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Drug features that contribute to the activity of quinolones against mammalian topoisomerase II and cultured cells: correlation between enhancement of enzyme-mediated DNA cleavage in vitro and cytotoxic potential.
Elsea SH, McGuirk PR, Gootz TD, Moynihan M, Osheroff N
(1993) Antimicrob Agents Chemother 37: 2179-86
MeSH Terms: Animals, Anti-Infective Agents, CHO Cells, Cattle, Cell Death, Cells, Cultured, Cricetinae, DNA, Fluoroquinolones, Humans, Nucleic Acid Conformation, Quinolones, Structure-Activity Relationship, Topoisomerase II Inhibitors
Show Abstract · Added March 5, 2014
CP-115,953 [6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid] is a novel quinolone that is highly active against topoisomerase II in vitro and in mammalian cells in culture (M. J. Robinson, B. A. Martin, T. D. Gootz, P. R. McGuirk, M. Moynihan, J. A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase II and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type II enzyme with a potency that was approximately 600-fold greater than that of the antimicrobial quinolone ciprofloxacin and approximately 50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase II was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type II enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase II rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase II to a cellular poison.
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14 MeSH Terms
Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.
Hubbard SC, Walls L, Ruley HE, Muchmore EA
(1994) J Biol Chem 269: 3717-24
MeSH Terms: Animals, Asparagine, Base Sequence, CHO Cells, Clone Cells, Cricetinae, DNA Primers, Glycoproteins, Glycosylation, Kanamycin Kinase, Molecular Sequence Data, Moloney murine leukemia virus, Mutagenesis, Insertional, Neuraminic Acids, Oligosaccharides, Phenotype, Phosphotransferases (Alcohol Group Acceptor), Polymerase Chain Reaction, Promoter Regions, Genetic, Repetitive Sequences, Nucleic Acid, Restriction Mapping, Transfection, Wheat Germ Agglutinins
Show Abstract · Added May 29, 2014
Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.
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23 MeSH Terms
Synthesis of heat shock proteins following oxidative challenge: role of glutathione.
Sierra-Rivera E, Meredith MJ, Voorhees GJ, Oberley LW, Eisert DR, Freeman ML
(1994) Int J Hyperthermia 10: 573-86
MeSH Terms: Animals, CHO Cells, Carrier Proteins, Cricetinae, Electroporation, Glutathione, Glutathione Disulfide, HSC70 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, NADP, Oxidation-Reduction, RNA, Messenger, Sulfhydryl Compounds, Vitamin K
Show Abstract · Added March 5, 2014
The relationship between glutathione metabolism, menadione sodium bisulphite oxidation of protein thiols, and the synthesis of hsc70 was investigated using CHO cells. A 30-min/37 degrees C exposure to menadione, a compound which redox cycles to produce superoxide anion radicals and hydrogen peroxide, resulted in rapid accumulation of hsc70 mRNA. PAGE and Western blot analysis indicated increased synthesis such that accumulation of hsc70 occurred. These changes were preceded by rapid oxidation of GSH to GSSG, followed by GSH depletion, and subsequent protein thiol oxidation. As a test of whether a correlation existed between GSH oxidation and depletion, protein thiol oxidation and hsp synthesis, cells were exposed to menadione in the absence and presence of glucose. Synthesis of hsc70 was increased in cells exposed to menadione in the absence of glucose compared with its presence. As a further test, cells were exposed to BSO/DEM in order to deplete GSH and then exposed to menadione. The synthesis of hsc70 following exposure to menadione was greatly increased in GSH-depleted cells compared with GSH-replete cells. Experiments were conducted to determine if electroporation of cells in GSSG containing buffer affected hsp synthesis. Electroporation in glucose-free buffer containing 3 mM GSSG did not affect hsp synthesis. We interpret these results to indicate that the inability to maintain glutathione in a reduced form during menadione redox cycling resulted in protein thiol oxidation. This, in turn, resulted in accumulation of hsc70 mRNA with a subsequent increase in the synthesis of hsc70.
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15 MeSH Terms
Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.
Ktistakis NT, Brown HA, Sternweis PC, Roth MG
(1995) Proc Natl Acad Sci U S A 92: 4952-6
MeSH Terms: ADP-Ribosylation Factors, Animals, Brefeldin A, CHO Cells, Carrier Proteins, Cell Fractionation, Cell Line, Cricetinae, Cyclopentanes, Dogs, Dose-Response Relationship, Drug, Enzyme Activation, GTP-Binding Proteins, Golgi Apparatus, Guanosine 5'-O-(3-Thiotriphosphate), Intracellular Membranes, Kinetics, Orthomyxoviridae, Phospholipase D, Protein Binding, Protein Synthesis Inhibitors
Show Abstract · Added March 21, 2013
ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.
