Mouse Models of XRCC1 DNA Repair Polymorphisms and Cancer

WC Ladiges

Department of Comparative Medicine, University of Washington, Seattle, WA, USA

DNA damage plays a major role in mutagenesis, that plays a major role in many cases of cancer. carcinogenesis and aging. A gene that is emerging as an Oxidatively modified DNA is present in many tissues essential element in the repair of both damaged bases and including tumor tissue (reviewed by De Bont and van single-strand breaks (SSB) is XRCC1. XRCC1 has been Larebeke, 2004). Oxidative stress occurs when the shown to have a large number of single-nucleotide production of ROS exceeds the body’s natural defense polymorphisms (SNPs),several of which are being mechanisms, causing damage to macromolecules such as increasingly studied in cancer epidemiology investigations, DNA. Any change in the efficacy of DNA repair will in part because of their relative high frequency in the alter the steady-state levels of oxidative DNA modifica- population. Although association trends with specific tions, which in turn affects the mutation rate and cancer types have occasionally been shown in a variety ultimately the cancer incidence. Oxidative base damage of ethnic backgrounds,there are often conflicting reports and single-strand breaks (SSB) are the most frequent that weaken any substantial conclusions. The functional types of DNA damage caused by ROS (Thompson and significance of these SNPs is still largely unknown. West, 2000), and if not repaired, can lead to much more XRCC1 is an excellent prototype to provide a forum for serious double-strand breaks that directly contribute to determining how epidemiological cancer association stu- the development of cancer. There is thus a great dies with DNA repair gene polymorphisms can be appreciation for molecular systems designed to repair validated or refuted. The focus is on the utilization of in damaged DNA. Base damage is handled by a process silico data and biochemical studies in cell lines and termed base excision repair, which consists of removal existing mouse models to help provide a framework for the by glycosylases followed by processing by XRCC1- development of new mutant mouse lines that mimic human assisted POL b-dependent short-patch and/or prolifer- polymorphisms. These mouse lines will provide the next ating cell nuclear antigen/POL b-dependent long-patch generation of mammalian tools for carcinogen exposure repair. SSB are repaired by activation of poly (ADP- studies relevant to human cancer and variations in ribose) polymerases (PARPs) and XRCC1, with sub- XRCC1,and provide the basis for investigating groups sequent recruitment of additional proteins involved in of genes and polymorphisms in an animal model. the repair process. A gene that is emerging as an Oncogene (2006) 25, 1612–1619. doi:10.1038/sj.onc.1209370 essential element in the repair of both damaged bases and SSB is XRCC1 (Figure 1). Keywords: DNA repair; polymorphisms; XRCC1; XRCC1 was the first human gene involved in SSB cancer; mouse models repair to be cloned (reviewed by Thompson and West, 2000). Cells lacking the gene product are hypersensitive to ionizing radiation, hydrogen peroxide, camptothecin and alkylating agents (reviewed by Caldecott, 2003). In addition, mutant cells show elevated frequency of Introduction spontaneous chromosomal aberrations and deletions, and null mutant mice exhibit an embryonic lethal DNA damage plays a major role in mutagenesis, phenotype indicating the importance of XRCC1 for carcinogenesis and aging. There are a number of genetic stability (Tebbs et al., 1999). A recent study has chemical events that lead to DNA damage including shown that XRCC1 is required for efficient DNA SSB hydrolysis, exposure to reactive oxygen species (ROS) repair (Brem and Hall, 2005). Interestingly, XRCC1 has and other reactive