Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity.

The production of glycoproteins with carbohydrates of defined structure and minimal heterogeneity is important for functional studies of mammalian carbohydrates. To facilitate such studies, several Chinese hamster ovary mutants that carry between two and four glycosylation mutations were developed. All of the lines grew readily in culture despite the drastic simplification of their surface carbohydrates. Therefore, both endogenous glycoproteins and those introduced by transfection can be obtained with specifically tailored carbohydrates. The lectin resistance properties of the mutants showed that each line expresses a novel array of cell surface carbohydrates useful for identifying specific roles for carbohydrates in cellular interactions. In addition, they showed that the epistatic relationships among different glycosylation mutations are not entirely predictable, providing insight into the complexity of the carbohydrate structures at the Chinese hamster ovary cell surface.     

Mutagenicity of vinyl chloride, vinylidene chloride and chloroprene in V79 Chinese hamster cells.

The mutagenicity of vinyl chloride, vinylidene chloride (1,1-dichloroethylene) and chloroprene (2-chloro-1,3-butadiene) was tested in V79 Chinese hamster cells in the presence of a 15 000 x g liver supernatant from phenobarbitone-pre-treated rats and mice. Mutations in terms of 8-azaguanine and ouabain resistance were induced in a dose-related fasion by exposure to vapour of vinyl chloride in the presence of liver supernatant from phenobarbitone-pretreated rats. Vapours of vinylidene chloride and chloroprene induced a dose-related toxicity in the presence of liver supernatant from phenobarbitone-retreated rats, but these two compounds were not mutagenic in V79 Chinese hamster cells under the present assay conditions. The results are discussed with regard to the metabolic activation of the compounds and to the correlation with their carcinogenicity in man and experimental animals.     

Species-specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes

The secretory cells of the oviductal epithelium secrete a high- molecular-weight glycoprotein (OGP). OGPs from different mammalian species show similar immunological characteristics, their cDNAs show high homologies, and they associate with the zona pellucida of oviductal oocytes in vivo. The purpose of this study was to determine the effect of OGP obtained from different species on the binding of hamster sperm to hamster oocytes. Hamster oocytes were inseminated (30 min) in the presence or absence of homologous or heterologous OGPs, and sperm bound/oocyte were counted after removing loosely attached sperm. Ovarian oocytes had an average of 2.9 ± 0.6 sperm bound/oocyte, whereas oviductal oocytes had 36.3 ± 2.7. Hamster OGP (0.1 mg/ml) significantly increased sperm binding to ovarian oocytes twofold and had no effect on sperm bound/oviductal oocytes. Human OGP (0.5 mg/ml) significantly decreased sperm binding to ovarian oocytes (0.9 ± 0.3 sperm bound/oocyte). This effect was dose dependent for oviductal oocytes and could be blocked by preincubating human OGP with a specific antibody to human OGP. The presence of baboon and cow OGP during the insemination of hamster oviductal oocytes also resulted in a significant decrease in sperm bound/oocyte, whereas the addition of hamster OGP to hamster oviductal oocytes had no effect. These results show that homologous OGP enhances sperm binding to the ZP, whereas heterologous OGP inhibits that effect. Thus, our results suggest that OGP plays a role in the species-specific characteristics of sperm/ZP interaction, and that one must use a homologous system (OGP and gametes from the same species) to study the biological effect of OGP. Mol Reprod Dev 46:201–207, 1997. © 1997 Wiley-Liss, Inc.     

Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells

The clastogenic activity of some pyrolysates of carbohydrates was examined in cultured Chinese hamster V79 cells. These pyrolysates include levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). LG-I did not induce a significant number of chromosome aberrations at doses up to 8000 micrograms/ml. In contrast, the related compound LG-II induced aberrations and reduced mitosis in a dose-dependent fashion at around 1/2000 of the LG-I doses. Both furan derivatives, FF and HMF, and both glyoxal derivatives, GL and MGL, induced a significant number of chromosome aberrations and a significant lowering of mitotic activity. Among these compounds, FF and MGL showed stronger clastogenic activity than HMF and GL, respectively. DG slightly but positively induced chromosome aberrations. TZ was one of the most potent clastogens among the compounds examined in this study, showing the highest incidence of aberrant cells with many exchanges at doses inducing a significant lowering of mitotic activity. The results of this study indicate the need for a re-evaluation of the thermal decomposition of carbohydrates as a source of genotoxic contaminants.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III activity

