Induction of mutations resistant to 6-thioguanine by fast neutrons in cultured Chinese hamster cells

A study was made of induction of mutations, resistant to 6-thioguanine (TGr), and reproductive death of Chinese hamster cells after irradiation by fission-spectrum fast neutrons (mean energy of 0.75 MeV) with doses of 10-130 cGy. A high relative biological effectiveness (RBE) of fast neutrons was shown. The maximum RBE values (13-16) were within the dose range inducing minimum mutagenic and lethal effects. RBE decreased with the dose increase. Inspite of high mutagenic effectiveness of neutrons, estimated according to TGr mutation frequency per cell per dose unit, their relative mutagenic effectiveness, estimated per cell per one lethal event, did not substantially differ from that of X-radiation.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Factors affecting the growth of Chinese hamster cells in halt selection media

Factors affecting the efficiency of selection of “reverants” of salvage pathway mutants in media containing amethopterin have been examined. Our V79 Chines hamster cell line was found to require a significantly higher level of thymidine for optimal growth in such media than has been reported for other cell lines. Hypoxanthine (but not glycine) was also required for reversal of amethopterin toxicity, but levels did not differ significantly from those reported elsewhere. Growth in HAT was also dependent on plating density and serum batch. Our modification (VHAT) was compared with published HAT recipies in back selection reconstruction experiments. A sharp fall in EOR (efficiency of recovery) of wild type cells from mixtures with mutants at plating densities greater than 3500 cells/cm² (10⁵ cells/6 cm dish) was observed for VHAT. EOR with other HAT recipes was lower still, and was affected also by the particular mutant used in the mixture.EMS induced “revertants” were isolated from three 8AZ^r mutants by plating in VHAT. All. revertants were however amethopterin resistant, they were also 8AZ resistant and the mobility of residual HGPRT (as measured by polyacrylamide gel electrophoresis) was similar to that of their 8AZ^r parents i.e. dissimilar from that in wild type. The modal chromosome number of V79 wild type cells was 21. No significant deviation from this mode was detected in any of the mutant lines examined. The data indicate that the recovery of colonies in HAT from 8AZ^r mutants does not necessarily indicate that a back mutation in the structural gene for HGPRT has occurred. Thus, the frequency of HAT⁺ colonies cannot be taken as a direct indication of reversion frequencies.     

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.     

Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells

Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.     

Methionine metabolism in BHK cells: selection and characterization of ethionine resistant clones.

The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo-B12. Clones resistant to 30 mug/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 X 10(-5). An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone. Measurement of the specific activity of menadione reductase, B12 methyltransferase and ATP: L-methionine S-adenosyl-transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback-inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase. These results suggest that the pathway of re-use of S-adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.     

Resistance to copper toxicity of cultured hepatoma cells. Characterization of resistant cell lines

A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium. The EC50, that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 h, increased 4-fold over that for wild type cells in the most resistant cell line. These cells were also resistant to zinc, cadmium, and mercury toxicity, but not to nickel or cobalt. The amount of copper in the soluble protein pool of the resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained. Associated with copper accumulation was the production of an 18-kDa cysteine-rich protein which complexes a significant amount of the metal. It is suggested that resistance to copper toxicity is due to sequestration of the metal by this protein. When resistant cells were removed from the copper-enriched environment, cellular copper levels rapidly fell to that observed for wild type cells, but no reduction in either the EC50 or the level of the cysteine-rich protein was noted. This suggests that a permanent change responsible for copper resistance had occurred which is maintained in the absence of the metal.     

Taxol resistant mutants of Chinese hamster ovary cells: genetic biochemical, and cross-resistance studies

The effects of the microtubule inhibitor taxol on the growth and viability of Chinese hamster ovary (CHO) cells have been examined. Stable mutants which are between seven to 11-fold more resistant to taxol have been selected in a single step from ethyl methanesulfonate-mutagenized CHO cells. The two taxol-resistant mutants (Tax^R-1 and Tax^R-2) which have been studied in detail exhibit novel and strikingly different cross-resistance/collateral sensitivity patterns to various microtubule inhibitors. For example, the Tax^R-1 mutant exhibits increased resistance to vinblastine, but in comparison to the parental cells, it shows enhanced sensitivity toward colchicine, colcemid, stegnacine, and griseofulvin. However, the sensitivity of this mutant toward other unrelated compounds, e.g., puromycin, daunomycin, etc., remained largely unaltered. The specific pattern of cross-resistance/collateral-sensitivity of this mutant toward various microtubule inhibitors suggests that the genetic lesion in this mutant may be affecting a microtubule-related component. The Tax^R-2 mutant, in contrast, is highly resistant to various microtubule inhibitors including colchicine, colcemid, stegnacine, maytan-sine, vinblastine, and podophyllotoxin. This mutant also exhibits greatly increased cross-resistance to daunomycin, puromycin, ethidium bromide, and VM-26 (compounds which do not inhibit microtubule assembly), and shows reduced cellular uptake of ³H-daunomycin indicating that the genetic lesion in this mutant nonspecifically affects the membrane permeability of various drugs. The cell hybrids formed between Tax^R-1 (or Tax^R-2 mutant(s)) and a taxol-sensitive cell line exhibit intermediate levels of resistance to the drug, indicating that the Tax^R phenotypes of both these mutants behave codominantly under these conditions.     