metabolites. These events result from been shown to have a large number of single-nucleotide metabolic, endogenous processes, or are triggered by polymorphisms (SNPs), several of which are being exposure to exogenous chemicals. It is now becoming increasingly studied in cancer epidemiology investiga- apparent that mutations due to DNA damage are tions, in part because of their relative high frequency in caused by endogenous factors that are modulated by the population. Although association trends with exogenous factors, and it is probably this combination specific cancer types have occasionally been shown in a variety of ethnic backgrounds, there are often conflicting reports that weaken any substantial conclu- Correspondence: Dr WC Ladiges, Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA. sions. Consequently, the functional significance of these E-mail: [email protected] SNPs is still largely unknown. This paper will use XRCC1 mouse models WC Ladiges 1613 XRCC1 as a prototype to provide a forum for how known enzymatic activity has been attributed to epidemiological cancer association studies with DNA XRCC1, but three interactive domains have been repair gene polymorphisms can be validated or refuted. identified (Figure 2), plus a nuclear localization signal The focus will be on the utilization of in silico data and and a phosphorylation site for Ck2 (Loizou et al., 2004). biochemical studies in cell lines and existing mouse The N-terminal domain (NTD) is the site for POL models to help provide a framework for the develop- b binding (Dianova et al., 2004). It is also the site for ment of new mutant mouse lines that mimic human direct binding to both gapped and nicked DNA, and polymorphisms. These mouse lines will provide the next gapped DNA complexed with POL b (Marintchev et al., generation of mammalian tools for environmental 1999). XRCC1 has two BRCT domains, which are exposure studies relevant to human cancer. weakly conserved motifs first identified in BRCA1, and which mediate protein interactions (Zhang et al., 1998; Taylor et al., 2002; Beernink et al., 2005). BRCT1 is an XRCC1 structure interactive site for PARP whereas BRCT2 is an interactive binding site for Lig3. The linker area between Human XRCC1 is composed of 17 exons and spans a the NTD and BRCT1 has been suggested to be the area genomic distance of 32 kb (Thompson and West, 2000). for binding with other proteins including Apex, OGG1 It is located on chromosome 19q 32–133. Mouse (Marsin et al., 2003) and PCNA (Fan et al., 2004). XRCC1 maps to a similar region on chromosome 7 and spans 26 Kb. The exon positions are the same for mouse and human, and there is a high degree of XRCC1 polymorphisms and cancer association studies homology of amino-acid residues (MGI database). No An extensive review of the epidemiology will not be presented, as there are several published documents that address this issue very well. Rather, an attempt will be made to speculate on association trends and how this Carcinogen information can be used to make decisions for investigating functional aspects of the XRCC1 gene. The Nickerson lab reports 27 gene variations for XRCC1 using a panel of 90 human lymphoid cell lines Oxidative stress (http://egp.gs.washington.edu/). Of these, three are 50 UTR’s, 12 are insertion/deletions, four are synon- Antioxidant enzymes ymous SNPs, and eight are nonsynonymous SNPs (Figure 3). Only two polymorphisms have been studied DNA in any detail because they occur with a high frequency in damage DNA single strand break the population. R399Q is most frequent followed by R194W. This frequency allows lower cohort numbers to POLβ be studied thus providing OR data that can be analyzed Parp to a significant level. A third polymorphism, R280H, has Lig3 also been studied but less frequently because of its lower prevalence in the population. Ck2 XRCC1

