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1.
Biochem Biophys Res Commun ; 282(3): 823-31, 2001 Apr 06.
Article in English | MEDLINE | ID: mdl-11401538

ABSTRACT

We describe here the cloning and characterization of the 5' flanking region of the human Bone Morphogenetic Protein-7 (BMP-7) gene from a 3.3 kb genomic DNA fragment. Functional analysis by transient transfection using the luciferase reporter gene indicated that this region had a low basal promoter activity in human Wilm's tumor derived renal (G401) and rat osteoblast (ROS 17/2.8) cell lines. Sequential deletion analysis of the promoter revealed sequences whose presence correlated with decreased expression of the reporter gene. Coexpression of transcription factors involved in epithelial/mesenchymal interactions during kidney and eye development dramatically stimulated the expression of the reporter gene from the putative BMP-7 promoter. Finally, a subset of agents that upregulated the expression of the reporter gene from the cloned promoter were also shown to increase the expression of the endogenous BMP-7 in G401 and ROS cell lines in vitro.


Subject(s)
Bone Morphogenetic Proteins/genetics , Transforming Growth Factor beta , Amino Acid Sequence , Animals , Base Sequence , Bone Morphogenetic Protein 7 , Bone and Bones/drug effects , Bone and Bones/metabolism , Cell Line , Cloning, Molecular , DNA Primers/genetics , Dinoprostone/pharmacology , Gene Expression/drug effects , Gene Silencing , Genes, Reporter , Humans , Luciferases/genetics , Molecular Sequence Data , Parathyroid Hormone/pharmacology , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Homology, Nucleic Acid , Species Specificity , Transfection
2.
J Bone Joint Surg Am ; 83-A Suppl 1(Pt 1): S70-8, 2001.
Article in English | MEDLINE | ID: mdl-11263669

ABSTRACT

BACKGROUND: The vasculature is an important component in the musculoskeletal system, and vascularization is a key event in the development of normal cartilage and bone formation. Blood vessels deliver nutrients, oxygen, and precursor cells to maintain the structural and functional integrity of joints and soft and hard tissues. Therefore, agents that help to inhibit proliferation and retain the phenotype of vascular smooth muscle cells (SMCs) are of critical importance. In this study, we examined the capacity of bone morphogenetic protein-7 (BMP-7) to inhibit the proliferation of SMCs and maintain their phenotype. METHODS: A thymidine-incorporation assay was used to monitor the proliferative activity of SMCs on stimulation with platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), agents known to be stimulatory for these cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and enzyme-linked immunosorbent assay (ELISA) were used to monitor the modulation of various genes and gene products. Immunolocalization of SMC specific markers was also performed. RESULTS: BMP-7 inhibited both serum-stimulated and growth factor-induced (PDGF-BB and TGF-beta1) SMC growth, as measured by 3H-thymidine uptake and cell number, in primary human aortic smooth muscle (HASM) cell cultures. The addition of BMP-7 stimulated the expression of developmentally regulated as well as SMC-specific markers, namely, Id-1 and Id-2, alpha-actin, and SMC-specific heavy-chain myosin, as examined by semiquantitative and quantitative RT-PCR and by Northern blot analysis. Additionally, BMP-7 exhibited anti-inflammatory activity by downregulating intercellular adhesion molecule-1 (ICAM-1) expression. The collagen type III/I ratio that becomes lower with the transdifferentiation of SMCs into myofibroblasts is maintained in BMP-7-treated cultures compared with untreated controls. Studies on the mechanism of action indicate that BMP-7 treatment induces cyclin-dependent kinase-2 inhibitor, p21, which was inhibited during PDGF-BB-induced proliferation of SMCs. Finally, BMP-7 upregulates the expression of the inhibitory Smads, Smad6 and Smad7, which are known to inhibit TGF-beta superfamily signaling. CONCLUSIONS: These results suggest that BMP-7 maintains the expression of the vascular SMC phenotype. Thus, BMP-7 may prevent vascular proliferative disorders and potentially could act as a palliative agent following damage to the vasculature. CLINICAL RELEVANCE: In musculoskeletal disorders in which the vasculature plays an important role, BMP-7 may be of benefit as an anti-inflammatory and anti-proliferative agent for vascular endothelium and help maintain vascular integrity.


