Toxicity of 4-nitroquinoline 1-oxide in Chinese hamster ovary cells: influence of cell density and of position in the cell cycle

Various factors influencing toxicity of 4-nitroquinoline 1-oxide (4-NQO) in Chinese hamster ovary cells were determined. Cell density during 4-NQO treatment and volume of treatment medium had a great effect on cell survival indicating that not the 4-NQO concentration per se, but the amount of 4-NQO per cell determines the toxic effect. When the cell-cycle response for 4-NQO-induced cell killing was measured in synchronous cells, a characteristic age response was seen in wild-type cells with greatly increased sensitivity in late G1 to early S and resistance increasing through the S-phase. In contrast, a UV-hypersensitive mutant, which is also more sensitive to 4-NQO showed only minor cell-cycle variations in its response to 4-NQO. Therefore, it appears that the cell-cycle pattern observed in the wild-type cells is associated with DNA repair.     

Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with [32P]orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.     

Patterns of protein synthesis during the cell cycle of Chinese hamster ovary cells

The protein synthesis patterns at various stages of the cell cycle of Chinese hamster ovary cells were examined by labelling cells with [35S]methionine and then separating the proteins by isoelectric focussing and two-dimensional, nonequilibrium pH gradient gel electrophoresis. We have observed a number of proteins which display quantitative differences in synthesis at specific cell cycle stages and of these the alpha- and beta-tubulins have been identified. A few proteins appear to be uniquely synthesized at specific times during the cell cycle. These include the histones and a modified version of them, which are synthesized only in S phase, and a pair of 21 kilodalton (kDa), pI 5.5 proteins, which appear only in late G2 and mitosis. We have also identified a 58-kDa, pI 7.5 protein which is present at all cell cycle stages except during late G2. This protein appears to have the same temporal properties as a 57-kDa protein called "cyclin" originally described in sea urchin embryos.     

Effect of cadmium on cell cycle progression in Chinese hamster ovary cells

Chinese hamster ovary K1 (CHO K1) cells are very sensitive to cadmium (Cd) toxicity. They were used to investigate the effect of Cd on cell cycle progression. Cells were cultured with 0.1, 0.4, 1 or 4 microM Cd for various time intervals. There was no difference in growth rate when less than 0.4 microM Cd was given within 24 h. A dose-dependent reduction of cell proliferation was observed when more than 0.4 microM of Cd was given. The cells were pulse-labeled with 5-bromodeoxyuridine (BrdU), and the labeled cells were cultured in the presence of increasing concentrations of Cd. Cell cycle progression was retarded as a function of Cd concentration. G2/M arrest was observed when the BrdU-labeled cells were treated with 1 microM Cd for 8h, whereas cells receiving 4 microM Cd stopped at the S phase within 4 h. Cell cycle analysis of cells treated with Cd for 24 h showed that G2/M arrest occurred only when cells received 0.8 to 2 microM Cd. Despite the occurrence of G2/M arrest in the Cd treatment, only a limited proportion of the cells were blocked in the M phase. However, the increase in M phase cells coincided with an elevation in the cyclin-dependent kinase 1 activity. To examine whether Cd acts on cells at a specific cell stage, they were synchronized at the G1 or G2/M phase then treated with 1 microM Cd for 12 h. The cells were blocked at the G2/M and G1/S phase, respectively. This finding indicates that Cd toxicity is global and not cell phase specific. We also investigated the involvement of Cd-induced reactive oxygen species (ROS) with the occurrence of G2/M block and found a lack of correlation between cell cycle arrest and ROS production. We measured the Cd content that caused G2/M arrest from a series of Cd treatments and determined the ranges of cumulative Cd concentrations that could result in cell cycle arrest.     

