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1.
Appl Environ Microbiol ; 73(15): 4805-12, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17557860

ABSTRACT

Human beta-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of alpha- and beta-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using alpha-1,6-mannosyltransferase-deficient (och1Delta) yeast as the host. Genes encoding the alpha- and beta-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (alphaalpha) and HexB (betabeta). A total of 57 mg of beta-hexosaminidase isozymes, of which 13 mg was HexA (alphabeta), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the beta-subunit. The purified HexA was treated with alpha-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 +/- 0.1 and 1.7 +/- 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the beta-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.


Subject(s)
Recombinant Proteins/biosynthesis , Saccharomycetales/enzymology , Sandhoff Disease/enzymology , Tay-Sachs Disease/enzymology , beta-N-Acetylhexosaminidases/biosynthesis , Amino Acid Sequence , Biotechnology/methods , Cells, Cultured , Fibroblasts/enzymology , Hexosaminidase A , Hexosaminidase B , Humans , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Saccharomycetales/genetics , Sandhoff Disease/therapy , Tay-Sachs Disease/therapy , beta-N-Acetylhexosaminidases/genetics , beta-N-Acetylhexosaminidases/therapeutic use
2.
Lab Invest ; 87(6): 540-7, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17401435

ABSTRACT

An essential coagulation factor, tissue factor (TF), is rapidly expressed by human monocytes when exposed to a variety of agonists, such as lipopolysaccharide or tumor necrosis factor (TNF). We previously found that 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its potent synthetic analogs downregulate TF and upregulate thrombomodulin expression on monocytic cells, counteracting the effects of TNF at the level of transcription. The human TF gene has characteristic binding sequences for activator protein-1 (AP-1) (c-Jun/c-Fos), nuclear factor-kappaB (NF-kappaB), Sp-1, and early growth response factor-1 (Egr-1). In this study, we investigated the regulatory mechanisms by which 1,25(OH)(2)D(3) inhibits TNF-induced TF expression in human monocytic cells. 1,25(OH)(2)D(3) reduced basal and TNF-induced TF activities. Gel-shift assay and luciferase assay with the respective reporter vectors showed that 1,25(OH)(2)D(3) reduced basal and TNF-induced activities of the nuclear proteins AP-1 and NF-kappaB, but not Egr-1. 1,25(OH)(2)D(3) inhibited TNF-induced phosphorylation of c-Jun without affecting phosphorylation of the other pathways. On the other hand, 1,25(OH)(2)D(3) directly inhibited nuclear binding and activities of NF-kappaB in the nucleus without affecting phosphorylation of the NF-kappaB activation pathway. These results indicate that 1,25(OH)(2)D(3) suppresses basal and TNF-induced TF expression in monocytic cells by inhibition of AP-1 and NF-kappaB activation pathways, but not of Egr-1. Our results may help to elucidate the regulatory mechanisms of 1,25(OH)(2)D(3) in TF induction, and may have physiological significance in the clinical challenge to use potential 1,25(OH)(2)D(3) analogs in antithrombotic therapy as well as immunomodulation and antineoplastic therapy of leukemia.


Subject(s)
Dihydrotachysterol/analogs & derivatives , Monocytes/metabolism , NF-kappa B/antagonists & inhibitors , Thromboplastin/antagonists & inhibitors , Transcription Factor AP-1/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Cells, Cultured , Dihydrotachysterol/pharmacology , Gene Expression Regulation/drug effects , Humans , Monocytes/drug effects , Thromboplastin/genetics
3.
Clin Chim Acta ; 378(1-2): 142-6, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17204262

ABSTRACT

BACKGROUND: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically. METHODS: Cultured fibroblasts from I-cell disease patients were double-stained with a monoclonal antibody to lysosome-associated membrane protein-1 (LAMP-1) and that to GM2 ganglioside, or a series of lectins that specifically bind to sugar moieties. RESULTS: The patients' cells were granularly stained with the antibody to GM2 ganglioside and the lectins including Maakia amurensis, Datura stramonium, and concanavalin A. Their localization was coincident with that of LAMP-1. CONCLUSIONS: GM2 ganglioside and various kinds of glycoconjugates having sialic acidalpha2-3galactose, galactosebeta1-4N-acetylglucosamine and mannose residues accumulate in enlarged lysosomes in I-cell disease fibroblasts.


