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At least 19 recordsLinked to original sources
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↗
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↗
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↗
PubMed1990

Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells.

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.

Animals↗
PubMedDate not supplied

[The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions].

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.

Animals↗
PubMed1988

Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector.

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.

Amino Acid Sequence↗
PubMed1987

Cytotoxicity, mutations and SCEs induced by methylating agents are reduced in CHO cells expressing an active mammalian O6-methylguanine-DNA methyltransferase gene.

Alkylation at the O6 position of guanine leading to miscoding during DNA replication has been shown to correlate with mutagenesis both in bacteria and mammalian cells. The widely used Chinese hamster ovary cells (CHO) are unable to remove O6-methylguanine (O6-meG) due to the absence of O6-meG DNA methyltransferase (MT) activity. Recently Ding et al. [Mol. Cell. Biol. (1985) 5, 3293-3296] transfected CHO cells with human liver DNA obtaining a line provided with a function for the repair of O6-meG. We confirmed the presence of MT activity in this particular clone (14,300 molecules/cell). We used this MT-proficient cell line as compared with the original MT-deficient CHO cell line to analyse the relevance of repair of this lesion on cell killing, ouabain resistance (ouar) mutations and sister chromatid exchanges (SCEs) induced by methylating agents. MT-proficient cells were more resistant than MT-deficient ones to the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). Furthermore a lower number of MNNG-induced SCEs were found in MT-proficient CHO than in MT-deficient cells. Similar ouar mutation frequencies were recorded in the two cell lines after 4-nitroquinoline-1-oxide (4NQO) treatment showing that the differences in cytotoxicity and mutagenesis are restricted to treatment with alkylating agents.

4-Nitroquinoline-1-oxide↗
PubMedDate not supplied

Effects of verapamil and diltiazem on hyperthermic cell death in CHO cells.

Non-toxic concentrations of verapamil (0.05 and 0.075 mM) and diltiazem (0.10 and 0.25 mM) sensitize CHO cells to heat killing at 44 degrees C. These drugs sensitize by reducing both the shoulder and the slope of the exponential survival curve. Exposure to verapamil or diltiazem in conjunction with the heat sensitizer procaine HCl at 44 degrees C reduced cell survival below that observed for procaine plus heat. The mechanism by which verapamil and diltiazem sensitize CHO cells to heat killing is not understood.

Animals↗
PubMed1992

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.

Animals↗
PubMed1986

Cell surface glycoproteins of CHO cells. II. Surface distribution and pathway of internalization.

The surface distribution and pathway for internalization of the major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent trinitrobenzenesulfonate. Molecules, haptenized with trinitrophenol groups, the majority of which are in a group of high molecular weight acidic glycoproteins (HMWAG), were labelled at 4 degrees C with anti-dinitrophenol immunoglobulins coupled to fluorescein isothiocyanate (FITC), horseradish peroxidase, or colloidal gold and either immediately fixed for mapping their distribution or followed intracellularly after warming to allow endocytosis to proceed. The distribution of label on the CHO cell surface was non-random with a large proportion arranged in clusters from 100 to 300 nm in diameter. Antibody label was concentrated heavily on microvilli, and about 10% of the molecules were always associated with clathrin-coated pits. Upon warming the cells to 37 degrees C, HMWAG were internalized immediately into smooth-membraned tubules (less than 80 nm luminal diameter) that appeared to connect with vesicles (less than 300 nm luminal diameter) located in the cortical cytoplasm. By 60 min, labelled antibody was located within larger vesicles (greater than 300 nm luminal diameter) that had a morphology characteristic of multivesicular bodies and not lysosomes. There was no evidence for entry of labelled molecules into either electron-dense, secondary lysosomes or into the Golgi cisternae, suggesting that neither compartment is involved in the major pathway of cell surface endocytosis. Our results are consistent with the view that the majority of plasma membrane protein are internalized as small discrete domains by a pathway very similar to that described by others for adsorptive endocytosis.

Animals↗
PubMed1991

Induction of mutations at the thymidine kinase locus in CHO cells by restriction endonucleases.

Induced mutation frequencies were measured at the tk locus (encoding for the enzyme thymidine kinase) following treatment of Chinese hamster ovary cells (CHO KI) with two restriction endonucleases (REs), PvuII and EcoRI, which generate 'blunt-ended' and 'cohesive-ended' DNA double-strand breaks (dsb), respectively. Electroporation was used to introduce these enzymes into the cells. Restriction endonucleases generating blunt-ended dsb have been shown to mimic the action of ionising radiation in causing chromosome aberrations, cell killing, mutations and oncogenic transformation. Here we show that the tk locus, PvuII induced an approximately 11-fold higher mutation frequency than EcoRI at the same enzyme concentrations. There are four PvuII and six EcoRI restriction sites in the Chinese hamster thymidine kinase gene. Hence the higher mutation induction by PvuII, despite the lower number of restriction sites than EcoRI in the tk gene, suggests that blunt-ended dsb represent more effective and critical mutagenic DNA lesions than the cohesive-ended type. In this respect, our results are similar to those we obtained previously for chromosomal aberrations and for cell killing. Results from the present study suggest that mutations could arise from unrepaired or misrepaired dsb possibly via induction of chromosomal deletions or stable exchanges between chromosomes.

Animals↗
PubMed1988

SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents.

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either sarin (GBI, GBII) at 1.4 X 10(-3) M or soman (GD) at 1.1 and 2.2 X 10(-3) M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 X 10(-3) M and cyclophosphamide (CP) at 10(-4) M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.

Animals↗
PubMed1989

Preferential loss or inactivation of chromosome Z4 APRT allele in CHO cells.

In CHO cells, heterozygotes for the adenine phosphoribosyltransferase (APRT) locus arise spontaneously at high frequencies. However, such heterozygotes always yield APRT- mutants at low spontaneous rates. In an attempt to determine whether differences in the genomic environments of the two CHO APRT alleles might render one gene more susceptible to high-frequency spontaneous inactivation or deletion, we have mapped the functional APRT allele in four different spontaneous APRT heterozygotes. In each case, the functional APRT gene was found to reside on the Z7 chromosome; it was always the Z4 APRT allele that had been lost or inactivated. Two of these heterozygotes were shown to be physically hemizygous while the other two retained two copies of the APRT gene, indicating that the high-frequency event can involve either spontaneous deletion or inactivation.

Adenine Phosphoribosyltransferase↗
PubMed1986

Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.

Animals↗
PubMed1978

Effect of extreme amino acid starvation on the protein synthetic machinery of CHO cells.

When CHO cells are incubated under conditions of extreme amino acid starvation, effected by withdrawal of an amino acid from the medium together with genetic or chemical interference with the activity of the corresponding aminoacyl-tRNA synthetase, there is a rapid and profound decline in the functional capacity of the protein synthetic machinery. The effect was observed for all amino acids tested including leucine, asparagine, histidine, methionine and glutamine. This decline in protein synthetic potential appears to be due to a progressive permanent inactivation of the specific aminoacyl-tRNA synthetase concerned, as shown by a decline in the amount of cellular, specific aminoacyl-tRNA and a decline in the cell-free enzyme activity, measured after reversal of the starvation conditions. When cells are left for more than several hours under these starvation conditions, they shrink in size, lose viability and eventually disintegrate, with anomalous rapidity. We suggest that the progressive loss of protein synthetic capacity of the cells is the prime cause of these subsequent events. If the starvation conditions are reversed before cell death, regeneration of the protein synthetic potential occurs rapidly but requires protein synthesis itself, implying the existence of strong control mechanisms for cellular aminoacyl-tRNA synthetase activities.

Amino Acids↗