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At least 19 recordsLinked to original sources
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↗
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↗
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↗
PubMed1978

Ultrastructural localization of transcription sites and of RNA distribution during the cell cycle of synchronized CHO cells.

The ultrastructural localization of [3H]uridine-labelled RNA synthesized in the course of the cell cycle of synchronized CHO cells is studied using high resolution autoradiography combined with a differential staining for nucleoproteins. It is shown that sites of RNA transcription can already be visualized on the periphery of chromosomes of apparently late metaphase-early anaphase cells with no visible association with the reforming nuclear membrane. In interphase cells they are associated with the border of intranucleolar chromatin or condensed nucleoplasmic chromatin, wherever this localization is made possible by the degree of chromatin dispersion. In cells labeled for 1 or 3 h, the rate of RNA synthesis is higher in S and G2 than in G1. When cells fixed immediately after a [3H]uridine pulse are compared with those post-incubated for 13 h in isotope-free medium, there is a clear difference in intensity of labelling between the nucleus and the cytoplasm. However, the localization pattern of radioactive RNA in the nucleus is similar for all incubation periods as well as for all phases of interphase. The groups of interchromatin granules are generally labeled weakly with radioactivity associated rather with the periphery of their clusters, or remain unlabelled. These results are discussed in the context of other recent findings concerning the distribution of RNA and RNP-structures in the nucleus.

Animals↗
PubMed1979

The effect of membrane-fluidizing agents on the adhesion of CHO cells.

Treatment of CHO cells with drugs which are known to increase membrane lipid fluidity reduced the cells' ability to adhere to protein coated substrates, The concentrations of local anesthetics, nonionic detergents or aliphatic alcohols required to reduce CHO cell adhesion by 50% were similar to those reported to block nerve conduction, indicating that these drugs can affect the membrane at physiologically significant concentrations. Nonionic detergents and aliphatic alcohols, but not local anesthetics, caused increases in the fluidity of CHO plasma membranes (measured by fluorescence polarization) at concentrations which inhibited cell adhesion. The adhesion versus temperature profile had a sigmoidal shape, suggesting that a temperature dependent cooperative process such as a lipid phase transition, might be involved. However, the temperature profile for CHO membrane fluidity manifested no discontinuities, indicating the absence of any discrete phase transitions of the lipid matrix. This observation, coupled with the result that the inhibition of CHO cell adhesion produced by low temperatures was not relieved by drugs which can increase membrane fluidity, suggests that the reduced adhesion seen at low temperature is probably not due to reduced lipid fluidity.

Anesthetics, Local↗
PubMed1992

Poly-D-lysine in G2 potentiates chromosome damage induced by X-rays and mitomycin C in CHO cells.

A number of reports suggest that the role of radiation-induced G2 arrest is to allow repair of potentially lethal damage in the cells before it comes to mitosis. Though the exact nature of the damage undergoing repair during the delayed G2 is not known, the yield of chromosomal aberrations observed in metaphase seems to be a good parameter to predict reproductive death of cells. In a previous paper, we have shown that poly-D-lysine, acting in a fashion reminiscent of that of caffeine in mammalian cells, is able to induce a premature onset of mitosis concomitant with an increase in the frequency of chromosomal aberrations in mutagen-treated plant cells. Cultured CHO cells were pre-exposed to either X-rays or mitomycin C and given different doses of the polycationic compound during G2 in order to analyze any effect on the frequency of chromatid-type aberrations as well as any modification of cell-cycle kinetics. A potentiation of chromosome damage and a premature arrival at mitosis were observed for both mutagens, though the effect was more evident in X-irradiated cells.

Animals↗
PubMed1992

Chemically defined medium for the production of biologically active substances of CHO cells.

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.

Animals↗
PubMed1992

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065.

WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G2 in WR-1065 treated Chinese hamster ovary cells grown in alpha-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with 137Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in alpha-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G2 population and a decrease in the G1 population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.

Animals↗
PubMed1975

Alpha-amanitin resistance: a dominant mutation in CHO cells.

Hybrids of CHO cells were constructed consisting of either a 1:1 or 1:2 ratio of alpha-amanitin-resistant and sensitive cells, respectively. The resistance of such hybrids to killing by the drug was similar but slightly less than that of the resistant parent. The hybrids contained both resistant and wild-type RNA polymerase II, in amounts related to the expected gene dosage. The alpha-amanitin marker therefore is expressed codominantly.

Amanitins↗
PubMed1992

Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.

Animals↗
PubMed2026

Improving recombinant protein productivity in CHO cells via multi-omics data integration.

