Mutagenesis vol. 18 no. 6 pp. 521-525,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Brca2 (XRCC11) deficiency results in enhanced mutagenesis
gorzata Z. Zdzienicka1,2,31Department of Toxicogenetics – MGC, Leiden University Medical Center, Leiden, The Netherlands and 2Department of Molecular Cell Genetics, the Ludwik Rydygier University of Medical Sciences, Bydgoszcz, Poland
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Abstract |
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Brca2 deficiency is associated with chromosomal instability and an increased risk of breast and other cancers. To examine the effect of Brca2 deficiency on mutagenesis, we measured the spontaneous mutation rate at the endogenous hprt gene in the Brca2-deficient Chinese hamster cell mutant V-C8. A 4.3-fold increase was found in the spontaneous mutation rate at this locus, indicating the importance of Brca2 in the prevention of mutagenesis. In addition, following exposure to IR, a 2.3-fold increase in mutant frequency per Gy was found for V-C8 in comparison with wild-type V79. These data suggest a potential risk from ionizing radiation for BRCA2 patients.
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Introduction |
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Human tumours contain numerous mutations by the time they are clinically detectable. The spontaneous background mutation rate is thought to be insufficient to account for this number of mutations. It has, therefore, been hypothesized that an early event in tumorigenesis is the development of a mutator phenotype: an increase in mutation rate caused by, for example, chromosomal instability (Lengauer et al., 1998
; Loeb, 2001).
A gene that has been found to play an important role in maintaining chromosomal integrity is the BRCA2 gene. Germline mutations in BRCA2 are associated with about one-third of breast cancer families and are known to considerably increase the risk of breast and ovarian cancer, as well as prostate, pancreas and male breast cancer (The Breast Cancer Linkage Consortium, 1999). The BRCA2 gene encodes a large protein of 3418 amino acids (Tavtigian et al., 1996) that is involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) (for reviews see Powell et al., 2002; Venkitaraman, 2002). Brca2 is able to interact with the central protein of HR, Rad51, via its so-called BRC repeats (Wong et al., 1997). The Rad51 protein is a homologue of the Escherichia coli RecA protein and facilitates strand invasion of the broken DNA into a homologous double-stranded DNA molecule by forming nucleoprotein filaments (reviewed in Thompson and Schild, 2001). Recent work has suggested that both the BRC repeats of Brca2, as well as the C-terminus, are involved in the orderly assembly of the Rad51 nucleoprotein filament, thereby regulating Rad51 function in HR-based repair (Pellegrini et al., 2002; Yang et al., 2002). Consequently, Brca2 deficiency leads to impaired DSB repair by HR (Moynahan et al., 2001; Tutt et al., 2001; Xia et al., 2001). Instead, DSBs were found to be repaired via an erroneous single-strand annealing pathway, leading to deletions (Larminat et al., 2002; Tutt et al., 2001). Besides hypersensitivity to agents that induce DSBs, such as ionizing radiation (IR) (Sharan et al., 1997; Abbott et al., 1998), sensitivity to DNA cross-linking agents has also been reported for Brca2-deficient cells (Yu et al., 2000; Kraakman-van der Zwet et al., 2002). The repair of DNA interstrand cross-links is thought to involve the HR machinery and requires the Fanconi anaemia (FA) proteins (Dronkert and Kanaar, 2001). FA is a heterogeneous, recessively inherited cancer susceptibility disorder and at least eight genes are involved: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF and FANCG (reviewed in Grompe and D’Andrea, 2001). FA patients are especially prone to develop acute myeloid leukaemia, but squamous cell carcinomas are also common (Alter, 1996). Recently, germline mutations in the BRCA2 gene have been found to be responsible for complementation group D1 of FA (FA-D1) (Howlett et al., 2002). The identification of the FANCD1 gene as BRCA2 suggests that BRCA2 might have a bridge function between the FA proteins and the HR machinery, but this remains to be clarified.
Brca2-deficient cells show a high frequency of spontaneous chromosomal aberrations (CA) (Tutt et al., 1999; Yu et al., 2000; Kraakman-van der Zwet et al., 2002). This is already suggestive of a higher spontaneous mutation rate, although the cytogenetic analysis to quantify CA does not take into account the viability and growth capability of the aberrant cells. Recently, using a LacZ plasmid transgene system in mice, it has been demonstrated that Brca2-deficient mouse embryos indeed acquire spontaneous mutations at a higher rate, which was exacerbated by exposure to IR (Tutt et al., 2002). We have recently shown that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11, which is also hypersensitive to cross-linking agents, is deficient in Brca2 (Kraakman-van der Zwet et al., 2002). Here we have used the endogenous hypoxanthine phosphoribosyltransferase (hprt) gene as a target to analyse the effect of Brca2 deficiency in V-C8 cells on spontaneous mutagenesis. We found that both the spontaneous mutation rate at the hprt locus as well as the IR-induced mutant frequency were increased in V-C8 cells in comparison with wild-type parental V79 cells, in agreement with the results published by Tutt et al. (2002).
