Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA

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Abstract

Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2 – 16 month old NER deficient XPA−/−, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA−/−, lacZ mice were comparable to XPA+/−, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA−/− mice after about 15 months of age.

Main

Accumulation of mutations in the DNA of the genome has been implicated as a causal factor in both carcinogenesis and ageing. To prevent high mutation loads, organisms have been equipped, during evolution with a network of genome stability systems, e.g., cell cycle arrest, DNA repair. Together, these often highly conserved functional pathways act to limit cancer risk and enable longevity. By using a transgenic mouse model, which harbors chromosomally integrated copies of the lacZ reporter gene as part of a plasmid, we have recently demonstrated, that during ageing somatic mutations accumulate in the liver but not in the brain (Dollé et al., 1997).

Nucleotide excision repair (NER) is considered a major DNA repair mechanism in mammalian cells. This system entails multiple steps that employ a number of proteins to eliminate a broad spectrum of structurally unrelated lesions such as UV-induced photoproducts, chemical adducts, as well as intra-strand crosslinks and some forms of oxidative damage (Bootsma, 1998). Deficiency in NER has been shown to be associated with human inheritable disorders, such as xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and trichothiodystrophy (TTD). These disorders are characterized by UV-sensitivity, genomic instability and various signs of premature ageing (Bootsma, 1998). Xeroderma pigmentosum patients can be classified into at least seven complementation groups (XPA-XPG), with most patients found in XPA. Its deficiency causes a complete block in NER and most severe symptoms, including a more than 2000-fold increased frequency of skin cancer and accelerated neurodegeneration (Hoeijmakers, 1994; Cleaver and Kraemer, 1995). The product of the XPA gene, possibly in combination with replication protein A, is involved in DNA damage recognition, i.e., the pre-incision step of NER, in both global and transcription-coupled NER (Sugasawa et al., 1998).

Mice deficient in the NER gene XPA have been generated by gene targeting in embryonic stem cells These NER-deficient mice were demonstrated to mimic the phenotype of humans with xeroderma pigmentosum, that is, increased sensitivity to UVB induced skin cancer (De Vries et al., 1995; Nakane et al., 1995). This was found to be associated with an almost complete lack of UV-induced NER, measured as unscheduled DNA synthesis in cultured fibroblasts, and a much lower survival of fibroblasts after UV-irradiation or treatment with DMBA (De Vries et al., 1995). However, at early age NER-deficient mice do not show spontaneous abnormalities. These mice develop normally, indistinguishable from wild-type mice (De Vries et al., 1997a). At older age, i.e., from about 15 months onwards, XPA−/− mice show an increased frequency of hepatocellular adenomas (De Vries et al., 1997a), which is a common pathological lesion in older mice (Bronson and Lipman, 1991). The lack of spontaneous abnormalities in young XPA−/− mice might be due to the fact, that under normal conditions mice have only limited exposure to NER-mediated DNA damage. Hence, at early age NER appears to be dispensable. This is in contrast to base excision repair, the complete inactivation of which is lethal (Gu et al., 1994).

In order to test the hypothesis that loss of NER causes accelerated mutation accumulation, preceding the onset of accelerated tumor formation and senescent deterioration XPA-deficient mice were crossed with the lacZ transgenic mice previously used to monitor mutation accumulation in liver and brain during ageing (Dollé et al., 1997). In the hybrid XPA−/−, lacZ mice, mutant frequencies were analysed in liver and brain at 2, 4 and 9 – 16 months. Figure 1 shows the effect of the XPA-deficiency on the mutant frequency as compared to the XPA+/−, lacZ littermates and the parental lacZ animals, against the normal background established earlier for the lacZ mice (Dollé et al., 1997). A statistical analysis of the data is given in Tables 1 and 2. Mutant frequencies of XPA+/−, lacZ and lacZ parental strain animals, analysed in parallel in this study, were not significantly different in liver and brain. Therefore these mice were considered as one group (`XPA-proficient' mice) and their mutant frequencies were compared to mutant frequencies of the XPA−/−, lacZ hybrids.