Subject(s)
Fibroblasts/chemistry , G(M2) Ganglioside/analysis , Lysosomal Membrane Proteins/analysis , Mucolipidoses/metabolism , Cells, Cultured , Female , Histocytochemistry , Humans , Infant , Lectins/analysis , Lysosomes/enzymology , Microscopy, Electron , Mucolipidoses/pathology , Skin/ultrastructure
4.
J Biochem ; 140(2): 193-200, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16829682

ABSTRACT

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.


Subject(s)
Abnormalities, Multiple/metabolism , Connective Tissue Diseases/genetics , Elastin/metabolism , Fibroblasts/metabolism , Tropoelastin/metabolism , Abnormalities, Multiple/genetics , Adolescent , Animals , Asian People , Cattle , Cell Proliferation , DNA, Complementary/genetics , Elastin/genetics , Female , Fibroblasts/cytology , Gene Expression Regulation , Humans , RNA, Messenger/metabolism , Skin/cytology , Transduction, Genetic , Tropoelastin/genetics
5.
Clin Chim Acta ; 372(1-2): 154-7, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16753138

ABSTRACT

BACKGROUND: A unique adult male patient who developed cardiomyopathy was first suspected to have cardiac Fabry disease based on the pathological findings in heart tissues obtained on biopsy, but the alpha-galactosidase activity in his leukocytes was normal and no mutation was detected in the coding region of the alpha-galactosidase gene. We identified accumulated materials in the myocardium of this patient. METHODS: Pathological and biochemical analyses were performed using the autopsied heart tissues as samples. RESULTS: Although numerous lamellar and concentric inclusion bodies were ultrastructurally found in the autopsied myocardium, the alpha-galactosidase activity in the heart tissues was not decreased. Lipid analysis revealed the accumulation of phospholipids including phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, but not globotriaosylcereamide or gangliosides. CONCLUSIONS: We found that a large amount of phospholipids accumulated in the myocardium of a patient with idiopathic cardiomyopathy, and electron microscopic findings of lamellar and concentric inclusion bodies in cardiomyocytes. A cardiac phospholipid storage disorder should be considered as an important candidate disease on differential diagnosis of myocardiac disorders including cardiac Fabry disease.


Subject(s)
Cardiomyopathies/metabolism , Phospholipids/metabolism , Autopsy , Cardiomyopathies/enzymology , Cardiomyopathies/pathology , Chromatography, Thin Layer , Humans , Male , Middle Aged , alpha-Galactosidase/metabolism
6.
J Hum Genet ; 51(4): 341-352, 2006.
Article in English | MEDLINE | ID: mdl-16532254

ABSTRACT

We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant alpha-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant alpha-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.


Subject(s)
Drug Delivery Systems , Fabry Disease/therapy , Lysosomes/enzymology , Saccharomyces cerevisiae/enzymology , alpha-Galactosidase/administration & dosage , alpha-Galactosidase/pharmacology , Animals , Carbohydrate Sequence , Cells, Cultured , Chromatography, High Pressure Liquid , Culture Media/chemistry , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fibroblasts/drug effects , Glycosphingolipids/metabolism , Humans , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/therapeutic use , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Distribution , alpha-Galactosidase/biosynthesis , alpha-Galactosidase/chemistry , alpha-Galactosidase/genetics , alpha-Galactosidase/isolation & purification , alpha-Galactosidase/pharmacokinetics
7.
J Hum Genet ; 51(3): 180-188, 2006.
Article in English | MEDLINE | ID: mdl-16372133

ABSTRACT

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.


Subject(s)
Fabry Disease/drug therapy , Isoenzymes/therapeutic use , alpha-Galactosidase/therapeutic use , Animals , Cells, Cultured , Disease Models, Animal , Fabry Disease/pathology , Fibroblasts/drug effects , Fibroblasts/ultrastructure , Humans , Isoenzymes/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Recombinant Proteins , alpha-Galactosidase/pharmacology
8.
J Hum Genet ; 50(9): 460-467, 2005.
Article in English | MEDLINE | ID: mdl-16180049

ABSTRACT

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl-beta-D-glucosamine (beta-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl-beta-D-glucosamine-6-sulfate (beta-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human beta-hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated beta-hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.