Figure 1 Schematic overview of the response of XRCC1 and its R194W interactive protein partners to a DNA single-strand break induced A number of cancer association studies have been by an environmental carcinogen and oxidative stress. reported. Based on a recent review by Goode et al.

NTD NLS Brct1 Ck2 Brct2

84-183 183-310. PCNA, Ape 315-403 514-530 538-629 POLβ Ogg1, MutyH? Parp1 phosphorylation Lig3 Parp2 PNK

Figure 2 Human XRCC1 domains and locations of binding sites with interactive protein partners.

Oncogene XRCC1 mouse models WC Ladiges 1614

NTD NLS Brct1 Ck2 Brct2

P161L R399Q T576S

P309S

V72H R194W T304A

R280H

Figure 3 Human XRCC1 domains and locations of the eight nonsynonymous (change in amino-acid residue) single-nucleotide polymorphisms (SNP) as identiﬁed by the Nickerson lab (http://egp.gs.washington.edu).

(2002), six studies reported a reduced risk of cancer hemizygosity of the XRCC1 gene in CHO cells accounts associated with the R194W SNP. These were all case for the relative ease of isolating mutants with pheno- control studies with each having greater than 200 cases. types of sensitivity to agents, such as methyl methane- This association was confirmed by a more recent meta- sulfonate (MMS), that produce high levels of SSB. One analysis of 38 case control studies in Chinese popula- mutant clone, designated as EM9, was shown to carry a tions (Hu et al., 2005). A Russian study of more than frameshift mutation that results in truncated polypep- 2000 cases reported that R194W was associated with a tide lacking two-thirds of the normal sequence. EM9 is reduced risk of lung cancer among subjects in the thus a null mutant with genetic characteristics compar- highest quartile of pack-years of smoking (Hung et al., able to mouse cells carrying a knockout mutation. 2005). These results are somewhat surprising since a Takanami et al. (2005) made plasmid constructs common perception is that a change in amino-acid expressing XRCC1 wild-type, R194W or R280H and structure would be deleterious to function and result in transfected them into EM9 CHO cells. These cells were an increased risk of cancer. There are a number of then exposed to MMS and assayed for survival. The possible explanations. First, the epidemiological studies EM9 cells had low survival but the R194W-transfected could be misleading due to inaccurate responses. In the cells had an increased survival similar to that of case of R194W, the evidence is fairly consistent over a transfected wild-type cells. Therefore, this in vitro large range of studies suggesting some type of valid functional study does not contradict the epidemiology inverse relationship. A second consideration is that there studies. is no direct association, but only linkage to another relevant polymorphism as part of a haplotype, or to another causative gene. Cellular functional studies will R280H helpresolve this issue. Thirdly, the change in amino-acid The R280H SNP has been studied less than R194W and structure could enhance protein binding. The 194 R399Q because of lower frequency of the allele and the position is within possible binding domains of several requirement of a larger sample size to attain significant interactive protein partners including PCNA, Ape1 and power analysis. Hu et al. (2005) did a meta-analysis of several glycosylases such as Ogg1 and MutyH. This area eight case–control studies, which showed a borderline is rich in proline, serine, arginine and lysine residues so significantly increased cancer risk in individuals carrying the change to tryptophan would represent a transition a histidine allele compared to individuals with the from positively charged arginine to a hydrophobic arginine/arginine genotype. In contrast, Hung et al. tryptophan, which could affect binding and DNA repair (2005) reported a reduced risk of lung cancer among efficiency. It is interesting that in silico predictions subjects carrying a histidine genotype and in the highest indicate a possibly damaging effect and an intolerant quartile of pack-years of smoking compared to indivi- protein as the result of the presence of the W allele. duals homozygous for the arginine allele. The small What this contrasting evidence means is not known, number of studies limits any positive conclusions but except more functional studies are needed. Lastly, it is suggest an association depending on environmental possible that the change in amino-acid structure results interaction. in an upregulation of another gene that enhances the Of more interest is a recent study by Takanami et al. DNA repair process. (2005) showing that the R280H variant transfected into Chinese hamster ovary (CHO) cell lines have been an MMS sensitive CHO cell line null for the XRCC1 used to study functional aspects of XRCC1 null and gene product did not restore resistance. MMS is an variant alleles in vitro (Shen et al., 1998). The alkylating chemical that generates SSB in DNA, which