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Materials and methods |
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Cells and culture conditions
The V-C8 mutant cell line derived from Chinese hamster V79 cells has been described previously (Zdzienicka and Simons, 1987
; Overkamp et al., 1993; Verhaegh et al., 1995; Kraakman-van der Zwet et al., 2002). Cells were routinely cultured in 10 cm dishes (Greiner) in Ham’s F10 medium (Life Technologies) supplemented with 15% newborn calf serum (Life Technologies), 100 U/ml penicillin and 1 mg/ml streptomycin, without hypoxanthine or thymidine. Cells were maintained at 37°C in a 5% CO2 atmosphere, humidified to 95–100%.
Measurement of the spontaneous hprt mutation rate
Cultures of V79 and V-C8 cells in exponential growth were trypsinized and 8 cultures for V-C8 and 12 cultures for V79 were propagated from inocula of 500 and 100 cells, respectively. The cells were grown on 15 cm dishes to a population of >107 cells, after which the frequency of hprt mutations was determined in each cell culture and the remainder of the cells propagated further.
To determine the frequency of hprt mutants in each culture, 105 cells were seeded per 10 cm dish (20 dishes in total) in medium containing 5 µg/ml 6-thioguanine (6-TG). In addition, 300 and 500 cells for V79 and V-C8, respectively, were seeded per 10 cm dish (5 dishes in total per culture) to determine the cloning efficiency. After 8–12 days, the cells were rinsed with 0.9% NaCl and stained with 0.25% methylene blue and colonies were counted. The mutant frequencies were calculated by correcting the observed frequencies for the corresponding cloning efficiencies:
mutant frequency = total 6-TG-resistant colonies/(total seeded cells x cloning efficiency)
The remaining cells of each culture were propagated further in such a way that they never reached confluence, to keep the cells in logarithmic growth phase. Each time the cells were trypsinized, 2 x 107 cells of each culture were propagated further. After four rounds of propagation the frequency of hprt mutants was assessed again as described above. For determination of the mutation rate, the generation increase in the V-C8 and V79 populations was calculated as follows:
total population expansion = 2(number of generations)
number of generations = log(total population expansion)/log(2)
For this, we assumed that the number of generations equals the number of population doublings. The mutation rate per cell per generation was calculated by dividing the increase in mutant frequency by the increase in generations.
IR-induced mutation frequency at the hprt locus
For the determination of X-ray-induced mutability, 1 x 106 cells were grown to a population of 
2 x 107 cells for each experiment. Cells were either mock treated or irradiated with 1, 2, 3 or 4 Gy for V-C8 cells or with 2, 4 or 6 Gy for V79 cells (dose rate 2.8 Gy/min, 200 kV, 4 mA, 0.78 mm Al). Following (mock) irradiation, cells were: (i) seeded at 300 cells/10 cm dish (5 dishes/dose) to determine clonogenic survival; (ii) propagated at 5 x 105 cells (1 and 2 Gy) or 106 cells/15 cm dish (3 and 4 Gy) for V-C8 cells and 3.5 x 105 cells (2 and 4 Gy) or 106 cells/15 cm dish (6 Gy) for V79 cells (3 dishes/dose) for expression of induced mutations. After 4 days, cells were trypsinized, mixed per group and seeded again at a density of 3.5 x 105 (V79) or 5 x 105 cells (V-C8) per 15 cm dish (3 dishes/group). After an additional 4 days, the cells were seeded for selection of hprt mutants in medium containing 5 µg/ml 6-TG (105 cells/10 cm dish, 10 dishes/group) and in normal medium to determine the cloning efficiency (300 cells/10 cm dish, 5 dishes/group). After 8–12 days, the cells were rinsed with 0.9% NaCl and stained with 0.25% methylene blue and colonies were counted. The mutant frequencies at each dose of X-rays were calculated by correcting the observed frequencies for the corresponding cloning efficiencies, as described above. The induced mutant frequencies at each dose of X-rays were determined by subtraction of the background frequency found without irradiation. Five independent experiments were performed.