Subject(s)
Cell Culture Techniques/methods , Gangliosidoses, GM2/metabolism , Recombinant Proteins/pharmacology , Sandhoff Disease/enzymology , Schwann Cells/cytology , beta-N-Acetylhexosaminidases/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Fibroblasts , Genotype , Humans , Immunohistochemistry , Lysosomes/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Schwann Cells/drug effects , Schwann Cells/enzymology
9.
Leuk Res ; 29(2): 197-203, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15607369

ABSTRACT

Effects of Notch activation on retinoic acid (RA)-induced differentiation and apoptosis were investigated. NB4, an acute promyelocytic leukemia (APL) cell line, undergoes neutrophilic differentiation and apoptosis by RA. Notch activation induced by a recombinant Notch ligand, Delta-1, did not affect the growth by itself. Treatment with RA plus Delta-1 made part of NB4 cells monocyte-like shaped and reduced the apoptosis. Similar phenomenon was also observed in primary APL cells. RA treatment induced cleavage of caspase-8 and PARP in NB4. Delta-1 suppressed the RA-induced cleavage of them, which may be a possible mechanism through which Delta-1 suppressed the RA-induced apoptosis.


Subject(s)
Apoptosis/drug effects , Cell Differentiation/drug effects , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Neutrophils/cytology , Tretinoin/antagonists & inhibitors , Antigens, CD/metabolism , Caspase 8 , Caspases/drug effects , Caspases/metabolism , Cell Differentiation/physiology , Cell Line, Tumor , Cell Lineage/drug effects , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/metabolism , Ligands , Monocytes/physiology , Neutrophils/drug effects , Neutrophils/metabolism , Poly(ADP-ribose) Polymerases/drug effects , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptors, Notch , Tretinoin/metabolism , Tretinoin/pharmacology
10.
Int J Mol Med ; 14(5): 861-6, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15492857

ABSTRACT

The Notch signaling pathway plays an important role in the regulation of self-renewal and differentiation of hematopoietic progenitors. Tumor necrosis factor (TNF)-alpha induces apoptosis through activation of caspase pathway. A monoblastic leukemia cell line, U937, undergoes apoptosis following stimulation with TNF-alpha. We found that Notch activation induced by a recombinant Notch ligand, Delta-1, reduced the TNF-alpha-induced growth suppression and apoptosis in U937 cells. As the molecular mechanism involved, we showed Delta-1 stimulation partially suppressed the sequential activation of caspase-8, caspase-3, and, PARP induced by TNF-alpha. The TNF-alpha-induced activation of c-Jun N-terminal kinase (JNK), p38, and NF-kappaB was not affected by Delta-1 stimulation. The cells needed to be exposed to Delta-1 prior to TNF-alpha stimulation to reduce the suppressive effect of TNF-alpha. Therefore, we thought that Delta-1 stimulation might reduce the expression of TNF-receptor (R) 1 and proteins to modulate the activation of caspases such as FLIP and XIAP. However, Delta-1 stimulation did not affect their expression. The precise mechanism by which Notch signaling suppresses caspase activation has yet to be determined. This is the first report to show the relationship between Notch activation and TNF-R1 signaling. The findings suggest possible mechanisms by which Notch activation supports cell survival.


Subject(s)
Apoptosis/drug effects , Caspases/metabolism , Cell Division/drug effects , Receptors, Cytokine/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Cell Survival , Enzyme Activation/drug effects , Humans , Signal Transduction , U937 Cells
11.
Int J Mol Med ; 14(2): 223-6, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15254769

ABSTRACT

It is known that Notch activation promotes the self-renewal of hematopoietic cells. However, we have previously found that the growth of a myeloid leukemia cell line, OCI/AML-6, was suppressed by Notch activation induced by stimulation with a recombinant Notch ligand, Delta-1 protein. We recently found that the growth of another leukemia cell line, THP-1, was also suppressed by the ligands Delta-1 and Jagged-1. In this study, we tried to clarify the cellular and molecular mechanism of the growth suppression induced by Notch activation. Flow cytometric analysis showed that Delta-1 stimulation increased the expression of differentiation markers such as CD11b and CD13 while it decreased the expression of CD117 (c-KIT), a marker for primitive cells in THP-1 cells. In OCI/AML-6 cells, Delta-1 stimulation decreased the expression of CD11b and CD14 and increased CD34 expression. Namely, Delta-1 showed the opposite effects on the differentiation markers of each cell line. Delta-1 stimulation did not increase the binding of annexin V, a marker for apoptotic cells in either cell line. Since the growth of myeloid cells is regulated by MAP kinase and JAK/STAT pathways, we investigated the effects of the ligand stimulation on these pathways. Delta-1 stimulation did not induce the phosphorylation of ERK1/2 and STAT3 proteins in either cell line. Pre-exposure to Delta-1 did not affect the phosphorylation of ERK1/2 and STAT3 induced by G-CSF in OCI/AML-6 cells, either. Namely, it is thought that these pathways are not involved in the growth suppression caused by Notch ligands. Our study revealed several findings on Notch function. However, the precise mechanism remains to be elucidated.


