Stability of tk-/- mutants of L5178Y mouse lymphoma cells in Fischer's medium

The phenotypic stability of over 2000 large- and small-colony trifluorothymidine-resistant (TFTres) variants of L5178Y/tk(+/-)-3.7.2C cells has been examined. All except 4 of 488 spontaneously arising small-colony variants analyzed (0.8%) retained the TFTres phenotype when rechallenged with TFT after growth for several generations in its absence. All of 558 spontaneous large-colony variants, and 440 small-colony or 487 large-colony variants arising from 13 different mutagens showed similar stability. These results attest to the completeness of TFT selection in the mouse-lymphoma assay when used at 1 microgram/ml in Fischer's medium supplemented with heat-inactivated serum and, together with previous cytogenetic and molecular studies, justify considering essentially all such TFTres variants as stable mutants. The implications of these results for those versions of the mouse lymphoma assay that fail to optimize the recovery and scoring of small-colony mutants is discussed.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line

The cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line was carried out, utilizing G-banded metaphase chromosomes, to provide a karyotypic basis for the precise delineation of induced rearrangements in TK-/- mutants. Band-pattern measurements were used to construct ideograms which represent the position, number, size and staining intensity of the chromosome bands. The TK+/-3.7.2C cell line has been shown to provide quantitation of forward mutations induced at the autosomal thymidine kinase (TK) locus in this cell line. Chromosome analysis of the TK+/-3.7.2C cell line and derived TK-/- mutants has become important in demonstrating that the TK+/-----TK-/- assay may detect and distinguish between chromosomal events and smaller, perhaps point-mutation, events in mutant colonies.     

Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells

Three classes of TFTr variants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells can be identified--large colony (lambda), small colony (sigma), and tiny colony (tau). The sigma and lambda mutants are detectable in the routine mutagenesis assay using soft agar cloning. The tau mutants are extremely slow growing and are quantitated only in suspension cloning in microwells. Variants of all three classes have been analyzed in the process of evaluating the usefulness of the thymidine kinase locus in L5178Y/TK+/- mouse lymphoma cells for detecting induced mutational damage. 150 of 152 variants from mutagen treated cultures and 163 of 168 spontaneous mutants were TFTr when rechallenged approximately 1 week after isolation (3 weeks after induction). All of the 41 mutants assayed for enzyme activity were TK-deficient. The sigma and tau phenotypes were found to correlate with slow cellular growth rates (doubling time greater than 12 h), rather than from effects of the TFT selection or mutagen toxicity. Cytogenetic analysis of sigma mutants approximately 3 weeks after induction shows an association between the sigma phenotype and readily observable (at the 230-300 band level) chromosomal abnormalities (primarily translocations involving that chromosome 11 carrying the functional TK gene) in 30 of 51 induced mutants studied. Using an early clonal analysis of mutants (approximately 2 weeks after induction) 28 of 30 sigma mutants showed chromosome 11 rearrangements. All lambda mutants studied (17 of 17 evaluated 3 weeks after induction and 8 of 8 evaluated 2 weeks after induction) showed normal karyotypes (at the 230-300 band resolution level), including the chromosome 11s. These observations support the hypothesis that sigma (and likely tau) mutants represent chromosomal mutations and lambda mutants represent less extensive mutations affecting the TK locus. The inclusion of sigma mutants in the total induced mutant frequency, as well as distinguishing them as a separate subpopulation of TK-deficient mutants, is, therefore, essential in obtaining maximum utility of the information provided by the L5178Y/TK+/- mouse lymphoma assay.     

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants

Mouse lymphoma cells of the L5178Y TK+/- -3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK+/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle.

An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated. Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant. One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II. Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division. The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C. Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.     

The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.     

Mutagenic action of alkylating agents on prophage lambda

The lethal and mutagenic effects of 7 alkylating agents: N-nitroso-N-methylurea (NMU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), nitrogen mustard (HN2), mitomycin C (MC), bifunctional acridine mustard (AM)--and of cyanate (KNCO) on heat inducible lambda cI857 prophage were studied. After treatment of lysogenic cells with mutagens, prophage was heat-induced either immediately or after 90 min incubation in nutrient broth and c mutants forming clear plaques at 32 degrees C were scored. NMU (0.02 M) when immediately induced with heat, induces c mutants very efficiently (maximal yield 10%) not only in the wild-type cells but also in repair-deficient mutants recA13, lexA102, uvrA6 umuC36, recF143, xthA9, polA1, uvrD3 and uvrD502. These data show that NMU-induced mutations are fixed as replication errors due to mispairing modified bases. After delayed heat induction, the prophage survival enhances and the frequency of c mutations declines considerably in host cells of all repair genotypes tested. Carbamoylation is not involved in the mutagenic action of NMU, because KNCO (0.02 M) has a very slight lethal effect and does not induce mutations. MNNG (100 micrograms/ml) and EMS (0.1 M) also induce mutations by replicative mechanism, because maximal yield of c mutations does not depend on RecA+ and is about 15 and 2%, respectively. MMS is a mutagen of the repair type, since its mutagenic action is suppressed by recA mutation of the host. NH2 only inactivates prophage, but does not induce mutations. MC (50 micrograms/ml) and AM (150 micrograms/ml) induce mutations rather inefficiently (the maximal yield 0.1 and 0.3%, respectively) both in recA+ and recA- hosts. The mutagenic action of these agents is probably due to intercalation.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