Oncogene XRCC1 mouse models WC Ladiges 1615 require XRCC1 for efficient repair. The results by the environmental challenges were different in the two Takanami et al. (2005) suggest that the function of studies, the contrasting results raise some questions XRCC1 may be compromised by the histidine allele. By about whether the R399Q polymorphism is completely using the comet assay, they showed that there was a innocuous. significant delay in SSB repair at 60 min in CHO cells with the histidine genotype compared to CHO cells with Other SNPs the arginine genotype. They also showed a depletion of There are five other nonsynonymous SNPs identified by NAD(P)H in the histidine-transfected CHO cells treated the Nickerson lab (http://egp.gs.washington.edu). These with MMS, suggesting that activation of PARP1 may be all have prevalence rates of less than 5%, and have not associated with XRCC1 function. been investigated epidemiologically. Only one of these, P161L, has an in silico prediction of probably damaging and intolerant. This is not surprising since it is within R399Q the POL b binding domain (Figure 3) and could disrupt The R399Q SNP has been the most studied of the the interactive function of this important gene product. XRCC1 variations and one of the most frequently Additional studies of P161L would be of interest. studied SNPs among all DNA repair gene variations. In Another SNP, Y576S, is predicted to be probably a review by Goode et al. (2002), R399Q showed damaging but tolerant and occurs within the BRCT2 associations in different directions for different cancers. domain where XRCC1 binds with Ligase 3 (Figure 3). For example, there was a decreased risk for nonmela- This would also be of interest to study further. noma skin cancer, esophageal cancer and bladder cancer but increased risk for lung cancer and stomach cancer. In the meta-analysis review by Hu et al. (2005), there Other polymorphisms was no overall association of genotype to cancer risk or The Nickerson lab has identified 12 insertion/deletion 0 cancer protection. However, when stratified to ethnic variations and three 5 UTR variations (http://egp.gs. background, significantly increased risks were found in washington.edu). These have not been studied. Asian and African subjects. In the Eastern European study by Hung et al. (2005) of more than 2000 case controls subjects, there was no association of lung Point mutations affect XRCC1 function cancer risk with the R399Q variant. These epidemiological results therefore do not provide any conclusive Three point mutations have been studied in CHO cell evidence of a direct association of the XRCC1 R399Q lines and shown to have an impact on XRCC1 function. SNP with cancer. V86R falls within the POLb binding domain (Figure 4) This conclusion is further supported by a functional and completely abolishes binding of the XRCC1 NTD study by Taylor et al. (2002) using transfected CHO cells to POLb (Dianova et al., 2004). The mutation is located deficient in XRCC1 gene product. Cells with the R399Q in the five-stranded beta sheet of the core beta sandwich, genotype showed no difference in sensitivity to MMS and disrupts a key hydrophobic interaction with the than cells transfected with Q399R. There was also no POL b thumb loopwithout adversely affecting the difference in ability to repair MMS-induced SSB in the folding of the NTD. The end result is a decreased two isoforms as determined by the comet assay. efficiency of base excision repair. The L360R point However, in another study, Au et al. (2003) used mutation occurs within the BRCT1 domain (Figure 4) cytogenetic irradiation and UV light challenge assays and inhibits correct folding of the BRCT1 domain to generate data suggesting that exposure of whole- (Taylor et al., 2002). The third point mutation, W385D, blood cells from individuals with the R399Q genotype also occurs in the BRCT1 domain, and allows an results in a significant increase in chromosomal dele- increased sensitivity to MMS and other alkylating tions. The implication is that chromosomal deletions are agents (Taylor et al., 2002), and interferes with PARP1 the result of deficient DNA repair that would facilitate binding (El-Khamisy et al., 2003). These point muta- genetic instability and increase the risk for mutations tions do not occur naturally, but are useful in studying leading to cancer. Even though the model systems and specific aspects of XRCC1 function.

NTD NLS Brct1 Ck2 Brct2

V86R L360R W385D

Figure 4 Human XRCC1 domains and locations of three point mutations shown to have an adverse effect on XRCC1 function (Taylor et al., 2002; Dianova et al., 2004).

