Mouse Models for Xeroderma Pigmentosum Group a and Group C Show Divergent Cancer Phenotypes

Research Article

Mouse Models for Xeroderma Pigmentosum Group A and Group C Show Divergent Cancer Phenotypes

Joost P.M. Melis,1 Susan W.P. Wijnhoven,1 Rudolf B. Beems,1 Marianne Roodbergen,1 Jolanda van den Berg,1 Hojin Moon,2 Errol Friedberg,3 Gijsbertus T.J. van der Horst,4 Jan H.J. Hoeijmakers,4 Jan Vijg,5 and Harry van Steeg1

1National Institute of Public Health and the Environment (RIVM), Laboratory for Health Protection Research, Bilthoven, the Netherlands; 2Department of Mathematics and Statistics, California State University, Long Beach, California; 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; 4MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands; and 5Buck Institute for Age Research, Novato, California

Abstract modifications (e.g., oxidative DNA damage). Nucleotide excision The accumulation of DNA damage is a slow but hazardous repair (NER) has a very broad lesion spectrum and is responsible phenomenon that may lead to cell death, accelerated aging, for the removal of bulky, DNA helix–distorting adducts (2–7). and cancer. One of the most versatile defense mechanisms The autosomal recessive disorder Xeroderma pigmentosum (XP) against the accumulation of DNA damage is nucleotide is an elucidative example of the influence of a DNA repair defect on excision repair, in which, among others, the Xeroderma cancer predisposition. XP patients exhibit extreme UV sensitivity pigmentosum group C(XPC)and group A (XPA) proteins are and are predisposed to skin cancer by a 1,000-fold higher risk (8, 9). involved. To elucidate differences in the functions of these two Until now, seven complementation groups (XPA through XPG) plus À À À À proteins, comprehensive survival studies with Xpa / , Xpc / a variant form (XPV) were identified. XP disorders arise from a and wild-type control female mice in a pure C57BL/6J deficiency in one or more of these XP proteins, which belong to the À À background were done. The median survival of Xpc / mice NER pathway, with the notable exception of the XPV protein, which showed a significant decrease, whereas the median survival is involved in translesion synthesis of UV-damaged DNA (10). À À À À À À of Xpa / mice did not. Strikingly, Xpa / and Xpc / mice NER can be subdivided into two subpathways: global genome also showed a phenotypical difference in terms of tumor NER (GG-NER), which covers the complete genome, and transcrip- À À spectrum. Xpc / mice displayed a significant increase in lung tion-coupled NER (TC-NER), which focuses on repair of the tumors and a trend toward increased liver tumors compared transcribed strand of active genes (11, 12). XPC is associated with À À with Xpa-deficient or wild-type mice. Xpa / mice showed a the GG-NER whereas XPA plays a role in both GG-NER and TC-NER. significant elevation in liver tumors. Additionally, Xpc- The XPC protein, in complex with the HR23Bprotein, is responsible deficient mice exhibited a strong increase in mutant frequency for DNA damage recognition (6, 13). Following detection of À À in lung compared with Xpa / mice, whereas in both models distorted helix structures, the XPC/HR23Bcomplex will initiate mutant frequency is increased in liver. Our in vitro data the GG-NER process (14). Subsequently, the XPC/HR23Bcomplex À À displayed an elevated sensitivity to oxygen in Xpc / in mouse will dissociate from the damaged DNA strand when the transcrip- À À embryonic fibroblasts (MEF) when compared with Xpa / tion factor TFIIH in combination with the XPA and RPA protein set and wild-type fibroblasts. We believe that XPCplays a role in the verification of the DNA damage in motion (15). XPC-HR23Bis the removal of oxidative DNA damage and that, therefore, dispensable for TC-NER; the CSA and CSBproteins, together with À À Xpc / mice display a significant increase in lung tumors and RNA polymerase II stalled at a lesion, fulfill the role of recognition a significant elevation in mutant frequency in lung, and factor in this pathway. As in GG-NER, DNA damage verification in Xpc-deficient MEFs show greater sensitivity to oxygen when TC-NER requires the presence of the TFIIH-XPA-RPA complex (16). À À compared with Xpa / and wild-type mice. [Cancer Res When TC-NER components or the more common elements in 2008;68(5):1347–53] NER (e.g., XPA) are affected, very complex clinical features are observed (17). Patients with a defect in genes unique to GG-NER Introduction (like XPC) exhibit fewer clinical symptoms besides cancer. In general, deficiencies in the TC-NER pathway are related to Cancer remains one of the main causes of death nowadays in neurodegenerative disorders, whereas defects in the GG-NER both men and women and is accompanied by a kaleidoscope of pathway are designated as more cancer prone (18–21). unsolved questions about the induction and progress of this The XPC is the most common type of the XP disease in North disease. An important factor in the development of cancer is the America and Europe (22). This form is only defective in the GG- accumulation of somatic DNA damage (1). Normally, several NER pathway. XPA patients are disrupted in both their GG-NER sophisticated defense mechanisms are active to repair the modified and TC-NER pathways. Mutant frequency analyses at the Hprt DNA to prevent mutations and damage accumulation. Base locus in mouse models of these two forms of XP previously excision repair (BER), for example, will remove most small base uncovered striking differences. Hprt mutant frequencies in the À À spleen of Xpc / mice in a mixed genetic background were highly elevated in comparison not only with their wild-type controls but Requests for reprints: Harry van Steeg, Laboratory for Health Protection À/À Research, National Institute of Public Health and the Environment, P.O. Box 1, 3720 also with Xpa , both in a pure C57BL/6J background (23). This BA Bilthoven, the Netherlands. Phone: 31-30-274-2102; Fax: 31-30-274-4446; E-mail: indicates that knockout mouse models of Xpc and Xpa may also [email protected]. XpcÀ/À I2008 American Association for Cancer Research. exhibit different spontaneous phenotypes. mice of a mixed doi:10.1158/0008-5472.CAN-07-6067 background (25% 129, 75% C57BL/6J) were shown to exhibit a www.aacrjournals.org 1347 Cancer Res 2008; 68: (5). March 1, 2008