Dose-rate effects of ethyl methanesulfonate on survival and mutation induction in cultured Chinese hamster cells

The dose-rate effects of ethyl methanesulfonate (EMS) on the survival and induction of mutations in Chinese hamster Don cells were investigated. The most effective time of exposure to EMS for reducing the surviving fraction of cells was 4 h, shorter and longer exposure times being less effective. The threshold or minimal concentration of EMS giving a surviving fraction of 0.5 was 0.05 mg/ml. The minimal effective time of exposure to EMS for cell death was 1 h. Corrected survival curves showed that longer exposure times at lower dose rates of EMS had less cytotoxic effect than shorter exposure times at higher dose rates.After exposure of Don cells to various doses of EMS for various times, the frequencies of mutations resistant to 6-thioguanine (6TG) were measured. An exposure time of 4 h produced a lower mutation frequency than shorter or longer exposure times that resulted in the same surviving fraction of cells. An exposure time of 20 h produced the highest induced mutation frequency.This system using cultured Chinese hamster cells should be useful as a sensitive procedure for detecting the mutagenic actions of chemicals.     

Isolation and characteristics of Chinese hamster cells resistant to colchicine

Several ClchR clones of CHO-K1 have been isolated by a single- and multistep selection. They are distinct from each other both in the level of colchicine resistance and in the phenotypic stability of this feature. Fluctuation tests showed that the generation of drug resistant variants in the wild type population was random and did not depend on the action of selective agent. The rate of spontaneous occurrence of these variants was approximatley 1.79 x 10(-6) per cell per generation. Treatment with MNNG enhanced the frequency of ClchR variants by 100 fold. Cytotoxic effect of Clch on resistant cells has been potentiated by non-ionic detergent Tween 80. All the stable resistant clones appeared to be cross resistant to unrelated drugs such as actinomycin D, ethydium bromide and aminopterine . These two observations allow to suggest the alteration of membrane permeability as a mechanism of resistance to Clch . Genetical mechanisms of Clch -resistance of cells are discussed.     

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.     

Puromycin resistance in Chinese hamster cells: Genetic and biochemical studies of partially resistant,unstable clones

Resistance to 10 μg/ml of puromycin has been analyzed in V79 Chinese hamster cells. Clones that were isolated in 10 μg/ml of puromycin and subsequently cultivated in its absence consistently lost their resistance. One clone was analyzed in detail by recloning in the presence and absence of puromycin, and it was found that non-puromycin cultivated sublones also lost their resistance and regained inhibition profiles similar to the V79 parent. Reconstruction experiments between sensitive and resistant cells demonstrated that the yield of mutants was not affected by metabolic cooperation. The mutation rate was calculated to be 1 × 10^?7 per cell per generation, and was the same within the limits of statistical error in a colchicine-produced polyploid derivative of the V79 line. Although a number of resistant clones were found to have polyploid karyotypes, the polyploid V79 line was not more resistant to puromycin, nor did it possess a higher frequency of puromycin resistant cells. Studies employing radiolabeled puromycin established that resistance was due to a lowered uptake of puromycin and that an inverse relationship existed between resistance level and uptake rate.     

Genetic and biochemical analysis of mutation(s) affecting ricin internalization in Chinese hamster ovary cells

We have previously reported the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in the internalization of ricin (Ray, B., and Wu, H.C. (1982) Mol. Cell. Biol. 2, 535-544). These mutants also do not exhibit the enhancement of ricin internalization by nigericin pretreatment at a low concentration, which is observed in the wild-type CHO cells. An analysis of somatic cell hybrids between the mutant and the toxin-sensitive wild-type CHO cell line shows that all of the phenotypes associated with the toxin resistance mutation are dominant in the hybrid cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]palmitic acid-labeled cell extracts from the mutant and toxin-resistant hybrid cell lines has revealed an increased incorporation of [3H] palmitic acid into two proteins with apparent molecular weights near 30,000 in the mutant and hybrid cells as compared to that in the wild-type cell line. Our studies indicate that these two fatty acyl proteins might be related to a dominant mutation(s) which results in a decreased uptake of ricin.     

Boron deficiency in cultured pine cells : quantitative studies of the interaction with ca and mg

A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (K_diss = 450 ± 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition—a result consistent with B having a primary role in cell wall biosynthesis.     

Podophyllotoxin resistance: A codominant selection system for quantitative mutagenesis studies in mammalian cells

Mutants resistant to the microtubule inhibitor podophyllotoxin (Pod^R), a codominant marker, can be readily selected in various mammalian cell lines such as, CHO, HeLa, mouse L cells, Syrian hamster cells (BHK₂₁) and a mouse teratocarcinoma cell line OC15. In CHO cells, the recovery of Pod^R mutants is not affected by cell density (up to 1 × 10⁶ cells per 100-mm diameter dish), and after treatment with the mutagen ethyl methanesulfonate maximum mutagenic effect is achieved after a relatively short expression time (40–48 h). The frequency of Pod^R mutants in various cell lines increased in a dose-dependent manner in response to treatment with the mutagens ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine. The Pod^R selection system thus provides a new genetic marker which should prove useful in studies of quantitative mutagenesis in mammalian cells.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein

The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.     

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.

Plasma membranes were prepared from cultured Chinese hamster ovary cells utilizing a two-phase polymer system and were characterized by enzymatic and chemical assay, and by electron microscopy. The usual degree of purification of presumptive membrane markers such as Na+-K+ ATPase (ATP phosphohydrolase, EC 3.6.1.3) ranged from three-to eightfold. Gel electrophoresis in SDS revealed several polypeptides and two glycopeptides which were enriched in the plasma membrane fraction.