Oncogene XRCC1 mouse models WC Ladiges 1616 XRCC1 mouse model development body weight as measured at the time of weaning and followed through adulthood. The hypomorph genotype XRCC1 null mouse line can easily be distinguished from littermate controls by XRCC1 gene knockout in mice results in embryonic visual observation. The cause of this low body weight lethality, demonstrating it is essential for development has not yet been determined but gross prosection (Tebbs et al., 1999). However, it is problematic to study suggests they have decreased body fat. We have any adult phenotype that may be the result of XRCC1 determined that decreased energy intake is not a factor deletion. It is possible, however, to see if the absence of as they eat similar amounts of chow as control one allele has any functional affect, that is a haploin- littermates. sufficiency phenotype. We are currently conducting Regarding incidence of cancer in XRCC1 hypo- several studies in this regard. We are also developing morphs, we have not yet evaluated enough mice an inducible knockout mouse that will be useful in pathologically to make any conclusions about specific temporal and tissue-specific deletion studies to bypass tumor types. So far, we have seen relatively little the embryonic lethality. It is of interest in this regard evidence of any tumors, but most of our mice are less that we have established that XRCC1 transgenic over- than 8 months of age. We have, however, seen several expression using the strong promoter PKG is embryonic testicular tumors in retired male breeders, but do not lethal (unpublished data) indicating that there is a know the significance of this. What we do know is that delicate balance between excess gene product and we have confirmed the observations of Tebbs et al. absence of gene product. (2003) that there is no robust lethal tumor phenotype (or any other lethal phenotype) in young adult animals. It will be of interest to see what the tumor spectrum will XRCC1 hypomorph mouse line be in older mice as we follow their lifespan and Tebbs et al. (2003) recently generated an XRCC1 pathologically evaluate end of life lesions. We know hypomorph mouse line. We are using this mouse model that our C57BL/6 aging colony has an average end as a prototype to examine phenotypic outcomes under of lifespan of 27 months with greater than 50 % conditions of low gene product expression. Mice have incidence of lymphomas plus a variety of epithelial approximately 10% protein levels compared to wild- and soft tissue tumors and malignancies (unpublished type control mice as determined by densitometry read- observations). This will be used as a background for ings from Western blots. The strategy in developing the comparison. mouse was based on using endogenous promoters to drive transgenic expression in a natural manner. Several promoter regions were screened for protein expression Development of XRCC1 mutant mouse lines and one selected with exceptionally low levels. The We are in the process of developing a number of mouse transgenic line that was developed was then crossed with lines representative of selected SNPs and point muta- the XRCC1 null mutant mouse line to rescue the tions, including R194W and R280H SNPs and V86R embryonic lethality. The double mutant thus provides a and L360R point mutations (Table 1). These are gene- model of reduced XRCC1 protein and can be studied targeted insertion mutant mice, that is knock-ins. The for adult phenotypes in relation to environmental and strategy is to duplicate the human variant sequence in carcinogen exposures. the mouse genome by constructing vectors containing We now have the XRCC1 hypomorph colony the homologous mouse variants targeted to the correct established in our lab (as the result of a gracious gift genomic location. This of course requires that the from Dr Robert Tebbs), and can make several sequence is conserved from human to mouse. Fortu- preliminary observations. First, breeding fecundity does nately, there is a high degree of homology in human and not seem to be appreciably affected, since pregnancies mouse XRCC1. We have used a C57BL/6 ES cell line to and litter sizes are similar to littermate controls. generate targeted ES cell clones for making chimeric Secondly, the hypomorphs are consistently smaller than mice, which are then tested for germline transmission littermate controls with an average of 25% reduction in and subsequent genotyping and phenotyping.

Table 1 Current status of XRCC1-induced mutant mouse models Induced mutation Developed by Comments

Knockout Tebbs et al. (1999) Embryonic lethal Conditional knockout Ladiges et al., unpublished Under development Transgenic (endogenous promoter) Tebbs et al. (2003) 10% gene product expression Transgenic on the knockout background (double mutant) Tebbs et al. (2003) Viable hypomorph with small body size phenotytpe Ladiges et al., unpublished R194W SNP (knockin) Ladiges et al., unpublished Viable line, not yet characterized R280H SNP (knockin) Ladiges et al., unpublished Viable line, not yet characterized R399Q SNP (knockin) Ladiges et al., unpublished Under consideration V86R point mutation (knockin) Ladiges et al., unpublished Chimeras established L360R point mutation (knockin) Ladiges et al., unpublished Chimeras established Transgenic, PKG promoter Ladiges et al., unpublished Embryonic lethal

