Growth Of Chinese Hamster Ovary Cells Research Articles

TRANSFECTION OF THE MULLERIAN INHIBITING SUBSTANCE GENE INHIBITS LOCAL AND METASTATIC TUMOR-GROWTH

Mullerian Inhibiting Substance (MIS), a gonadal growth factor important in sexual differentiation, has antiproliferative activity against several human carcinoma cell lines. In this study, we examine the effect of MIS-transfection on the growth characteristics of Chinese hamster ovary (CHO) and human ocular melanoma (OM431) cells, compared to wild-type lines and a CHO line transfected with a noncleavable, inactive MIS mutant. MIS-transfection inhibited proliferation of CHO cells in double-layer agarose, tumor spheroid, and murine subrenal capsule assays, as well as growth of CHO and OM431 cells in pulmonary metastasis studies. These results anticipate further study of targeted gene therapy of certain human tumors with MIS gene constructs.

Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a “growth factor”. Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207–214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO 4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.

The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.

N-(5,5-diacetoxypent-1-yl)doxorubicin: a new intensely potent doxorubicin analogue.

N-(5,5-Diacetoxypent-1-yl)doxorubicin (DAPDOX) (3), a new, water-soluble analogue of doxorubicin, has been synthesized by coupling doxorubicin with 5-oxopentane-1,1-diacetate in the presence of NaBH3CN. This analogue was designed to be converted to the corresponding aldehyde, N-(5-oxopent-1-yl)doxorubicin, in the presence of carboxylate hydrolases, enzymes that are ubiquitous in tissue. DAPDOX had a half-life of several days in 0.05 M phosphate or 0.05 M acetate buffer solution at pH 4.0. However, in 0.05 M phosphate buffer at pH 7.4 in the presence of 20 unit equiv of porcine liver carboxylate esterase, the half-life of DAPDOX was less than 1 min. N-(5-acetoxypent-1-yl)doxorubicin (4), which should give rise to N-(5-hydroxypent-1-yl)doxorubicin on esterase-mediated hydrolysis, and N-(pent-1-yl)doxorubicin (5), were also prepared for comparative biological studies. DAPDOX was 150 times more potent than doxorubicin at inhibiting the growth of Chinese hamster ovary (CHO) cells in culture. The compound retained the same degree of potency against a CHO subline 100-fold resistant to doxorubicin (CHO/DOX) that expressed elevated levels of P-glycoprotein. Compounds 4 and 5, on the other hand, were no more effective than doxorubicin at inhibiting the growth of CHO cells and were 4-7-fold less potent against the CHO/DOX subline. DAPDOX is representative of a new structural class of doxorubicin analogues with unique chemical and biological properties.

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.

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism.

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.

Effects of High Pressure and Metal Salts on Cell Growth

The effects of chromate, vanadate, nickel, manganese, copper and barium, both separately and in combination with high pressure helium gas, have been studied in an epithelial-like cell line — Chinese hamster ovary (CHO) cells, using inhibition of cell growth as the cytotoxic endpoint. High pressure (175 bar) by itself reduced the net growth of CHO cells to 71.7 ± 5.5% (mean ± SD) of normobaric control growth. The combined effects of high pressure and metal salts were additive for the lowest concentrations of chromate, copper, manganese and barium, more than additive for the lowest concentration of nickel, and less than additive for vanadate and the highest concentrations of chromate, copper, manganese and barium. A ranking of the normobaric growth inhibition of the metal salts, estimated as the concentration giving 50% inhibition, was Cr>V>Mn>Ni>Cu>Ba. The growth inhibition of barium was less than 50% at a concentration of ImM BaCl2. The combined exposure to high pressure and the metal salt did not change the toxicity ranking.

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.

Recombinant human interferon-gamma production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l −1 for ammonia and 5.0 g l −1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 × 10 6 ml −1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l −1 glucose and 0.29 g l −1 glutamine, and no glucose feed).

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene.

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.

Effect of polyphenols on the growth and enzymes of some animal cells.