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Cancer Research high prevalence of lung tumors (24), but this has not been studied 45 or 50 of their wild-type controls (referred to in the text as C57BL/6J 1 and in a pure C57BL/6J background; also, a clean comparison with C57BL/6J 2) were monitored during their entire life span. The health state of À À Xpa / mice has not yet been made. the mice was checked daily, beginning at the day of weaning. Individual To refine and expand our knowledge on human XPA and XPC, animals were weighed biweekly to determine live weights. During the entire À À À À experiment, animals were kept in the same stringently controlled (specific Xpa / and Xpc / mice were used. To investigate the phenotypic pathogen-free) environment, fed ad libitum, and kept under a normal day/ differences between these mice, we carried out a more comprehen- night rhythm. The microbiological status of the cohorts was monitored sive study with both mouse models in a pure C57BL/6J background. every 3 months. Animals from the longevity cohort were removed from the We compared the life span of the two models and determined the study when found dead or moribund. Complete autopsy was done on pathology with focus on tumor development. In addition, to help animals of all cohorts; a total of 45 different tissues were isolated from each explain the differences in tumor outcomes, we carried out mutation animal and stored for further histopathologic analysis (see below). In analyses in several organs. Our data suggest that Xpc-deficient mice addition, a selective set of 20 different tissues were snap frozen in liquid lacZ are more sensitive to (oxidative) DNA-damaging agents in the lung N2 for molecular analyses (e.g., mutant frequency analyses). Total compared with the Xpa-deficient mice and wild-type controls. These animal weights as well as various organ weights were determined at time of findings support evidence provided in various studies that XPC, death or when killed. Histopathology. Organ samples (45 organs and tissues) of each animal in addition to its function in GG-NER, is also involved in the repair XpcÀ/À were preserved in a neutral aqueous phosphate-buffered 4% solution of of oxidative DNA damage. Therefore, mice seem to be more formaldehyde. Tissues required for microscopic examination were pro- sensitive to oxidative stress and lung tumor development than their cessed, embedded in paraffin wax, sectioned at 5 Am, and stained with H&E. À/À NER-deficient counterparts, Xpa mice. Detailed microscopic examination was done on nine major organs of all female mice from the longevity cohort and on all gross lesions suspected of being tumors or representing major pathologic conditions. For each animal, Materials and Methods histopathologic abnormalities, tumors as well as nonneoplastic lesions, À À À À Mice. The generation and characterization of Xpa / and Xpc / mice were recorded using the PATHOS pathology data acquisition software, and have been described before (25, 26). To obtain a genetically homogenous if possible, cause of death was established. À À À background, Xpa- and Xpc-deficient mice were back-crossed more than Hprt mutant frequency analyses. Xpc / , Xpc+/ mice and their wild- 10 times with C57BL/6J animals (Harlan). To offer the future possibility to type controls were sacrificed at 13 or 52 weeks and spleens were isolated to monitor genomic instability, the heterozygous mutant mouse strains as well determine spontaneous Hprt mutant frequencies. All mice were in pure as C57BL/6J controls were crossed with pUR288-lacZ C57BL/6J transgenic C57BL/6J background. The number of mice within one genotype and age mouse line 30, homozygous for lacZ integration on chromosome 11 (27). In group varied between 4 and 10. the second round of breeding, double heterozygous mice were intercrossed Priming and cloning of T lymphocytes was done in RPMI 1640 as to obtain homozygous mice carrying one locus of the integrated copies described by Tates et al. (29) with some minor modifications (30). Mouse of the lacZ marker, used in the third breeding round to generate the T lymphocytes were isolated from the spleen by rubbing the spleen through experimental animals for the aging and cross-sectional studies as described a sterile 70-Am nylon mesh (Falcon Cell Strainer, 2350). Subsequently, cells below. Mice were genotyped by a standard PCR reaction using DNA isolated were frozen in RPMI 1640 supplemented with 10% DMSO and 40% fetal from tail tips. Primers to amplify the wild-type and targeted Xpa and bovine serum (FBS) by using a Cryomed freezing apparatus (Forma Xpc alleles, as well as primer sequences for lacZ determination, have Scientific). When required, frozen cells were thawed and immediately stored previously been described (27, 28). The experimental setup of the studies on ice. Stimulated T lymphocytes were cultured and selected for Hprt was examined and agreed on by the institute’s Ethical Committee on deficiency in the presence of lethally irradiated (30-Gy X-rays) mouse Experimental Animals according to national legislation. lymphoblastoid Sp2/0 feeder cells (29). Cloning efficiencies and mutant Experimental design. Female mice were marked and randomized at the frequencies were calculated as described (29). Further details on the day of birth in different groups (i.e., longevity cohorts or cross-sectional procedure were described by Wijnhoven et al. (30). cohorts in which the mice were sacrificed at fixed time points). Cross- LacZ mutant frequency analyses. From a selected set of snap frozen À À À À sectional cohorts of Xpa / , Xpc / mice and their C57BL/6J controls were tissues, DNA was extracted with a phenol/chloroform/iso-amyl alcohol sacrificed at a fixed age of 13, 52, 78, and 104 weeks. The interim cohorts mixture (25:24:1). Complete protocols for plasmid rescue and mutant consisted of at least 15 female mice per time point and genotype. In the frequency determinations with the pUR288 model have been described À À À À longevity cohorts, a total of 45 Xpa / and 50 Xpc / female mice and elsewhere (31). Briefly, between 10 and 20 Ag of genomic DNA were digested

