ABSTRACT
Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower. However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.
Subject(s)
Prolactin/metabolism , Recombinant Proteins/metabolism , Animals , CHO Cells/drug effects , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cricetinae , Cricetulus , Drug Evaluation, Preclinical/methods , Folic Acid Antagonists/pharmacology , Glycosylation , Humans , Lymphoma , Methotrexate/pharmacology , Prolactin/genetics , Prolactin/pharmacology , Protein Engineering/methods , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
The human homologue of the recently cloned murine IL-13 binding protein (IL-13R alpha1) was cloned from a cDNA library derived from the carcinoma cell line CAKI-1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL-13R alpha1, and 27% identical to the human IL-13R alpha2. CHO cells expressing recombinant hIL-13R alpha1 specifically bind IL-13 (Kd approximately 4 nM) but not IL-4. Co-expression of the cloned cDNA with that of IL-4R alpha resulted in a receptor complex that displayed high affinity for IL-13 (Kd approximately 30 pM), and that allowed cross-competition of IL-13 and IL-4. Electrophoretic mobility shift assay showed that IL-13 and IL-4 were able to activate Stat6 in cells expressing both IL-4R alpha and IL-13R alpha1, while no activation was observed in cells expressing either one or the other alone.
Subject(s)
Antigens, CD/genetics , Receptors, Interleukin/genetics , Amino Acid Sequence , Animals , Antigens, CD/metabolism , CHO Cells , Cloning, Molecular , Cricetinae , Gene Expression Regulation , Humans , Interleukin-13 Receptor alpha1 Subunit , Molecular Sequence Data , Receptors, Interleukin/metabolism , Receptors, Interleukin-13 , Receptors, Interleukin-4 , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , STAT6 Transcription Factor , Signal Transduction , Trans-Activators/metabolism , Tumor Cells, CulturedABSTRACT
Cytokines locally delivered to the site of a tumor boost both specific and nonspecific host anti-tumor defenses. Interleukin (IL)-13 is a recently described cytokine produced by mouse type 2 helper T lymphocytes. The aim of this study was to evaluate the inhibition of tumor growth induced by IL-13 delivered locally within or around transplanted tumor cells in mice. We observed that local administration of IL-13 at the site of transplanted tumor cells in vivo had potent inhibitory effects on growth of both immunogenic (P815 mastocytoma, H-2d) or nonimmunogenic (3LL lung carcinoma, H-2b) tumor cells. Mice injected with transfected P815 cells secreting large amounts of IL-13 rejected the P815 tumor and developed systemic specific anti-tumor immunity leading to long-lasting specific anti-tumor protection. Less efficient anti-tumoral effects were obtained with the nonimmunogenic 3LL tumor model when local administration of IL-13 was achieved by co-inoculating xenogeneic chinese hamster ovary (CHO) IL-13 cells. Several local injections of CHO IL-13 cells were needed to obtain rejection of 3LL tumors and no induction of long-lasting anti-3LL memory was obtained. Several studies were performed to elucidate the IL-13 anti-tumoral effects. Experiments with nude mice indicated that Il-13 can also stimulate nonspecific anti-tumor defenses. The histological examination of P815 IL-13 cells undergoing rejection showed monocytic cells and neutrophils infiltrating the tumor. Studies indicated that IL-13 administered in vitro did not directly stimulate the cytotoxicity of peritoneal macrophages and natural killer cells. However, experiments with Boyden chemotaxis chambers indicated that IL-13 was chemotactic for macrophages. Finally, preliminary experiments in vitro suggest that IL-13 improved antigenic presentation of P815 membranes. Thus, anti-tumor effects of IL-13 in vivo most probably result from pleiotropic effects including recruitment of nonspecific cells and improved stimulation of immune-specific anti-tumor effectors.
Subject(s)
Genetic Therapy/methods , Interleukin-13/genetics , Neoplasms, Experimental/therapy , Transfection/physiology , Animals , Chemotaxis, Leukocyte , Humans , Interleukin-13/biosynthesis , Interleukin-13/therapeutic use , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Nude , Neoplasms, Experimental/immunology , Tumor Cells, CulturedABSTRACT
SR 142801 is the first potent and selective non-peptide antagonist of the tachykinin NK3 receptor. It inhibited [MePhe7]NKB binding to its receptor from various species, including humans. SR 142801 was a competitive antagonist of [MePhe7]NKB-mediated contractions of guinea-pig ileum and inhibited the acetylcholine release following the activation of the guinea-pig ileum tachykinin NK3 receptor. In vivo, SR 142801 potently inhibited the turning behaviour induced by intrastriatal injection of senktide in gerbils, and appears as a powerful tool for investigation of the physiological and pathological role of NKB and its NK3 receptor.
