John C. Hozier

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.

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.

Chromosome 11 aberrations in small colony L5178Y TKPsu−/− 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.

Cytogenetic distinction between the TK+ and TK− chromosomes in the L5178Y TK+ / − 3.7.2C mouse-lymphoma cell line

We have analyzed the banded metaphase karyotypes of the L5178Y TK+ / − 3.7.2C mouse-lymphoma cell line as well as a class of slow growing (σ) TK+ / − mutants which show chromosome 11 rearrangements. We have discovered a centromeric heteromorphism in the chromosomes 11 (the known location of the thymidine kinase gene in the mouse) which, together with the types of chromosome rearrangements thus far catalogued for TK+ / − → TK− / − mutagenesis, allows us to propose a cytogenetic distinction between the TK-competent (TK+) and TK-deficient (TK−) chromosome.

High resolution G-banded chromosomes of the mouse.

High resolution G-banded mouse chromosomes were prepared using an actinomycin D and acridine orange pretreatment protocol, resulting in late prophase mouse chromosomes which reveal over twice the number of bands as compared with mid metaphase. These elongated chromosomes, described here in detail and used to construct a precise schematic representation of the late prophase banding patterns, should be generally useful in high resolution mouse chromosome analysis.

Methapyrilene is a genotoxic carcinogen: Studies on methapyrilene and pyrilamine in the L5178Y/TK+/− mouse lymphoma assay

Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/-----TK-/- mouse lymphoma assay (MLA) in the presence of aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK-/-) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK-/- mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.

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.

Sequence analysis of tka−-1 and tkb+-1 alleles in L5178Y tk+/− mouse-lymphoma cells and spontaneous tk−/− mutants

The mouse-lymphoma mutagenesis assay detects forward mutations at the heterozygous thymidine kinase (tk-1) locus in L5178Y tk+/- 3.7.2C cells. This assay of genotoxicity is widely used to quantitate the mutagenic potential of a variety of chemical and physical agents. A NcoI heteromorphism between the tka- and tkb+ alleles allows the use of Southern blotting to broadly detect two major categories of mutations. These consist of deletions of the functional allele, characterized by absence of a 6.3-kb tk-hybridizing band, and apparent point mutations, indistinguishable from wild-type on blots. Rarely, Southern blots reveal a partial deletion of tkb. The variety of lesions recorded at the heterozygous tk-1 locus may be representative of events important in mammalian carcinogenesis and may include a greater range of mutagenic events than can be observed at hemizygous test loci. To further assess the ability of the mouse-lymphoma assay to detect a variety of mutations and to allow identification of point mutations, we have sequenced the entire tk-1 coding region from both tka- and tkb+ alleles of L5178Y 3.7.2C mouse-lymphoma cells. Sequences were obtained using PCR amplified double-stranded DNA templates prepared from cytoplasmic RNA from the heterozygous cell line. The two alleles were found to differ by a single TA to GC transversion, altering one amino acid in the deduced amino acid sequence. 4 spontaneous mutants were also sequenced and demonstrated a variety of mutations, including a 6-base pair in-frame deletion, a CG to GC transversion upstream of the start codon, a mutant apparently lacking expression of the tkb allele, and a mutant with apparent wild-type coding sequence for both tka- and tkb+ alleles. The diverse nature of the mutants isolated from L5178Y cells suggests that the mouse-lymphoma mutagenesis assay is capable of detecting a number of mutation types, enhancing the utility of the assay in studying the range of genetic lesions important in human disease. The lesions produced are readily analyzed using a combination of Southern blotting and sequence analysis.

Binding of lectins to mitotic chromosomes and interphase nuclear substructures

Abstract We have investigated binding of the lectins wheat germ agglutin (WGA) and concanavalin A (Con-A) to mitotic chromosomes and interphase nuclear substructures using fluorescein and rhodamineconjugated lectins. Standard cytogenetic preparations of human lymphocyte chromosomes were incubated with WGA. Mitotic chromosomes and interphase nuclei are brightly fluorescent. Con-A did not detectably bind chromosomes prepared in this way. Mitotic chromosomes display a distinctive banding pattern with WGA similar to quinacrine banding. We also studied binding to interphase “nuclear scaffolds”, which are prepared by treating isolated Hela cell nuclei with DNAse l and high salt. WGA binds nuclear scaffolds internally while Con-A binds to the periphery. The specific binding of lectins to chromosomal structures derived from these two different methods indicates that glycosides are true chromosomal components and that glycoproteins may have a role in chromosome organization.

Evidence for chromosomal replicons as units of sister chromatid exchanges.

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 μm with a peak value around 0.3 to 0.6 μm regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 μm was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 μm in size. In addition, we have found that this chromosomal unit of replication or “chromosomal replicon” does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 μm in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.