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At least 19 recordsLinked to original sources
PubMed2005

Inhibition of proteinase 3 (PR3) by suramin and fetal calf serum (FCS): effect of PR3 and suramin on Chinese hamster ovary cells (CHO-cells).

BACKGROUND: Proteinase 3 (PR3) is a lysosomal protease that is stored in azurophilic granules neutrophilic granulocytes and monocytes. A number of inhibitors for this proteinase are reported. Comprehensive studies on the inhibitory effect of suramin and heat treated fetal calf serum (deltaFCS) on PR3 have not been reported. It has been reported that PR3 is able to destroy the cytoskeletal integral proteins, but we have not find any reports which showed the effect of this protease on Chinese hamster ovary cells (CHO-cells) in culture medium. Suramin has proven to be useful as an antitumor drug, but there was not any report on the effect of suramin on CHO-cells. METHODS: The effects of various concentrations of deltaFCS (from 0.5% up to 10%) and suramin (from 0.8 microM up to 100 microM) on PR3 and different concentrations of suramin (from 0.8 microM up to 1000 microM) on CHO-cells were investigated. Data analysis were performed by, Kolmogorov-Smirnov test, ANOVA test and Tukey HSD post tests. RESULTS: Results showed that deltaFCS and suramin have an inhibitory effect on PR3 and these effects increased with increasing the concentration significantly (p < 0.01). PR3 with the concentration of 2.2 Unit/ml has no effect on CHO-cells. Although suramin with the concentration of less than 125 microM cell growth retarded for only a few hours, but with the concentration of 125 to 250 microM inhibit the cell growth for a week, and after that cells gain normal growth gradually. Suramin with concentration of more than 500 microM inhibited the cell growth completely. CONCLUSIONS: Although suramin reversibly inhibit the PR3 activity but in concentration of less than 250 microM it had no long-term effect on CHO-cells. Therefore it can be used in the investigation of proteases. There were unknown components in deltaFCS, which cause the inhibition of PR3 activity. This finding is very important in PR3 production in culture medium. However CHO-cells are resistant to PR3 and suramin in low concentration.

Animals↗
PubMed1989

[Effect of expression of exogenous PDGF-A chain on growth and transformation of CHO cells].

CHO cells were transfected with plasmid pSV2-PDGF-A (containing human PDGF-A cDNA) by calcium phosphate method. Twenty transfected cell lines were obtained after G418 selection. The selected 2 cell lines At1 and Aot7), with prominent changes in morphology and growth behaviour, showed transcription of PDGF-A chain mRNA much higher than CHO cells, strong fluorescent PDGF-specific reaction, appearing that PDGF-like proteins were synthesized in cytoplasm of these cells. At1 and Aot7 cells not only had increased growth rate, but also formed large colonies in soft agar and grew into fibrosarcomas in nude mice. These results suggested that the expression of exogenous PDGF-A gene might cause the uncontrolled growth and malignant transformation of CHO cells.

Animals↗
PubMed1989

[Expression of exogenous platelet-derived growth factor B chain gene in CHO cells].

CHO cells were transfected with plasmid pSM-1 (containing human c-sis cDNA) singly or co-transfected with pSV 2 neo DNA by calcium phosphate method. After low serum or G418 selection several cell lines with expression of platelet-derived growth factor (PDGF) were obtained. One among them, FB5, was of the highest PDGF expression and showed the following biological characteristics when compared with CHO cells: (1) a prominent change in morphology from spindle to round in shape: (2) increase of growth rate; (3) growth in low serum (2%) medium as a semisuspension culture; (4) growth on soft agar to larger colonies; (5) synthesis of PDGF in cytoplasm identified by immunofluorescent method; (6) the conditioned medium stimulated DNA synthesis of NRK cells; (7) RNA dot hybridization showing high transcription of PDGF mRNA; (8) southern blot showing integration of human c-sis gene was still stable after 7 months. These results indicated that intergration of exogenous c-sis gene and its high expression might cause CHO cells to high growth rate and even transformation. The establishment of this stable transformed cell line, FB5 is thought to be a good model for further study on the function of PDGF in cell growth control and cell transformation.