Subject(s)
Piperidines/pharmacology , Receptors, Neurokinin-3/antagonists & inhibitors , Animals , Behavior, Animal/drug effects , Dose-Response Relationship, Drug , Gerbillinae , Guinea Pigs , In Vitro Techniques , Neurokinin B/analogs & derivatives , Neurokinin B/antagonists & inhibitors , Neurokinin B/metabolism , Rats , Vasoconstriction/drug effectsABSTRACT
An expression system combining a unit for the expression of the gene of interest reinforced by the hepatitis B virus X transactivator and a selectable gene weakened by the insertion of an A+T-rich sequence derived from the 3'-untranslated region of the granulocyte-macrophage colony-stimulating factor mRNA is described. This vector allows rapid one-step isolation of highly productive Chinese hamster ovary clones.
Subject(s)
Culture Techniques/methods , Globins/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Interleukin-2/biosynthesis , Recombinant Proteins/biosynthesis , Transfection/methods , 3T3 Cells , Animals , CHO Cells , Cloning, Molecular/methods , Cricetinae , Genetic Vectors , HeLa Cells , Hepatitis B Antigens/biosynthesis , Human T-lymphotropic virus 1/genetics , Humans , Introns , L Cells , Mice , Promoter Regions, Genetic , RNA, Messenger/metabolism , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Trans-Activators/biosynthesis , Viral Regulatory and Accessory ProteinsABSTRACT
The mononuclear cell surface protein IA4 was originally identified in our lab using a mAb selected because of its strong reactivity with three lymphoblastoid variant cell lines which are HLA class I deficient, are LAK susceptible, and form a high number of conjugates with LAK effectors. We previously cloned the cDNA of the IA4 protein, coding for a 267-amino-acid type III integral membrane protein, with four transmembrane domains and three possible N-glycosylation sites. The IA4 protein belongs to the tetra span transmembrane (TST) new family of surface molecules, which also includes CD9, CD37, CD53, CD63, and TAPA-1. IA4 antigen was recently recognized as belonging to a new cluster of differentiation CD82 (International CD Workshop, Boston 1993). The IA4 antigen expression pattern at the surface of immune cells from normal donors was studied. On T lymphocytes, IA4 was barely detectable on resting cells and increased 3.5- to 7-fold following PHA or PHA+PMA stimulation. This IA4 increased expression is correlated with the morphologic change in blast cells and with the expression of activation markers such as CD2 and MHC class II antigens, therefore suggesting that IA4 is an activation marker on T lymphocytes. The expression of IA4 was low on circulating resting monocytes collected by elutriation. However, these monocytes, cultured in medium alone or with GM-CSF, acquired the morphology of macrophage and simultaneously overexpressed MHC Class II, CD14, and IA4 antigens, suggesting that IA4 is a differentiation marker for macrophages, whatever the culture conditions, either adherent (plastic culture dishes) or nonadherent (Teflon culture bags). IA4 stable transfectants of the murine mastocytoma cell line P815 were obtained and used to generate a new mAb. Competitive epitope binding studies have shown that IA4 antigen presents a dominant epitope recognized by most of the mAb prepared either in our lab or elsewhere. This dominant epitope is not shared by any of the other antigens of the TST family. Using this new mAb we were able to biochemically characterize the IA4 antigen as a 28-kDa protein, highly N-glycosylated with different patterns on various cells.
Subject(s)
Antigens, CD/analysis , Antigens, Surface/analysis , Leukocytes, Mononuclear/immunology , Membrane Glycoproteins , Proto-Oncogene Proteins , Animals , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Surface/genetics , Antigens, Surface/immunology , Cell Communication , Cell Line , Chromosome Mapping , Epitopes , Humans , Immunoblotting , Kangai-1 Protein , Killer Cells, Lymphokine-Activated/immunology , Lymphocyte Activation , Macrophages/immunology , Mice , Mice, Inbred DBA , T-Lymphocytes/immunologyABSTRACT
We have isolated a cDNA (NC28) transcribed from a mRNA which is transiently induced in U937 promonocytic cells by PMA and super-induced by cycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human monocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant MCP-3 protein shows chemotactic activity for monocytes but not for neutrophils. However the secreted MCP-3 protein differs from MCP-1 in being N-glycosylated. The 3' noncoding regions of MCP-3 and MCP-1 mRNAs are more diverged (44%), allowing specific cDNA probes to be made, and indicating that the two genes are evolutionarily distant. Sequence comparisons of the 3' noncoding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 genes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by monocytes, with MCP-1 mRNA being expressed at levels 2-4 times that of MCP-3 mRNA. However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulation, MCP-3 mRNA is expressed only at very low levels in these cells. The cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRNA levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased by IFN-gamma, although IL-6 mRNA is not induced. They are also increased by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and MCP-3 mRNAs are thus co-ordinately regulated in monocytes in response to a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.