Figure 1. Survival curves of female Xpc À/À, Xpa À/À and their wild-type control cohorts. C57BL/6 1 is the control cohort for Xpa À/À. C57BL/6 2is the control cohort for Xpc À/À. Median survival of the cohorts: C57BL/6 1, 103 wk (light green); C57BL/6 2, 102.5 wk (dark green); Xpc À/À,94wk(P = 0.0023; red); Xpa À/À, 94.5 wk (P = 0.6023; blue). For further details, see Materials and Methods.

Cancer Res 2008; 68: (5). March 1, 2008 1348 www.aacrjournals.org

Table 1. Tumor incidences and P values for difference between mutant and its wild-type group

C57BL/6J 1 XpaÀ/À P C57BL/6J 2 XpcÀ/À P

No. animals (absolute) 40 31 29 38 All organs Tumor-bearing animals 30 (75) 21 (68) 24 (83) 23 (61) Liver Hepatocellular tumor 0 (0) 3 (10) 0.0002 2 (7) 5 (13) * Lung Bronchioloalveolar tumor 0 (0) 2 (6) 1 (3) 6 (16) 0.02 Pituitary Pars distalis adenoma 20 (50) 8 (26) * 20 (70) 9 (24) 0.01 All organs c Pituitary adenomas excluded 18 (45) 19 (61) 0.001 21 (72) 23 (61)

NOTE: Data are absolute values with percentages in brackets. Statistics: poly-k test (poly-3 test and Peto test generally gave similar significances, although exact P values may differ). À À À À *Approaches significance with poly-3 test only (P = 0.08 for Xpc / liver and P = 0.054 for Xpa / pituitary). cNot reaching significance with poly-k test, but P = 0.05 (positive trend) by the Peto test in this case.

