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.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.     

Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures

Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size.     

Comparative induction of gene mutations and chromosome damage by 1-methoxy-1,3,5-cycloheptatriene (MCHT), 2. Results using L5178Y mouse lymphoma cells to detect both gene and chromosome damage; validation with ionizing radiation, methyl methanesulphonate, ethyl methanesulphonate and benzo[a]pyrene

A variation of the mouse lymphoma (L5178Y TK+/(-)-3.7.2c) assay has been developed using a microtiter cloning technique instead of the standard agar method. The cell line has been used to detect both gene mutations (at the Na+/K+ ATPase and thymidine kinase loci) and chromosome damage (micronucleus induction) in the same experiment. The system was validated using gamma-irradiation (a known clastogen), 2 direct-acting mutagens, ethyl and methyl methanesulphonate and an indirect-acting mutagen, benzo[a]pyrene. Using the assay, 1-methoxy-1,3,5-cycloheptatriene was shown to be a clastogenic mutagen in the presence of S9, since a clear dose-dependent increase in micronuclei was observed, mainly small colony thymidine kinase mutants were observed, and no ouabain-resistant mutants were induced, a profile very similar to gamma-irradiation. The results suggest that metabolic activation potential explains the results in the accompanying paper (Asquith et al., 1990). The implications for mutagenicity testing are discussed.     

Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK^+/+). TK-deficient (TK^?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK^+/? revertants arising in TK^?/? cultures should possess TK enzyme identical with one of those present in the original TK^+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK^+/? lines, each derived in turn from separate TK^?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) K_m′s; and (4) ATP-Mg²⁺ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.     

High-resolution cytogenetic characterization of the L5178Y TK+/- mouse lymphoma cell line

High-resolution chromosome preparations from L5178Y TK+/- 3.7.2C mouse lymphoma cells were obtained using acridine orange in the cell harvest procedure. With this technique it is possible to visualize over 500 bands in elongated mouse lymphoma cell chromosomes as compared to the approximately 230 bands visualized in metaphase preparations. High-resolution lymphoma cell chromosomes are described, and chromosome rearrangements carried in the cell line are characterized by ideograms representing the position, number, size, and relative staining intensity of the G-band patterns. Use of elongated chromosomes of mouse lymphoma TK+/- mutants should facilitate analysis of the cytogenetic effects associated with TK+/- ----TK-/- mutagenesis.     

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.     

Do trifluorothymidine-resistant mutants of L5178Y mouse lymphoma cells re-express thymidine kinase activity following 5-azacytidine treatment?

TFT is an effective selective agent for TK-deficient mutants of L5178Y TK+/- -3.7.2C mouse lymphoma cells. Mutants can be classified by colony size into small colonies (many of which show readily observable chromosome abnormalities associated with chromosome 11--the location of the TK gene) and large colonies (which may represent events affecting only the expression of the TK gene). The precise nature of the induced damage causing the loss of the TK-enzyme activity for both mutant type is not known and is currently under investigation. The hypomethylating agent 5-azacytidine can be utilized to investigate the possibility that mutants might be the result of a suppressed rather than an altered TK gene. Mutant cell lines are treated with 5-azacytidine and then evaluated for re-expression of the TK enzyme as measured by resistance to THMG. In these studies, 11 mutants have been evaluated. None of the 11, including 10 small-colony mutants (6 with chromosome 11 translocations) and 1 large-colony mutant, show a high conversion to TK competency following 5-azacytidine treatment.     

Mutagenicity of actinomycin D in mammalian cells due to clastogenic effects

Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism.     

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.     

Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells

The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.     

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.     

Micronucleus, chromosome aberration, and small-colony TK mutant analysis to quantitate chromosomal damage in L5178Y mouse lymphoma cells

In testing the hypothesis that the small-colony thymidine kinase-deficient mutants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells represent an estimate of the clastogenicity of test chemicals, we have been performing gross aberration analysis. The present study was initiated to determine if the cytokinesis block method of micronucleus analysis could be performed in mouse lymphoma cells and to compare 3 different endpoints of clastogenicity: the number of metaphases with aberrations, number of binucleates with micronuclei, and small-colony TK mutant frequency. In this study, 12 compounds having varying clastogenic potencies were evaluated. As would be expected, the 3 endpoints vary in the relative magnitude of the quantitated response. This difference likely results from the types of clastogenic damage detected by each endpoint. Of the 3 endpoints tested, only the small-colony TK mutant frequency measures events compatible with long-term cell survival.     

Isolation and characterization of mutants at the APRT locus in the L-5178Y TK+/TK- mouse lymphoma cell line

2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.     

Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history

We have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments we show that the major proportion (93%) of small-colony (sigma) mutants studied have chromosome 11 rearrangements (the chromosome containing the thymidine kinase gene) while large-colony (lambda) mutants do not have detectable chromosome rearrangements. In addition, we find among the chromosome abnormalities in sigma mutants a significant proportion (34%) with dicentric chromosomes involving chromosome 11. These potentially unstable chromosome rearrangements may help to explain the karyotypic instability and heterogeneity among chromosome 11 aberrations previously noted in sigma mutants when they are analyzed later in their clonal history.     

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.     

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells

Microtitration methods for assaying cell survival and mutation frequency to ouabain resistance, 6-thioguanine resistance and 1-β-d-arabinofuranosyl cytosine resistance in L5178Y mouse lymphoma cells were compared to the standard agar cloning technique. The two methods gave essentially similar results for untreated cells, and after treatment with ethyl methanesulphonate and 4-nitroquinoline 1-oxide. Potential advantages of the microtitration method as a routine assay system are discussed.     

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.     

Spontaneous Mutation Rates in Mammalian Cells: Effect of Differential Growth Rates and Phenotypic Lag

We calculated a spontaneous rate of 26-37 x 10(-6) mutations per cell division for L5178Y MOLY (mouse lymphoma) cells at the thymidine kinase locus (tk+/(-)----tk-/-) using a procedure that isolated and segregated cells during expression. This rate was 50 times higher than when cells expressed the mutant phenotype in suspension. The higher mutation rates obtained with the in situ procedure suggest that many of the mutants, whether expressed or unexpressed, grew more slowly than wild-type cells prior to selection with trifluorothymidine (TFT), implying that the slow growth phenotype is expressed earlier than the TFTr (TFT-resistant) phenotype. The loss of mutants was not restricted to cells forming small colonies; the mutation rate for cells forming large colonies was more than ten times higher using the in situ procedure. In this new procedure, the cells expressed spontaneous mutations while growing in semisolid medium for up to 3 days without TFT. Mutants were then selected in situ by adding an overlay of TFT and the visible colonies were analyzed after 11 days. Cells with spontaneous mutations at the tk locus required approximately 30 hr for the more rapidly expressing cells with new mutations to be detected. Most of the TFTr colonies selected after 60 hr of growth in semisolid medium represented independent mutations that had accumulated during the first 30 hr.(ABSTRACT TRUNCATED AT 250 WORDS)