Animals↗
PubMed1999

Reduced levels of catalase activity potentiate MPP+-induced toxicity: comparison between MN9D cells and CHO cells.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to be toxic by inducing oxygen free radicals in the mammalian nervous system, especially in the nigrostriatal dopaminergic system. The present study was designed to compare the toxic effects of MPP+, the active metabolite of MPTP, in MN9D neuronal cells that exhibit relatively low levels of catalase activity, as compared to CHO cells, which exhibit high levels of catalase activity. The survival of the MN9D cells in the presence of 250 microM MPP+ was less than 10%, whereas CHO cells exhibited 70% survival at the same concentration of MPP+. The ED50 values of MPP+ in MN9D and CHO cell lines were 60-600 microM, respectively. MN9D cells contain less catalase, an enzyme believed to be involved in the detoxification of free radicals compared to CHO cells. The catalase activity was 2 Units/mg protein in MN9D cells and 30 U/mg protein in CHO cells. The catalase activity in CHO cells increased with increasing MPP+ concentrations from 100-500 microM, however, it decreased at 1 mM MPP+. In contrast, catalase activity in MN9D remained the same at all MPP+ concentrations. When the CHO cells were pre-treated with 10-25 mM 3-aminotriazole (3-AT), which inhibits catalase activity, and exposed to MPP+ at various concentrations, they became susceptible to MPP+. It is evident from these data that the differential susceptibility to MPP+ in these two cell lines are due to differences in catalase activity. In addition, the inhibition of constituentive catalase activity in CHO cells by 3-AT treatment enhances their susceptibility. In conclusion, the study demonstrates that catalase activity represents an important defence mechanism in MPTP-induced toxicity.

1-Methyl-4-phenylpyridinium↗
PubMed2002

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells.

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.

Animals↗
PubMed1976

Evidence for a four micron replication unit in CHO cells.

CHO cells in culture were synchronized by mitotic selection, allowed to reattach to plastic flasks, and reach S phase in the presence of fluorode-oxyuridine at concentrations known to completely block the synthesis of thymidylate. The cells were released from the block with 3H-thymidine for pulses of 4, 8, 12, 24 and 40 min and DNA fiber autoradiographs prepared. An analysis of the spacing between origins of replication indicates that sites are available at intervals of about 4 mum along most of the DNA. Chain growth proceeds at about 1,000 nucleotides per minute and some of the closely situated sites become continuous, labeled segments after 8-12 min. However, unlabeled segments are still present between the replicated segments after 40 min. The data may be interpreted as evidence for regularly spaced initiation sites which are available in CHO cells, even though only one in 10-15 of these may be utilized for initiation each cycle under normal growth conditions in these cultures.

Animals↗
PubMed2000

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor.

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.

Animals↗
PubMed1995

The kinetics of chromosome and DNA damage by streptonigrin in CHO cells.

CHO cells were pulse-treated for 20 min with increasing doses of streptonigrin (SN). First division cells analyzed 18 h after the end of treatment showed a combination of chromosome and chromatid aberrations with a typical dose-response curve. The frequency of SCEs in second division cells (24 h harvesting time) also exhibited a positive correlation with the SN dose. A high incidence of chromatid aberrations was detected in second and third division metaphases. This indicates that SN has a persistent clastogenic action that lasts for at least three cell cycles after the end of treatment. The kinetics of DNA damage and repair was studied by the alkaline unwinding and single cell gel methods. It was found that a pulse treatment with SN elicited a triphasic response characterized by repair-damage-repair. It is proposed that SN forms stable complexes with the DNA. These complexes by a continuous cycling redox process would produce active oxygen species inducing direct damage to DNA.

Animals↗
PubMed1999

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.

Animals↗
PubMed1995

Molecular structural analysis of 417 HPRT mutations induced by restriction endonucleases in Chinese hamster ovary (CHO) cells.

