Single-step Selection Research Articles

Isolation and uptake characteristics of adenovirus transformed cell revertants resistant to the antiproliferative effects of methylglyoxal bis(guanylhydrazone)

Four independent variant cell lines resistant to the toxic action of methylglyoxal bis(guanylhydrazone), an anticancer drug and inhibitor of polyamine synthesis, have been isolated by single step selection from an adenovirus type 2-transformed rat brain cell line. Drug-resistance was accompanied by loss of tumorigenic potential in athymic nude mice. MGBG resistance was attributable to decreased drug uptake.

Mutants of embryonal carcinoma cells defective in the expression of embryoglycan.

Embryonal carcinoma cells defective in the expression of developmentally regulated carbohydrate epitope of teratocarcinoma cells (TEC-1) were isolated from mutagenized P19X1 and P19S1801A1 cells by a single-step selection technique using monoclonal antibody TEC-01 conjugated to plant toxin ricin. Three independently isolated mutant cell lines were characterized in detail. Analysis of the expression of the TEC-1 epitope in somatic cell hybrids constructed between wild-type and mutant cells and between two mutant cell types revealed that the mutant phenotypes are recessive and that the mutants belong to, at least, two complementation groups. Each mutant cell line exhibited a unique binding pattern of four monoclonal antibodies and five lectins, and different properties of large glycopeptides were distinguished by Sephadex G-50 column chromatography. The combined data suggest that our mutants identify three genes involved in the synthesis of embryoglycan, one of which appears to be the regulatory or structural gene for fucosyltransferase. One mutant cell line was completely deprived of embryoglycan and several carbohydrate structures typical of early embryonic and embryonal carcinoma cells; however, the cells were similar to parental cells in their morphology, their ability to form aggregates when cultured in suspension, their ability to differentiate into neuron-like cells after treatment with retinoic acid, and their ability to form tumors composed of embryonal carcinoma cells. Thus, embryoglycan is not required for the expression of a number of properties of the embryonal carcinoma phenotype.

Resistance to toxic effects of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells.

A spontaneously arising clone, stably resistant to the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdU), was isolated from unmutagenized V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The hmdUr cells were selected in the continuous presence of 30 microM hmdU, a concentration which reduces the plating efficiency of wild-type cells to less than 1% after a 24-h exposure. A line of human HeLa cells were found to be intrinsically resistant to concentrations of hmdU as high as 100 microM. All of the hmdUr cells were found to grow normally in HAT medium, which requires the expression of thymidine kinase activity; be sensitive to the toxic effects of high concentrations of 5-bromo-2'-deoxyuridine, another thymidine analog; have unaltered hmdU nucleotide metabolism, as measured by HPLC analysis of acid-soluble cell extracts; and have decreased levels of hmdU incorporation into DNA. Although high concentrations of 5-hydroxymethyluracil (hmUra) were found to be nontoxic for both wild-type and hmdUr cells, the resistance phenotype could be suppressed by exposing the cells to hmdU and high concentrations of hmUra simultaneously.

Tunicamycin-resistant Leishmania mexicana amazonensis: expression of virulence associated with an increased activity of N-acetylglucosaminyltransferase and amplification of its presumptive gene.

Tunicamycin at 10 micrograms/ml inhibits the growth and infectivity of the parasitic protozoan Leishmania mexicana amazonensis. Tunicamycin-resistant variants of this parasite were produced by gradual acclimatization of cells to increasing concentrations of the drug up to 80 micrograms/ml and a single-step selection of ethyl methanesulfonate-pretreated or differentiating leishmanias with the drug at 10 micrograms/ml. Prolonged exposure to the drug increases stability of drug resistance of those resistant to 10 micrograms/ml. Tunicamycin-resistant cells contain amplified DNA, which hybridizes in proportion to the cells' degree of drug resistance with Alg 7, a cloned DNA probe apparently encoding yeast N-acetylglucosaminyltransferase. This enzyme from all variants remained sensitive to inhibition by tunicamycin, but its specific activity was up to 15-fold higher than that of the wild type. Thus, amplification of the gene encoding this enzyme appears to result in its overproduction in the variants, accounting for their resistance to tunicamycin. The tunicamycin-resistant cells are more virulent to mice than their parental wild type. Thus, leishmanial virulence may be related to amplification or expression of gene(s) encoding enzymes involved in the regulation of N-glycosylation of parasite proteins.

Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells

A glyphosate-tolerant variant of cultured tomato cells (Lycopersicon esculentum × L. peruvianum hybrid) was isolated via a single-step selection. Growth of the variant in suspension culture was essentially unaffected by 10 mM glyphosate, 100 times the concentration needed to significantly reduce the growth rate of wild type cells. When treated with glyphosate, variant cells accumulated much less shikimic acid than did the wild type cells. In analyses of 5-enolpyruvyl-shikimic acid 3-phosphate (EPSP) synthase activity in two separate experiments, the variant cells had 8 and 13 times higher specific activity than the wild type cells. The enzyme activities from the two types of cells were equally inhibited by glyphosate. These results suggest that the glyphosate tolerance of the variant results from overaccumulation of a glyphosate-sensitive EPSP synthase. Attempts to regenerate fertile plants from the variant cells were unsuccessful, but abnormal shoots were regenerated and callus from leaves of these shoots retained the tolerance to glyphosate.

Biochemical and cross-resistance studies with HeLa cell mutants resistant to cardiac glycoside SC4453. Regulation of the resistant form of Na+/K+-ATPase in the mutant cells.

In HeLa cells, stable mutants which are between 25-to about 200-fold resistant to the cardiac glycoside derivative SC4453 (a digoxin analog which contains a pyridazine ring in place of a lactone ring in the C-17 position) have been isolated after a single step selection in the presence of the drug. Based on their cross-resistance pattern towards various cardiac glycosides, the mutants resistant to SC4453 (SCR mutants) appear to be of two different kinds and they differ from the two classes of ouabain-resistant mutants described previously (Gupta, R. S., and Chopra, A. (1985) J. Biol. Chem. 260, 6843-6850). One type of SCR mutants (designated as group C) exhibit a high degree of cross-resistance to all cardiac glycosides and their genins (viz. ouabain, digitoxin, digoxin, digoxigenin, convallatoxin, gitoxin, strophanthidin, and bufalin). In contrast, the second type of SCR mutant (group D) exhibit considerable resistance to only SC4453, digoxin, and digoxigenin, but showed very little or no cross-resistance to the other cardiac glycosides examined. The cross-resistance of the mutants towards cardiac glycosides was highly specific as they exhibited no cross-resistance towards a large number of other structurally and functionally related compounds (viz. ethacrynic acid, sanguinarine nitrate, penicillic acid, methyl quinolizinum bromide, 5,5'-diphenylhydantoin, deoxycorticosterone, vanadium pentoxide, and adriamycin). The cellular uptake of 86Rb in the mutant cells was found to be resistant to specific cardiac glycosides. Studies on the sensitivity of plasma membrane Na+/K+-ATPase to cardiac glycosides show that about 10-15% of the enzymic activity in the mutant cells was highly resistant to inhibition by the specific drugs to which the mutants exhibit increased resistance. Very interestingly, when the mutant cells are grown in cardiac glycoside-containing medium, the resistant form of the enzyme accounts for about 50-60% of the total enzyme. These results show that both classes of SCR mutants are affected in Na+/K+-ATPase and that the amount of the resistant enzyme in the mutant cells is regulated in response to cardiac glycosides.

Resistance to beta-lactamase-stable cephalosporin antibiotics in strains of Enterobacteriaceae and Pseudomonas aeruginosa isolated from blood cultures.

A total of 224 strains of Enterobacteriaceae and Pseudomonas aeruginosa isolated from blood cultures were screened for resistance to cefuroxime and cefotaxime. Seven of 200 strains of Enterobacteriaceae were resistant to either cefuroxime or cefotaxime, while five of 24 strains of Ps. aeruginosa were resistant to cefotaxime. A further 32 resistant variants were isolated by use of a single step selection procedure, without prior mutagenesis, from strains which were sensitive on initial screening. From the total of 44 resistant strains and variants isolated, 31 were found to produce elevated levels of chromosomal beta-lactamases. Of these 31, five strains of Proteus sp. produced significant amounts of beta-lactamase only when induced in the presence of cefuroxime or cefotaxime, while the remaining 26 strains also produced high levels of beta-lactamase in the absence of an inducer. Thirteen resistant strains and variants (9 Ps. aeruginosa, 3 P. mirabilis, 1 K. aerogenes) did not produce detectable levels of beta-lactamase in either the presence or absence of induction and presumably owed their resistance to another mechanism.

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.

In vitro antibacterial activity of BMY-28142, a new extended-spectrum cephalosporin.

