Mutagenesis vol. 19 no. 6 © UK Environmental Mutagen Society 2004; all rights reserved.
Baseline levels of chromosome instability in the human lymphoblastoid cell TK6
Department of Radiation Oncology, University of Washington, 1959 NE Pacific, Box 356069, Seattle, WA 98195, USA, 1Department of Radiation Oncology, Case Western Reserve University, Cleveland, OH 44106, USA and 2Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
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Abstract |
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Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK–/–), measured in 44 independently isolated clones, was 2.73 ± 0.78 x 10–6/cell, while that for slow growth mutants was 2.39 ± 0.52 x 10–6/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04±0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.
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Introduction |
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There are many different in vitro cell model systems that have been used to study mechanisms underlying mutation formation and to analyze potential genotoxicity of a variety of different agents. One of the more often studied human cell lines is TK6 (Liber and Thilly, 1982
; Penman et al., 1983; Call et al., 1986; Jensen and Thilly, 1986; Penman and Crespi, 1987; Kodama et al., 1989; Kronenberg and Little, 1989a,b; O'Brien et al., 1990; Whaley et al., 1990; Whaley and Little, 1990). TK6 is a human B lymphoblastoid cell line that was isolated from a spontaneous immortalized lymphoblast culture WIL2 in 1968 (Levy et al., 1968). TK6 was isolated from WIL2 through a series of mutagenic and selection procedures to produce a cell line heterozygous for thymidine kinase (TK), thus providing an additional marker for mutation analysis (Liber and Thilly, 1982). TK6 is a relatively stable p53-normal immortal cell line (Xia et al., 1995) and it shows low gene and chromosome mutation frequencies (Liber and Thilly, 1982; Jensen and Thilly, 1986); TK–/– mutant frequencies range from 1–4 x 10–6 mutants/viable cell, while the reported frequency of unstable chromosome aberrations is 0.01/cell. In comparison, the average HPRT mutant frequencies in primary human cells can range from about 1.5 to almost 40 x 10–6 mutants/cell, with an average of between 1 and 10 x 10–6 (Jones et al., 1993; Park et al., 1995). Chromosome aberration frequency is usually reported to be 
0.01/cell in normal primary human cells and, as is the case for TK6 cells, almost all of the aberrations observed are chromatid breaks. Thus it would appear that TK6 cells are as genetically stable for these gene and chromosome mutation end-points as most non-transformed blood cells.
Recent studies of ours have challenged this view of the inherent genetic stability of TK6 cells. While both normal human cells and TK6 cells show low levels of dicentric chromosomes, the frequencies and types of dicentrics in TK6 differ from those reported for primary human cell lines (Tucker et al., 1994). In addition, the characteristics of dicentrics observed in TK6 are unusual in that they represent end-to-end fusions and often show telomere signals at the sites of fusion (Schwartz et al., 2001, 2003), suggesting that telomere biology might be altered in TK6 cells. We previously reported that TK6 cells show delayed elevated frequencies of dicentric chromosomes and non-reciprocal chromosome translocations following radiation exposure or p53 inactivation (Evans et al., 2001, 2002, 2003; Schwartz et al., 2001, 2003). In contrast, prior exposure to radiation or p53 loss has little if any effect on the delayed appearance of TK gene mutations or on the delayed appearance of other types of unstable chromosome aberrations (Evans et al., 2001, 2002, 2003). Our earlier studies determined baseline frequencies of chromosome alterations in a limited number of cells (50/clone). The small number scored limited our ability to make accurate measures of these low aberration frequencies in individual clones. Our previous work also only examined normal growth TK mutant frequencies, not slow growth mutant frequencies (Liber and Denault, 1991), which might be a better marker of instability. In order to understand the nature of spontaneous genome instability in TK6 cells, we expanded our study to score larger numbers of cells for cytogenetic abnormalities and examined slow growth as well as normal growth TK mutants. Our results provide more precise information on the frequencies and types of genetic alterations in TK6 and suggest that TK6 cells differ from normal primary cells in factors that specifically influence end-to-end chromosome fusion and non-reciprocal chromosome translocation frequencies.
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Materials and methods |
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Cell culture
TK6 cells were maintained in suspension at 37°C in an atmosphere of 5% CO2/95% air in RPMI medium containing 10% heat-inactivated horse serum (Sigma Chemical Co., St Louis, MO). The cell density was maintained at 2–10 x 105 cells/ml. As in previous studies (Evans et al., 2001
, 2002, 2003; Schwartz et al., 2001, 2003), we used clones isolated from TK6 in order to detect potentially low frequency events that also might affect cell growth. For clone isolation, cells were plated into 96-well plates (0.2 ml/well) at a cell concentration of 1 cell/ml. Cells were cultured for 10–12 days in standard conditions (37°C, 5% CO2, 100% humidity). Single colonies were isolated and expanded in culture for later analysis. All analyses were done approximately 35 generations after initial plating.