Gene amplification in a single cell cycle in Chinese hamster ovary cells

We have employed Chinese hamster ovary cells synchronized by mitotic selection to study the replication and amplification of the dihydrofolate reductase gene. Using bromodeoxyuridine to differentially label newly replicated DNA, we show that the dihydrofolate reductase gene is replicated during the first 2 h of S phase, a time when, at most, 10% of the total genome has been replicated. We find that a 6-h inhibition of DNA synthesis by hydroxyurea beginning 2 h after the initiation of S phase markedly increases the frequency with which cells become resistant to a 100-fold increment in methotrexate. When DNA synthesis resumes following removal of the hydroxyurea, virtually all of the DNA replicated prior to inhibition, including the dihydrofolate reductase gene, is rereplicated. Analysis of the dihydrofolate reductase enzyme content of cells 24 h after treatment with hydroxyurea using the fluorescence-activated cell sorter reveals a subset of cells with elevated dihydrofolate reductase. It is this subset that contains additional copies of the dihydrofolate reductase gene and from which emerge highly methotrexate-resistant cells. We propose that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome. As cells are subsequently placed under selection, a number of processes, including recombination events and loss of nonselected DNA sequences occur, resulting in what appears as differential gene amplification.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.     

Stimulation of plasminogen activator production by dimethyl sulfoxide in Chinese hamster ovary cells

The production of plasminogen activator activity in an auxotrophic mutant of the Chinese hamster ovary cell line was found be greatly stimulated by low concentrations of dimethyl sulfoxide. The production of both cell-associated and excreted plasminogen activator activities was stimulated maximally by dimethyl sulfoxide at a concentration of 2.5%. The stimulation of plasminogen activator activity production was found to be completely inhibited by actinomycin D and cycloheximide but not by mitomycin C, implying that new protein and RNA syntheses were required for this process. Using specific antibodies against plasminogen activator, the presence of a tissue-type plasminogen activator could only be detected in dimethyl sulfoxide treated cells. The dimethyl sulfoxide induced plasminogen activator production was observed only in a mutant auxotrophic for adenosine, glycine, and thymidine but not in wild-type cells. The ability of dimethyl sulfoxide to induce the synthesis of plasminogen activator was lost when the cells were hybridized with another complementary auxotrophic mutant. This implies that the ability of dimethyl sulfoxide to stimulate the production of plasminogen activator may be related to the auxotrophic mutation in this cell.     

Cell membrane permeability during the cell generation cycle in Chinese hamster ovary cells

Summary Changes in the permeability of the cell membrane in cultured Chinese hamster ovary cells at different stages of the cell cycle were investigated. These were followed by measuring the intracellular retention of fluorescein molecules produced by the enzymatic hydrolysis of fluoresceindiacetate in the cytoplasm of CHO cells. Rate constants for the permeation of fluorescein have been calculated.     

Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells

Cytotechnology - Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production...     

Levels of filamentous and globular actin in Chinese hamster ovary cells throughout the cell cycle

Synchronous Chinese hamster ovary (CHO) cells were obtained by mitotic selection and the levels of globular (G) actin, filamentous (F) actin, and cytoskeletal-associated F-actin were determined as cells progressed through the cell cycle. Total actin levels remained quite constant when expressed as a percent of the total protein. An increase in F-actin occurred upon plating the mitotic cells, but this increase was shown to be a result of attachment to the substratum, since cells which remained attached during the second mitosis failed to show these changes. No large variation in the levels of either F-actin or cytoskeletal-associated F-actin occurred throughout the cell cycle. Therefore, changes in the morphology of the CHO cells which are accompanied by a reorganization of actin-containing microfilaments during the cell cycle are not accompanied by significant changes in the size of the monomeric actin pool.     

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.     

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.     

Estimation of Chinese hamster ovary cell density in packed-bed bioreactor by lactate production rate

A method is described for estimating recombinant Chinese hamster ovary (rCHO) cell density in a packed-bed bioreactor by lactate production rate. The lactate production rate, which depended on both the cell numbers and cell growth rate, was modeled by segregating the cell population into two parts: one growing at a maximum specific growth rate and another non-growing. The individual cell in each part had the same lactate production rate. The established rate equation of lactate production matched the experimental data reasonably well and could be used to estimate the cell growth in the batch culture with microcarriers. Furthermore, in the perfusion culture of rCHO cells in a packed-bed bioreactor, the final cell density, 1.3×10¹⁰ cells l^–1, estimated by lactate production rate, was comparable to the direct sample counting of 1.2×10¹⁰ cells l^–1, showing that lactate production rate method would be useful in tracing the cell growth in packed-bed bioreactors.     

Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells

Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the