The in vitro activity of BMY-28142 was compared with that of cefotaxime, ceftazidime, moxalactam, and imipenem against 639 clinical isolates and a number of in vitro-selected resistant mutants. BMY-28142 was the most potent compound against the members of the family Enterobacteriaceae with a MIC for 90% of the strains of 0.12 micrograms/ml. The activity against Pseudomonas aeruginosa was comparable to that of ceftazidime and imipenem. Strains of staphylococci were moderately susceptible to BMY-28142 (MIC required to inhibit 90% of strains, 4 micrograms/ml), but Streptococcus faecalis isolates were resistant. The activity of the five compounds was inoculum dependent for several gram-negative species. By a single-step selection procedure, resistant mutants were selected from strains of Citrobacter freundii, Enterobacter cloacae, and P. aeruginosa. The mutant frequencies with the cephalosporins, including BMY-28142, ranged between 10(-6) and 10(-8). BMY-28142 was the most active cephalosporin against these resistant organisms, most of them strong beta-lactamase producers. It inhibited all mutants of C. freundii and E. cloacae at 2 micrograms/ml and all mutants of P. aeruginosa at 32 micrograms/ml. Imipenem on the other hand was as active on all of these resistant organisms as on the parent strains.

Isolation and partial characterization of lectin-resistant F9 cells.

Cytotoxic plant lectins have been used for the single-step selection of mouse embryonal carcinoma cell mutants with altered expression of surface glycoconjugates. Following mutagenesis, several F9 and OTF9-63 cell lines resistant to the lectins from Triticum vulgaris or Ricinus communis were obtained. At least five distinct lectin-resistant (LecR) phenotypes have been identified on the basis of their relative sensitivities to four different plant lectins and their altered lectin-binding properties. None of the mutant types exhibits a significant change in the ability to bind a monoclonal antibody against the stage-specific embryonic antigen, SSEA-1. All of the mutants form aggregates when cultured in bacteriological petri dishes and appear to differentiate into endoderm-like cells following exposure to retinoic acid. However, two of the LecR cell lines exhibit an altered morphology when grown on a plastic substratum.

O6-Ethylguanine is not the only ethylated adduct responsible for the induction of mutations in Syrian hamster embryo (SHE) cells

Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5–6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10−4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5–6 × 10−8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dihydrofolate reductase genes.

Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12,13,20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximately equal to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximately equal to 1 microgram/ml) is approximately equal to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.

Tunicamycin-resistant mutations in mouse FM3A cells

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 μg/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1–1.2 μg tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10 −4 to 10 −5 in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 μg N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 × 10 −6 per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D 10 value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [ 3H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variant-specific antigens by selection of antigen-loss variants with cytolytic T cell clones.

Mouse mastocytoma P815 tumor cell variants that express new individual antigens were subjected to immunoselection in vitro with specific cytolytic T lymphocyte (CTL) clones. From one variant (P35) a number of resistant clones were isolated after a single-step selection. These immunoselected clones (P35.iscA-) proved to have acquired a stable and complete resistance to lysis by the selecting CTL, but remained sensitive to lysis by a CTL clone that recognizes a common P815 antigen. However, when these P35.iscA- clones were used to stimulate in vitro spleen cells from mice immunized with variant P35, the cytolytic activity was significantly higher on P35 and P35.iscA- targets than on the original P815 clone (P1). This indicated that P35.iscA- cells, which had lost a P35-specific determinant (P35A), had nevertheless retained another P35-specific determinant (P35B). CTL clones directed against the residual P35B determinant were then isolated. When P35 cells were submitted to selection with anti-P35B CTL, resistant clones (P35.iscB-) were obtained that were still lysed by anti-P35A and anti-P815 CTL clones. Tum- variant P35 therefore carries at least two specific determinants that can be lost independently. These results show the possibility of using selection with CTL clones to separate different components of complex cell surface antigens. The correlation between antigen expression and tumorigenicity was also examined. Antigen-loss P35.iscA- clones were found to be more tumorigenic than variant P35 which has a greatly reduced tumorigenic capacity compared to the original P815 tumor. This correlation suggests that the P35 determinant that was no longer expressed by the P35.iscA- clones is recognized on the original P35 tum- variant as a tumor rejection antigen.

Hormone-induced caulogenesis in long-term tobacco cell lines and its effect on nuclear DNA content

Manipulation of exogenous auxin and cytokinin levels during sequential subculture in vitro has been used to induce caulogenesis in several long-term tobacco cell lines. Concurrently, tissue samples at various stages of caulogenesis have been examined for nuclear DNA content. While a variety of hormone regimes permitted caulogenesis, extremely high (122.95 μM) cytokinin levels and extremely low (0.285 μM) auxin levels generally gave optimal response. For three lines, caulogenesis was accompanied by a progressive decrease in nuclear DNA content beyond that due to the loss of polyploid cells. In one line, however, DNA content remained stable during regeneration, perhaps reflecting the acquisition of a stably adapted aneuploid karyotype. Both caulogenic response and amount of nuclear DNA were affected by changes in the culture medium. The progressive nature of the observed changes in DNA content is inconsistent with a single-step selection for euploid competent cells. Alternative models postulating either progressive selection for euploidy, or the regulation of karyotype are proposed to explain the results.

