Showing papers on "Cell culture published in 1987"
TL;DR: A simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells are described and linear DNA is almost inactive in mammalian cells.
Abstract: We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
5,481 citations
Journal Article•
TL;DR: The results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.
Abstract: We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.
1,886 citations
Book•
01 Jan 1987
TL;DR: The results allowed us to assess the importance of knowing the carrier and removal status of canine coronavirus, as a source of infection for other animals, not necessarily belonging to the same breeds.
Abstract: Production of teratocarcinomas from embryos transplanted to extra-uterine sites / Ivan Damjanov, Andrea Damjanov, and Davor Solter -- Cell culture methods and induction of differentiation of embryonal carcinoma cell lines / Michael A. Rudnicki and Michael W. McBurney -- Isolation of genetic variants and fusion hybrids from embryonal carcinoma cell lines / Martin L. Hooper -- Embryo-derived stem cell lines / Elizabeth J. Robertson -- Production and analysis of chimaeric mice / Allan Bradley -- Introduction of DNA into embryonic stem cells /Robin H. Lovell-Badge -- Experimental analysis of teratocarcinoma cell multiplication and purification of embryonal carcinoma-derived growth factor / John K. Heath -- Cell lines from human germ cell tumours / Peter W. Andrews, J. Wolter Oosterhuis, and Ivan Damjanov.
1,541 citations
TL;DR: Results identify T3-epsilon as a cell surface protein involved in the transduction of activation signals and can both activate and inhibit T-cell function.
Abstract: A monoclonal antibody (145-2C11) specific for the murine T3 complex was derived by immunizing Armenian hamsters with a murine cytolytic T-cell clone. The antibody is specific for a 25-kDa protein component (T3-epsilon) of the antigen-specific T-cell receptor. It reacts with all mature T cells and can both activate and inhibit T-cell function. These results identify T3-epsilon as a cell surface protein involved in the transduction of activation signals.
1,509 citations
TL;DR: The scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells, and might be involved in epithelial migration.
Abstract: Various factors are known to regulate cell growth and differentiation, but less is known of agents which affect movement and positioning, particularly in epithelial-mesenchymal interactions. Cultured human embryo fibroblasts release a protein with a relative molecular mass (Mr) of approximately 50,000 (50K) that affects epithelial cells by causing a disruption of junctions, an increase in local motility and a scattering of contiguous sheets of cells. To investigate specificity, a range of cells has been examined for the ability to produce the factor and for sensitivity to its action. Most freshly isolated normal epithelia and epithelia from cell lines of normal tissue, but not epithelia from tumour cell lines or fibroblasts, were sensitive to scatter factor. In contrast, production of the factor, as identified by activity and by chromatography, was restricted to embryonic fibroblasts and certain variants of 3T3 and BHK21 cells and their transformed derivatives. We conclude that the scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells. The factor might be involved in epithelial migration, such as occurs in embryogenesis or wound healing.
1,346 citations
TL;DR: In this paper, a simple yet quantitative method for assessing glutamate mediated central neuronal cell injury in cortical cell culture has been proposed; the magnitude of LDH efflux in the cultures correlates in a linear fashion with the number of neurons damaged by glutamate exposure.
1,330 citations
TL;DR: There is evidence that foreign DNA can be specifically delivered to cells by a soluble carrier system that takes advantage of receptor-mediated endocytosis, and that competition by a 10-fold excess of ASOR prevented gene transformation by the ASOR X poly-L-lysine X DNA complex.
1,225 citations
TL;DR: Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes, and there was no need for the monocytes to be stimulated but the cells did require the presence of serum.
Abstract: Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-β2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human γ-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.
1,129 citations
TL;DR: The high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage‐forming cells makes them probable candidates for the role of osteogenic stem cells.
Abstract: Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies When transplanted in diffusion chambers after 20-30 cell doublings in vitro, the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells
1,025 citations
TL;DR: A new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins is demonstrated and a functional basis for the role of their overexpression in the development of human malignancies is provided.
Abstract: A wide variety of human tumors contain an amplified or overexpressed erbB-2 gene, which encodes a growth factor receptor-like protein. When erbB-2 complementary DNA was expressed in NIH/3T3 cells under the control of the SV40 promoter, the gene lacked transforming activity despite expression of detectable levels of the erbB-2 protein. A further five- to tenfold increase in its expression under influence of the long terminal repeat of Moloney murine leukemia virus was associated with activation of erbB-2 as a potent oncogene. The high levels of the erbB-2 product associated with malignant transformation of NIH/3T3 cells were observed in human mammary tumor cells that overexpressed this gene. These findings demonstrate a new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins and provide a functional basis for the role of their overexpression in the development of human malignancies.