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Results |
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Mutation rate in V-C8 cells
To examine the effect of Brca2 deficiency on spontaneous mutagenesis, we used Brca2-deficient hamster V-C8 cells to measure the spontaneous mutation rate at the endogenous hprt locus. Several independent, large populations of V-C8 mutant cells and parental wild-type V79 cells were grown. The mutant frequency of each culture was measured at the beginning of the culture and after four rounds of subculturing (on average 10.2 or 10.6 generations later, for V-C8 and V79 cells, respectively). To determine the number of hprt mutants, cells were plated in 6-TG-containing medium. The mutation rates were calculated as described in Materials and methods. Data presented in Table I show that the spontaneous mutation rate at the hprt gene for V-C8 cells was 26 ± 13 x 10–7 mutations/cell/generation, versus 6 ± 3 x 10–7 for V79 cells. Thus, the rate of spontaneous mutations at this locus was 4.3-fold higher in V-C8 cells and this difference was statistically significant (P < 0.001, Wilcoxon test). These results indicate that a deficiency of the Brca2 protein indeed leads to an increase in the rate of spontaneous mutation formation.
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For the calculation of mutation rates, it was assumed that the number of generations equalled that of population doublings. However, it is not unlikely that, because of chromosomal instability, more cell death occurs for V-C8 than for V79 cells. The doubling time for V-C8 was 34 h, while that for V79 was 18 h. This longer doubling time for V-C8 could very well be due to cell loss. If we assume that the generation time of V-C8 cells would actually be the same as for V79, i.e. 18 h, the number of generations for V-C8 would be 19 instead of 10.2. Then the mutation rate would be lower: 14 ± 7 x 10–7 mutations/cell/generation. However, this is still significantly higher than (twice) that of V79 cells (P < 0.022, Wilcoxon test). Thus, these recalculations show that, considering possible cell death, Brca2 deficiency indeed causes an increase in spontaneous mutation rate.
The molecular natures of the spontaneous hprt mutations in V-C8 cells have been published previously (Kraakman-van der Zwet et al., 2002). Summary data on the mutation spectrum of V-C8 cells are shown in Figure 1, together with those of V79 cells (Zhang et al., 1992). The mutation spectrum of V-C8 is dominated by deletions (52%), which are mainly large deletions of one or more exons, while base substitutions were found in only 10% of mutants. This mutation spectrum differs significantly from that of V79 cells (Zhang et al., 1992). In V79 cells, mainly base substitutions have been found (43% transversions, 21% transitions), while deletions were only found in 13% of hprt mutants. The mutation rate data (Table I) together with the mutation spectrum data (Figure 1) allowed us to calculate the mutation rate for specific mutation types. For V-C8 cells, the deletion rate is 14 x 10–7/cell/generation, while for V79 cells it is 1 x 10–7. The base substitution rate for V-C8 cells is 3 x 10–7/cell/generation, while for V79 cells it is 4 x 10–7. Therefore, the rate at which base substitutions occur in V-C8 cells is similar to that of wild-type V79 cells, while the rate of deletions is much higher in V-C8 than in V79 cells (14-fold increase). This suggests that Brca2 especially prevents the occurrence of deletions.
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IR-induced mutant frequency in Brca2-deficient V-C8 cells
In V-C8 cells, the frequency of erroneous HR-based repair has been found to be increased in comparison with V79 cells (Larminat et al., 2002
). Therefore, IR-induced mutagenesis might also be increased in these cells. To determine the IR-induced mutability following exposure to different doses of X-rays, both V79 and V-C8 cells were propagated for expression of induced mutations at the hprt locus, as described in Materials and methods. The induced mutant frequencies were calculated by subtraction of the background frequency found without irradiation (mean values 2.0 and 6.0 x 10–5, median 0.6 and 6.5 x 10–5, for V79 and V-C8 cells, respectively). The results presented in Figure 2 show the individual data points of the observed induced hprt mutant frequencies of V79 and V-C8 cells following exposure to different doses of X-rays. We found a 2.3-fold increase in mutability per Gy in V-C8 cells (comparison of the slopes of the lines calculated by linear regression analysis). Despite the large variation between individual experiments, the difference between the regression lines for V-C8 and V79 was statistically significant (P < 0.01).
