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.     

The RNA polymerase II of an α-amanitin-resistant Chinese hamster ovary cell line

Amal, an α-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to α-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to α-amanitin and more thermolabile than the wild-type enzyme.     

Surface polypeptides of the cultured Chinese hamster ovary cell.

The organization of the plasma membrane of logarithmically growing Chinese hamster ovary (CHO) suspension cells has been probed using surface label techniques in conjunction with subcellular fractionation and sodium dodecyl sulfate gel electrophoresis. Five components of apparent molecular weights 137,000, 121,000, 97,000, 67,000, and 57,000 have been shown to be exposed at the outer surface of the cell. These components fully meet the criteria of being (a) reactive with two or more surface label reagents, (b) enriched in a purified plasma membrane fraction, and (c) sensitive to proteolytic digestion of intact cells. Three other components of molecular weights 200,000, 44,000 and 30,000 are also reactive with certain surface label reagents, but fail to meet other criteria for cell surface components. Two polypeptides of molecular weights 180,000 and 37,000 are substantially enriched in the plasma membrane fraction, but are unreactive with surface label reagents. The organization of the CHO cell membrane and the applicability of surface label techniques to cultured cell systems are discussed.     

Messenger RNA synthesis in synchronized Chinese hamster ovary cells

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

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.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors

Summary Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K _m for dCTP and the apparent K _i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.Abbreviations ara-A 9--d-arabinofuranosyl adenine - ara-C 1--d-arabinofuranosyl cytosine - aph aphidicolin     

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.     

Human mutant cell lines with altered RNA polymerase II

A human fibrosarcoma cell line, HT-1080-6TG-9AM, resistant to α-amanitin at concentrations up to 10 μg/ml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled α-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50-to 100-fold. This functional test demonstrated that the biochemical basis for the resistance of the cells to α-amanitin is due to an alteration of RNA polymerase II.     

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.     

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

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

Subcellular distribution of aminoacyl-transfer RNA synthetases in Chinese hamster ovary cell culture

Chinese hamster ovary cells grown in cell culture were broken and fractionated by differential centrifugation. Four principal fractions: nuclear and membrane, microsomal, postribosomal, and supernatant were obtained. The distribution of aminoacyl-tRNA synthetases in these four fractions was determined for all twenty amino acids.It was shown that there is a differential distribution of synthetases. Activities specific for eight amino acids: Ala, Ser, Gly, Cys, His, Arg, Thr and Pro were found mainly in the supernatant fraction. Activities specific for eleven amino acids: Asp, Asn, Glu, Gln, Ile, Leu, Lys, Met, Phe, Tyr and Val were found mainly in the postribosomal fraction. Four activities were found at significant levels in the microsomal fraction: Asp, Phe, Lys and Pro. The nuclear and membrane fraction contained activity for Lys, His, Asp and Thr.Changes in aminoacyl-tRNA synthetase activities in various fractions from preparations made by breaking cells with a membrane-dissociating detergent showed that some of the aminoacyl-tRNA synthetase activities may be membrane bound.     

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.     

Inhibition of Chinese hamster ovary cell DNA synthesis by hydrogen peroxide

The DNA synthesis inhibitory effect of hydrogen peroxide has been examined under a number of experimental conditions. Results have indicated that the effect of the oxidant is more pronounced when the treatment is performed at 37 degrees C than at 4 degrees C and in low density as compared to high density cultures. In addition, similar levels of inhibition were achieved by measuring the incorporation of radiolabelled thymidine in the presence of, or following treatment with, the oxidant. Although early events seem to be responsible for the decreased rate of DNA synthesis, it would appear that hydrogen peroxide does not alter thymidine extracellularly and/or decrease the transport of the nucleoside across the plasma membrane, which may actually be slightly augmented. Thus, the previously illustrated results may represent an underestimate of the actual capacity of the oxidant to reduce DNA synthesis. This inference is further supported by the fact that the effect of hydrogen peroxide appears markedly enhanced in cells preloaded with the radiolabelled precursor. A temporal relationship seems to exist between the steady state level of DNA single strand breaks and the extent of DNA synthesis inhibition by hydrogen peroxide. The oxidant has no effect on DNA chain elongation. In conclusion, data presented in this paper suggest that early events, involving selective effects on replicon initiation, mediate the DNA synthesis inhibitory effect of hydrogen peroxide.     

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.     

Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans.

Chinese hamster ovary lines with two mutations, one causing accumulation of Man5GlcNAc2-P-P-dolichol and a second resulting in defective N-acetylglucosaminyltransferase I activity, synthesize asparagine-linked glycans with the structure Man3GlcNAc2. As a result, the asparagine-linked glycans produced by these lines are smaller and less heterogeneous than those produced by other currently available animal cell lines.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. II. Segregational analysis and DNA transfection of the aphr gene

Genetic characterizations of the Chinese hamster ovary cell mutants resistant to the DNA polymerase inhibitors (aphidicolin, ara-A and ara-C) have been described. Resistance to all three inhibitors showed dominance among the progeny of somatic cell crosses between the wild type and mutant parents. Analysis of the segregation of the drug-resistant character among 566 hybrid progeny from somatic crosses between the wild type (aphs, ara-As, and ara-Cs) and the triple mutants (aphr, ara-Ar, ara-Cr) showed the involvement of at least three unlinked genes in controlling the expression of the resistance to different DNA polymerase inhibitors. The mutant (aphr) DNA was used to transfect aphidicolin resistance to recipient mouse Ltk- cells either directly or in combination with the plasmid pTK2 DNA. The aphidicolin resistance of the transfected cells was found to be a stable phenotype and could be used in multiple rounds of transfection, indicating the chromosomal integration of the transfecting gene.