Cytogenetic analysis
For cytogenetic analysis, exponentially growing cells from each clone were incubated starting at a density of 4 x 105 cells/ml at 37°C for 24 h, after which time colcemid was added to a final concentration of 0.2 mM to arrest the cells in metaphase. Cells were harvested 2 h later by centrifugation and slides were prepared from the fixed cell suspensions. Giemsa stained cells (100 metaphases/clone) were analyzed for ploidy changes and for chromatid-type breaks.
A second set of slides from each clone were analyzed for dicentric chromosomes using centromere-specific staining. Slides were treated with 100 µg/ml RNase A (Amresco) at 37°C for 1 h and then with 1 mg/ml pepsin (Sigma) in 0.01 N HCl for 10 min at 37°C. Slides were fixed in 1% formaldehyde in phosphate-buffered saline containing 50 mM MgCl2. After fixation, slides were dehydrated in 70, 80 and 95% ethanol for 2 min each and allowed to dry. After drying, the slides were denatured for 2 min in 70% formamide, 2x SSC at 72°C and then dehydrated in ethanol again. After drying, centromere probes (20 ng/slide) in 70% formamide, 2x SSC were applied to the slide. The slides were coverslipped and incubated at room temperature for 2 h. After incubation, slides were washed in 70% formamide, 2x SSC for 15 min at 29°C and then for 5 min in TNT buffer (0.1 M Tris, pH 8.0, 0.15 M NaCl, 0.02% Tween 20). Excess liquid was removed from the slides and Antifade reagent (Prolong; Molecular Probes, Eugene, OR) containing 1 µg/ml DAPI was added to the slides. The slides were coverslipped and analyzed. Images of dicentric chromosomes were captured using a CCD camera and IPLab software (Scanalytics, Alexandria, VA). Multicentric chromosomes were identified and scored for the presence of more than one clear centromere signal. A minimum of 300 cells were scored for each TK6 clone.
A third set of slides was used to analyze chromosome aberrations involving chromosome 1 or 2. Slides were denatured in 70% formamide, 2x SSC at 72–75°C for 5 min, followed by dehydration in 70, 80 and 95% ethanol (1 min each). Whole chromosome probes (Vysis, Downer's Grove, IL) were denatured at 75°C for 5 min. The slides were placed on a 42°C slide warmer and the denatured probe mixture was applied to the slides. Slides were then coverslipped, sealed with rubber cement and incubated at 37°C overnight in a humidified chamber. The slides were washed in 0.4x SSC, 0.3% Igepal CA630 (Sigma, St Louis, MO) for 2 min at 72–75°C followed by a 1 min wash in 2x SSC, 0.1% Igepal CA630. Slides were then air dried in the dark. After drying completely, the slides were mounted with the same antifade reagent used for the centromere stained slides. Five hundred cells were analyzed for each chromosome probe. Translocations, deletions and numerical changes in chromosome 1 or 2 were scored in cells with at least two clear chromosome signals.
Gene mutation analysis
All analyses were done 35 generations after the initial plating. The method of Furth et al. (1981) was used to determine mutant fraction (MF) and plating efficiency. The method takes into account Poisson distributions of cells/well. Mutation frequencies at the TK locus were determined in the following way. Cells were grown for 2 days in CHAT (10 µM deoxycytidine, 200 µM hypoxanthine, 0.2 µM aminopterin and 17.5 µM thymidine), to remove pre-existing TK–/– mutants from the population, followed by 1 day's growth in standard growth medium plus THC (CHAT without aminopterin) (Liber and Thilly, 1982). After CHAT/THC treatment, cultures were maintained in suspension for 14 days. Four times over this time period aliquots of cells were sampled to assay for MF. The fractions of TK–/– mutants were determined by seeding 20 000 cells/well in the presence of 2.0 µg/ml trifluorothymidine in 96-well plates. Cells from each culture were also plated at the same time at an average of 1 cell/well (5 cells/ml) without trifluorothymidine to determine plating efficiency. All plates were incubated for 10–11 days prior to scoring colonies to determine number of early arising TK–/– mutants. These were defined as normal growth mutants. Rates of accumulation of normal growth mutants can be determined from these data by measuring the slope of mutant frequency versus time. A significant fraction of TK–/– mutants grow slowly, with doubling times of 24–42 h (Liber and Denault, 1991). Therefore, after the initial colony count at 10–11 days, the mutation plates were refed by addition of fresh trifluorothymidine medium to each well and incubated for an additional 7–9 days to observe the appearance of any late appearing slow growth mutants. Slow growth mutant frequencies were calculated from five measurements over 2 weeks, but since slow growing mutants are diluted from the culture due to faster growing wild-type cells, these measurements reflect steady-state frequencies and cannot be used to calculate a rate.
