Evidence for a receptor-mediated endocytosis of Alu I in Chinese hamster ovary cells

Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.     

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.     

Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.     

Binding of concanavalin A to Chinese hamster ovary cells after storage in liquid nitrogen.

The binding of Concanavalin A to the surface of Chinese Hamster Ovary (CHO) cells after storage in liquid nitrogen was studied by a number of techniques. Electron microscope cytochemistry revealed that cells frozen at 1.7 °C/min resulting in high viability had a Con A positive surface coat similar to non-frozen control cells whereas those that had been frozen at 200 °C/min, resulting in low viability, exhibited a range of staining patterns with many cells showing a loss of the coat. However, the binding of I¹²⁵-labeled Con A to the frozen cells revealed no overall loss of binding sites as compared to non-frozen cells although those frozen at 1.7 °C/min appeared to have two types of binding sites whereas the cells frozen at 200 °C/min had only one. Agglutination studies showed that the frozen cells had reduced levels of agglutinability.     

Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.     

Deoxycytidine kinase-deficient mutants of Chinese hamster ovary cells are hypersensitive to DNA alkylating agents

Chinese hamster ovary cell strains deficient in deoxycytidine kinase activity were selected by isolating mutants resistant to high concentrations of the analogue arabinosyl cytosine. Mutants isolated were deficient in the pool of dCTP, supporting earlier a suggestion that the deoxycytidine kinase may play a role in the turnover and maintenance of the dCTP pool. Consistent with earlier observations that increased intracellular levels of dTTP relative to dCTP lead to increased sensitivy to monofunctional DNA alkylating agents, deoxycytidine kinase-deficient mutants showed a 2–5-fold increase in sensitivity to the cytotoxic and mutagenic effects of one agent, ethyl methanesulfonate (EMS). The survival of the two kinase-deficient strains after mutagen treatment was clearly related to dCTP level as the strain with lowest dCTP was most sensitive to EMS. Thus hypersensitivity to this class of DNA damaging agents can result from cellular mutations decreasing the intracellular level of dCTP.     

Rabbit brain glucose transporter responds to insulin when expressed in insulin-sensitive Chinese hamster ovary cells

Transfection of Chinese hamster ovary cells with the expression vector containing rabbit brain HepG2-type glucose transporter cDNA resulted in a dramatic over-expression (approximately 10-fold) of glucose transporter as assessed by either immunoblotting with antipeptide antibody against rabbit brain glucose transporter or photoaffinity labeling with [3H]cytochalasin B. 2-Deoxyglucose uptake was also increased 4-fold in the transfected cells, while no increase in transport activity or transporter amount was observed in cells that were transfected with the expression vector alone without glucose transporter cDNA. Significantly, insulin (10(-7) M) increased 2-deoxyglucose uptake in both control and transfected cells, but the increased amount of the transported 2-deoxyglucose by insulin in the transfected cells was 4.2-fold greater than that in control cells, indicating that the expressed rabbit brain HepG2-type glucose transporter responded to insulin. In addition, we have recently demonstrated that the HepG2-type glucose transporter exists in rat adipocytes and responds to insulin in a fashion similar to a majority of other types of glucose transporters (Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., and Takaku, F. (1988) J. Biol. Chem. 263, 13432-13439). In contrast, insulin did not stimulate glucose transport activity in HepG2 cells or IM-9 lymphocytes that have a significant amount of the HepG2-type glucose transporter. Thus, the results in this study further support the notion that insulin regulation of glucose transport activity depends on a tissue-specific signaling mechanism.     

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

Summary The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-pheny-lindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.     

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.     

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Caffeine sensitization of Chinese hamster ovary cells to heat killing

The effects of the methylxanthine, caffeine, on heat sensitization was investigated using Chinese hamster ovary (CHO) cells. Caffeine sensitized CHO cells to heat killing by reducing both the shoulder and the slope of the 44 degrees C survival curve. Heating was performed in suspension by addition of cells to preheated spinner flasks containing caffeine. Changes in intracellular free calcium levels, [Ca2+]i, were measured at 37 degrees C using the luminescent probe aequorin. Caffeine (1-5 mM) induced a transient increase in [Ca2+]i at 37 degrees C. The transient increase in [Ca2+]i was reduced 15-fold when 5 mM caffeine was added to aequorin-loaded cells suspended in Ca(2+)-free Hanks' balanced salt solution. However, 5 mM caffeine sensitized the cells to the same extent when they were suspended in either Ca(2+)-containing or Ca(2+)-free Hanks' balanced salt solution. The mechanism of heat sensitization by caffeine is still unknown.     

Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.