with HindIII for 1 h in the presence of magnetic beads (Dynal) recoated C57BL/6J controls (C57BL/6J 1, 103 weeks). This difference in with lacI-lacZ fusion protein. The beads were washed thrice to remove the significance is mainly caused by the fact that a certain fraction À À unbound mouse genomic DNA. Plasmid DNA was subsequently eluted from (F10%) of the Xpa / animals survive extremely long (see Fig. 1). the beads by isopropyl-L-thio-B-D-galactopyranoside and circularized with Survival curves of both wild-type control studies show a very T4 DNA ligase. Next, ethanol-precipitated plasmids were used to transform E. coli C D À similar median survival and shape of the curve. ( lacZ, galE ) cells. One thousandth of the transformed cells was f plated on the titer plate (with X-gal) and the remainder on the selective Of all groups 30 or more animals were histopathologically plate (with p-gal). The plates were incubated for 15 h at 37jC. Mutant examined. Neoplasms and inflammation (mainly ulcerative derma- frequencies were determined as the number of colonies on the selective titis) are the most common cause of demise in all groups, ranging À/À plates versus the number of colonies on the titer plate (times the dilution from 72% in wild-type controls to 87% in Xpc mice (data not factor of 1,000). Each mutant frequency is based on at least 300,000 shown). Occasionally more than one pathologic condition might recovered plasmids. have contributed to death, in which case the most pronounced Statistical evaluation. Incidences of tumors were analyzed with the condition was taken as cause of demise. Incidences of major tumor À À À À method of Peto (SAS; ref. 32) and with the poly-3 and poly-k method (33). types at the time of death are listed in Table 1. Xpa / and Xpc / These tests for statistical analysis of tumor incidences take differences in mice lived shorter than their respective wild-type controls. survival of the various groups into account. The poly-3 and poly-k tests were Accordingly, lower tumor incidences were expected in the mutant done by Dr. H. Moon (California State University, Long Beach, CA). Cell culture. Primary mouse embryonic fibroblasts (MEF) were isolated animals because they had less time to develop tumors. Statistical from E13.5 day embryos, all in C57BL/6J background, and genotyped as methods that take differences in survival into account were there- previously described (27, 28). MEFs were cultured as described before (34) in fore used in this case to evaluate the significance of differences in DMEM (Life Technologies, Inc.) supplemented with 10% FBS (fetal calf tumor incidences. Nevertheless, some tumor types were increased À À À À À À serum, Biocell), 1% nonessential amino acids (Life Technologies), penicillin in either Xpc / or Xpa / mice. Xpc / mice show a significant A A j (0,6 g/mL), and streptomycin (1 g/mL) at 37 C, 5% CO2. MEFs were increase (P = 0.02) in bronchioalveolar lung tumors and near À/À cultured 3 days per passage at 3% or 20% O2. Cell survival was determined significant elevation (P = 0.054) in hepatocellular tumors. Xpa by blue/white screening using trypan blue stain 0.4% (Life Technologies; mice solely show a significant increase (P < 0.01) in hepatocellular 1:1), counting a minimum of 200 cells per sample. A minimum of three tumors when compared with their matching wild-type control different embryos were used per genotype, plus a technical replica of all À À group. Furthermore, female Xpc / mice also show a significant samples was used. increase in acidophilic macrophage pneumonia (P = 0.032, data À À not shown), which is not apparent in Xpa / mice. Results The percentage of tumor-bearing NER-deficient animals (all À À À À To determine the average life span of Xpc / and Xpa / mice, types together) is surprisingly lower than the corresponding NER- 45 to 50 mutant females and corresponding C57BL/6J control mice proficient control groups. In addition to the shorter life span, the were followed during aging. The survival curves for the longevity strong and significant decrease in benign pituitary adenomas in À À À À À À À À cohorts of female Xpc / , Xpa / and both matching wild-type both Xpc / and Xpa / mice is mostly responsible for this lower control groups are depicted in Fig. 1. The median survival of female percentage in tumor-bearing animals. The increase in number À À Xpc / mice (94 weeks) was significantly reduced (P = 0.0023, of tumor-bearing animals (excluding the pituitary adenomas and Kaplan-Meier with log-rank test) compared with the median taking into account the survival distribution) reaches significance À À À À survival of their female wild-type C57BL/6J controls (C57BL/6J 2, for Xpa / mice (P < 0.01) and approaches significance for Xpc / À À 102.5 weeks). Female Xpa / median survival (94.5 weeks), on the animals (P = 0.05) using the Peto test. other hand, was not significantly reduced (P = 0.6023, Kaplan-Meier Although a variety of nonneoplastic changes were observed in with log-rank test) compared with the median survival of its female the organs of mutant as well as wild-type mice, there were no www.aacrjournals.org 1349 Cancer Res 2008; 68: (5). March 1, 2008