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Results |
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Chromosome alterations
Eleven individual clones were analyzed for cytogenetic changes. Both unstable and stable chromosome aberrations were studied, and the results are summarized in Table I. The most frequent unstable aberration observed was a chromatid break. Chromatid break frequencies ranged from 0 to 18% per clone. Almost all cells with breaks had a single break, with the average frequency of breaks/cell being 0.04 ± 0.01. This result is higher than that reported by Jenson and Thilly (1986)
, but our result reflects in part the ‘outlier’ clone 8-0-2. If the other 10 clones are considered separately, there is no significant difference between our results and theirs.
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Dicentric chromosomes were the only other unstable chromosome aberration observed (Figure 1A). A total of 23 dicentrics were observed in the 3995 cells scored. Dicentric frequencies per cell ranged from 0 to 1.7% per clone, with 7 out of the 11 clones (64%) having cells with dicentrics. The average dicentric frequency/cell was 0.006 ± 0.002, a value 10-fold higher than the 0.0006 reported by Tucker et al. (1994)
. One cell had 2 dicentrics. All the other cells with dicentrics had a single dicentric. The dicentrics were not associated with any accompanying acentric fragment, suggesting that they represented end-to-end joining between telomeres. Previous studies have demonstrated that many of these dicentrics contain telomere signals at their junctions (Schwartz et al., 2003). All of the dicentrics were found in diploid cells. There was no apparent correlation between chromatid break frequency and dicentric frequency per cell, although the average chromatid break frequency per clone was significantly higher (P = 0.04) in the three clones with dicentric frequencies more than one standard deviation higher than the mean.
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There were a variety of different stable chromosome alterations observed, although all at very low frequencies. The most frequent alteration observed was polyploidy. Eight out of the 11 clones had polyploid cells. The percentage of polyploid cells per clone ranged from 0 to 2%. The average was 0.9 ± 0.2%. Two of the polyploid cells were triploid, while 31 were tetraploid. There was one cell with a chromosome number greater than 4n. There was no apparent relationship between ploidy and either chromosome breaks or dicentric chromosomes. This contrasts with studies on p53-deficient lymphoblastoid cells of similar origin to TK6 (Schwartz et al., 2001
). There were also changes in the numbers of chromosomes 1 and 2 that were independent of ploidy variations. About 0.5% of all the cells scored had additional chromosomes 1 or 2. There was no relationship between chromosome 1 and chromosome 2 aneuploidy. Clones with higher levels of chromosome 1 aneuploidy did not show a corresponding increase in chromosome 2 aneuploidy. Neither was there a relationship between dicentric frequencies and either chromosome 1 or chromosome 2 aneuploidy. Stable chromosome translocations were rare. Only three reciprocal translocations (Figure 1B) involving chromosome 1 out of 5500 cells scored were observed. No chromosome 2 reciprocal translocations were observed. Non-reciprocal translocations (NRT) (Figure 1C and D) were more common. Four NRT involving chromosome 1 and four NRT involving chromosome 2 were observed. All the NRTs were observed in clones containing dicentric chromosomes, but there was no obvious relationship between dicentric frequency and NRT frequency per cell. In most of the cells containing NRTs the NRT was associated with a deletion in the chromosome from which the translocated piece was derived (Figure 1C). In at least one cell the apparent NRT was associated with a duplication in the chromosome from which the translocation was derived (Figure 1D). The NRT in this cell was also distinguished from the other NRTs by its much larger length. Seven obvious deletions of part of either chromosome 1 or chromosome 2 were observed. One cell had a chromosome 1 insertion.
Plating efficiencies and TK–/– mutant frequencies
Plating efficiencies and the frequency of trifluorothymidine-resistant mutants were determined for 44 independent clones of TK6 (Figure 2). Plating efficiencies ranged from 0.03 to >1.00. The values >1.0 are likely due to cell clumping. The mean ± SEM plating efficiency for this group of clones was 0.64 ± 0.02. Normal growth mutant frequencies ranged from 0 to >15 x 10–6/viable cell (mean 2.73 ± 0.78 x 10–6/viable cell), while slow growth mutant frequencies ranged from 0 to 15 x 10–6/viable cell (mean 2.39 ± 0.52 x 10–6/viable cell). Estimates for the rate of mutation formation were made by making multiple analyses of mutant frequency over time (five measurements over 2 weeks). The results (Table I) show little or no change in normal growth mutant frequency with time in culture. As mentioned in Materials and methods, slow growing mutants are diluted from the culture due to faster growing wild-type cells. Therefore, slow growth mutant data cannot be used to calculate a rate.
