Mutagenesis vol. 19 no. 4 pp. 263-268,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Loss of P53 heterozygosity is not responsible for the small colony thymidine kinase mutant phenotype in L5178Y mouse lymphoma cells
University of North Carolina Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, USA, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA, 2Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA and 3Toxicology and Molecular Biology Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA
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The mouse lymphoma L5178Y Tk+/– 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk–/– mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/– cells or Tk–/– mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/– 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The L5178Y/Tk+/– 3.7.2C mouse lymphoma assay (MLA) is a well-characterized in vitro system used for the study of somatic cell mutation. This assay detects forward mutation of the wild-type thymidine kinase (Tk1) allele located on chromosome 11 such that mutant cells can form colonies in the presence of the toxic pyrimidine analog trifluorothymidine (TFT). TFT-resistant colonies have a bimodal size distribution (Clive et al., 1979
; Clive and Moore-Brown, 1979; Moore et al., 1985a) and the frequency of both sizes of colonies (large and small) can be increased after exposure of Tk1+/– cells to mutagenic chemicals (Moore et al., 1985a). Tk mutants differ in their growth kinetics; immediately after isolation of the mutant clone, cells from the small colony mutants grow slowly while cells from large colony mutants grow at normal rates (Moore et al., 1985b).
Because small colony but not large colony Tk mutants grow slowly, one must assume that they result from genetic events affecting more than just the Tk1+ allele. As we published earlier (Moore and Doerr, 1990), small colony mutants would be expected to fall into at least one of three general types: (i) a multi-locus deletion affecting the Tk1+ allele and other linked loci, the alteration of which affects growth rate; (ii) multiple mutations, one affecting the Tk1+ allele and one or more multi-locus events not involving the Tk1+ allele and resulting in slow growth; (iii) multiple point mutations affecting both the Tk1+ allele and another allele(s) causing a mutation(s) that makes the cell grow slowly. For mutants falling into the first class, it has generally been assumed that there must be at least one gene (an ‘essential’ gene), located somewhere on chromosome 11, that is required for normal growth. The simultaneous deletion (or loss due to mitotic recombination) of Tk1+ and this ‘essential’ gene would make the cells grow slowly. So far, this ‘essential’ gene (or genes) has not been identified.
In 1997, Mitchell proposed a new hypothesis for the bimodal size distribution of mutant colonies. The hypothesis was formulated based on evidence (Storer et al., 1997) that mouse lymphoma L5178Y/Tk+/– 3.7.2C cells were heterozygous for P53 (properly referred to, in the mouse, as Trp53) gene, which is also located on chromosome 11. Mitchell postulated (i) that the Trp53– allele is located on chromosome 11b (which also harbors the Tk1+ allele) in 3.7.2C mouse lymphoma cells, (ii) that large colony mutants retain the dominant negative Trp53 heterozygous genotype and (iii) that small colony mutants have undergone loss of heterozygosity at both Tk1 and Trp53. In her hypothesis, she postulated that three possible genotypes could result in small colony mutants: Trp53+/+/Tk–/–, Trp53+/+/Tk–/0 and Trp53+/0/Tk–/0. Assuming that the cells are heterozygous for Trp53, induction of the first two genotypes requires that both a mutant Trp53 allele revert to a functional wild-type allele and the functional Tk1 allele mutate to a non-functional allele (either by an intragenic mutation or deletion of the allele) (Mitchell, 1997). While this might occur with some very potent mutagens, it is generally unlikely that both the Tk1 and Trp53 genes would simultaneously mutate within a single cell. To explain the third genotype (Trp53+/0/Tk1–/0), Mitchell proposed that many terminal deletions result in simultaneous elimination of the Trp53– allele and the Tk1+ allele. She argued this possibility by citing Blazak et al. (1989), who cytogenetically examined both large and small colony mutants. Mitchell inferred from those data that the observed aberrations affecting chromosome 11b included deletion of the region containing both Trp53 and Tk1+. Blazak observed that cells that contained chromosome 11b damage to the distal region showed the translocation of whole or parts of other chromosomes to this region and that the amount of the translocated chromatid remained constant from cell to cell (Blazak et al., 1989).
Subsequent to the publication of Storer et al. (1997), we conducted additional analysis of the Trp53 alleles in a number of different L5178Y mouse lymphoma cell sublines (including 3.7.2C) and determined that these cells are not heterozygous for Trp53. In fact, 3.7.2C cells have no wild-type Trp53 alleles (Clark et al., 1998; Hess et al., 2003). Because we sequenced exons 4–8 while Storer only sequenced exons 5–9, we were able to identify that the allele thought to be wild-type by Storer et al. was actually mutated. As reported by Hess et al. (2003), all of the L5178Y sublines that we have subsequently analyzed show a missense mutation in exon 5 on chromosome 11a causing an amino acid change from a cysteine to an arginine (seen also by Storer et al., 1997) and a nonsense mutation in exon 4 changing the codon encoding a glutamine to a stop codon (Clark et al., 1998) on chromosome 11b.