À À À À obvious genotype-specific pathologies typical for Xpc / or Xpa / mice. The observed changes belonged to the normal background pathology of C57BL/6J mice and generally occurred to about the same degree in all groups. Because mutant animals died slightly earlier than wild-type mice, it may well be that these spontaneous À À À À aging lesions occurred slightly earlier in Xpc / and Xpa / mice than in wild-type animals. However, this aspect was not explicitly investigated in this study. Mutant frequency analyses. A comprehensive analysis of À À spontaneous Hprt mutant frequency was done with Xpc / , À Xpc+/ mice and their wild-type controls in a pure C57BL/6J background (Fig. 2). At the age of 52 weeks, Hprt mutant frequencies À (0.9 Â 10 6) in wild-type mice were in the same range as previously À À À reported (23). At this age, mutant frequencies in Xpc / and Xpc+/ mice were elevated 15- and 3-fold, respectively, when compared with their age-matched wild-type controls. In addition, 13-week-old À À Xpc / mice already exhibited a 5-fold increase in mutant frequency compared with the 52-week-old wild-type control animals. In addition, we determined mutant frequencies in several tissues À À À À of C57BL/6J, Xpc / , and Xpa / mice using the lacZ recovery system. Samples were taken from spleen, liver, and lung of 13-, 52-, and 78-week-old animals. Results are shown in Fig. 3. Table 2 depicts the P values of the compared genotypes in all three tissues at different ages. Mutant frequencies increased over time in spleen, À À À À liver, and lung of Xpc / mice. In Xpa / and C57BL/6J wild-type mice, an increase over time was visible in liver and lung, but not in spleen. Mutant frequencies in spleen showed a significant increase À À in mutants in 52- and 78-week-old Xpc / mice compared with À À their age-matched Xpa / and wild-type samples (Fig. 3A). Samples À À of Xpa / exhibited a similar lacZ mutant frequency pattern in the spleen as the wild-type samples over the entire life span. The liver À À À À of Xpc / and Xpa / mice showed a strong increase in mutant frequency compared with that of wild-type at 52 and 78 weeks of À À age (Fig. 3B). Xpc / liver samples exhibited slightly higher values À À on average than Xpa / samples at those time points. À À The mutant frequency in Xpc / lung samples was significantly elevated in comparison with wild-type controls at all time points. À À Xpa / mice only exhibited this increase at 13 and 52 weeks of age.

Figure 3. LacZ mutant frequencies including SDs of Xpc À/À, Xpa À/À and their wild-type controls at ages 13, 52, and 78 wk in spleen (A), liver (B), and lung (C). The numbers of biological replicas are between 5 and 6. For further details, see Materials and Methods.

À À At 78 weeks, the mutant frequency of Xpa / lungs was À À comparable to that of wild-type controls. Strikingly, Xpc / lungs showed a significant elevation in mutant frequency at ages of 52 and 78 weeks as compared with their NER-deficient counterpart À À À À Xpa / . A 2-fold increase was visible in Xpc / lung samples at 78 weeks in relation to both wild-type controls and Xpa-deficient samples (see Fig. 3C). Cell survival under oxygen exposure. In view of the increased tumor incidence and elevated spontaneous mutant frequency in À À lung, we suspected that the Xpc / animals were more susceptible to oxidative stress. To directly test this hypothesis, we investigated Figure 2. Hprt mutant frequencies including SDs of Xpc À/À, Xpc +/À and their wild-type controls at ages 13 and 52wk in spleen. The numbers of biological the effects of exposure to different levels of oxygen, 3% and 20%, À/À À/À replicas are between 4 and 10. For further details, see Materials and Methods. on Xpa , Xpc and wild-type MEFs. Results are shown in

