected with a retroviral construct containing the major splice variant. Over-expression of Pref-1A in HT-29 cells led to a marked resistance to butyrate-induced differentiation and growth inhibition. Our data indicate that Pref-1, a protein that suppresses differentiation and promotes colonocyte growth, may account in part for the sensitivity of A/J mice to AOM-induced carcinogenesis. In addition, detection of Pref-1 in a human colon tumor cell line suggests that it may also participate in human colon tumorigenesis.


Subject(s)
Azoxymethane/pharmacology , Cell Differentiation/drug effects , Intercellular Signaling Peptides and Proteins/physiology , Intestinal Mucosa/cytology , Alkaline Phosphatase/metabolism , Animals , Butyrates/pharmacology , Calcium-Binding Proteins , Carcinogens/pharmacology , Caspase 3/metabolism , Colon , DNA Primers , Intercellular Signaling Peptides and Proteins/genetics , Intestinal Mucosa/drug effects , Intestinal Mucosa/physiology , Male , Mice , Mice, Inbred A , Mice, Inbred AKR , Reverse Transcriptase Polymerase Chain Reaction
16.
Int J Hematol ; 79(4): 328-33, 2004 May.
Article in English | MEDLINE | ID: mdl-15218959

ABSTRACT

Using the intra-bone marrow injection (IBMI) technique, we recently identified human cord blood-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid reconstituting ability. In this study, we further investigated the hematopoietic stem cell (HSC) characteristics of these cells in terms of proliferative and migratory potentials. The absolute numbers of CD45+ and CD34+ cells generated by 1 CD34- SRC are significantly higher than those generated by 1 CD34+ SRC. It is interesting that CD34- SRCs have significantly higher migratory and proliferative abilities than CD34+ SRCs. Moreover, only 2 CD34- SRCs transplanted to primary recipients consistently showed secondary reconstituting capacity. This finding suggested the more homogenous nature of CD34- SRCs than that of the population of CD34+ SRCs. These results provided further evidence that CD34- SRCs are functionally different from CD34+ SRCs and that they are a distinct class of primitive HSCs.


Subject(s)
Cord Blood Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Regeneration , Severe Combined Immunodeficiency/pathology , Animals , Antigens, CD34/analysis , Bone Marrow/physiology , Cell Count , Cell Division , Cell Movement , Hematopoietic Stem Cells/physiology , Humans , Mice , Mice, SCID , Transplantation, Heterologous
17.
Biochem Biophys Res Commun ; 308(4): 927-32, 2003 Sep 05.
Article in English | MEDLINE | ID: mdl-12927808

ABSTRACT

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.


Subject(s)
Cell Nucleus/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Proto-Oncogene Proteins/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Line , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport , Humans , Luciferases/metabolism , Mice , Microscopy, Fluorescence , Plasmids/metabolism , Precipitin Tests , Protein Binding , Proto-Oncogene Proteins c-mdm2 , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , Two-Hybrid System Techniques , Ubiquitin/metabolism
18.
J Cell Sci ; 116(Pt 9): 1775-86, 2003 May 01.
Article in English | MEDLINE | ID: mdl-12665558

ABSTRACT

Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk+ cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk+ cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk+ cells contained both albumin+ and cytokeratin 19+ cells, indicating that a majority of colony-forming Dlk+ cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition, numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk+ cells under this condition. Furthermore, 7% of the colony-forming Dlk+ cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk+ cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk+ cells differentiated into hepatocytes in vivo. These results indicate that Dlk+ cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.


Subject(s)
Hepatocytes/cytology , Hepatocytes/metabolism , Membrane Proteins/metabolism , Repressor Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Animals , Base Sequence , Calcium-Binding Proteins , Cell Division , Cell Separation/methods , DNA, Complementary/genetics , Fetus/cytology , Fetus/metabolism , Gene Expression , Gestational Age , Hepatocytes/transplantation , Intercellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Microscopy, Electron , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation
19.
Blood ; 101(5): 1777-83, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12406868

ABSTRACT

Mouse long-term hematopoietic reconstituting cells exist in the c-Kit+Sca-1+Lin- (KSL) cell population; among them, CD34(low/-) cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34(low/-)c-Kit+Sca-1+Lin- (34-KSL) cells with the HES-1 gene, which encodes a basic helix-loop-helix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34-KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34(low/-) profile in the bone marrow KSL population in each recipient mouse at ratios 3.5- and 7.8-fold those produced by nontransduced 34-KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34-KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34-KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells.


Subject(s)
Hematopoietic Stem Cells/cytology , Homeodomain Proteins/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Lineage , Cell Survival , Cells, Cultured/cytology , Genes, Reporter , Green Fluorescent Proteins , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/classification , Homeodomain Proteins/genetics , Luminescent Proteins/analysis , Mice , Mice, Inbred C57BL , Phenotype , Radiation Chimera , Recombinant Fusion Proteins/physiology , Transcription Factor HES-1 , Transduction, Genetic
20.
Dev Growth Differ ; 44(1): 63-75, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11869293

ABSTRACT

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.


Subject(s)
Hematopoiesis/physiology , Neoplasm Proteins , Proto-Oncogene Proteins/physiology , Receptors, Cytokine , Signal Transduction , Thrombopoietin/physiology , Animals , Rats , Receptors, Thrombopoietin , Signal Transduction/physiology , Xenopus laevis
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