The effect was investigated of some polyphenol compounds on the growth and intracellular enzyme activity of human-derived cells and Chinese hamster ovary (CHO) cells. Quercetin, a mutagen, inhibited the growth of serum-free cultured human-human hybridomas (SI102 and HB4C5) and a human histiocytic lymphoma cell line (U-937), but did not affect the growth of CHO cells. Glycosides of quercetin such as quercetin-4'-glucoside (Q-4'-G), quercetin-3, 4'-glucoside (Q-3, 4'-G) and rutin, and other polyphenol compounds (catechin and epicatechin) had no significant inhibiting effect on the growth of human-derived cells or CHO cells. These compounds slightly promoted the growth of human-derived cells. Most of the polyphenols used increased the activity of a drug-metabolizing enzyme, NADPH-cytochrome C reductase, in the U-937 cells and CHO cells, this effect being more marked in the CHO cells than in the U-937 cells. Quercetin markedly reduced the activity of catalase in the human-derived cell lines, while it slightly activated catalase in the CHO cells. Rutin, Q-4'-G, Q-3, 4'-G, catechin and epicatechin produced no significant change in catalase activity. Quercetin also reduced the activity of glutamic oxaloacetic transaminase in the U-937 cells.

Effects of phenols on growth rate and adenosine uptake of CHO cells

The use of Chinese hamster ovary (CHO) cells as a test system for predicting the toxicity of xenobiotics is described. Compared to eukaryotic microorganisms this system is two to three times more sensitive but needs more care in handling. In the study of the effects of various phenol derivatives on CHO cell growth and on an important functional membrane protein, the nucleoside transport system, a good correlation was observed. In addition, in both cases the effects correlated very well with the octanol/water partition coefficient, making quantitative structure-activity predictions possible. The results suggest that there is a common mechanism of action of these lipophilic xenobiotics. They are accumulated in the hydrophobic region of the lipid bilayer of the plasma membrane, where enzymes and transport systems are located, which are important for cell viability. These high concentrations of the chemicals lead to an inhibition of functional membrane proteins, with the consequence that cell growth stops.

Genetic and biochemical studies on biosynthesis and physiological roles of phosphatidylserine in mammalian cells

The base-exchange enzymes which catalyze exchange of free serine, choline, and ethanolamine with polar head groups of phospholipids are known to occur in eukaryotic cells. To understand the physiological roles of the base-exchange reactions, a rapid autoradiographic in situ enzyme assay has been developed for detecting the base-exchange activities in Chinese hamster ovary (CHO) cell colonies immobilized on filter paper or polyester cloth and obtained several mutants strikingly defective in the choline-exchange activity. CHO cell mutants that required exogenously added phosphatidylserine for cell growth were also isolated by using the replica technique with polyester cloth. Biochemical characterization of these mutants has revealed the following results. 1) There are at least two kinds of serine-exhange enzyme in CHO-K1 cells ; one of the enzymes (serine-exchange enzyme I) can catalyze the base-exchange reaction of phospholipids with serine, choline and ethanolamine, and the other enzyme (serine-exchange enzyme II) does not use choline as a substrate. 2) Phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions : phosphatidylcholine→phosphatidylserine→phosphatidylethanolamine→phsophatidylserine. The three reactions are catalyzed by serine-exchange I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively. 3) Serine-exchange enzyme I is essential for the growth of CHO cells. In addition, we have found that exogenous phosphatidylserine can be efficiently incorporated into CHO cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs.

Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32Pi and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.

Simplification of the CHO/HGPRT mutation assay through the growth of Chinese hamster ovary cells as unattached cultures

A novel technique for the growth of Chinese hamster ovary (CHO) cells as unattached cells on nontissue culture plates was applied to the CHO/HGPRT mutation assay, using EMS and MNNG as mutagens. The subculturing procedures for the unattached cultures involved less time and effort than those for the conventional attached cultures since trypsinization was not required for cell detachment. No significant difference in the maximum mutation frequency was observed for cells grown as unattached or attached cultures during the expression period. The optimum expression time was, however, shorter for the unattached cells (6 days) than for the attached cells (9 days). No selection for or against the mutant population was observed when mutant and wild-type cells were co-cultivated as unattached cultures, indicating that the procedure does not affect the quantitativeness of the mutation assay. The growth of CHO cells as unattached cells during the expression period thus could decrease the cost and effort involved in the use of the CHO/HGPRT mutation assay in the screening of potential mutagens/carcinogens.

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 mound (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 heterogenieity 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.

Phosphorylation of high-molecular-weight membrane protein species in Chinese-hamster ovary cells in culture: effect of 6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate plus testosterone.

Growth of Chinese-hamster ovary cells in [(32)P]phosphate and [(3)H]leucine and subsequent assay of the plasma membranes reveals phosphorylation in two protein regions corresponding to molecular weights of 280000 and 195000. Culture in the presence of the 6-N,2-O'-dibutyryl derivative of cyclic AMP plus testosterone does not stimulate [(32)P]-phosphate incorporation, but determines a modification in the qualitative pattern of phosphorylation.