Trifluorothymidine resistance and colony size in L5178Y/TK +/- cells treated with methyl methanesulfonate

L5178Y/TK +/- cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft-agar medium containing trifluorothymidine (TFT). Dose-dependent and consistent increases in the frequency of TFTR cells were observed after each of the 48-240-hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft-agar colonies from either MMS-treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFTR colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small-colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small-colony-derived cell lines revealed that, with one exception, small-colony morphology was not a heritable trait. While all large- and some small-colony-derived stocks from MMS-treated cells were of the phenotypically stable TK-/- type; spontaneous small TFTR colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS-treated cell lines several generations after isolation as small TFTR colonies. These results suggest that delayed MMS cytotoxicity in TK +/- cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small-colony TFTR variants.     

Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate

The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.     

Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells.

The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 micrograms/ml AsA, EMS-induced 6TG-resistant mutations were reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsA and EMS which had previously been incubated at 37.0 degrees C for 2 h. In pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.     

Temperature- and dose-dependent internalization of concanavalin A in monolayer culture

When rat hepatoma cells (R117-21B) were incubated for 20 h at 37 degrees C with 125I-labeled concanavalin A at low concentrations (0.5-10 micrograms/ml), only 20-30% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside, but at high concentrations (50-500 micrograms/ml), 60-80% of the cell-associated radioactivity was released. At 4 degrees C, the cell-associated radioactivity decreased with the increase in concentration of concanavalin A, and more than 80% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside. These results suggest that the amount of cell-associated concanavalin A is related to the physicochemical state of the plasma membrane, which can be altered by the incubation temperature or by the concentration of concanavalin A, the transitional concentration being 5-10 micrograms/ml.     

The utilization of trifluorothymidine (TFT) to select for thymidine kinase-deficient (TK−/−) mutants from L5178Y/TK+/− mouse lymphoma cells

Trifluorothymidine (TFT), a thymidine analog, was analyzed for its ability to select for thymidine kinase-deficient (TK^−/−) mutants. In comparison with BUdR, the traditional selective agent for TK^−/− cells, it was determined that TFT at 1/50th the dose (1μg/ml vs. 50 μg/ml) is a more effective and versatile selective agent for TK^−/− mutants arising from the TK^+/−-3.7.2C heterozygote of L5178Y mouse lymphoma cells. Since TFT acts more rapidly than BUdR, it can be utilized in procedures (such as the analysis of the phenotypic lag) requiring the fast arrest of cell division. Reconstruction analyses of effective TK^−/− mutant recovery indicate that TFT can be used to recover mutants from significantly higher densities of TK^+/− cells than can BUdR. In addition, TK^−/− mutants can attain larger colony size in TFT than in BUdR where severe stunting of growth occurs at high TK^−/− cell densities. 190 of 194 isolated TFT-resistant large and small colony mutants (both spontaneous and induced)     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Quantitative studies of increased cell-to-substratum adhesion at low temperature

The cell-to-substratum adhesion of an established epithelial cell line cultured for 24 h on glass coverslips was determined at 4 degrees C, 8 degrees C and 37 degrees C using a miniaturised parallel-plate shearing apparatus. The measurements of the minimum shear necessary to dislodge the cells (minimum distraction force, MDF) demonstrated a three- to fourfold increase in the adhesion of the cells at 4 degrees C (6.17 Pa) compared to that at 37 degrees C (1.36 Pa). At 8 degrees C the MDF was 2.31 pascals. Part of the adhesion was resistant to mild trypsinisation. Trypsin-resistant adhesion (TRA) was stabilised by low temperature, and by treatment with concanavalin A (50 micrograms ml-1) or colchicine (200-400 microM). The effects of con A (140 micrograms ml-1) and low temperature (4 degrees C) were additive, giving a combined MDF of greater than 9.27 pascals. On the basis of their different temperature and protease susceptibility it is suggested that trypsin-sensitive adhesion (TSA) and TRA represent separate functional classes of cell-to-substratum attachment corresponding to 'frictional' and 'tractional' adhesion, respectively.     

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.     

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.