Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Variability in the Stability and Productivity of Transfected Genes in Chinese Hamster Ovary (CHO) cells

In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these two traits, we would need to further our understanding of the factors affecting them. These would include integration site of the gene, gene copy number, cell phenotypic variation and cell environment. As these factors play different parts in the development process, they lead to variable productivity and stability of the transfected gene between clones, the well-known phenomenon of “clonal variation”. A study of this phenomenon and how the various factors contribute to it will thus shed light on strategies to improve productivity and stability in the production cell line. Of the four factors, the site of gene integration appears to be one of the most important. Hence, it is proposed that work is done on studying how different integration sites affect the productivity and stability of transfected genes in the development process. For the study to be more industrially relevant, it is proposed that the Chinese Hamster Ovary dhfr-deficient cell line, CHO-DG44, is used as the model system.

Genotoxicity of Brosimum gaudichaudii measured by the Salmonella/microsome assay and chromosomal aberrations in CHO cells

The root bark of Brosimum gaudichaudii Trecul (Moraceae) is popularly used for treatment of vitiligo. In the present study the mutagenic activity of the aqueous and methanolic extract as well as of the n-butanolic fraction of this medicinal plant were evaluated using Salmonella typhimurium assays, TA100, TA98, TA102 and TA97a strains, while the clastogenic effect in Chinese hamster ovary (CHO) cells in the G(1)/S, S and G(2)/S phases of the cell cycle. The results showed mutagenic activity of the aqueous extract against TA102 in the presence of S9, and of methanolic extract, with and without metabolic activation. TA100 mutagenicity was only observed for the methanolic extract in the absence of S9. The n-butanolic fraction did not present mutagenic activity. In CHO cells only the methanolic extract induced a significant increase of chromosomal aberrations in the G(1)/S and S phases, whereas a decrease in the mitotic index was observed in the G(1)/S and G(2)/S phases. No clastogenicity was observed for the aqueous extract. The furocoumarins (psoralen and bergapten) presented in the extracts might contribute to the mutagenicity. The lower activity of the aqueous extract was probably due to the presence of smaller amount of furocoumarins compared to the methanolic extract. (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.

Genotoxicity and cytotoxicity of glass ionomer cements on Chinese hamster ovary (CHO) cells

Glass ionomer cements are widely used in dentistry as restorative materials and adhesives for composite restorations. However, the results of genotoxicity studies using these materials are inconclusive in literature. The goal of this study was to examine the genotoxic and cytotoxic potential of three different glass ionomer cements available commercially (Ketac Cem, Ketac Molar and Vitrebond) by the single cell gel (comet) assay and trypan blue exclusion test, respectively. For this, such materials were exposed to Chinese hamster ovary (CHO) cells in vitro for 1 h at 37 degrees C. Data were assessed by Kruskall-Wallis nonparametric test. The results showed that the powder from Ketac Molar displayed genotoxicity only in the maximum concentration evaluated (100 mu g/mL). In the same way, the liquid from Vitrebond at 0.1% dilution caused an increase of DNA injury. Significant differences (P < 0.05) in cytotoxicity provoked by all powders tested of glass ionomer cements were observed for exposure at 1000 mu g/mL concentration. With respect to liquids of glass ionomer cements evaluated, the major toxic effect on cell viability was produced at 10%, beginning at the dilution of 0.5% for Vitrebond. Taken together, we conclude that some components of glass ionomer cements show both genotoxic and cytotoxic effects.

Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells

Objective. In the current study, the potential DNA damage associated with exposure to a number of antimicrobial endodontic compounds was assessed by the single cell gel (comet) assay in vitro.Study design. Chinese hamster ovary (CHO) cells were exposed to formocresol, paramonochlorophenol, calcium hydroxide, or chlorhexidine at final concentration ranging from 0.01% to 1%.Results. Formocresol, paramonochlorophenol, and calcium hydroxide, as well as chlorhexidine in all concentrations tested did not contribute to the DNA damage.Conclusion. These findings are clinically relevant since they represent an important contribution to the correct evaluation of the potential health risk associated with exposure to dental agents.

Genotoxicity and cytotoxicity of mineral trioxide aggregate and regular and white Portland cements on Chinese hamster ovary (CHO) cells in vitro

Objective. Recently, mineral trioxide aggregate (MTA) and Portland cement have been used in dentistry as root-end-filling materials. However, the reported results concerning the biocompatibility of these materials are inconsistent. The goal of this study was to examine the genotoxicity and cytotoxicity of MTA and Portland cements in vitro by the single-cell gel (comet) assay and trypan blue exclusion test.Study design. Chinese hamster ovary (CHO) cells were exposed to MTA and regular and white Portland cements at final concentration ranging from 1 to 1000 mu g/mL for 1 h at 37 degrees C.Results. All compounds tested did not show genotoxic effects in all concentrations evaluated. No significant differences (P > .05) in cytotoxicity were observed for all compounds tested.Conclusions. Taken together, our results suggest that MTA and Portland cements are not genotoxins and are not able to induce cellular death.

Angiotensin II AT(1) receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT, receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2(+) influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation. (c) 2005 Elsevier B.V. All rights reserved.

Expression and function of $\beta$-tubulin isotypes in Chinese hamster ovary (CHO) cells

The availability of isotype specific antisera for $\beta$-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of $\beta$-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor $\beta$-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. Their stoichiometry is approximately 1:1:0.7:0.2. All three isotypes assemble into both cytoplasmic and spindle microtubules, and are similar in their responses to cold, colcemid, and calcium induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that $\beta$-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid resistant mutants with a demonstrable alteration in $\beta$-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist. Under various conditions of the cell growth, the relative proportion of each expressed isotype does not significantly seem to change except in the early G1 phase of the cell cycle. At this time the synthesis of isotype V increases more than two fold relative to isotype I and IV, while at the same time, total $\beta$-tubulin synthesis is decreased about 60-70%. ^

Cell-cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells.

FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell-cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1-Ig2-Ig3 or the Ig2-Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a β-sheet, which is part of a larger β-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell-cell fusion.

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

Antibody Recognition Of Plasmodium Falciparum Infected Red Blood Cells By Symptomatic And Asymptomatic Individuals In The Brazilian Amazon.

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Mutagenicity and Antimutagenicity of Baccharis dracunculifolia Extract in Chromosomal Aberration Assays in Chinese Hamster Ovary Cells

Baccharis dracunculifolia De Candole (Asteraceae), a native plant from the Brazilian ""cerrado"", is widely used in folk medicine as an anti-inflammatory agent and for the treatment of gastrointestinal diseases. B. dracunculifolia has been described as the most important plant source of propolis in southeastern Brazil, which is called green propolis due to its color. The aim of the present study was to evaluate the mutagenic and antimutagenic effects of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE) on Chinese hamster ovary cells. On one hand, the results showed a significant increase in the frequencies of chromosome aberrations at the highest Bd-EAE concentration tested (100 mu g/mL). On the other hand, the lowest Bd-EAE concentration tested (12.5 mu/mL) significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that Bd-EAE has the characteristics of a so-called Janus compound, that is, Bd-EAE is mutagenic at higher concentrations, whereas it displays a chemopreventive effect on doxorubicin-induced mutagenicity at lower concentrations. The constituents of B. dracunculifolia responsible for its mutagenic and antimutagenic effects are probably flavonoids and phenylpropanoids, since these compounds can act either as pro-oxidants or as free radical scavengers depending on their concentration.

Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells

The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and now cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.