Figure 1
figure1

Spontaneous mutant frequencies in (a) liver and (b) brain of XPA−/−, lacZ hybrid mice (closed triangles) as compared to XPA+/−, lacZ (open triangles), the lacZ parental strain (open circles) and the lacZ parental strain as reported by Dollé et al. (1997) (small closed circles) as a function of age. XPA−/− animals were originally made in the 129 strain, then bred into C57BL/6 for 10 generations and subsequently crossed with the lacZ animals which had been made directly in C57BL/6 (Dollé et al., 1997). Mutant frequencies were determined after rescuing lacZ-containing plasmids from genomic DNA. Briefly, about 30 μg genomic DNA was digested with HindIII followed by separation of linear plasmid copies using magnetic beads coated with the lacI repressor protein. Plasmids were circularized with T4 DNA ligase, precipitated and electrotransformed into E. coli (ΔlacZ, galE) cells. Plasmids with a mutation in the lacZ gene were selected in a positive selection system on plates containing the lactose analog Phenyl β-D-Galactoside. Mutant frequencies were calculated as the number of mutants versus the number of total transformants

Table 1 Table 1
Table 2 Table 2

In liver of 2-month-old mice, mutant frequencies of the XPA−/−, lacZ animals were still comparable to those for the XPA-proficient animals. This is consistent with earlier data on young adult XPA−/− mice (De Vries et al., 1997b). In 4-month-old XPA−/−, lacZ mice, mutant frequencies were found to be significantly increased by a factor of two. A further, albeit smaller increase was observed between 4 and 9 – 16 months. At the latter age level mutant frequencies were in the range of the maximum level reported earlier for 25 – 34-month-old lacZ mice, with a similar high individual variation (Figure 1a; Dollé et al., 1997). In brain, mutant frequencies were not found to increase over the age levels studied, which is in keeping with the lack of an age-related increase reported previously for the lacZ mice (Figure 1b; Dollé et al., 1997).

Mutant spectra were analysed from liver of 2-month and 9 – 16-month-old XPA−/−, lacZ mice. The results, shown in Figure 2, demonstrate that there is an about equal fraction of size change versus point mutations, but a higher level of mouse-sequence mutations in the 9 – 16-month old mice. Mouse-sequence mutations are size change mutations due to genome rearrangement events involving the 3′ mouse flanking region. These large mutations are assumed to have the highest impact on cell functioning and genome stability and have previously been found to increase with age only after 25 months (Dollé et al., 1997).

Figure 2
figure2

Mean frequencies of no-change mutants, size-change mutants and mouse-sequence mutants in liver of 2 and 9 – 16 month old XPA−/−, lacZ hybrids. Mutant colonies were grown overnight in LB medium, plasmids were mini-prepared and digested with AvaI and HindIII. To identify mouse-sequence mutations the restriction patterns were transferred to nylon membranes and hybridized with total mouse genomic DNA. A total of 60 – 100 mutants from each of four animals per age group were characterized

The results of this present study indicate that a deficiency in the NER gene XPA causes an early accelerated accumulation of somatic mutations in liver, but not in brain. The increase in mutant frequency in liver is in keeping with the higher incidence of spontaneous liver tumors several months later (De Vries et al., 1997a). Therefore, it is tempting to conclude that the higher mutant frequencies in liver predict an organ-specific predisposition to spontaneous cancer. The difference between liver and brain could be due to a higher proliferative activity or a more rapid accumulation of DNA damage associated with detoxification processes in the former organ. In the corresponding human syndrome, xeroderma pigmentosum, internal tumor development is rare, but neurological abnormalities are frequently observed (Bootsma, 1998). In XPA-deficient mice such abnormalities have thus far not been found. Phenotypical differences between XPA-deficient mice and human XPA might be due to tissue-dependent variation in the levels of endogenous DNA damaging species such as oxygen free radicals. In humans this may lead to deleterious effects in brain, whereas in mice it may cause a higher frequency of spontaneous liver tumors. Humans with XP rarely survive beyond the third decade of life, a consequence of the dramatic increase in sunlight induced skin cancer (Cleaver and Kraemer, 1995). Skin cancer does not occur in rodents, which have a fur that cannot be penetrated by UV and are also kept under conditions not permitting exposure to sunlight. This explains the lack of such a phenotype of the XPA mutation at early ages. In the young animals NER could be essentially redundant, but become increasingly important at later ages. Indeed, it has been repeatedly argued that loss of redundancy in, e.g., cell number, gene copy number, functional pathways, could be responsible for the gradual loss of individual stability and increased incidence of disease associated with ageing (Strehler and Freeman, 1980). This would be especially true for loss of redundancy in genome stabilization pathways, which have been hypothesized to represent both cancer suppressing and longevity assurance systems (Hart et al., 1979). The XPA mutant mice seem to be a suitable model to test this hypothesis.

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Acknowledgements

This work was supported by NIH grants PO1 1801 AG10829-01, 1 P30 AG13314-01 and 1 RO1 ES/CA 08797-01. We thank JM LaPlante for critical reading of the manuscript.

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Correspondence to Jan Vijg.

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Giese, H., Dollé, M., Hezel, A. et al. Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA. Oncogene 18, 1257–1260 (1999) doi:10.1038/sj.onc.1202404

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Keywords

  • nucleotide excision repair
  • XPA, mutation accumulation
  • lacZ reporter gene
  • ageing

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