Oncogene XRCC1 mouse models WC Ladiges 1617 The rationale for selecting the R194W SNP for mouse the glycosylases including MPG and OGG1. We have model development is based on epidemiological studies, found that these are available from a variety of sources in silico predictions, and experiments in CHO cell lines. and research labs. To our knowledge, there was not a Overall, there is a suggestion that the W allele may be null mutant mouse model available for ligase 3, but an protective of cancer, or at least may alter the molecular ES cell line was available from Bay Genomics (http:// mechanisms involved in increasing the efficiency of baygenomics.ucsf.edu/). Using this gene trapped ES cell repairing damaged DNA. The objective is to develop a line, we have generated null mice for ligase 3, which are mouse line that can be challenged in a manner similar to viable but not yet fully characterized. the hypomorphs. A targeting vector has been constructed (Figure 5) and targeted C57/BL6 ES cell clones used to generate chimeras, which are currently being evaluated. We have made similar progress with the Strategies for carcinogen challenge studies second SNP we selected for mouse model development, R280H. The rational is similar to that for R194W, The concept is that variants will affect DNA repair gene except that the overall trend is suggestive of an increased function in an adverse manner allowing damaged DNA risk for cancer. We expect to have both of these mouse to replicate and form cancer-causing mutations. The lines available in the coming months. The rationale for reverse could also be true, that is, the variant could be selecting V86R and L360R point mutations is based on associated with an increased efficiency of the DNA reported affects on XRCC1 function. We are especially repair gene and increase resistance to cancer. Even interested in the interaction of POL b with XRCC1 at though most polymorphisms are only mildly penetrant the NTD, and are developing the V86R mutant mouse and may not have much impact singly, we are starting line to see if this mutation disrupts binding as in CHO with individual variant mutant mice in our prototype cells, and whether it might have any effect on the repair gene, XRCC1, in order to determine what effect if any of abasic sites and SSB and the development of cancer each variant may have on XRCC1 function. We can phenotypes. The development of the L360R mutant then proceed to examine combinations and gene mouse model is geared toward providing an in vivo interactions, as suggested in a recent review (Kelada system to investigate the critical nature of the BRCT I et al., 2003). binding domain for XRCC1 function, since this point mutation has been shown to disrupt specific structural Single mutant mice aspects of this domain in CHO cells. Of specific interest These mice are suitable for general or tissue-specific here is the impact on XRCC1 function, if binding to carcinogen exposure studies. Studies with single mutant PARP1 in prevented, and the consequences for tumor mice will need to be well justified and designed since the development, especially under conditions of toxic or chances of positive results may be low. Several scenarios carcinogenic exposure. are possible. First, it may be advantageous to screen with a general mutagen to compare tumor rates, and Other related mouse lines possibly other phenotypes. Ethyl nitroso urea (ENU) We are developing other mouse lines as complementary has been used extensively in mice and induces a variety to investigating XRCC1 function in DNA repair and of tumor phenotypes within 10–12 months following tumorigenesis. We are especially interested in mutant neonatal exposure. Another strategy would be to mice representing partner proteins for XRCC1, or genes identify a specific target tissue and challenge with an that are integral to the BER or SSBR pathway. A appropriate carcinogen. For example, evidence suggest- number of these have already been developed, such as ing that XRCC1 R194W is protective of lung cancer null mutants for POL b, Ape1, PARP1 and several of might indicate a challenge study with a lung tissue-

Xrcc1 R194W Knock-In 42943 41621 42587 35942 47238 48293 39150 42750 48000

E5 E6 E7 E8 E9 E16 E17 Genome

SNP Linearization 42567 Site Vector E6 E7 E8 E16 Neo TK LoxP LoxP

3.6kb 5.3 kb

Figure 5 XRCC1 R194W targeting vector strategy (constructed by Vector Biolabs, Philadelphia, PA, USA). Boundaries of homologous arms are located within introns, at least 100 bpaway from the nearest exon. Exon with SNP is placedvery close to Neo cassette (but at least 100 bpaway) in order to ensure the cross pointof homologous recombination of right arm is on the right side of the SNP.

Oncogene XRCC1 mouse models WC Ladiges 1618 specific carcinogen, such as urethane. Another example cal studies have suggested that the incidence of cancer would be to investigate the differential sensitivity to the phenotypes may be associated with the presence of one colonotrophic carcinogen, azoxymethane, since several or more polymorphisms in other genes. This precludes epidemiological studies have suggested an association the requirement that mutant mice for these other genes with incidence of colon cancer and XRCC1 polymorph- are available. The Comparative Mouse Genomics isms. A third example would be a challenge study with Center Consortium (CMGCC) has developed a number bleomycin, since lymphocytes from XRCC1 R194W of these mouse lines (http://www.niehs.nih.gov/cmgcc/), individuals have been shown to be have fewer chromatid many of which are available. An example of a double breaks in in vitro exposure experiments with bleomycin mutant of interest would be XRCC1 and XPD. Triple compared to lymphocytes from individuals with the mutant mice could also readily be generated to represent wild-type genotype (Wang et al., 2003). Bleomycin three polymorphisms from three separate genes. The mimics radiation exposure and causes pulmonary breeding strategy is intense but straightforward. fibrosis in mice. A third area of interest in developing multimutant Finally, it is of interest to pursue the functional mice is the combination of SNP mutations with point significance of XRCC1 polymorphisms in mutant mice mutations. Mice for XRCC1 point mutations have not by exposing them to ionizing radiation. Exposure to yet been characterized and validated so it is premature ionizing radiation has been linked to cancers of the to plan specific experiments. However, one of the thyroid, breast and lung as well as leukemias in humans rationales for making these mice is to provide a tool (Ron, 1998), and is known to cause DNA damage for investigating consequences of inhibiting the binding requiring the base excision pathway (Wallace, 1994). of interactive protein partners. For example, the Not only is there relevance to individuals exposed to low XRCC1 V86R point mutation has been shown to dose ionizing radiation (Aka et al., 2004), but a recent completely block the binding of POL b in CHO cells study suggests that XRCC1 polymorphisms may be (Dianova et al., 2004), and we will soon be able to associated with ineffective radiotherapy in some cancer determine if this is also the case in mice. If this is the patients (Moullan et al., 2003). case, then we could cross V86R mice with R280H mice and determine the affect of R280H in the absence of Multimutant mice POL b activity in repair of abasic sites or SSB when Since our central theme involves a prototype gene that is challenged with a mutagenic alkylating agent such as responsive to the repair of oxidatively damaged DNA, it MMS. is of interest to ask the question of how XRCC1 polymorphisms affect repair function under conditions of suboptimal antioxidant activity. A number of studies have suggested a correlation with such genes as Conclusions gluthathione transferase (GST). For example, a recent study by Geisler et al. (2005) found that patients with XRCC1 has been shown to have a large number of the GST-T1 functional genotype were three times as SNPs, and several have been extensively evaluated in likely to die from squamous cell carcinoma, whereas the cancer epidemiology association investigations because XRCC1 399 glutamine genotype was predictive of of their relative high frequency in the population. disease recurrence. Another recent study by Kirk et al. Association trends with specific cancer types have been (2005) showed that the GST-M1 null phenotype and the shown in a variety of ethnic backgrounds, but conflict- XRCC1 399 AG genotype were significantly associated ing reports have prevented definite conclusions. As the with hepatocellular carcinoma in patients with hepatitis functional significance of these SNPs is still largely B infection and chronic aflatoxin ingestion. These unknown, new model systems are needed to help studies suggest that further functional investigations determine their relevance in cancer susceptibility. The may be of value in determining the role of these development of new mutant mouse lines that mimic polymorphic gene combinations in environmentally human polymorphisms will provide a new generation of related cancer. A typical experiment might consist of mammalian tools for carcinogen exposure studies crossing a specific XRCC1 SNP mutant mouse line with relevant to human cancer. a specific GST mutant mouse line and conducting a carcinogen challenge. Once the mice are available, it is Acknowledgements only a matter of doing the intercross breeding to make the multimutant line. Studies were supported by NIEHS Grant U01 ES11045, A second relationshipthat is of interest is the effect of (Ladiges PI), and NIEHS Grant P30 ES07033 (Eaton PI; other DNA repair gene polymorphisms. Epidemiologi- Ladiges Core PI).

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