1,021 citations
TL;DR: It is shown that cosecretion of TGF-beta and its monocyte/macrophage-derived binding protein may modulate growth factor action and that monocytic expression of other growth factor genes, such as the B chain of platelet-derived growth factor, is not constitutive and requires activation.
Abstract: Alveolar macrophages activated with concanavalin A and peripheral blood monocytes activated with lipopolysaccharide secrete type beta transforming growth factor (TGF-beta). There is minimal TGF-beta secretion in unactivated monocytes, even though TGF-beta mRNA is expressed in these cells at a level similar to that in activated, lipopolysaccharide-treated cultures. U937 lymphoma cells, which have monocytic characteristics, also express mRNA for TGF-beta. Freshly isolated monocytes, both control and lipopolysaccharide-treated, secrete an acid-labile binding protein that inhibits TGF-beta action. We conclude the following: (i) that expression of TGF-beta mRNA is unrelated to monocyte activation, (ii) that secretion of TGF-beta is induced by monocyte activation, and (iii) that cosecretion of TGF-beta and its monocyte/macrophage-derived binding protein may modulate growth factor action. In contrast, monocytic expression of other growth factor genes, such as the B chain of platelet-derived growth factor, is not constitutive and requires activation.
TL;DR: A cDNA encoding the CD2 antigen has been isolated by a highly efficient technique based on transient expression in COS cells and adherence of cells expressing surface antigen to antibody-coated dishes.
Abstract: A cDNA encoding the CD2 antigen has been isolated by a highly efficient technique based on transient expression in COS cells and adherence of cells expressing surface antigen to antibody-coated dishes. COS cells expressing a CD2 cDNA isolated by this method readily formed rosettes with sheep as well as human and other mammalian erythrocytes. Pretreatment of transfected COS cells with anti-CD2 antibody, or pretreatment of human erythrocytes with anti-LFA-3 antibody, abolished rosette formation.
Journal Article•
TL;DR: The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure and does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthetic, or ADP ribosylation reactions.
Abstract: L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.
TL;DR: A model system for cytokine-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected promonocyte clones was established and can be used to delineate the potential mechanisms whereby HIV-1 infection regulates cellular gene expression.
Abstract: A model system for cytokine-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected promonocyte clones was established. The parent promonocyte cell line U937 was chronically infected with HIV-1 and from this line a clone, U1, was derived. U1 showed minimal constitutive expression of HIV-1, but virus expression was markedly up-regulated by a phytohemagglutinin-induced supernatant containing multiple cytokines and by recombinant granulocyte/macrophage colony-stimulating factor alone. Recombinant interleukin-1 (IL-1), IL-2, interferon-gamma, and tumor necrosis factor-alpha did not up-regulate virus expression. Concomitant with the cytokine-induced up-regulation of HIV-1, expression of membrane-bound IL-1 beta was selectively induced in U1 in the absence of induction of other surface membrane proteins. This cytokine up-regulation of IL-1 beta was not seen in the uninfected parent U937 cell line. These studies have implications for the understanding of the mechanism of progression from a latent or low-level HIV-1 infection to a productive infection with resulting immunosuppression. In addition, this model can be used to delineate the potential mechanisms whereby HIV-1 infection regulates cellular gene expression.
TL;DR: A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described, capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.
Abstract: A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.
TL;DR: Analysis of normal skin biopsies using in situ hybridization and immunohistochemistry demonstrates the in vivo presence of TGF-α messenger RNA and protein in the stratified epidermis, suggesting that a mechanism of auto-induction exists.
Abstract: Transforming growth factor-alpha (TGF-alpha) is a polypeptide which is structurally related to epidermal growth factor (EGF) and binds to the EGF receptor TGF-alpha synthesis occurs in a variety of neoplastic cells and during early fetal development but has not been reported in normal cells of the adult organisms TGF-alpha has therefore been regarded as an embryonic growth factor which is inappropriately expressed during neoplasia Here we report that primary cultures of normal human keratinocytes synthesize TGF-alpha Furthermore, we show that addition of EGF or TGF-alpha to these cultures induces TGF-alpha gene expression, suggesting that a mechanism of auto-induction exists Analysis of normal skin biopsies using in situ hybridization and immunohistochemistry demonstrates the in vivo presence of TGF-alpha messenger RNA and protein in the stratified epidermis
TL;DR: This work uses gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse5.