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Discussion |
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We found that Brca2-deficient V-C8 cells displayed a 4.3-fold higher spontaneous mutation rate at the endogenous hprt locus than wild-type parental cells (Table I), indicating a role of Brca2 in the prevention of spontaneous mutagenesis. Our data are in agreement with recently reported in vivo data on Brca2-deficient mouse embryos carrying a LacZ plasmid transgene, which showed a 2.3-fold increase in the spontaneous LacZ mutation rate in these embryos compared with wild-type embryos (Tutt et al., 2002
). In contrast, in a previous study of FA-D1 lymphoblasts, now known to be homozygous for a mutation in BRCA2 (Howlett et al., 2002), no significant difference in the frequency of spontaneous hprt mutants was found between the FA-D1 and wild-type lymphoblasts (Papadopoulo et al., 1990). In addition, after induction of DNA monoadducts and/or interstrand cross-links, the hprt mutant frequency was lower for FA-D1 than for wild-type lymphoblasts (Papadopoulo et al., 1990), while our data, as well as the data of Tutt et al. (2002), showed an increased frequency of mutations in response to IR. This discrepancy could be due to differences in the apoptotic response, which is more prominent in cells of lymphoid origin. However, it might also be due to the different types of lesions that were introduced in the FA-D1 cells.
We have previously shown that the majority of spontaneous mutations in V-C8 cells at the hprt locus are deletions (Kraakman-van der Zwet et al., 2002), while in wild-type V79 cells mainly base substitutions are found (Zhang et al., 1992) (Figure 1). Considering the mutation rate, we calculated that deletion mutations occur at a 14-fold higher rate in V-C8 cells than in wild-type V79 cells, whereas the rates of acquiring base substitutions were similar. In nearly all V-C8 deletion mutants examined one or more exons of the hprt gene were missing, in about half of which the entire hprt gene was lost (Kraakman-van der Zwet et al., 2002). A bias to deletions in the spectrum of spontaneous mutations has also been demonstrated in the above mentioned FA-D1 lymphoblasts (Papadopoulo et al., 1990), as well as in Brca2-deficient mouse embryos (Tutt et al., 2002). Consistent with our data, the deletions found in these studies also concerned large deletions/rearrangements. It has been demonstrated that DSB repair by HR is impaired in Brca2-deficient cells and that instead, an error-prone homology-directed repair pathway is used (Moynahan et al., 2001; Tutt et al., 2001; Larminat et al., 2002). In addition, previous data have shown reduced fidelity of DSB repair and V(D)J recombination in FA-D1 cells (Escarceller et al., 1997; Smith et al., 1998). Taken together, these results provide an explanation for the increased frequency of spontaneous deletions. It seems that in the ‘Others’ category of hprt mutants (Figure 1), spliced mutants are most prevalent in wild-type V79 cells, whereas in Brca2-deficient V-C8 cells no detectable amounts of hprt cDNA could be obtained for most mutants while all exons were present in the genomic hprt DNA (Zhang et al., 1992; Kraakman-van der Zwet et al., 2002). However, to draw any conclusions more hprt mutants should be analysed and the exact molecular nature of these mutations established.
In summary, our data obtained with hamster V-C8 cells give additional evidence for enhanced spontaneous as well as IR-induced mutagenesis after loss of Brca2 function, in agreement with the data observed using Brca2-deficient mouse embryos (Tutt et al., 2002). Increased IR-induced mutagenesis would increase the tumour risk to BRCA2 patients, as they are exposed to IR in screening mammography and radiation therapy. However, Tutt et al. did not find an increase in mutagenesis following IR in Brca2+/– mouse embryos, suggesting that heterozygous tissues in BRCA2 patients would respond normally to IR. On the other hand, loss of the remaining wild-type BRCA2 allele is inherent in the course of the pathological process in BRCA2 patients (Collins et al., 1995), keeping tissues at constant risk (Cornelis et al., 1995). IR could, therefore, accelerate mutation acquisition and tumorigenesis in cells in which the second allele has already been lost. Thus, IR in mammography screening and radiation therapy forms a potential risk for BRCA2 mutation carriers.
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Acknowledgements |
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The authors would like to thank W.J.I. Overkamp and G. Giangirolami for their skilful technical assistance and H. Vrieling for providing the primers for PCR of hprt cDNA and genomic DNA. This work was supported by grant 9.0.14 from the J.A. Cohen Institute (Interuniversity Research Institute for Radiopathology and Radiation Protection, The Netherlands), by NWO grant 901-01-190 and by Dutch Cancer Foundation (KWF) grant UL 2002-2739.
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3To whom correspondence should be addressed at: Department of Toxicogenetics – MGC, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 71 5276175; Fax: +31 71 5276173; Email: m.z.zdzienicka{at}lumc.nl
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Received on May 6, 2003; accepted on September 1, 2003.
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