The fact that the cells are actually Trp53–/– negates the possibility that small colonies result because the cells mutate to be either Trp53+/0 or Trp53+/+. However, we were interested in the possibility that the Trp53 gene might still be involved in the small colony phenotype. We wanted to determine if, in fact, any of the terminal deletions were large enough (as proposed by Mitchell, 1997) to extend into or past the Trp53 gene. Therefore, we analyzed a number of small colony mutants by Southern blot analysis to determine whether the Tk1+ allele had been deleted. Mutants showing deletions of the Tk1 gene were then evaluated for the presence or absence of a series of microsatellite markers. These markers map to locations along the entire length of chromosome 11 (see Figure 1) and are heterozygous in Tk+/– 3.7.2C cells (Liechty et al., 1998).
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Materials and methods |
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Growth of cell clones
Thirty-one small colony mutants that had been frozen and stored in liquid nitrogen were quickly thawed, cultured according to the procedures of Turner et al. (1984
) and expanded to 50–100 x 106 cells. The small colony mutants 925–975 were generated in a previous experiment in which mouse lymphoma L5178Y/Tk+/– 3.7.2C cells were exposed to 0.6–8.0 µg/ml bleomycin sulfate. Small colony mutants 540 and a subclone of 540 (540F) were obtained from cells exposed to pyrimethamine (Hozier et al., 1981). Mutant G4 is a small colony mutant obtained after exposure to 
-radiation and has been characterized previously as having two copies of the chromosome 11 carrying the Tk1– allele and no copies of the chromosome 11 carrying the Tk1+ allele (Applegate and Hozier, 1989; Applegate et al., 1990), i.e. it appears to have resulted from a non-disjunction event. In addition, cells from a growing Tk+/– 3.7.2C mouse lymphoma culture were also expanded for molecular characterization.
DNA preparation
DNA was prepared using standard proteinase plus detergent digestion followed by phenol:chloroform extraction and dialysis. Briefly, 50 x 106 cells were resuspended in TEN (10 mM Tris–HCl, pH 7.8, 25 mM EDTA, 150 mM NaCl) (Sigma, St Louis, MO). An equal volume of 3% sarcosyl, 50 mM EDTA, pH 8 (Sigma) and 1 mg/ml proteinase K (Gibco BRL, Grand Island, NY) was added to the sample, which was incubated overnight at 55°C. The following day, the sample was sheared through a 21 gauge needle five times before addition of RNase (Sigma) to 150 µg/ml and incubated for 1 h at 55°C. The DNA solution was then extracted three times with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1) (Amresco, Solon, OH) each for 1 h at room temperature. The aqueous layer of the final extraction was then dialyzed against TE (10 mM Tris–HCl, pH 8, 1 mM EDTA) (Sigma). The concentration of DNA was determined by spectrophotometry and the 260 nm/280 nm ratio for all samples was between 1.7 and 2.0.
Microsatellite PCR amplification
Primers for amplification of microsatellite sequences were purchased from Applied Genetics Laboratory (Melbourne, FL). Microsatellite sequences were chosen so that the distal (D11MIT62), mid (D11MIT4) and proximal (D11MIT59, D11MIT128 and D11MIT42) regions of mouse chromosome 11 were represented. Microsatellite sequences were amplified from 20 µl reactions comprising 400 ng DNA, 200 µM each dNTP (Gibco BRL), 0.1 µM each primer and 0.5 U Taq DNA polymerase (Perkin Elmer). Reactions were performed according to the following program: a hot start at 80°C for 3 min, followed by 30 cycles of 94°C denaturation for 1 min, 55°C for 1 min and 72°C for 1 min, followed by a 72°C extension time for 10 min. Amplified PCR products were electrophoresed on a 3% Metaphor gel (FMC Bioproducts, Rockland, ME), stained with 0.5 µg/ml ethidium bromide and visualized on a UV transilluminator.