Abstract: Two recent developments suggest a route to predetermined alterations in mammalian germlines. These are, first, the characterization of mouse embryonic stem (ES) cells that can still enter the germline after genetic manipulation in culture and second, the demonstration that homologous recombination between a native target chromosomal gene and exogenous DAN can be used in culture to modify specifically the target locus. We here use gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse. This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulating mammalian genomes in predetermined ways.
TL;DR: The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo.
Abstract: Cultures greatly enriched in granule cells from early postnatal cerebellum (P8) were grown in a medium containing fetal calf serum. Under the conditions used, nerve cells died, usually within a week, unless the K+ concentration in the medium was greater than or equal to 20 mM. The requirement for elevated [K+]e was manifested by about 3 d in vitro, and after this time continuous exposure to high [K+]e was essential for the survival of the granule cells. The initial morphological and biochemical maturation of the granule cells was similar in the presence and the absence of elevated [K+]e, suggesting that the dependence on depolarizing conditions develops in parallel with the expression of the differentiated characteristics of the cells. The positive effect of elevated [K+]e on granule cell survival was not influenced by preventing bioelectric activity in the cultures with TTX and xylocaine. On the other hand, depolarization-induced transmembrane Ca2+ flux was essential in securing the maintenance of the granule cells. Depolarized nerve cells were compromised when Ca2+ entry was blocked by elevated Mg2+, EGTA, or organic Ca2+ antagonists, while dihydropyridine Ca2+ agonists [BAY K 8644, (+)-(S)-202 79 1 and CGP 28392] were potent agents preventing nerve cell loss in the presence of 15 mM [K+]e, which was ineffective on its own. Calmodulin inhibitors (1 microM trifluoperazine or calmidazolium) blocked the beneficial effect of K+-induced depolarization on granule cells. The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo through excitatory amino acid receptors, including N-methyl-D-aspartate receptors, involving Ca2+ entry and the activation of a Ca2+/calmodulin- dependent protein kinase.
TL;DR: This work cloned full-length cDNA encoding E-cadherin from F9 cells and introduced it into L fibroblasts deficient in the molecule, which acquired strong Ca2+-dependent aggregating activity and were morphologically transformed so as to form colonies in which cells were tightly connected to each other.
Abstract: E-cadherin is a cell surface glycoprotein responsible for Ca2+-dependent intercellular adhesion between epithelial cells1; it is also called uvomorulin2, L-CAM (ref. 3), cell-CAM 120/80 (ref. 4) or Arc-1 (ref. 5). Because blocking the action of E-cadherin by monoclonal antibodies causes dispersion of compact cell colonies1, this molecule is thought to be an important factor for maintenance of multicellular systems. To demonstrate directly that E-cadherin is involved in cell–cell adhesion, we cloned full-length cDNA encoding E-cadherin from F9 cells and introduced it into L fibroblasts deficient in E-cadherin. These L cells acquire strong Ca2+-dependent aggregating activity by expressing the E-cadherin derived from the introduced cDNA, and were morphologically transformed so as to form colonies in which cells were tightly connected to each other.
TL;DR: This work establishes a link between B cell phenotype and the accompanying pattern ofEBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.
Abstract: Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.
TL;DR: The purified PTH-related protein had a specific biological activity approximately equal to 6 times greater than that of bovine PTH(1-34) and may have a role in the syndrome of humoral hypercalcemia of malignancy.
Abstract: A protein with biological activities similar to parathyroid hormone (PTH) has been purified from serum-free culture medium obtained from a human lung cancer cell line (BEN). A major protein band of 18 kDa was obtained on NaDodSO4/polyacrylamide gels, with faint bands at 35 kDa and 67 kDa. Biological activity was associated only with the 18-kDa band. Amino acid sequence analysis of the material purified by HPLC revealed that 8 of the 16 residues were identical with those of human PTH. Antibody raised to a corresponding synthetic peptide recognized the PTH-related material but showed less than 1% cross-reactivity with human PTH amino-terminal peptides. BEN cells contained PTH DNA, but not PTH messenger RNA, indicating involvement of another gene. The purified PTH-related protein had a specific biological activity approximately equal to 6 times greater than that of bovine PTH(1-34). The PTH-related protein may have a role in the syndrome of humoral hypercalcemia of malignancy.