Cancer Res 2008; 68: (5). March 1, 2008 1350 www.aacrjournals.org

À À Fig. 4. Xpc / fibroblasts subjected to 3% oxygen level seemed to and 55%, respectively). High incidences of ulcerative dermatitis in À À be slightly more sensitive than Xpa / and wild-type MEFs in the control C57BL/6J mice are considered an inevitable byproduct of À À first passage. Survival of Xpc / fibroblasts was 68% after 3 days handling the animals during their entire life span and are a result of À À of culturing (first passage in Fig. 4), whereas Xpa / and wild-type a Staphylococcus aureus infection. In addition, the type of diet fibroblasts exhibited 80% and 82% survival, respectively. After two seems to play a role in the severity of this skin phenotype. These more passages, at 3% oxygen pressure, 62% survival was visible in findings are, however, clearly in line with our earlier findings (35) À À À À cultured Xpc / fibroblasts. The survival of Xpa / and wild-type and those of others (36, 37) working particularly with female MEFs, at this point and under 3% oxygen pressure, was 79%. When, C57BL/6J mice. after the initial passage of 3% oxygen, the fibroblasts were cultured To our surprise, the percentage of tumor-bearing animals was for two more passages at 20% oxygen pressure, a severe decrease higher in NER-proficient mice than in their NER-deficient counter- À À in survival was exhibited in Xpc / fibroblasts. The percentage of parts. This observation is, next to the longer life span observed in survival dropped to a mere 39%. This dramatic decrease was not wild-type mice, mostly attributable to a dramatic decrease in À À À À observed for Xpa / and wild-type (67% and 74% survival, pituitary tumor development in Xpc / mice (from 70% to 24% À À respectively). occurrence) and in Xpa / mice (from 51% to 26% occurrence). A similar strong reduction in pituitary tumor development was TTD Discussion observed in another NER-deficient mouse model, Xpd (35). Here Previous results obtained in separate studies in which patterns of a significant decrease from 50% to 9% occurred. Interestingly, no survival, tumor formation, and mutation accumulation were deter- such a decrease in pituitary tumor development was found in the mined in Xpc- and Xpa-deficient mice were difficult to interpret TC-NER–deficient Csb mouse model (data not shown). Apparently, because of variation in genetic background and the use of different a defect in GG-NER is accompanied by suppression of a specific set mutational reporter genes. Here we carried out a side-by-side of tumor types. Further studies are needed to substantiate this comprehensive study of these end points in the same genetic hypothesis. background using both the Hprt and lacZ mutational target genes. A distinct difference between the two NER-deficient mouse À À NER-deficient mouse models lacking a functional XPA or XPC models is the observed tumor spectrum. Female Xpa / mice protein show phenotypic differences when compared with their exhibit a significant increase in hepatocellular adenomas compared À À wild-type controls. A significant reduction in spontaneous survival with wild-types. Female Xpc / mice also show an elevation in the was observed in our comprehensive longevity studies for female number of hepatocellular neoplasms, which approaches signifi- À À Xpc-deficient mice in a pure C57BL/6J background. Xpa-deficient cance. Additionally, female Xpc / mice do show a significant female mice also showed a decrease in survival when compared increase in bronchioalveolar neoplasms compared with their pro- À À with their wild-type control, although not statistically significant. ficient controls. Such an increase is absent in female Xpa / mice. À À À À Previously reported survival studies of Xpc / mice in a mixed Our results showed that female Xpc / mice in a pure C57BL/6J background did not exhibit a decrease in survival (24). Female background are susceptible to lung cancer and support the previous À À À À À À Xpa / and Xpc / mice in our studies reached an average median findings of the lung cancer susceptibility in Xpc / mice in the age of 94.5 and 94 weeks, respectively, whereas their matching wild- more sensitive mixed background (24). However, in this previous type controls attained a median survival of 103 and 102.5 weeks. study, the lung tumors observed and diagnosed as adenomas and Both the wild-type control cohorts show a similar survival pattern adenocarcinomas seemed to be more malignant and were and a comparable median age of 50% survival. As in humans, a accompanied by a higher incidence of atypical hyperplasia. Possibly, deficiency in one of these NER proteins leads to a reduced life span, depending on the genetic background and spontaneous (oxidative) and therefore XPA and XPC prove to be part of delicate and DNA damage levels, tumors will progress earlier to a more important processes that have a substantial effect on survival. malignant state. Next, several studies provide information that À À À À Additional pathologic analyses of female Xpa / and Xpc / Xpc polymorphisms in humans may also contribute to genetic mice showed that the cause of death in many of these animals was susceptibility for lung cancer (38–40). Exposure of Xpc-deficient not accountable to the presence of neoplasms. Another main cause mice (in a mixed genetic background) to the harmful genotoxic of death of the NER-deficient mice was in fact the occurrence of carcinogen 2-acetylaminofluorene (2-AAF) resulted in a significant inflammation, mainly ulcerative dermatitis. For the C57BL/6J elevation in the number of liver and lung tumors compared with À À À À 1 group (which was simultaneously executed with the Xpa / wild-type animals (18, 41). In contrast, Xpa / mice in a pure À À study) and Xpa / , high incidences of inflammation were apparent C57BL/6J background only show an elevation in liver tumors after À À (50% and 61%, respectively). The C57BL/6J 2 and the Xpc / exposure to 2-AAF, and no increase in lung tumors was apparent in À À studies showed a somewhat lower incidence of inflammation (24% that study (18). The occurrence of lung tumors in Xpc / mice in

Table 2. P values of mutant frequency comparisons between genotypes, depicted per age per tissue

Spleen 13 wk 52wk 78 wk Liver 13 wk 52wk 78 wk Lung 13 wk 52wk 78 wk

Xpc vs WT 0.016 0.012 Xpc vs WT 0.002 Xpc vs WT 0.04 0.00003 0.0001 Xpc vs Xpa 0.0098 0.007 Xpc vs Xpa Xpc vs Xpa 0.02 0.0005 Xpa vs WT Xpa vs WT 0.04 0.002 Xpa vs WT 0.003 0.003

NOTE: Open cells represent nonsignificant (P > 0.05) differences.