Probe preparation
Two LB agar stab bacterial artificial chromosome (BAC) cultures were purchased from BACPAC resources (Children’s Hospital, Oakland, CA). BAC RP23-5O23 contains the Trp53 gene, while BAC RP23-101J23 contains the Tk1 gene. First, the clones were streaked on LB agar plates containing 20 µg/ml chloramphenicol. After incubation at 37°C for 2 days, a single colony was picked and put into a 10 ml starter culture which also contained 20 µg/ml chloramphenicol. After incubation at 37°C with vigorous shaking (250 r.p.m.) for 2 days, the culture was then transferred to a 1 l LB broth culture containing 20 µg/ml chloramphenicol and cultured at least overnight until the bacteria density was high enough for harvesting. Plasmid isolation kits (Qiagen, Valencia, CA) were used for DNA isolation and their protocol was followed. The nick translation kits were purchased from Vysis (Downers Grove, IL) and their protocol was followed. Briefly, 1–3 µg DNA, 2.5 µl of 0.2 mM Spectrum Green dUTP, 5 µl of 0.1 mM dTTP, 10 µl of dNTP mix, 5 µl of 10x nick translation buffer and 10 µl of nick translation enzyme were mixed in a microfuge tube and incubated at 15°C for 8 h, then the reaction was stopped by heating to 70°C for 10 min. Then the nick translation product (10–30 µl), 3 µl of COT-1 DNA, 0.5 µl of salmon sperm DNA, 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol were mixed thoroughly and incubated on dry ice for 15– 30 min. After incubation, the mixture was centrifuged (12 000 r.p.m.) at 4°C for 30 min, then the supernatant was discarded and the pellet was dried in a vacuum centrifuge for 5–8 min. Then 3 µl of nuclease-free water and 7 µl of hybridization buffer were added and the probes were stored at –20°C at least overnight before hybridization.
Chromosome metaphase preparation
L5178Y/Tk+/– 3.7.2C mouse lymphoma cells were cultured in Fischer’s medium with glutamine (Quality Biologicals, Gaithersburg, MD) and 10% horse serum (Invitrogen, Carlsbad, CA) at 37°C, 5% CO2. When the cell density reached 1 x 106/ml, colcemid (final concentration 0.05 µg/ml) was added to block the cells in metaphase. After incubation for 30 min, cells were washed and then incubated with 19:1 0.075 M KCl:1% sodium citrate (v/v) for 20 min at 37°C. The cells were fixed using 3:1 methanol:acetic acid (v/v) and chromosome metaphase spreads were made.
Fluoresence in situ hybridization (FISH)
Whole chromosome paint for mouse chromosome 11 was purchased from Cambio (Cambridge, UK) and their protocol was followed. For hybridization with the point probes, the following procedures were performed. Briefly, slides were denatured in 70% formamide/2x SSC at 78°C for 7 min and then dehydrated in an ethanol series (70, 80 and 100%) for 2 min each. After dehydration, 11 µl of probe mix was immediately applied to the hybridization area and the coverslips were sealed with rubber cement. After incubation at 37°C for 72 h, the slides were washed three times in 50% formamide/2x SSC at 45°C for 5 min each, then three times in 0.1x SSC at 60°C for 5 min each. The slides were air dried and then Anti-fade/4',6-diamidino-2-phenylindole mixture (DAPI) (Vector Laboratories, Burlingame, CA) was applied. The slides were observed under a Zeiss Axioplan 2 microscope and photographed using Applied Imaging Cytovision Genus version 2.7 software. At least 20 metaphase spreads of good hybridization quality were imaged to evaluate the position of each locus-specific and the whole chromosome probe.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Small colony mutant induction is associated with chemicals that have clastogenic activity, while large colony mutant induction occurs with exposure to chemicals that cause point mutations (Hozier et al., 1981,1985
; Doerr et al., 1989; Applegate et al., 1990; Moore and Doerr, 1990). It should be noted that many chemicals induce both point mutations and multiple locus lesions and increase the frequency of both small and large colony mutants (Moore et al., 1985a, 1989). Extensive research has been performed to characterize the two types of mutant colonies (Hozier et al., 1981, 1982, 1985, 1992; Moore et al., 1985b; Blazak et al., 1989; Applegate et al., 1990). The elucidation of mutational mechanisms has been facilitated because the cell line carries two chromosomes 11, with distinctly different centromeres (Hozier et al., 1982). The Tk1+ allele is located on the distal end of the large centromere chromosome 11 (11b) while the Tk1– allele maps to the distal end of the small centromere chromosome 11 (11a) (Hozier et al., 1991). Southern blot analysis has also revealed a NcoI restriction fragment polymorphism in the Tk1 alleles of 3.7.2C cells (Applegate et al., 1990). In mutants in which the inactivated Tk1+ allele is not deleted, a 6.3 kb restriction fragment is present on the Southern blots. These mutants are assumed to have a point mutation in the Tk1+ allele. Those mutants that had lost the 6.3 kb restriction fragment had lost the Tk1+ allele (see Figure 2).