TL;DR: A new, direct and rapid technique that employs scrape-loading to introduce a low molecular weight fluorescent dye, Lucifer yellow CH, into cells in culture and allows the monitoring of its transfer into contiguous cells, showing effective blockage of the dye transfer at non-cytotoxic doses.
TL;DR: Exogenous addition to bulk cell cultures of small antisense oligomers inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry and Interestingly, c- myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.
Abstract: Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in GO, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated1–4. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells3. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible5–7, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific anti-sense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis8,9. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region10. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically11. We report here that the same c-myc complementary oligonuelectide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.
TL;DR: Primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro and it was found that lipoproteins contained in these cells may function to redistribute lipid and regulate cholesterol homeostasis within the brain.
TL;DR: A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells after treatment with IL-1 or TNF.
Abstract: A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.
TL;DR: In experiments designed to assess the role of the HER2 gene in oncogenesis, overexpression of unaltered HER2 coding sequences in NIH 3T3 cells resulted in cellular transformation and tumorigenesis.
Abstract: The HER2 gene encodes a cell-surface glycoprotein with extensive homology to the epidermal growth factor receptor. Recently it was found to be amplified in about 30% of primary human breast malignancies. In experiments designed to assess the role of the HER2 gene in oncogenesis, we found that overexpression of unaltered HER2 coding sequences in NIH 3T3 cells resulted in cellular transformation and tumorigenesis.
TL;DR: The findings indicate that overexpression of the erbB‐2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities.
Abstract: Amplification of the erbB/EGF receptor and a structurally related gene, designated erbB-2, have previously been detected in a variety of human tumors In a series of human mammary tumor cell lines, analysis of transcripts of these genes revealed elevated levels of one or the other in more than 60% of tumors analyzed Eight cell lines demonstrated erbB-2 mRNA levels ranging from 4- to 128-fold above those of normal controls erbB-2 expression was evaluated in comparison to the expression level of actin observed in these cell lines There was no evidence of an aberrantly sized erbB-2 transcript in any of these lines Immunoblot analysis indicated elevation in levels of the 185-kd product of the erbB-2 gene expressed by these cells In four lines erbB-2 gene amplification in the absence of an apparent gene rearrangement was demonstrated In a representative cell line of this type, SK-BR-3, the amplified erbB-2 gene copies were located in an aberrant chromosomal location Four additional cell lines, which demonstrated 4- to 8-fold overexpression of erbB-2 mRNA, did not exhibit gene amplification In a representative cell line of this type ZR-75-1, an apparently normal chromosomal location was found for the erbB-2 gene Our findings indicate that overexpression of the erbB-2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities
TL;DR: EGF receptor overexpression appears to amplify normal EGF signal transduction, and high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.
TL;DR: Using polyclonal monospecific antisera to recombinant protein and synthetic peptides, it is demonstrated that the IP-10 protein is associated, in vivo, with a delayed-type hypersensitivity response.
Abstract: An IFN-gamma-inducible protein, IP-10, has previously been described to belong to a gene family of chemotactic and mitogenic proteins, associated with inflammation and proliferation. Biochemical characterization of this predicted protein has been pursued through the development of polyclonal monospecific antisera to recombinant protein and synthetic peptides. These reagents establish that the IP-10 protein is secreted from a variety of cells (endothelial, monocyte, fibroblast, and keratinocyte) in response to IFN-gamma. Posttranslational processing occurs in the biosynthesis of this protein, resulting in a 6-7-kD species, which may reflect COOH-terminal cleavage. Pulse-chase studies indicate that this processing is a rapid event in the primary cell lines studied, completed in the 30-min labeling period. A model is presented for the processing and secondary structure of this protein. In an accompanying study, Kaplan, et al. using these antisera, demonstrate that the IP-10 protein is associated, in vivo, with a delayed-type hypersensitivity response.
Journal Article•
TL;DR: The Saos-2 cells possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.
Abstract: This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4–7 µmol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nm, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D 3 to 3.2S receptor protein with an apparent K d of 0.21 nm; the numbers of sites per cell were 3,300 at 50,000 cells/cm 2 and 1,800 at 280,000 cells/cm 2 . The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around M r 40,000 in nonreduced gels and M r 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.