A biochemical basis for the LEC10 mutant phenotype of Chinese hamster ovary cells has been identified. Independent LEC10 mutants, originally selected for resistance to the toxicity of ricin, have been shown to exhibit reduced binding of 125I-ricin at the cell surface. Although this is indicative of structural changes in cell-surface carbohydrates, labeling of plasma membranes with galactose oxidase/[3H]borohydride revealed no significant differences between mutant and parental cells. Alterations in the carbohydrates synthesized by LEC10 cells were, however, resolved by lectin-affinity chromatography of glycopeptides from the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC10. LEC10/VSV glycopeptides contain a fraction which is not bound to concanavalin A-Sepharose but is strongly retarded on E-PHA (erythroagglutinin from Proteus vulgaris)-agarose. In contrast, CHO/VSV glycopeptides or those from a LEC 10 revertant (R.LEC 10/VSV) do not contain carbohydrates with these properties. High-field 1H NMR spectroscopy of the novel LEC10/VSV carbohydrates showed that they are complex, biantennary structures containing N-acetylglucosamine in beta(1,4)-linkage to the beta-linked core mannose residue. The presence of these structures correlates with the expression of the enzyme responsible for the addition of this "bisecting" GlcNAc residue, UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III (GlcNAc-TIII). Parental Chinese hamster ovary cells and the LEC10 revertant possess no detectable GlcNAc-TIII activity. The combined evidence suggests that the LEC10 mutation induces the expression of the GlcNAc-TIII enzyme in Chinese hamster ovary cells.     

Novel glycosylation-defective baby hamster kidney cells.

The plant lectin wheat germ agglutinin (WGA) has previously been used to select more than ten different glycosylation-defective phenotypes in a variety of mammalian somatic cells. Three WGA-resistant phenotypes have now been obtained spontaneously from baby hamster kidney (BHK) cells. These mutant BHK cells exhibit a pattern of cross resistance and sensitivity to multiple plant lectins, suggesting that the cell surface carbohydrates of these cells are altered. Two WGA-resistant BHK phenotypes appear similar to WGA-resistant CHO cells that lack terminal sialic acid and galactose residues on their cell surface carbohydrates. The third WGA-resistant BHK cell phenotype has not previously been seen in WGA-resistant mammalian cells.     

In vitro metabolism and biological activity of all-trans-retinoic acid and its metabolites in hamster trachea

The in vitro metabolism of all-trans-[11,12-3h]retinoic acid to several more polar compounds has been demonstrated in a hamster tracheal organ culture system. The production of these metabolites is dependent on the presence of tissue. The physiological significance of these compounds is shown by the cochromatography of several of the in vitro formed metabolites synthesized from [carboxy-14C]retinoic acid with metabolites isolated from the intestine and urine of hamsters previously injected with 0.1 to 1.5 microgram of [3H]retinoic acid. One of the metabolites shows about one-tenth the biological activity of all-trans-retinoic acid when tested in a hamster tracheal organ culture assay. This biologically active metabolite is converted by the hamster trachea in vitro to a biologically inactive metabolite.     

Ethanolamine inhibits choline uptake in the isolated hamster heart

The effect of exogenous ethanolamine on phosphatidylcholine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [Me-3H]choline in the presence of 0.05-0.5 mM ethanolamine. Incorporation of label into phosphatidylcholine was decreased 26-63% at 0.1-0.5 mM ethanolamine. Similar decreases in the labelling of the metabolites of the CDP-choline pathway were observed at these ethanolamine concentrations. The observed decrease in phosphatidylcholine labelling at 0.1-0.5 mM ethanolamine was attributed to an inhibition of labelled choline uptake by ethanolamine. The inhibitory role of ethanolamine to choline uptake was examined by comparison to hemicholinium-3. Both compounds inhibited choline uptake in a competitive manner. Intracellular choline, phosphocholine and CDP-choline concentrations were not altered under all experimental conditions. It can be concluded that exogenous ethanolamine has no immediate effect on the rate of phosphatidylcholine biosynthesis in the isolated hamster heart. The reduced labelling of phosphatidylcholine in the presence of ethanolamine is a direct result of the reduction of labelled choline taken up by the heart.     