Subject(s)
Chemotactic Factors/genetics , Cytokines , Monocyte Chemoattractant Proteins , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Cells, Cultured , Chemokine CCL7 , Cloning, Molecular , DNA/genetics , Gene Expression Regulation , Humans , Mice , Molecular Sequence Data , Monocytes/immunology , RNA, Messenger/genetics , Rats , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
The discovery of new cytokines normally relies on a prior knowledge of at least one of their biological effects, which is used as a criterion either for the purification of the protein or for the isolation of the complementary DNA by expression cloning. However, the redundancy of cytokine activities complicates the discovery of novel cytokines in this way, and the pleiotropic nature of many cytokines means that the principal activities of a new cytokine may bear little relation to that used for its isolation. We have adopted an alternative approach which relies on differential screening of an organized subtracted cDNA library from activated peripheral blood mononuclear cells, using the inducibility of lymphokine messenger RNAs by anti-CD28 as a primary screening criterion. The ligation of the CD28 antigen on the T lymphocyte by a surface antigen, B7/BB-1, expressed on activated B lymphocytes and monocytes is a key step in the activation of T lymphocytes and the accumulation of lymphokine mRNAs. Here we report the discovery by molecular cloning of a new interleukin (interleukin-13 or IL-13) expressed in activated human T lymphocytes. Recombinant IL-13 protein inhibits inflammatory cytokine production induced by lipopolysaccharide in human peripheral blood monocytes. Moreover, it synergizes with IL-2 in regulating interferon-gamma synthesis in large granular lymphocytes. Recent mapping of the IL-13 gene shows that it is closely linked to the IL-4 gene on chromosome 5q 23-31 (ref. 4). Interleukin-13 may be critical in regulating inflammatory and immune responses.
Subject(s)
B-Lymphocytes/immunology , Interleukins/genetics , Interleukins/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Base Sequence , CD28 Antigens , DNA/blood , DNA/genetics , DNA/isolation & purification , Gene Library , Genetic Variation , Humans , Inflammation/immunology , Interleukin-13 , Molecular Sequence Data , RNA, Messenger/genetics , Receptors, Cell Surface/immunology , Sequence Homology, Amino AcidABSTRACT
The IA4 mAb was identified among a series of antibodies raised in BALB/c mice after immunization against a HLA class I-deficient, lymphokine-activated killer (LAK)-susceptible EBV-B lymphocyte line. The IA4 antibody was selected because of its high expression, in the range of 10(5) to 25 x 10(5) sites/cell, on several B lymphocyte lines (EBV-transformed or Burkitt) and monocytic lines such as HL60 and U937, and because its expression was correlated with both target susceptibility to LAK lysis and reduced expression of HLA class I surface Ag on two pairs of EBV-B-transformed cell lines (721/721.134 and MM/10F2). Despite the strategy followed to raise the mAb and the correlation mentioned above, no direct role of the IA4 molecules in LAK susceptibility has been established, since the IA4 molecule is poorly expressed on the sensitive targets Daudi and K562; moreover, the IA4 antibody did not affect reproducibly the in vitro killing of positive target cells by LAK effectors. The IA4 antibody was poorly immunoprecipitating and the surface molecule recognized was identified by gene cloning following an expression strategy using a U937 cDNA library transfected in COS cells, and a screening strategy based on membrane expression of IA4 molecule. The IA4 cDNA is virtually identical to "R2," a mRNA species previously identified in activated human T cells by subtractive hybridization. The IA4 cDNA contains an open reading frame coding for a protein 267 amino acids long with four potential transmembrane domains and one large external hydrophilic domain of about 110 amino acids, possibly glycosylated. The encoded protein belongs to a family of surface molecules, the tetra spans transmembrane protein superfamily, all displaying the four transmembrane domains, expressed on various cell types including lymphocytes (CD9, CD37, CD53, TAPA-1), melanoma cells (ME491), and intestinal cells (CO-029). These molecules have been reported to be involved in cell activation and cell death. Surprisingly, the Schistosoma mansoni Ag Sm23 displays significant homologies with this family. The IA4 molecule is a widely distributed surface marker expressed on circulating lymphocytes and monocytes, newborn thymocytes, and the cell lines mentioned above. The IA4 molecule expression is up-regulated upon cell activation. Weakly expressed on resting peripheral T and B lymphocytes and large granular lymphocytes (NK), its expression roughly doubles after activation by PHA, staphylococcus aureus Cowan I, and IL-2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Histocompatibility Antigens Class II/immunology , Killer Cells, Lymphokine-Activated/immunology , Signal Transduction/immunology , Amino Acid Sequence , Antigens, Surface/immunology , Base Sequence , Burkitt Lymphoma/immunology , Calcium/metabolism , Cell Line , Dose-Response Relationship, Immunologic , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/genetics , Humans , Interleukin-4/pharmacology , Lymphocyte Activation/immunology , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Tumor Necrosis Factor-alpha/pharmacology , Up-RegulationABSTRACT
A recombinant plasmid was constructed permitting the efficient synthesis of human growth hormone (hGH) in monkey cells. The plasmid contains the cDNA sequence of the hGH precursor and controlled by the SV40 early promoter, an intron, and the poly(A)-addition site of the mouse alpha-globin gene. To permit selection of transformed cells, a selectable marker (xanthine-guanine phosphoribosyl transferase; XGPRT) was also introduced into this plasmid. Transformation of an established monkey kidney cell line (VERO) permitted the isolation of cell lines excreting hGH. One of these strains (VEH 1) excreted hGH up to 5.6 micrograms per 10(6) cells per 24 h during exponential growth.