Subject(s)
Bone Morphogenetic Proteins/physiology , Muscle, Smooth, Vascular/physiology , Transforming Growth Factor beta , Animals , Bone Morphogenetic Protein 7 , Cells, Cultured , Down-Regulation/physiology , Enzyme-Linked Immunosorbent Assay , Humans , Intercellular Adhesion Molecule-1/physiology , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/physiology
3.
J Cell Physiol ; 184(1): 37-45, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10825232

ABSTRACT

Vascular proliferative disorders are characterized by migration and proliferation of vascular smooth muscle cells (SMCs), loss of expression of SMC phenotype, and enhanced extracellular matrix synthesis (e.g., type I collagen). We report here that bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor-beta (TGF-beta) superfamily, is capable of inhibiting both serum-stimulated and growth factor-induced (platelet-derived growth factor [PDGF-BB] and TGF-beta1) cell growth as measured by (3)H-thymidine uptake into DNA synthesis and cell number in primary human aortic smooth muscle (HASM) cell cultures. Concomitantly, addition of BMP-7 stimulates the expression of SMC-specific markers, namely alpha-actin and heavy chain myosin as examined by RT-PCR and Northern blot analyses. The collagen type III/I ratio that becomes lower with the transdifferentiation of SMCs into myofibroblasts is also maintained in BMP-7-treated cultures as compared to untreated controls. Studies on the mechanism of action indicate that BMP-7 treatment inhibits cyclin-dependent kinase 2 (cdk-2) that was stimulated during PDGF-BB-induced proliferation of SMCs and upregulates the expression of the inhibitory Smad, Smad6, which was shown to inhibit TGF-beta superfamily signaling. These results collectively suggest that BMP-7 maintains the expression of vascular SMC phenotype and may prevent vascular proliferative disorders, thus potentially acting as a palliative after damage to the vascular integrity.


Subject(s)
Bone Morphogenetic Proteins/pharmacology , Gene Expression Regulation/drug effects , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Actins/genetics , Aorta , Apoptosis/drug effects , Becaplermin , Bone Morphogenetic Protein 7 , Bone Morphogenetic Protein Receptors, Type II , Cell Division/drug effects , Cells, Cultured , Humans , Muscle, Smooth, Vascular/drug effects , Myosin Heavy Chains/genetics , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-sis , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta/physiology
4.
J Clin Invest ; 102(1): 202-14, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9649574

ABSTRACT

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.


Subject(s)
Acute Kidney Injury/drug therapy , Bone Morphogenetic Proteins/pharmacology , Ischemia/drug therapy , Kidney/blood supply , Transforming Growth Factor beta , Animals , Apoptosis , Bone Morphogenetic Protein 7 , Bone Morphogenetic Proteins/genetics , Growth Substances/genetics , Humans , Intercellular Adhesion Molecule-1/analysis , Kidney/drug effects , Male , RNA, Messenger/analysis , Rats , Rats, Wistar , Recombinant Proteins/therapeutic use
5.
Biotechnology (N Y) ; 12(9): 890-7, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7765352

ABSTRACT

The production of several single-chain Fv (sFv) antibody proteins was examined by three modes of mammalian cell expression. Our primary model was the 741F8 anti-c-erbB-2 sFv, assembled as either the VH-VL or VL-VH, and expressed alone, with C-terminal cysteine for dimerization, or as fusion proteins with carboxyl-terminal effector domains, including interleukin-2, the B domain of staphylococcal protein A, the S-peptide of ribonuclease S, or hexa-histidine metal chelate peptide. Constructs were expressed and secreted transiently in 293 cells and stably in CHO or Sp2/0 cell lines, the latter yielding up to 10 mg per liter. Single-chain constructs of MOPC 315 myeloma and 26-10 monoclonal antibodies were also expressed, as were hybrids comprising unrelated VH and VL regions. Our results suggest that mammalian expression is a practical and valuable complement to the bacterial expression of single-chain antibodies.


Subject(s)
Antibodies, Monoclonal/biosynthesis , ErbB Receptors/immunology , Immunoglobulin Fragments/biosynthesis , Interleukin-2/biosynthesis , Proto-Oncogene Proteins/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Transfection , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , CHO Cells , Cell Line , Chromatography, Affinity , Cricetinae , DNA Primers , Gene Expression , Humans , Immunoglobulin Fragments/isolation & purification , Interleukin-2/isolation & purification , Macromolecular Substances , Molecular Sequence Data , Plasmacytoma , Plasmids , Polymerase Chain Reaction , Receptor, ErbB-2 , Recombinant Fusion Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Restriction Mapping , Tumor Cells, Cultured
6.
Mol Immunol ; 29(12): 1487-91, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1454066

ABSTRACT

The role of inter-heavy and light chain disulfide bonds in the effector functions of human IgG1 was investigated. This was accomplished by mutating appropriate sites in IgG1 such that the disulfide bond pattern now resembled that of IgG4. The effector functions of the mutant antibody were then compared to native IgG1 and IgG4. The antibody-dependent cell cytotoxicity activity was completely abolished in the mutant and the complement-dependent cytotoxicity assay was reduced fifteen-fold. The results suggest that the inter-heavy and light chain disulfide bond pattern of an antibody molecule play a role in its effector functions.


