Cell cycle and cell protein content of Chinese hamster cells grown in culture with daily renewal of medium

Microspectrophotometric and autoradiographic procedures were used to determine cell protein content and cell cycle parameters of Chinese hamster V79 cells growing as monolayer cultures with daily renewal of medium. The frequency of G 1 cells decreased and then increased as the cultures grew. Cell size at mitosis and at the G 1/S boundary changed only slightly during growth. The changes were very similar to those reported earlier for cultures of the same cell line grown without renewal of medium, but changes in the frequency of G 1 were somewhat slower and those in cell size were much less marked when the medium was renewed daily. The results of the two studies taken together show that cell cycle parameters and cell size change even during periods of exponential growth, and that daily renewal of medium slows but does not prevent the changes. Both studies show that the onset of DNA synthesis is more sensitive to conditions in older cultures than is the onset of mitosis. Our results indicate that radiation from incorporated isotopes might seriously affect the results of the labeled mitoses method of cell cycle analysis, especially when cell crowding is involved.     

Cell cycle-dependent surface changes in Chinese hamster cells grown in suspension culture

The cell surface appears to play an important part in the control of cell replication. It has been demonstrated that the cell membrane undergoes cyclic changes in appearance which bear a relation to the cell cycle phase, irrespective of close intercellular contact. The surface of Chinese hamster (CHO) cells was investigated using the scanning electron microscope (SEM). The cells were synchronized in suspension culture and were sampled at frequent intervals during the cell cycle. During mitosis, the cells showed microvilli and few blebs. In early G 1 phase, profuse microvilli were seen. In late G 1 phase, blebs appeared and persisted in great numbers. During the synthesis of DNA in the S phase, blebs were observed in the early stages and then declined in number; in G 2 phase, the blebs appeared to be larger (1–2 μm) and more sparsely distributed than in late S phase. Some of these blebs were pedunculated and, in some instances, the diameter of the pedicles approximated the diameter of microvilli. Since the reasons for these changes are not understood, our long-range goal is to correlate the observed surface changes with internal biochemical events during the cell cycle.     

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.     

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.     

Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture

Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.Abbreviations ANOVA analysis of variance - CHO Chinese hamster ovary cells - CFA colony-forming ability - dhfr gene for dihydrofolate reductase - DHAP dihydroxyacetone phosphate - FBS fetal bovine serum - G-3-P glyceraldehyde-3-phosphate - GloI glyoxalase I - GloII glyoxalase II - GSH reduced glutathione - HPLC high-performance liquid chromatography - IMDM Iscove's modified Dulbecco's medium - MTX methotrexate - 2-MQ 2-methylquinoxaline - 5-MQ 5-methylquinoxaline - MEM minimal essential medium - P_i inorganic phosphate - PCA perchloric acid - o-PD o-phenylenediamine     

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.     

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K₁) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.     

High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium

A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l^–1 after 10 days. From a seeding density of 5.7 × 10⁵ cells ml^–1, the highest viable cell density of CHO2DS was 1.12 × 10⁷ cells ml^–1.     

Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account.     

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.     

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

A Chinese Hamster Ovary cell line, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell⁻¹ h⁻¹) and early decline (0.040 pg cell⁻¹ h⁻¹) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h⁻¹. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.     

Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.     

Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production

Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 degrees C compared to 37 degrees C. However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO (CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 degrees C in a repeated batch mode using spinner flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 degrees C. Specific growth rates of CHO-EPO and CHO-FSH cells at 32 degrees C, through adaptation, were increased by 73% and 20%, respectively. During adaptation at 32 degrees C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly, suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased specific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively. Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant protein production, since specific productivity decreases during adaptation to the low culture temperature.     

Nature of the iron requirement for Chinese hamster V79 cells in tissue culture medium

The Chinese hamster V79 cell line can be grown in medium containing iron instead of lactalbumin hydrolysate and containing defined low molecular weight components instead of peptone. A rather large amount of inorganic iron must be supplied for optimum growth. Dose-response curves done with commercially available transferrins from various species show that this Chinese hamster cell line grows well with human and rabbit transferrins but poorly with porcine, bovine, and chicken egg white (conalbumin) transferrins. An assay of Chinese hamster serum in the presence and absence of iron shows that hamster serum is better at providing the V79 cells with iron than human or rabbit transferrin. Thus, the nature of the iron requirement of V79 cells lies in the requirement for a specific transferrin.     

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of $\text{ATP}\text{10}{}^6$ cells were observed; a maximum of 3.2 nmoles of $\text{ATP}\text{10}{}^6$ cells was reached at 12 h (G 2 phase). The adenylate energy charge, $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.     

Properties of a hamster tumor cell line grown in a glutamine-free medium

Summary Hamster tumor cells requiring glutamine for survival and growth were used in this study. By gradually reducing the glutamine concentration over a period of 3 weeks, it was possible to establish a line which grew in the absence of glutamine. These cells, in contast to the glutamine-dependent cells, showed parallel alignment which is typical of normal hamster embryo fibroblasts. They were, however, readily distinguishable from normal cells because they grew to higher maximum density, were more refractile when examined by phase-contrast microscopy, and were more readily dispersed with trypsin. Injection of these cells into hamsters resulted in the development of slow growing solid tumors. When cells from these tumors were explanted into tissue culture, they were found to have reverted to glutamine dependence and to display the random crisscross growth pattern which was typical of the tumor cell line from which they were originally derived. This investigation was supported in part by Grant CA-02568 from the National Cancer Institute, Bethesda, Maryland 20014.