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Thirty-one small colony mutants resulting from treatment with bleomycin or
-irradiation were expanded and characterized molecularly. Restriction digestion with NcoI followed by Southern blot analysis of the bleomycin- and radiation-treated cells revealed that most small colony mutants lacked the 6.3 kb fragment (Figure 2 and Table I). Mutant 926 (Figure 2) is an exception in that it retained the 6.3 kb fragment. It was not further analyzed for polymorphic markers.
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Because most small colony mutants lack the 6.3 kb fragment, we can speculate that they represent large-scale DNA deletions which include and may extend beyond the Tk locus. To determine the extent of deletion in these mutants, genomic DNA was extracted and amplified using primers specific for polymorphic microsatellite sequences proximal (D11MIT62), mid (D11MIT4) and distal (D11MIT59, D11MIT128 and D11MIT42) of chromosome 11. Of the 31 mutants, three (928, 939 and G4) were homozygous for all chromosome 11 microsatellite sequences examined, indicating a loss of heterozygosity at each of these loci and possibly the entire chromosome homolog. G4 has previously been analyzed and determined by banded karyotype analysis to have two copies of chromosome 11a, i.e. these three mutants likely result from non-disjunction.
Further analysis of the bleomycin-induced small colony mutants revealed that most of them exhibited terminal deletions. One mutant (950) lacked polymorphisms at loci initiating from D11MIT59 (
64 cM) and extending distal to the end of chromosome 11, a deletion of 20% of the chromosome. Fifteen of the small colony mutants demonstrated homozygosity beginning at D11MIT128 (68 cM) and extending to the end of chromosome 11, a deletion of 15% of the chromosome. Eight mutants retained all of the microsatellite bands. Of note, mutants 930 and 964 lacked a polymorphism at D11MIT128 but retained the expected polymorphic patterns at all other microsatellite sequences examined. The lack of a polymorphism at just one microsatellite sequence may indicate deletion of that sequence, as well as loss of the Tk locus, indicating a complex rearrangement, or the conversion of one sequence to the other, resulting in homozygosity at this locus. Further analysis is required to determine the complexity of these mutants.
With the exception of mutants 928, 939 and G4, which demonstrated loss of heterozygosity at all microsatellite sequences tested, none of the small colony mutants analyzed by amplification of the microsatellite sequences possessed deletions that extended to the region of the chromosome where Trp53 is located (39 cM).
From our studies it is clear that Mitchell’s hypothesis is incorrect. In addition, we should note that in her paper Mitchell indicated an incorrect location for Trp53 on chromosome 11. Although Mitchell stated that the Trp53 gene is 35 cM from the Tk1 locus, she positioned the Trp53 locus at the distal end of chromosome 11, between the D and E regions. This is not the correct position. In the mouse the Trp53 locus is located at 39 cM on chromosome 11. This places Trp53 near the middle of chromosome 11, in the B region, nearly half a chromosome away from the Tk1 locus (Buchberg et al., 1989). Our in situ hybridization analysis (Figure 3) confirmed that the Trp53 gene in L5178Y mouse lymphoma cells is, in fact, in the middle of chromosome 11. Placing the Trp53 gene in the correct position shows that only a very large deletion could eliminate both the Tk1 and Trp53 genes. We found no such mutants. In the present set of mutants, no small colony mutant showed a deletion that extended to or beyond the Trp53 locus unless loss of heterozygosity was observed at all microsatellite sequences. We assume that these mutants are like G4 and have two copies of chromosome 11a and, therefore, result from non-disjunction rather than from deletion.
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From these analyses it appears that deletions >20% or so of chromosome 11 do not occur, may be rare or, alternatively, may not be compatible with cell survival and thus are not recovered in the assay. It is clear that newly induced mutations (deletions) of the Trp53 gene are not involved in the small colony phenotype. It is more likely that a locus closer than Trp53 to the Tk1 locus is responsible for the slow growth characteristics in those mutants resulting from large-scale chromosomal deletions.
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Acknowledgement |
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This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and approved for publication. Approval does not signify that the content necessarily reflects the views and policies of the Agency nor does mention of trade names of commercial products constitute endorsement or recommendation for use.
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Notes |
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4To whom correspondence should be addressed at: Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, HFT 120, 3900 NCTR Road, Jefferson, AR 72079, USA. Tel: +1 870 543 7050; Fax: +1 870 543 7393; Email: mmmoore{at}nctr.fda.gov
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References |
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