www.aacrjournals.org 1351 Cancer Res 2008; 68: (5). March 1, 2008

À À À À analyses on several tissues of wild-type, Xpa / , and Xpc / female mice. Initial spontaneous Hprt mutant analyses in spleen showed a strong increase of mutant frequency in T lymphocytes of À À À À 52-week-old Xpc / mice compared with their age-matched Xpa / À À and C57BL/6J controls. However, in an earlier study, Xpc / mice with a mixed background were used (23). More comprehensive Hprt analyses using Xpc-deficient mice in a pure C57BL/6J background were done here. The strong increase in mutant frequency compared with the wild-type control was reproducible in À À Xpc / in a pure C57BL/6J background, albeit lower than in a mixed background. The results obtained with lacZ mutant analyses À À show a more moderate response in spleen when Xpc / is compared with wild-type. This can be explained by the fact that the background level of the lacZ mutant analyses is higher than that of Hprt. Although Hprt analysis is a more sensitive method than lacZ analyses, its drawback is that it is only applicable to the spleen. Other previous studies conducted here showed an elevation of À À mutant frequencies in Xpa / in liver and kidney using lacZ analyses (43, 44). Our results of the lacZ mutant analyses show a striking increase À À of mutant frequency in lung tissue of Xpc / mice when compared À À with wild-type and Xpa / mice, especially at the age of 78 weeks. À À The fact that no severe increase in lung is observed in Xpa / mice À À but is distinctly present in Xpc / mice supports the hypothesis that the XPC protein might be involved in the removal of oxidative DNA damage, because the level of oxidative stress is higher in lungs compared with other tissues due to the constant exposure of À À this tissue to oxygen (45). In the spleen, Xpc / mice also show a significant increase in mutant frequency compared with wild- À À type and Xpa / at 52 and 78 weeks. In liver, on the other hand, À À À À the mutant frequencies of Xpa / and Xpc / liver are virtually equal over all time points. DNA damage in the liver most likely arises as a result of genotoxic bulky byproducts of metabolism. Compared with wild-type, 78-week-old NER-deficient mouse livers do show a significant elevation of mutant frequency. This illustrates the sensitivity of the NER-deficient mouse strains to À À DNA damage. Over time and in all investigated tissues, Xpc / mice show the highest mutant frequency, indicating an even higher À À sensitivity to DNA damage than Xpa / animals. The putative role in the removal of oxidative damage is À À supported by our in vitro data, in which Xpc / MEFs exhibit a severe decrease in survival when cultured at 20% oxygen compared with 3% oxygen pressure. We believe that the excess of oxidative damage is most likely the cause of death because oxygen pressure is the only variable that was changed in culturing. Xpc-deficient fibroblasts seem to be impaired in the removal of this damage. Xpc Figure 4. Survival of MEFs including SDs of Xpc À/À, Xpa À/À and their wild-type Even at a low oxygen level of 3%, growth of -deficient cells controls under different levels of oxygen pressure. Cells at the first passage seems to be more sensitive compared with Xpa-deficient and wild- were all cultured in 3% oxygen (A); subsequent passages were cultured in 3% type fibroblasts. (B)or20%(C) oxygen. À À The high mutant frequencies in Xpc / cells in oxygen-rich surroundings and the severe decrease in cell survival under 20% pure C57BL/6J background after exposure to 2-AAF has not yet been oxygen pressure are concurrent with the present understandings À À assessed. The difference in tumor spectrum between Xpc / and and recent discoveries about the XPC protein. Several recent À À Xpa / mice in our study indicates that the XPC protein is, besides studies provide information about the involvement of XPC in BER active in NER, possibly also involved other repair systems, most and the putative role in the repair of oxidative DNA damage. BER is likely including BER. This idea was recently also put forward by mainly responsible for mending the DNA damage caused by À À others (42). The outcome of an increase in lung tumors in Xpc / oxidative stress (46). XPC can physically and functionally interact mice could, therefore, point to an involvement of the XPC protein in with thymine DNA glycosylase, which plays a role in BER (47). the repair of oxidative DNA damage. XPC-HR23Balso has been assigned as a cofactor for BERof To analyze the mutation spectrum in repair-deficient mice in the 8-hydroxyguanine by stimulating the activity of its specific DNA different organs, we conducted additional mutant frequency glycosylase OGG1 (48). In addition, a recent study has shown a