Unstable aryl hydrocarbon hydroxylase-deficient variants of a rat hepatoma line.

The H-4-II-E-C3 line has high aryl hydrocarbon hydroxylase (AHH) activity. It was also found to be very sensitive to benzo(a)pyrene (BP) toxicity, and a single-step selection procedure for isolating BP-resistant (BPr) clones was designed. All the BPr variants tested had reduced AHH activities under both inducing and noninducing conditions. As H-4-II-E-C3 was propagated, it gradually declined in AHH activity and increased in cloning efficiency in BP. The spontaneous rate of origin of the BPr variants was approximately 3 x 10(-5) events per cell generation. However, the variants gradually increased in AHH activity and decreased in cloning efficiency in BP as they were propagated. Partially resistant variants also generated, at an appreciable rate, cells with even greater degrees of resistance to BP. The instabilities of the wild-type and variant cultures suggest that the variants arose by an epigenetic mechanism or by changes in gene dosage, rather than by point mutations.

Studies on the cross-resistance of Mycobacterium tuberculosis, strain H37Rv, to aminoglycoside- and peptide-antibiotics.

Various phenotypes of the resistance to aminoglycoside- and peptide-antibodies of Mycobacterium tuberculosis strain H37Rv were produced by single- and/or two-step selection of the parent strain. Mutants obtained by single-step selection with antibiotics were classified into ten phenotypes; one of single resistance, two of triple resistance, three of quadruple resistance, and four of sextuple resistance. There were two kinds of sextuple resistance (high resistance to enviomycin, viomycin, capreomycin, kanamycin, lividomycin). One was isolated from the parent strain by single-step selection and could be eliminated by mutation to isoniazid resistance, the other was obtained by two-step selections and was not eliminated by mutation to isoniazid resistance. Interaction between mutation to streptomycin resistance and mutation to quadruple resistance (4R phenotype) was observed. Streptomycin resistance interfered with the formation of the 4R phenotype and produced a different phenotype, KR instead of the 4R phenotype. The existence of mutation of the 4R phenotype did not usually interfere with mutation to streptomycin resistance, but a small portion of the mutants with the 4R phenotype were altered in their phenotype from 4R to KR after addition of the mutation to streptomycin resistance. This effect of the mutation to streptomycin resistance was not observed in mutants which already had a mutation to klR phenotype (mutation to low concentrations of kanamycin only).

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase ( aprt) and thymidine kinase ( tk) loci. Presumptive aprt +/− heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt +/− heterozygote with ∼50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase ( hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.

Mutagenicity testing in mammalian cells: II. Validation of multiple drug-resistance markers having practical application for screening potential mutagens

Chinese hamster ovary (CHO) cell lines heterozygous at both the adenine phosphoribosyltransferase ( aprt) and thymidine kinase ( tk) loci were used for single-step selection of spontaneous and induced mutants resistant to 8-azaadenine (AA r), 6-thioguanine (TG r), ouabain (OUA r), or 5-fluorodeoxyuridine (FUdR r). Mutation data are reported for direct mutagens (EMS, ethyl methane-sulfonate; MNNG, N-methyl- N′-nitro- N-nitrosoguanidine; NQO, 4-nitroquinoline 1-oxide) and promutagens (DMN, dimethylnitrosamine; BP, benzo[ a]-pyrene) activated by rat-liver homogenates. Optimal plating densities were established for AA r, TG r, OUA r and FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP were 2–4 d for AA r, 6–8 d for TG r, 1–3 d for FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP showed locus-specific differences in sensitivity. Of 61 clonal isolates resistant to AA and assayed for APRT activity, 87% had ⩽ 5% wild-type activity; of 30 TG r clones assayed, 83% had ⩽ 5% wild-type HGPRT activity. Of 42 FUdR r clones assayed, 98% had ⩽ 1% wild-type TK activity. 50 clones selected in medium containing FUdR displayed cross-resistance to 5-bromodeoxyuridine (BUdR) and trifluorothymidine (TFT) and all were sensitive to HAT (hypoxanthine-amethopterin-thymidine) medium. The tk locus showed the largest mutational response as a function of cell survival after mutagen treatment. The rapid expression kinetics for FUdR r and the possibility that the locus detects a broader spectrum of genetic lesions than the other drug-resistance markers are discussed in terms of a sensitive screening assay for detecting potential mutagens.