CHO cells were exposed to 11 different restriction endonucleases by electroporation and their mutagenicity was measured. Nine of them have one or more recognition sites within exons of the HPRT gene, whereas the remaining two cut in introns only. The mutagenic efficiency of the various enzymes varied markedly; mutagenicity of Sau3AI was considerably higher than that of the other enzymes. Neither cytotoxicity nor mutagenicity could be related to the number or location of recognition sites within the cDNA. A total of 417 independent restriction enzyme induced mutant clones were isolated from 20 separate experiments for molecular analysis; all nine exons of the HPRT gene were analyzed by a modified multiplex deletion screening method with polymerase chain reaction (PCR) amplification. Among spontaneously arising mutants, 70.8% showed no change in PCR pattern, indicating a small scale change (point mutation), whereas partial deletions were observed in 24.7%, and total deletions in 4.5% of mutant clones. In contrast, approximately 70% of restriction enzyme induced mutants showed partial or total deletions. There was no obvious relationship between type of break (blunt versus staggered ends), and the DNA structure of the mutations induced. For partial deletions, the distribution of breakpoints within introns appeared to occur at random, and did not correlate with the mutagenicity of a given enzyme. Thus, though DNA double-strand breaks appear to be important mutagenic lesions that can induce a high frequency of deletion mutants, no specific relationship of mutagenic potential to the type of breaks, their sites within the HPRT gene or the molecular structure of the mutations induced could be identified.

Animals↗
PubMed1987

Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells.

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.

Amino Acids↗
PubMed1987

Selenalysine utilization by CHO cells.

CHO cells allowed to grow in a medium containing selenalysine can utilize it for protein synthesis. Selenalysine is incorporated into cell proteins in substitution of lysine: a maximum of 5% of protein lysine can be substituted. Protein lysine substitution by selenalysine can be correlated to the reduced viability of cells grown in its presence.

Animals↗
PubMed1997

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells.

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.

Animals↗
PubMed1997

The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR.

The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl- conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation DeltaF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm) and the membrane capacitance (Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 micromol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and DeltaF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from -40 +/- 1.5 to -32 +/- 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 +/- 0.9 to 36 +/- 3.9 nS (n = 65). The conductance increase was mostly for Cl-, because under stimulated conditions a reduction in bath Cl- concentration depolarized these cells further and significantly from -26 +/- 1.8 to -10 +/- 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br- > Cl- > I- (n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of DeltaF508-CFTR in CHO cells induces a cAMP-activated Cl- conductance. The activation of this large conductance obviously proceeds with little if any exocytosis.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.

Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

[3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor.

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.

Animals↗
PubMed1995

Influence of ara A on the formation of dicentrics in irradiated quiescent CHO cells.

CHO K1-cells, arrested in G0/G1 phase were irradiated with graded doses of 150-kV X-rays, and the dicentric chromosome aberrations in the first metaphase after reincubation in full growth medium were evaluated. When 500 microM ara A was administered at least 1 h before irradiation, maximal enhancement of the dicentric yield was obtained under 'immediate plating' conditions as well as under 'delayed plating' conditions, and under both conditions only the linear component alpha D of the aberration yield was affected. Since ara A had no effect upon the number of induced dsbs, as measured with PFGE at doses up to 100 Gy, the action pathway of ara A is suggested to be the repair/misrepair process manifested in the linear component of the dicentric yield. By varying the time at which either ara A at 37 degrees C was administered before irradiation or ara A at 4 degrees C was added after irradiation, it was observed that the repair step sensitive to ara A was completed within about 5 min after irradiation. This is in agreement with other observations of ara A action upon a fast repair step. The conversion of DNA single-strand breaks or base damage sites into double-strand breaks as a consequence of polymerase beta inhibition by ara A would explain the present results.

Animals↗
PubMed2005

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells.

CHO cells were grown in the presence of 1 mu M CdCl(2) and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 x 10(6) cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be suppressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2-2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5-2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9-3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3-3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5-3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8-4. C).

Animals↗