Immunological and biochemical properties of metallothioneins of golden hamster,mouse and rat

Summary Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Interaction of leachable model compounds and their impact on Chinese hamster ovary cell cultivation

The presence of leachables in biopharmaceutical processes using single-use technologies (SUT) is well known. For the detection and quantification of the latter, extractable studies of SUT are very common nowadays. Although a mixture of compounds is regularly found in extractable studies, research has only been carried out regarding the effect of individual compounds on cell culture and the cumulative effect of a mix of leachables has not been investigated yet. In this study, a set of leachable model compounds (LMCs) was chosen and the effect of the LMCs on a Chinese hamster ovary DG44 cell line producing an IgG antibody was investigated concerning cell growth, cell cycle distribution and productivity. It was shown that even if worst-case concentrations were used, the LMCs solely impact cell growth. Additionally, interaction studies revealed that the inhibiting effect of the mix is lower than the expected cumulative effect. A strong antagonism between the antioxidant butylated hydroxytoluene and the plasticizer Tris(2-ethylhexyl)trimellitate was found using an isobologram analysis.     

A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Characterization of beta-D-N-acetylhexosaminidase isoenzymes in man-Chinese hamster somatic cell hybrids.

A series of man-Chinese hamster hybrids were investigated with the use of an anti-Chinese hamster hexosaminidase serum, a specific anti-human hex A serum and an anti-human hex B serum. The expression of human hex A was found to be dependent on the presence of hex B. A heteropolymeric molecule is formed independently of hex B, which consists of Chinese hamster and specific hex A moieties. It has an electrophoretic mobility nearly identical to hex A. A relationship between the absence and presence of the heteropolymeric molecule, mannosephosphate isomerase (MPI), and pyruvate kinase (PK-3), assigned to chromosome 15, was established. With respect to the two locus subunit model, the gene coding for the alpha subunit, specific for hex A, has been localized on chromosome 15.     

Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos

In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pH(i) and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.     

Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line

The carbohydrate structure of the major oligosaccharide of human interferon-beta (IFN-beta) synthesized by a genetically engineered Chinese hamster ovary cell line has been determined. Analysis of the glycopeptidase F-released carbohydrates by sequential exoglycosidase treatment, methylation analysis, and fast atom bombardment-mass spectrometry revealed that 95% of the IFN-beta oligosaccharides had the following structure: (Formula: see text). The remaining 5% of the carbohydrates are probably tri- or higher antennary oligosaccharide chains. The major oligosaccharide of the recombinant IFN-beta is remarkably homogeneous with respect to terminal galactose sialylation. NeuAc, which is alpha 2-3-linked to galactose in the human IFN-beta secreted by Chinese hamster ovary cells, can be re-incorporated with an alpha 2-6 linkage in vitro, into enzymatically desialylated IFN-beta using rat liver Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. The sugar chain is important for maintaining protein solubility as shown by the fact that IFN-beta protein precipitates after deglycosylation with glycopeptidase F.     

Desmosteryl sulfate and desmosterol in hamster epididymal spermatozoa

Desmosteryl sulfate composes 95% of the sterol sulfate fraction of hamster epididymal spermatozoa at a level of 10 mg/g dry weight. Desmosterol represents as much as 40% of the free sterol fraction of these cells. Only cholesterol was detected in the free sterol fraction from plasma and liver of the same animals. The present report details the evidence for proof of structure of the two compounds isolated.     

Inducible repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells

Preirradiation of Chinese hamster cells with low-level UV-light does not influence the efficiency of repair of gamma-radiation-induced DNA single-strand breaks. With fractionated gamma-irradiation, cycloheximide delivered during the interval between the two fractions reduces the number of DNA breaks (compared to that in cells affected by the same nonfractionated dose). The data obtained indicate the presence of an inducible component of repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells.