The cytotoxic effect of purine riboside on COS-7 cells.
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5'-Nucleotidase↗
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Activation of PPARalpha and PPARgamma by environmental phthalate monoesters.
Phthalate esters are widely used as plasticizers in the manufacture of products made of polyvinyl chloride. Mono-(2-ethylhexyl)-phthalate (MEHP) induces rodent hepatocarcinogenesis by a mechanism that involves activation of the nuclear transcription factor peroxisome proliferator-activated receptor-alpha (PPARalpha). MEHP also activates PPAR-gamma (PPARgamma), which contributes to adipocyte differentiation and insulin sensitization. Human exposure to other phthalate monoesters, including metabolites of di-n-butyl phthalate and butyl benzyl phthalate, is substantially higher than that of MEHP, prompting this investigation of their potential for PPAR activation, assayed in COS cells and in PPAR-responsive liver (PPARalpha) and adipocyte (PPARgamma) cell lines. Monobenzyl phthalate (MBzP) and mono-sec-butyl phthalate (MBuP) both increased the COS cell transcriptional activity of mouse PPARalpha, with effective concentration for half-maximal response (EC50) values of 21 and 63 microM, respectively. MBzP also activated human PPARalpha (EC50=30 microM) and mouse and human PPARgamma (EC50=75-100 microM). MEHP was a more potent PPAR activator than MBzP or MBuP, with mouse PPARalpha more sensitive to MEHP (EC50=0.6 microM) than human PPARalpha (EC50=3.2 microM). MEHP activation of PPARgamma required somewhat higher concentrations, EC50=10.1 microM (mouse PPARgamma) and 6.2 microM (human PPARgamma). No significant PPAR activation was observed with the monomethyl, mono-n-butyl, dimethyl, or diethyl esters of phthalic acid. PPARalpha activation was verified in FAO rat liver cells stably transfected with PPARalpha, where expression of several endogenous PPARalpha target genes was induced by MBzP, MBuP, and MEHP. Similarly, activation of endogenous PPARgamma target genes was evidenced for all three phthalates by the stimulation of PPARgamma-dependent adipogenesis in the 3T3-L1 cell differentiation model. These findings demonstrate the potential of environmental phthalate monoesters for activation of rodent and human PPARs and may help to elucidate the molecular basis for the adverse health effects proposed to be associated with human phthalate exposure.
3T3-L1 Cells↗
A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system.
The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.
Alleles↗
Formation of the human fibrinogen subclass fib420: disulfide bonds and glycosylation in its unique (alphaE chain) domains.
COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel alphaE chain and those of the more abundant subclass whose alpha chains lack alphaE's globular C-terminus. That region, referred to as the alphaEC domain, is closely related to the ends of beta and gamma chains of fibrinogen (betaC and gammaC). Transfection of COS cells with alphaE, beta, and gamma cDNAs alone results in secretion of the symmetrical molecule (alphaEbetagamma)2, also known as Fib420. Cotransfection with cDNA for the shorter alpha chain yielded secretion of both (alphabetagamma)2 and (alphaEbetagamma)2 but no mixed molecules of the structure alphaalphaE(betagamma)2. Exploiting the COS cells' fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the alphaE chain. No evidence from Cys --> Ser replacements was found for interchain disulfide bridges involving the four cysteines of the alphaEC domain. However, for fibrinogen secretion, the alphaE, beta, and gamma subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.
Animals↗
Expression of recombinant glutathione S-transferase pi, Ya, or Yb1 confers resistance to alkylating agents.
Increased levels of glutathione S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) mRNA, protein, and activity in tumor biopsy samples and in drug-resistant cultured cells are associated with resistance to anticancer drugs. We report that each of three full-length cloned GST cDNAs, that for pi (acidic), Ya (basic), and Yb1 (neutral), can confer drug resistance when expressed in cultured mammalian cells. In one approach, stably transfected mouse C3H/10T1/2 cells that express GST pi, Ya, or Yb1 were cloned and analyzed for drug resistance in colony-forming assays. Transiently transfected COS cells that were sorted on a fluorescence-activated cell sorter were used in the second approach to avoid interclonal variation in factors other than the recombinant GST and to show that reversion of transient GST expression correlated with loss of drug resistance. A sorting technique, developed to separate the 20% of the electroporated COS cell population that transiently expressed GST pi, Ya, or Yb1 from the nonexpressing population, was based on a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane. GST Ya conferred the greatest increase in resistance to chlorambucil and melphalan (1.3- to 2.9-fold), Yb1 conferred the greatest increase in resistance to cisplatin (1.5-fold), and pi conferred the greatest increase in resistance to a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene and 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and doxorubicin (1.5- and 1.3-fold) relative to controls. These resistance values to alkylating agents are commensurate with values observed clinically. Cytotoxicity curves representing recombinant GST+ populations were significantly different from their controls with P values ranging from 0.005 to 0.0001. No resistance to vinblastine was detected. Conferred drug resistance was proportional to the magnitude of GST Ya expression, and reversion of transient expression in GST Ya+ COS cell clones to a GST Ya- phenotype was associated with total loss of drug resistance.
Animals↗
Effects of heterologous expression of thioredoxin reductase on the level of reactive oxygen species in COS-7 cells.
Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.
Animals↗
Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.
Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.
Animals↗
Multiple GPI-anchored receptors control GDNF-dependent and independent activation of the c-Ret receptor tyrosine kinase.