Subject(s)
Leukemia, Myeloid/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Annexin A5/pharmacology , Apoptosis , CD11b Antigen/biosynthesis , CD13 Antigens/biosynthesis , Calcium-Binding Proteins , Cell Line, Tumor , Cell Proliferation , Enzyme Inhibitors/pharmacology , Flow Cytometry , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Immunoblotting , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Ligands , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Proto-Oncogene Proteins c-kit/biosynthesis , Receptors, Notch , Recombinant Proteins/metabolism , Serrate-Jagged Proteins , Time Factors
12.
Int J Mol Med ; 13(3): 419-23, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14767573

ABSTRACT

Notch signaling plays an important role in the regulation of self-renewal and differentiation of hematopoietic cells. Human monoblastic U937 cells undergo differentiation into macrophage-like cells, growth suppression, and apoptosis following stimulation with GM-CSF. We examined the effects of Notch activation induced by Notch ligands on GM-CSF-induced differentiation and apoptosis in U937 cells. Furthermore, the molecular mechanism of the effects was investigated. A recombinant Notch ligand, Delta-1 protein did not affect the growth of U937 cells by itself. GM-CSF-induced growth suppression and apoptosis of U937 cells were partially rescued by incubation with Delta-1. Delta-1 also reduced the GM-CSF-induced differentiation. Incubation with Delta-1 did not affect the expression of GM-CSF receptor. GM-CSF stimulation induced the phosphorylation of ERK1/2 and STAT5 and the cleavage of caspase-8, which were not affected by Delta-1 incubation, either. GM-CSF stimulation induced the cleavage of PARP, which is the key molecule for differentiation and apoptosis. We found that incubation with Delta-1 significantly suppressed the GM-CSF-induced cleavage of PARP. Taken together, we found that Notch activation induced by Delta-1 partially inhibited GM-CSF-induced differentiation, growth suppression, and apoptosis, along with reducing the GM-CSF-induced cleavage of PARP. These findings suggest one of the mechanisms by which Notch activation inhibits differentiation and apoptosis.


Subject(s)
Apoptosis/drug effects , Cell Differentiation/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Membrane Proteins/pharmacology , Milk Proteins , Poly(ADP-ribose) Polymerases/metabolism , Caspases/metabolism , DNA-Binding Proteins/metabolism , Enzyme Activation/drug effects , Humans , Intracellular Signaling Peptides and Proteins , Ligands , MAP Kinase Signaling System/drug effects , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Notch , Recombinant Proteins/pharmacology , STAT5 Transcription Factor , Trans-Activators/metabolism , U937 Cells
13.
Int J Oncol ; 22(5): 1073-9, 2003 May.
Article in English | MEDLINE | ID: mdl-12684674

ABSTRACT

The self-renewal and differentiation of hematopoietic progenitors are regulated by the interaction between Notch receptors and Notch ligands. Since AML originates from dysregulated hematopoietic progenitors, some abnormalities in the Notch system may be involved in the abnormal proliferation of AML cells. However, the significance of the Notch system in AML is not known. We examined the functional roles of Notch activation on the in vitro growth of seven human AML cell lines using three kinds of recombinant Notch ligand proteins, Jagged-1, Delta-1 and Delta-4. The ligands significantly affected the growth of two cell lines. In TMD7 cells, Delta proteins promoted the short-term growth, however, suppressing the self-renewal capacity and long-term growth. In OCI/AML-6 cells, Delta proteins suppressed the growth and self-renewal capacity while inducing differentiation into macrophage-like cells. We additionally found that Notch ligands needed to be immobilized on culture wells to affect the cells. These findings were in contrast to our hypothesis that Notch activation in AML cells leads to excessive self-renewal capacity and proliferation. If the Notch system in AML cells is precisely understood, the control of Notch activation will become a novel therapeutic approach for AML.


