losartan-potassium and Fibrosarcoma

Topics: Animals; Blood Protein Electrophoresis; Cell Line; Cell Line, Tumor; Cricetinae; Doping in Sports; Erythropoietin; Fibrosarcoma; Glycosylation; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Kidney; Mesocricetus; Molecular Weight; Polyethylene Glycols; Protein Processing, Post-Translational; Recombinant Proteins; Urinalysis

Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.

Topics: Butyrates; Cell Line, Tumor; Computer Simulation; Dose-Response Relationship, Drug; Erythropoietin; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Models, Biological; Neoplasm Proteins; Protein Engineering; Recombinant Proteins

The induced expression of xanthine-guanine phosphoribosyl transferase (XGPRT) by low concentrations (-2 pg/ml) of interferon-alpha (IFN-alpha) or IFN-beta, in the 2fTPGH cell line caused a 50% cytotoxicity when these cells were grown in medium containing 6-thioguanine. We extended the application of this sensitive, reliable, and easy bioassay to other members of the cytokine family. To activate the IFN signaling pathway, we made receptor chimeras, consisting of the IFN type I receptor intracellular and transmembrane domains, fused to either the interleukin-5 (IL-5) receptors or erythropoietin (Epo) receptor extracellular domains as model systems. 2fTGH cells, stably transfected with these receptor chimeras, responded to very low concentrations of IL-5 or Epo (IC50 values of approximately 15 pg and 3 pg/ml, respectively) and thus can be used as a very sensitive bioassay for both ligands. Background activity of IL-5, Epo, tumor necrosis factor (TNF), IL-6, or leptin on cells that did not carry the receptor chimeras was very low. This methodology can in principle be extended to any ligand that acts via clustering of its receptors.

Topics: Biological Assay; Coloring Agents; Culture Media; Enzyme Induction; Erythropoietin; Fibrosarcoma; Gentian Violet; Humans; Interferon alpha-2; Interferon-alpha; Interferon-beta; Interleukin-5; Interleukin-6; Leptin; Ligands; Membrane Proteins; Neoplasm Proteins; Pentosyltransferases; Receptor Aggregation; Receptor, Interferon alpha-beta; Receptors, Erythropoietin; Receptors, Interferon; Receptors, Interleukin; Receptors, Interleukin-5; Recombinant Fusion Proteins; Recombinant Proteins; Selection, Genetic; Sensitivity and Specificity; Signal Transduction; Thioguanine; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 x 10(6) cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Topics: Animals; Avian Sarcoma Viruses; Cells, Cultured; Chick Embryo; Chloramphenicol O-Acetyltransferase; CHO Cells; Cricetinae; Cytomegalovirus; Ducks; Embryo, Nonmammalian; Erythropoietin; Fibrosarcoma; Gene Expression Regulation; Genetic Engineering; Humans; Mice; Mice, Inbred Strains; Promoter Regions, Genetic; Quail; Recombinant Proteins; Simian virus 40; Terminal Repeat Sequences; Transfection

Recombinant adeno-associated virus encoding the monkey erythropoietin gene (rAAV-cm-Epo) was generated and tested for its potential to confer long-term expression of the gene product following intramuscular injection. A single intramuscular injection of 2 x 10(12) rAAV-cm-Epo particles into two baboons led to sustained high circulating Epo levels and a concomitant increase in hematocrit. The hematocrits reached 62 and 75% by week 10 (from pre-injection values of 38 and 40%, respectively) and remained elevated throughout the study period (28 weeks). Circulating Epo levels were also elevated throughout the study period. Our data demonstrate the potential for long-term gene expression in large animals by a single intramuscular injection of a recombinant adeno-associated virus (rAAV) vector.

Topics: Animals; Antibodies; Dependovirus; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Fibrosarcoma; Gene Transfer Techniques; Hematocrit; Humans; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Papio; Recombinant Proteins; Time Factors