Paraquat-resistant cell lines derived from Chinese hamster ovary cells

Two paraquat-resistant clones, PR-1 and PR-2, were selected from CHO K1 cells pretreated with ethyl methanesulfonate. PR-1 and PR-2, routinely cultured in a normal medium without paraquat, were six fold more resistant to paraquat than the parental CHO K1 cells. There was no difference in the uptake of [3H]paraquat among PR-1, PR-2, and CHO K1 cells. Both PR-1 and PR-2 cells showed no cross resistance to free radical generating agents and no increase in total activity of superoxide dismutase. The activities of paraquat-dependent NADPH oxidase and glucose-6-phosphate dehydrogenase were significantly reduced in PR-1 and PR-2 cells, hence the rate of paraquat radical formation will be limited. In addition, an elevation of glutathione levels in PR-1 cells or an increase in glutathione S-transferase activity in PR-2 cells may also play a certain role in protective mechanisms against the toxicity of paraquat.     

Verapamil hypersensitivity of vincristine resistant Chinese hamster ovary cell lines

Vincristine resistant CHO cell lines, obtained by prolonged selection in semi-inhibitory drug concentrations show considerable hypersensitivity to verapamil. Their D10 values are around 0.2 micrograms/ml compared to 23 micrograms/ml for unselected controls. Reversion of vincristine resistance during growth in vincristine free medium is correlated with reversal of verapamil sensitivity indicating that the two aspects of the cells' phenotype have a common underlying cause. The rate of uptake of calcium in the absence and presence of verapamil is similar in the vincristine resistant cells and the controls. The correlation of verapamil sensitivity with vincristine resistance is not a universal feature of CHO cell lines resistant to antimicrotubular drugs, since it was found that other resistant cell lines which have been selected by short term exposure to high drug concentrations were not verapamil hypersensitive.     

A block at Man5GlcNAc2-pyrophosphoryldolichol in intact but not disrupted castanospermine and swainsonine-resistant Chinese hamster ovary cells

A mutation in glycoprotein processing inhibitor-resistant (PIR) Chinese hamster ovary (CHO) cells was previously shown to result in a block at the Man5GlcNAc2 stage of the dolichol-oligosaccharide biosynthetic pathway (Lehrman, M.A., and Zeng, Y. (1989) J. Biol. Chem. 264, 1584-1593). These cells had normal mannose-P-dolichol synthase activity and were able to transfer the Man5GlcNAc2 oligosaccharides to protein. We have now characterized the mutation in greater detail. In PIR cells, biosynthesis of GDP-mannose and mannose-P-dolichol was normal, and pulse-chase analysis indicated that the rate of Man5GlcNAc2-P-P-dolichol formation in vivo was similar to that in parental CHO cells but without subsequent formation of larger intermediates. Cell fusion studies demonstrated that the PIR genotype was recessive and that PIR cells could complement the mutation in B4-2-1 cells, which fail to synthesize mannose-P-dolichol. In contrast to the results obtained with intact cells, incubation of membrane preparations of PIR cells with GDP-[3H]mannose resulted in the synthesis of intermediates containing up to 9 mannose residues, indicating that the cells contained active mannosyltransferases VI to IX. With a simplified assay for the formation of intermediates containing 6 to 9 mannoses, it was shown that physical disruption of PIR cells was able to eliminate the block at the pentamannosyl stage. Furthermore, although the temperature requirements of the reactions for the control CHO and PIR membranes were similar, Man5GlcNAc2-elongating activity in CHO membranes was inhibited by alkaline pH treatment, whereas this treatment irreversibly stimulated the activity in PIR membranes. Taken together, these results suggest that the PIR cells have a recessive defect, and that the missing gene product is required by mannosyltransferase VI in vivo for proper utilization of either mannose-P-dolichol or Man5GlcNAc2-P-P-dolichol. Since the defect was manifested in vivo but not in vitro, this requirement appears necessary for intact cells but not for disrupted cells or isolated membranes.     

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2

Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.     

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

Isolation of Chinese hamster ovary cell lines expressing human acyl- coenzyme A/cholesterol acyltransferase activity

We have previously reported the isolation of Chinese hamster ovary cell mutants deficient in acylcoenzyme A/cholesterol acyltransferase (ACAT) activity (Cadigan, K. M., J. G. Heider, and T. Y. Chang. 1988, J. Biol. Chem. 263:274-282). We now describe a procedure for isolating cells from these mutants that have regained the ability to synthesize cholesterol esters. The protocol uses the fluorescent stain Nile red, which is specific for neutral lipids such as cholesterol ester. After ACAT mutant populations were subjected to chemical mutagenesis or transfected with human fibroblast whole genomic DNA, two revertants and one primary transformant were isolated by virtue of their higher fluorescent intensities using flow cytofluorimetry. Both the revertants and transformant have regained large amounts of intracellular cholesterol ester and ACAT activity. However, heat inactivation experiments revealed that the enzyme activity of the transformant had heat stability properties identical to that of human fibroblasts, while the ACAT activities of the revertants were similar to that of other Chinese hamster ovary cell lines. These results suggest that the molecular lesion in the ACAT mutants resides in the structural gene for the enzyme, and the transformant has corrected this defect by acquiring and stably expressing a human gene encoding the ACAT polypeptide. Secondary transformants were isolated by transfection of ACAT mutant cells with primary transformant genomic DNA. Genomic Southern analysis of the secondary transformants using a probe specific for human DNA revealed several distinct restriction fragments common to all the transformants which most likely comprise part or all of the human ACAT gene. The cell lines described here should facilitate the cloning of the gene encoding the human ACAT enzyme.     

Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins

A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.     

Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines.

A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the alpha-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2- to 3-fold, 8- to 10-fold, and about 800-fold higher concentrations of alpha-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (KD) for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA polymearse II indicated that differences in alpha-amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different alpha-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the alpha-amanitin sensitivity and the [3H]amanitin binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to alpha-amanitin resistance involves structural changes in the gene coding for the alpha-amanitin binding subunit of RNA polymerase II. These changes appear to account for the alpha-amanitin-resistant phenotypes of these mutant cells.     

Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines.

RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.     

Selection and characterization of eight phenotypically distinct lines of lectin-resistant Chinese hamster ovary cell.

Clones resistant to the lectins phytohemagglutinin (PHA), wheat germ agglutinin (WGA), the agglutinin(s) from Lens culinaris (LCA), and ricin (RIC) have been selected from parental auxotrophic Chinese hamster ovary (CHO) cells. The sensitivity to other lectins of these cells and of CHO cells resistant to concanavalin A (ConA) has been determined, and their activity of UDP-N-acetyl-glucosamine glycoprotein N-acetyl-glucosaminyltransferase (GlcNAc-T) has been measured. At least 8 different phenotypes have been identified on the basis of this analysis, and complementation between 2 of them demonstrated.     

Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines.

Complementary DNA cloning, differential screening and Northern hybridization techniques were used to study differential gene expression in the wild-type Chinese hamster ovary (CHO) K1 cell line and its two X-ray sensitive mutants, xrs-5 and xrs-6. 11 species of mRNAs were found underexpressed in the two independently isolated mutants. The steady-state levels of those mRNAs are 3-26-fold less in the two mutants, depending on the particular species. 6 of the underexpressed mRNAs have been identified by comparing the sequences of the cloned cDNAs to the known sequences in GenBank. 4 of them code for the structural proteins of ferritin heavy chain, nonmuscle myosin light chain 3nm, ribosomal protein S17 and L7, respectively. The other two have strong homology with mouse B2 or retroviral sequences. The remaining 5 mRNAs did not show significant homology with any of the known sequences and apparently represent newly isolated species. The effect of 137Cs gamma-rays on the expression of the 11 mRNAs has been studied. Radiation inhibited the expression of the B2-like gene in the mutants but not in the wild-type CHO cells. The levels of the other 10 mRNAs were not affected by radiation. The underexpression of this group of genes in both xrs-5 and xrs-6 mutants seems to be related to their radiation-sensitive phenotype, although the specific gene responsible has not been identified. Two models are proposed to explain the mechanism of underexpression. It is suggested that a cellular factor or/and chromosome structural changes are involved.     

Isolation and characterization of vitamin A-sensitive Chinese hamster lung cell lines

Retinyl acetate (RA)-sensitive variants (RAs-2 and RAs-3) of V79 cell line were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The variants were stable and showed a 3- to 4-fold increase in sensitivity to RA compared to parental V79 cells. The RAs-2 clone was also sensitive to retinol and retinol palmitate. The RA-sensitivity behaves as a recessive trait in all hybrids of RAs-2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs-2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA-sensitive phenotype was observed: Gomori acid-phosphatase staining of RA-treated RAs-2 and V79 cells indicated that lysosomal membrane of RAs-2 cells was more labile than those of the parental V79 cells.     

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.     

Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.

Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.     

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines.

The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions.     

Establishment of human interleukin-4 producing Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were transfected with a human interleukin 4 (IL-4) expression plasmid in which human IL-4 cDNA is linked downstream of the human cytomegalovirus/human immunodeficiency virus chimeric promoter. The plasmid also contained a mouse dihydrofolate reductase (dhfr) gene, expression of which is directed by the SV40 early promoter. The resulting methotrexate-resistant, transformed cells constitutively secreted a high level of human IL-4. CHO cells producing human IL-4 were cultured on microcarriers in a perfusion cell culture system containing 1 l of culture medium, and a high level of human IL-4 (5 × 10⁴U ml⁻¹) was produced at a high cell density (1 × 10⁷cells per ml). Serum-free culture was also examined.     

Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurensis lectin binding in a new Chinese hamster ovary cell line

We report the characterization of two Chinese hamster ovary cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient cell mutant pgsA-745 with a cDNA library prepared from wild-type cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.