Subject(s)
Blood Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Leukemia, Myeloid, Acute/pathology , Membrane Proteins/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Blood Proteins/genetics , Calcium-Binding Proteins , Cell Division/drug effects , Cell Division/physiology , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Homeodomain Proteins/metabolism , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Kinetics , Ligands , Membrane Proteins/genetics , Proteins/genetics , Recombinant Proteins/metabolism , Serrate-Jagged Proteins , Tumor Cells, Cultured
14.
Thromb Res ; 112(5-6): 313-20, 2003.
Article in English | MEDLINE | ID: mdl-15041276

ABSTRACT

Tissue factor (TF) is expressed rapidly by human monocytes exposed to a variety of agonists such as lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Activation of both activator protein-1 (AP-1; c-Jun/c-Fos) and nuclear factor-kappaB (NF-kappaB) pathways is necessary for maximal induction of the TF gene. It has been demonstrated that activation of both AP-1 and NF-kappaB is correlated with the degradation of both phosphorylated c-Jun and inhibitor kappaB (IkappaB) by proteasome. The present study was designed to investigate whether various protease inhibitors, including proteasome inhibitors, affect TF expression in monocytic cells. Protease inhibitors, 3,4-dichloroisocoumarin (DCI), N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), and N-acetyl-Leu-Leu-norleucinal (ALLN) induced TF activity in monocytic cells in a dose- and time-dependent manner at the level of the transcription of the TF gene, which was mediated through inducing phosphorylation of both Jun-N-terminal kinase and p38. The early growth response-1 (Egr-1) pathway was not affected. The NF-kappaB pathway was not activated; rather it was inhibited. These results were distinct from the findings previously reported for LPS-stimulated cells. The present study demonstrated that some protease inhibitors might act as stress and induced TF expression with direct phosphorylation of JNK and p38, followed by phosphorylation and activation of AP-1 in monocytic cells. This evidence may help elucidate further regulatory mechanisms of TF induction, and might have physiological significance for the clinically challenged use of proteasome inhibitors. In addition to phosphorylation of JNK and p38, an unknown signal pathway needs to be clarified for TF induction.


Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Monocytes/metabolism , Protease Inhibitors/pharmacology , Thromboplastin/genetics , Transcription Factor AP-1/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Kinetics , Phosphorylation , Thromboplastin/drug effects
15.
Br J Haematol ; 117(2): 373-8, 2002 May.
Article in English | MEDLINE | ID: mdl-11972520

ABSTRACT

A novel human leukaemia cell line, designated TMD7, was established from blast cells of a patient with de novo acute myeloblastic leukaemia with trilineage myelodysplasia (AML/TLD). As seen in the original blast cells, TMD7 cells expressed CD7, CD13, CD33 and CD34 and showed an abnormal karyotype containing -5, -7, -8, der(16)t(10;16)(q22;q13). The cells proliferated without added growth factors. Growth was stimulated with the addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and interleukin 3. Differentiation was not observed with the addition of various cytokines. As a cell line derived from AML/TLD has not been reported, TMD7 will be a useful tool as a model of AML/TLD cells. Recently, it was reported that the Notch system has crucial roles to regulate the self-renewal and differentiation of haematopoietic stem cells. We found that TMD7 cells expressed Notch-1 and Notch-2 mRNA. The exposure to recombinant Delta-1 protein, which was one of the Notch ligands, significantly stimulated the growth of TMD7 cells. This is the first human cell line which was shown to proliferate in response to Delta-1, without artificially expressed Notch protein. Therefore, TMD7 will also be a useful tool to study the mechanism of the Notch-Notch ligand interaction.


Subject(s)
Leukemia, Myeloid, Acute , Membrane Proteins/pharmacology , Myelodysplastic Syndromes , Transcription Factors , Tumor Cells, Cultured , Calcium-Binding Proteins , Cell Division/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Intercellular Signaling Peptides and Proteins , Interleukin-3/pharmacology , Intracellular Signaling Peptides and Proteins , Karyotyping , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Male , Membrane Proteins/metabolism , Middle Aged , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Proteins/pharmacology , Receptor, Notch1 , Receptor, Notch2 , Receptors, Cell Surface/metabolism , Serrate-Jagged Proteins , Tumor Cells, Cultured/metabolism
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