The effects of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells

Glucose deprivation induces the major glucose regulated proteins (GRPs) in Chinese hamster ovary cells. When these cells are then returned to a glucose containing environment, GRP synthesis is repressed while concurrently other proteins, identified as heat shock proteins, are induced. The induction of the GRPs is found to mark precisely the onset of a decline in the cell's ability to survive a thermal stress while the expression of heat shock proteins, when glucose is restored, is paralleled by significant increases in survival protection or thermotolerance.     

Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells

In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 microM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids.     

Messenger RNA synthesis in synchronized Chinese hamster ovary cells

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.     

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 effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39°C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34°C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39°C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.     

High level synthesis of immunoglobulins in Chinese hamster ovary cells

The expression of lambda L and microH chain cDNA was examined in Chinese hamster ovary cells. Each cDNA was linked to a different, amplifiable, selectable drug marker gene, and expression was monitored in the presence of increasing concentrations of the selective drugs. Cells were obtained that produced greater than 60 micrograms/10(6) cells/48 h of assembled antibody. This Chinese hamster ovary cell-synthesized IgM was polymeric, and exhibited specific hapten binding and C fixation. The expression strategy employed here may prove useful for the future production of genetically engineered antibodies and other multi-subunit proteins.     

The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells

The frequency of mutations induced by ethyl methane sulfonate was compared in a pseudodiploid Chinese hamster cell strain and in a tetraploid substrain derived from it. The frequency of reverse mutations from glycine auxotrophy to glycine independence was similar in the two strains, as expected for a dominant phenotype. Forward mutation to 6-thioguanine-resistance was 25 fold lower in the tetraploid as compared to the diploid strain. The resistant mutants lack hypoxanthine phosphoribosyl transferase activity and their resistant phenotype is recessive in somatic cell hybrids. A combination of chromosomal segregation and mutation could account for the frequency of these recessive drug-resistant mutants in the tetraploid population.     

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.     

The effect of inhibitors of RNA and protein synthesis on dibutyryl cyclic AMP mediated morphological transformations of Chinese hamster ovary cells in vitro

Morphological transformation of cultured Chinese hamster ovary cells (CHO-Kl) produced by the addition and removal of dibutyryl cyclic AMP plus testosterone are unaffected by inhibition of either RNA synthesis or protein synthesis.     

Rate of synthesis and concentration of specific mRNA sequences in cultured Chinese hamster ovary cells compared to liver cells

With the aid of recombinant DNA molecules the concentration of nine different mRNAs in cultured Chinese hamster cells has been compared to liver cells. One clone in nine showed a marked increase (13-fold) in liver cells but the remainder were present at about the same level in the two cell types. The rate of RNA synthesis in the nucleus was not increased by 13-fold for this clone but only about 3-fold. These data show that quantitative hybridization to recombinant DNA can estimate mRNA concentrations and rates of synthesis accurately and suggest levels at which control operates.     

N-Carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N-carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug-resistant and parental wild-type cells required the same optimum conditions for enzyme activity. The Ki values for N-carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NC^R-30A cells and 2.3 mM for wild-type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the V_max values for NC^R-30A cells were 1.01 nmoles dCDP formed/5 × 10⁶ cells/hour and 1.83 nmoles dADP/5 × 10⁶ cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 × 10⁶ cells/hour and 1.00 nmoles dADP/5 × 10⁶ cells/hour. This approximate twofold increase in reductase activity at least partially accounts for a 2.6-fold increase in D₁₀ value for cellular resistance to N-carbamoyloxyurea exhibited by NC^R-30A cells. The NC^R-30A cell line was also cross-resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NC^R-30A cell line. The properties of N-carbamoyloxyurea-resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.     

The effect of hyperthermia on transmembrane potential in Chinese hamster ovary cells in vitro

The effect of elevated temperature on transmembrane potential was studied in Chinese hamster ovary cells in vitro using tetraphenylphosphonium cation (TPP+) and 3,3'-dipentyloxacarbocyanine [Di-O-C5(3)], two unrelated lipophilic cation probes that equilibrate across the plasma membrane according to the transmembrane potential. Uptake of TPP+ was measured using a tritium-labeled probe and the uptake of the fluorescent probe Di-O-C5(3) was measured by flow cytometry. The Nernst equation was used to calculate transmembrane potential. The absolute values obtained for transmembrane potential at 37 degrees C using the two probes were different, but qualitatively similar results were obtained using either probe in the hyperthermia studies. Transmembrane potential measured at 43 and 45 degrees C was at least 20% higher than that measured at 37 degrees C, and the difference was statistically significant (P = 0.025 and P less than 0.01, respectively). The hyperpolarization induced by exposure to 45 degrees C persisted temporarily after cells had been returned to 37 degrees C. The hyperpolarization at 37 degrees C associated with a previous exposure to hyperthermia was maximal after cells had been held at 45 degrees C for 2.0 min, and fell to normal levels after 15.0 min at 37 degrees C.     

Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.     

Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells

In a proline-requiring Chinese hamster ovary cell line, if both proline and serum are removed from the culture medium, net RNA synthesis is reduced to about 12 % of the unstarved control. This reduction in RNA synthesis is comparable to the stringent regulation of RNA in bacteria. A beta-globulin carbohydrate containing (3.5 % $\text{w}\text{w}$) protein factor was isolated and partially purified from fetal calf serum. The isolated serum factor is able to replace whole serum in stimulating cellular RNA synthesis and has an RNAase inhibitory effect in vitro. The effect of proline starvation and serum factor deprivation on RNA synthesis are independent and additive; each regulates about half of the total RNA synthesized. The regulation appears to affect the synthesis of all species of cytoplasmic RNA.     

High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells

Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).     

Cycloheximide resistance in Chinese hamster cells. III. Characterization of cell-free protein synthesis by polysomes.

Two clones were selected for mass cultivation from 18 phenotypically stable CHM-resistant CHO clones. The polysomes isolated from these two clones were compared with CHO wildtype polysomes and rat liver polysomes in a cell-free protein synthesis system for their ability to incorporate amino acids. CHM had an inhibitory effect on the protein synthesis activity of CHO wildtype and rat liver polysomes, but had no effect on the polysomes obtained from either of the mutant CHO clones.     

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.