Subject(s)
Disulfides/chemistry , Immunoglobulin G/immunology , Animals , Antibody-Dependent Cell Cytotoxicity , Antigen-Antibody Reactions , Base Sequence , Binding, Competitive , Dose-Response Relationship, Immunologic , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulin Heavy Chains , Immunoglobulin Light Chains , Molecular Sequence Data , Mutagenesis, Site-Directed , Restriction Mapping
7.
Hybridoma ; 11(5): 667-75, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1459589

ABSTRACT

The human hybridoma cell line HR78 secretes a human antibody of the IgM isotype directed against bacterial endotoxin. The cell line produces low levels of antibody and, more importantly, the antibody product is likely to be impure since two and perhaps more species of heavy chain are being synthesized as judged by cloning and expression studies. To address these issues, the cell line was used as a source of mRNA for the construction of cDNA libraries and the subsequent isolation of the sequences encoding heavy and light chains. Expression of these chains in a non-immunoglobulin producing murine hybridoma cell line resulted in a monoclonal antibody that bound antigen in a manner essentially identical to that observed for the parental antibody. Moreover, the level of expression of human IgM in selected clones was increased approximately 50-fold over the parental cell line and was comparable to or better than antibody expression by a typical murine hybridoma.


Subject(s)
Antibodies, Monoclonal/genetics , Immunoglobulin M/genetics , Lipid A/immunology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA/genetics , Gene Expression , Genetic Vectors , Humans , Hybridomas/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Molecular Sequence Data
8.
Hybridoma ; 10(2): 211-7, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1874515

ABSTRACT

The N-linked carbohydrate attachment site of human IgG1 Ab has been eliminated by site-directed mutagenesis. Effector functions of aglycosylated Ab was then compared to its native counterpart. Aglycosylated Ab failed to exhibit any ADCC activity, but a significant level of CDC activity was retained by the aglycosylated Ab. These observations differ from those reported previously. Serum half-life and biodistribution of aglycosylated Ab in mice were comparable to the native Ab. Together, these results show that some, but not all, effector functions of a human IgG1 Ab are affected by aglycosylation.


Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin G/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacokinetics , Antibody-Dependent Cell Cytotoxicity , Antigen-Antibody Reactions , Base Sequence , Binding Sites, Antibody , Binding, Competitive , Complement System Proteins/immunology , Glycosylation , Humans , Immunoglobulin G/genetics , Immunoglobulin G/pharmacokinetics , Melanoma, Experimental/metabolism , Metabolic Clearance Rate , Mice , Mice, Nude , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Processing, Post-Translational
10.
J Immunol ; 139(12): 4232-41, 1987 Dec 15.
Article in English | MEDLINE | ID: mdl-3121734

ABSTRACT

Murine/human chimeric gamma 1 and K Ig genes were cloned adjacent to the gene coding for methotrexate-resistant dihydrofolate reductase. These constructs were introduced into myeloma cells, and lines containing stably integrated genes were selected. The integrated Ig genes were then amplified by selection of the cells in increasing concentrations of methotrexate. The extent of gene amplification, mRNA accumulation, and production of Ig was studied in transfectomas containing introduced light chain genes, heavy chain genes, or both. When the light chain gene was introduced alone, it was expressed at low levels, but after selection with methotrexate, light chain expression was increased as much as 63-fold. In contrast, the transfected heavy chain genes were highly expressed, but production of the corresponding protein was increased a maximum of only fourfold by methotrexate treatment. Cellular toxicity of unassembled heavy chain monomer was not observed, even at amounts equivalent to 2% of total cellular protein. Cointroduction of the heavy and light chain constructs with subsequent amplification resulted in as much as 25-fold increase in secretion of intact antibody relative to unamplified cells. The results demonstrate that amplification of Ig genes can induce transfectomas to secrete antibody at nearly the rate of hybridomas.


Subject(s)
Gene Amplification , Gene Expression Regulation , Genes, Immunoglobulin , Tetrahydrofolate Dehydrogenase/genetics , Drug Resistance , Immunoglobulin gamma-Chains/biosynthesis , Immunoglobulin gamma-Chains/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Methotrexate/pharmacology , Multiple Myeloma , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection , Tumor Cells, Cultured
11.
J Immunol ; 137(3): 1066-74, 1986 Aug 01.
Article in English | MEDLINE | ID: mdl-3088107

ABSTRACT

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.


Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Neoplasm/genetics , Antibody Specificity , Chimera , Genetic Engineering , Animals , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred C57BL , Transfection
12.
Mol Cell Biol ; 2(10): 1187-98, 1982 Oct.
Article in English | MEDLINE | ID: mdl-6184609

ABSTRACT

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.


Subject(s)
Antigens, Viral , Cell Membrane/immunology , Polyomavirus/immunology , Antigens, Viral, Tumor , Cell Compartmentation , Cytoskeleton/enzymology , Molecular Weight , Phosphotyrosine , Protein Kinases/metabolism , Tyrosine/analogs & derivatives , Tyrosine/biosynthesis
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