Glial cell line-derived neurotrophic factor (GDNF) mediates neuronal survival through a receptor complex composed of the c-Retproto-oncogene and GFR alpha-1, a member of a family of GPI-anchored receptors. The extent of cross-talk between GDNF and GFR alpha receptors and its possible significance for c-Ret activation is presently unclear. Using chemical crosslinking we demonstrate here a specific interaction between GDNF and GFR alpha-2 expressed in COS cells, albeit of a lower affinity than the one between GDNF and GFR alpha-1. In addition, GFR alpha-2 mediated crosslinking of GDNF of c-Ret as well as ligand-dependent stimulation of c-Ret tyrosine phosphorylation. We also describe the isolation of a novel, more divergent member of the GFR alpha family, GFR alpha-3, which did not bind GDNF directly, but was able to mediate crosslinking of GDNF to c-Ret when both receptors were coexpressed in COS cells. Thus, all three GFR alpha receptors mediate GDNF binding to c-Ret with efficiencies GFR alpha-1 > GFR alpha-2 > GFR alpha-3. c-Ret showed high levels of constitutive tyrosine autophosphorylation upon overexpression in COS cells, which was inhibited in a dose-dependent manner by coexpression with any of the GFR alpha receptors, suggesting that GFR alpha s may also provide a gain control mechanism to increase the signal-to-noise ratio of the response to ligand. GFR alpha-2 showed a dynamic pattern of expression in rat brain, distinct from that of GFR alpha-1, characterized by high expression in cortex, basal forebrain, and specific layers of the olfactory bulb, and low or no expression in substantia nigra, cerebellum, and motor nuclei. GFR alpha-2, but not GFR alpha-3 mRNA expression was highly induced in several nuclei after stimulation with kainic acid. In contrast to GFR alpha-1 and GFR alpha-2, GFR alpha-3 expression in postnatal and adult brain was highly restricted. Developmentally regulated expression of GFR alpha-3 was, however, detected in several peripheral organs and ganglia. Together, these results indicate complementary roles for GFR alpha receptors in the regulation of c-Ret activity and the maintenance of distinct neuronal circuits in the central and peripheral nervous systems.
Amino Acid Sequence↗
Recombinant f1 phage particles can transfect monkey COS-7 cells by DEAE dextran method.
Recombinant f1 phage particles containing cDNA of the human urokinase type-plasminogen activator (u-PA) under the control of the simian virus 40 (SV40) early promoter were used for transfection of monkey COS-7 cells using a DEAE dextran method. The fibrinolytic activity of u-PA was detected in the culture medium of the 10(5) cells transfected with the phage particles containing single-stranded (ss) DNA of more than 0.2 ng. This finding will lead us to develop a simple and efficient method for expression cloning using mammalian cells.
Animals↗
Phospholipase C activation by VIP1 and VIP2 receptors expressed in COS 7 cells involves a pertussis toxin-sensitive mechanism.
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Animals↗
A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.
An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.
Amino Acid Sequence↗
Live imaging of glucose homeostasis in nuclei of COS-7 cells.
Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.
Algorithms↗
Cloning of murine and rat vascular cell adhesion molecule-1.
Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.
Amino Acid Sequence↗
Characterization of hepatitis B virus X gene: in vitro translation of mRNA from COS-1 cells transfected with the X gene.
To elucidate the biological role of the X region of HBV DNA, we constructed an expression plasmid pSV2HBx by inserting the StuI/BglII fragment of HBV DNA into the pSV2 vector. Although X region products were not identified in vivo in transfected COS-1 cells, transcripts were translated in vitro and a 17-k Da protein was immunoprecipitated with an antibody raised against a synthetic peptide corresponding to the carboxy terminus of the X region open reading frame. Among 68 HBV carrier sera tested, two were found to contain antibodies reactive to the in vitro translation product. These results suggest that the X region is a gene which can be actually expressed at some stage of HBV infection.
Animals↗
The hydrolysis of oestrone sulphate and dehydroepiandrosterone sulphate by human steroid sulphatase expressed in transfected COS-1 cells.
Whether the same or distinct steroid sulphatases (STS) are involved in the hydrolysis of alkyl and aryl steroid sulphates remains controversial. We have examined the ability of a placental steroid sulphatase to hydrolyse oestrone sulphate and/or dehydroepiandrosterone sulphate (DHA-S) by expressing the enzyme in COS-1 cells. Using either intact cells or broken cell preparations, the expressed sulphatase was found to hydrolyse both oestrone sulphate and DHA-S. The catalysis of oestrone sulphate and DHA-S by the expressed sulphatase was almost completely abolished by the steroid sulphatase inhibitor, oestrone-3-O-sulphamate. It is concluded that both alkyl and aryl steroid sulphates can be hydrolysed by the same steroid sulphatase.
Animals↗
Enzymatic activities of P-450(11 beta)s expressed by two cDNAs in COS-7 cells.
Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.
Animals↗
Expression of four mutant human ornithine transcarbamylase genes in cultured Cos 1 cells relates to clinical phenotypes.
Ornithine transcarbamylase (OTC) deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. To detect mutations in the OTC gene using genomic DNA, we have developed a method in which all exons and adjacent introns are amplified and sequenced. Although this approach detected mutations in many cases, the relationship between a mutation and the OTC phenotype was not firmly established. Therefore, we investigated the issue by expression analysis of mutant OTC cDNA in cultured cells. Four mutant OTC cDNAs were constructed, based on the reported cases, using our newly developed method. The normal (wild-type) human OTC cDNA was reproducibly expressed at high levels in these Cos 1 cells. Predicted OTC activities of mutant OTC cDNAs ranged from 0% to 8.9% of the normal level together with variable amounts of the enzyme protein. The predicted enzyme activities account for the clinical phenotype of the disease. Our observations confirm that these mutations are responsible for OTC deficiency in these patients.
Amino Acid Metabolism, Inborn Errors↗
Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment.
We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.
Animals↗