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Nine of the 11 clones analyzed for cytogenetic alterations were also studied for plating efficiency and mutant frequencies (Table I). There were no obvious differences in plating efficiencies, TK–/– mutant frequencies or rates of mutation when this group was compared with the larger group of 44 clones. There was also no obvious relationship between mutant frequencies and any of the other cytogenetic alterations observed in these clones.
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Discussion |
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These results confirm our previous observations of elevated baseline frequencies of dicentrics in TK6 cells (Evans et al., 2001
, 2002, 2003; Schwartz et al., 2001, 2003). While the frequency (0.006/cell) is low, it is nonetheless 10-fold higher than that reported by Tucker et al. (1994) in their study of human peripheral blood lymphocytes (T cells). The difference between TK6 cells and normal primary human cells is likely larger than 10-fold (Tucker et al., 1994), because in the Tucker study most of the dicentrics were observed in older individuals. Thus they might be due to some undocumented prior exposure to a clastogen during the individual's lifetime or some age-dependent change in these human peripheral blood T lymphocytes. Given the long life of some T lymphocyte subsets and the fact that they are in G0 for most of their lifespan (Michie et al., 1992), damaged cells might be expected to accumulate in vivo. Our use of actively dividing clones from an immortal cell line analyzed less than 40 generations after plating individual cells makes it unlikely that the dicentric frequencies we observed reflect normal age-dependent accrual of damage. Moreover, as the dicentrics in TK6 cells involve end-to-end fusions at sites of telomeres (Schwartz et al., 2001, 2003), the origin for these dicentrics is probably different from the normal break and recombination processes that underlie most commonly observed dicentrics (Cornforth, 1989). Further evidence for the unusual nature of TK6 cells comes from the analysis of stable chromosome translocations. The total frequency of translocations (0.007 translocations/cell) is similar to the results of Tucker et al. (1994), but balanced translocations predominate in human T lymphocytes (Johnson et al., 1998), while we observe primarily NRTs.
Our observations concerning chromosome instability in TK6 cells are consistent with other reports of karyotypic instability in TK6 cells (Yandell and Little, 1986; Kodama et al., 1989). In these studies the authors report evidence for karyotype evolution and low levels of chromosome duplications and translocations similar to what we observed in our study. Such changes are apparently not unusual for lymphoblastoid cell lines (Abruzzo et al., 1986).
Any small-scale genetic alterations associated with either dicentric or NRT formation probably involve only those sequences at or very near telomere sequences, as there was no evidence for any increased frequency of TK gene mutations in TK6 cells. The processing of DNA double-strand breaks, such as those that must result from the resolution of dicentric chromosomes, is usually associated with deletions and insertions (Jasin, 2000; Allen et al., 2003). Further analysis of genetic elements near telomeres could provide information on whether such sites are susceptible to elevated mutation rates in TK6 cells. The low gene mutation frequencies in TK6 cells contrasts with other leukemic and lymphoma cell lines, which have been reported to have much higher mutant frequencies (Seshadri et al., 1987).
Understanding long-term effects of mutagen exposure is complicated by the phenomenon of induced genome instability (Kronenberg, 1994; Kadhim et al., 1995; Morgan et al., 1996; Wright, 1997; Mothersill and Seymour, 1998; Loeb and Loeb, 2000). Exposure to different agents can lead to delayed appearance of gene and chromosome mutations. The basis for the phenomenon of induced instability is under intense study. Some have proposed that instability develops as a result of chromosome bridge breakage and fusion cycles, persistent DNA damage or through cycles of apoptosis that lead to the release of reactive oxygen species or other types of DNA-damaging agents (Kronenberg, 1994; Kadhim et al., 1995; Morgan et al., 1996; Wright, 1997; Mothersill and Seymour, 1998; Limoli et al., 1999, 2000, 2001; Loeb and Loeb, 2000; Grosovsky et al., 2001). Our own work suggests that instability can develop when normal cell cycle checkpoint responses are altered (Kaufmann et al., 1997; Schwartz et al., 2001, 2003). Under these circumstances the induction of genome instability could reflect a reduced ability to eliminate cells expressing high levels of DNA alterations, including those DNA alterations that develop spontaneously.
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Acknowledgments |
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The research described herein was jointly supported by the National Aeronautics and Space Administration and the National Cancer Institute through research grant CA 73931 (H.H.E.) and by the Low Dose Radiation Research Program, Biological and Environmental Research (B.E.R.) and US Department of Energy grant no. DE-FG03-02ER63365 (H.L.).
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3 To whom correspondence should be addressed. Tel: +1 206 598 4091; Fax: +1 206 598 6473; Email: jschwart{at}u.washington.edu
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Received on August 25, 2004; revised on October 10, 2004; accepted on October 11, 2004.
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