Chinese hamster ovary (CHO) cells represent the dominant host system for the production of recombinant therapeutic proteins. In recent decades, extensive research has focused on process/media optimization and cell line engineering to improve both the productivity and quality of biopharmaceutical proteins produced in CHO cells. Nevertheless, the inherent complexity of biological pathways and the heterogeneous cellular responses to different environmental conditions have posed substantial challenges to traditional methodologies. Recent advances in omics technologies have enabled comprehensive characterization of CHO cell physiology, providing multidimensional molecular and phenotypic insights that facilitate the enhancement of recombinant protein production. This review first summarizes the methodologies and advances in CHO omics research, including genomics, transcriptomics, proteomics, metabolomics, and epigenomics. It then examines contemporary approaches to integrate and analyze multi-omics data in CHO cells. The review further elucidates how these multi-omics datasets can be strategically applied across various developmental stages, including cell line selection, genetic engineering, expression vector design, and bioprocess optimization. Finally, we explore the transformative potential of integrating multi-omics with artificial intelligence and discuss promising future research directions in CHO cell studies. These emerging paradigms offer novel opportunities for data-driven cell engineering and bioprocess optimization in CHO-based biomanufacturing.

Bioprocessing↗
PubMed1992

Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-delta subspecies was markedly inhibited, whereas cell lines expressing PKC-alpha, PKC-beta II, and PKC-zeta subspecies were not significantly affected. Flow cytometric analysis indicated that all cell lines overexpressing PKC-delta subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrested cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-delta subspecies may play a role in the normal cell cycle progression.

Animals↗
PubMed1992

Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products.

cDNA encoding human PACAP precursor was expressed in non-neuroendocrine Chinese hamster ovary cells, CHO-K1, The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir-PACAP) into the conditioned medium was established. RP-HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP-related peptide (ir-PRP) was secreted into the cultured medium. The ir-PACAPs were confirmed to ahve biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.

Adenylyl Cyclases↗
PubMed1992

Ablation of stimulation of a cAMP-responsive promoter in CHO cell lines defective in their cAMP-dependent protein kinase system.

We have studied the requirement for an intact cAMP-dependent protein kinase (PKA) system to regulate cAMP-mediated gene transcription in Chinese hamster ovary (CHO) cells. Wild-type CHO cells and mutant CHO cell lines selected for their resistance to the growth inhibitory effect of 8-Br-cAMP and defective in their PKA system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (TAT) gene promoter. This segment of DNA contains no CRE-like sequences, yet wild-type transfectants exhibited a specific increase in TAT promoter activity following growth in medium containing 8-Br-cAMP. In CHO cell lines defective in their PKA, the transfected TAT promoter failed to respond to cAMP treatment. We conclude that an intact PKA system is necessary for the cAMP-mediated increase in TAT promoter activity in CHO cells and that there is no requirement for a CRE to see this effect.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1977

Clonal variation in colony morphology and growth of CHO cells cultured on agar.

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10-12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mount (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer). These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed. These observations reveal a previously unsuspected heterogeneity in an established line of cultured mammalian cells and provide a method for studying new classes of in vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.

Cell Division↗
PubMed1975

The interactions of benzo(a)pyrene with cell membranes: uptake into Chinese hamster ovary (CHO) cells and fluorescence studies with isolated membranes.

The interactions of benzo(a)pyrene (B(a)P) with the cell surface membrane were studied by measuring B(a)P uptake into intact mammalian cells and by determining B(a)P fluorescence in the presence of isolated cell surface membranes. It was found that 0.19 mu-g B(a)P were taken up by 10-6 Chinese hamster ovary (CHO) cells after 30 min exposure to a solution containing 0.59 mu-g/ml. Culture conditions were found to markedly alter B(a)P uptake. Low cell culture densities resulted in a four-fold increase in rate of B(a)P uptake per cell relative to confluent monolayer cultures. The uptake rate of B(a)P was reduced in the presence of bovine serum (BS) and, under some conditions, perylene. This information should be considered in the design of experiments on the biological effects of B(a)P. Another aspect of B(a)P membrane interaction was that the binding of B(a)P to cell surface membranes could be measured by fluorescence. The additional B(a)P fluorescence, found in the presence of cell surface membranes, was sufficiently large that the methods of data treatment used in the study of fluorescent probe-membrane interactions could be applied to get quantitative information on B(a)P-membrane interactions. It was found that 0.6 x 10-8 moles B(a)P were bound per mg membrane protein and that the apparent statistical dissociation constant for the complex was 3.8 x 10-7 M. The data suggest that the mechanism of uptake of B(a)P is probably passive diffusion.

Anilino Naphthalenesulfonates↗