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21 MeSH Terms
Use of monoclonal anti-light subunit antibodies to study the structure and function of the Entamoeba histolytica Gal/GalNAc adherence lectin.
McCoy JJ, Weaver AM, Petri WA
(1994) Glycoconj J 11: 432-6
MeSH Terms: Acetylgalactosamine, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Blotting, Western, CHO Cells, Chromatography, Affinity, Cricetinae, Entamoeba histolytica, Galactose, Lectins, Membrane Glycoproteins, Molecular Sequence Data, Protein Binding, Protozoan Proteins, Structure-Activity Relationship
Show Abstract · Added March 5, 2014
Adherence of Entamoeba histolytica trophozoites to host cells is mediated by a galactose (Gal) and N-acetylgalactosamine (GalNAc)-specific surface lectin. The lectin is a heterodimeric protein composed of heavy (170 kDa) and light (35-31 kDa) subunits linked by disulfide bonds. Polyclonal and monoclonal antibodies (mAb) raised against a light subunit-glutathione-S-transferase fusion protein were used to probe its structure and function. Four light subunit-specific mAb were produced which recognized distinct epitopes on five different light subunit isoforms. Immunoblots with these mAb demonstrated co-migration of light and heavy subunits when nonreduced trophozoite proteins were analysed by SDS-PAGE, indicating that the subunits do not exist free of the heterodimer in significant quantities. While anti-heavy subunit antibodies had previously been shown to alter adherence, anti-light subunit antibodies did not, suggesting that the heavy subunit contains the carbohydrate recognition domain.
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16 MeSH Terms
Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor.
Freeman ML, Borrelli MJ, Syed K, Senisterra G, Stafford DM, Lepock JR
(1995) J Cell Physiol 164: 356-66
MeSH Terms: Animals, CHO Cells, Cricetinae, DNA-Binding Proteins, Diamide, Drug Stability, Heat Shock Transcription Factors, Heat-Shock Proteins, Hot Temperature, Oxidation-Reduction, Protein Denaturation, Shock, Signal Transduction, Sulfhydryl Compounds, Transcription Factors
Show Abstract · Added March 5, 2014
The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.
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15 MeSH Terms
A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.
Rathmell WK, Chu G
(1994) Mol Cell Biol 14: 4741-8
MeSH Terms: Animals, Ataxia Telangiectasia, Azacitidine, Binding, Competitive, CHO Cells, Cell Line, Cell Nucleus, Cricetinae, DNA, DNA Damage, DNA Probes, DNA Repair, DNA-Binding Proteins, Genes, Immunoglobulin, Genetic Complementation Test, HeLa Cells, Humans, Kinetics, Mice, Mice, SCID, Nuclear Proteins, Recombination, Genetic, Skin
Show Abstract · Added October 17, 2015
We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.
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23 MeSH Terms
Integrin cytoplasmic domains mediate inside-out signal transduction.
O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, Quaranta V, Loftus JC, Shattil SJ, Ginsberg MH
(1994) J Cell Biol 124: 1047-59
MeSH Terms: Amino Acid Sequence, Animals, Base Sequence, CHO Cells, Cell Line, Conserved Sequence, Cricetinae, Cytoplasm, Energy Metabolism, Fibronectins, Humans, Integrins, Ligands, Molecular Sequence Data, Platelet Glycoprotein GPIIb-IIIa Complex, Receptors, Fibronectin, Recombinant Fusion Proteins, Signal Transduction, Transfection
Show Abstract · Added March 27, 2014
We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.
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19 MeSH Terms
Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.
Nanney LB, Mueller SG, Bueno R, Peiper SC, Richmond A
(1995) Am J Pathol 147: 1248-60
MeSH Terms: Animals, Antigens, CD, Burns, CHO Cells, Chemokine CXCL1, Chemokines, CXC, Chemotactic Factors, Cricetinae, Epidermis, Growth Substances, Humans, Intercellular Signaling Peptides and Proteins, Keratinocytes, Neoplasm Proteins, Receptors, Interleukin, Receptors, Interleukin-8B, Skin, Tissue Embedding, Wound Healing
Show Abstract · Added May 30, 2013
The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.
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19 MeSH Terms
A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells.
Hansen AP, Petros AM, Mazar AP, Pederson TM, Rueter A, Fesik SW
(1992) Biochemistry 31: 12713-8
MeSH Terms: Amino Acids, Animals, CHO Cells, Carbon Isotopes, Cells, Cultured, Cricetinae, Culture Media, Cysteine, Glutamine, Isotope Labeling, Magnetic Resonance Spectroscopy, Nitrogen Isotopes, Recombinant Proteins, Urokinase-Type Plasminogen Activator
Show Abstract · Added March 5, 2014
A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.
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14 MeSH Terms