Cancer Res 2008; 68: (5). March 1, 2008 1352 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Mouse Models for XP and Their Cancer Phenotypes deficient BER in XPC fibroblasts after oxidative DNA damage consequence of this engagement could explain the difference in À À À À induced by methylene blue plus visible light (49). Methylene blue tumor spectrum between Xpa / and Xpc / mice. and visible light produce oxidative DNA damage, among others 8-hydroxyguanine. Our results indicate the importance of the XPC protein in vivo, Acknowledgments where the absence of the protein is responsible for susceptibility to Received 10/31/2007; revised 12/19/2007; accepted 12/19/2007. lung tumors in mice compared with wild-type and their NER- Grant support: NIH/National Institute of Aging grant 1 PO1 AG-17242, National XpaÀ/À Institute of Environmental Health Sciences grant 1UO1 ES011044, Netherlands deficient counterpart . Mutant frequency analyses show an Organization for Scientific Research (NWO) through the foundation of the Research additional sensitivity in spleen compared with Xpa-deficient Institute Diseases of the Elderly, Dutch Cancer Society grant EUR 99-2004, and EC Xpa XpcÀ/À grant QRTL-1999-02002. animals. In accordance with deficiency, mice also The costs of publication of this article were defrayed in part by the payment of page show a high mutant frequency in liver. Therefore, our results, charges. This article must therefore be hereby marked advertisement in accordance together with the accumulating evidence provided by others, with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Piet de With, Gwen Intres, Conny van Oostrom, Coen Moolenbeek, support the theory of XPC involvement in BER or additional Christine Soputan, Sandra de Waal, Sisca de Vlugt, and Ewoud Speksnijder for their pathways and the removal of oxidative DNA damage. A subsequent skillful technical assistance.

References repair-deficient mice is associated with a defect in 34. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, global genome repair but not with transcription coupled Jacks T, Hannon GJ. Radiation-induced cell cycle arrest 1. Lengauer C, Kinzler KW, Vogelstein B. Genetic repair. DNA Repair (Amst) 2005;4:3–9. compromised by p21 deficiency. Nature 1995;377:552–7. instabilities in human cancers. Nature 1998;396:643–9. 19. de Vries A, van Oostrom CT, Hofhuis FM, et al. 35. Wijnhoven SW, Beems RB, Roodbergen M, et al. 2. Takata M, Sasaki MS, Sonoda E, et al. Homologous Increased susceptibility to ultraviolet-Band carcinogens Accelerated aging pathology in ad libitum fed Xpd(TTD) recombination and non-homologous end-joining path- of mice lacking the DNA excision repair gene XPA. mice is accompanied by features suggestive of caloric ways of DNA double-strand break repair have over- Nature 1995;377:169–73. restriction. DNA Repair (Amst) 2005;4:1314–24. lapping roles in the maintenance of chromosomal 20. van der Horst GT, van Steeg H, Berg RJ, et al. 36. Kastenmayer RJ, Fain MA, Perdue KA. A retrospective integrity in vertebrate cells. EMBO J 1998;17:5497–508. Defective transcription-coupled repair in Cockayne study of idiopathic ulcerative dermatitis in mice with a 3. Friedberg EC. DNA damage and repair. Nature 2003; syndrome Bmice is associated with skin cancer C57BL/6 background. J Am Assoc Lab Anim Sci 2006;45: 421:436–40. predisposition. Cell 1997;89:425–35. 8–12. 4. Friedberg EC. How nucleotide excision repair protects 21. Berg RJ, de Vries A, van Steeg H, de Gruijl FR. Relative 37. Lawson GW, Sato A, Fairbanks LA, Lawson PT. against cancer. Nat Rev Cancer 2001;1:22–33. 5. Balajee AS, Bohr VA. Genomic heterogeneity of susceptibilities of XPA knockout mice and their Vitamin E as a treatment for ulcerative dermatitis in nucleotide excision repair. Gene 2000;250:15–30. heterozygous and wild-type littermates to UVB-induced C57BL/6 mice and strains with a C57BL/6 background. 6. Thoma BS, Vasquez KM. Critical DNA damage skin cancer. Cancer Res 1997;57:581–4. Contemp Top Lab Anim Sci 2005;44:18–21. recognition functions of XPC-hHR23Band XPA-RPA in 22. Meira LB, Reis AM, Cheo DL, Nahari D, Burns DK, 38. Lee GY, Jang JS, Lee SY, et al. XPC polymorphisms nucleotide excision repair. Mol Carcinog 2003;38:1–13. Friedberg EC. Cancer predisposition in mutant mice and lung cancer risk. Int J Cancer 2005;115:807–13. 7. van der Wees C, Jansen J, Vrieling H, van der LA, Van defective in multiple genetic pathways: uncovering 39. Bai Y, Xu L, Yang X, et al. Sequence variations in DNA Zeeland A, Mullenders L. Nucleotide excision repair in important genetic interactions. Mutat Res 2001;477: repair gene XPC is associated with lung cancer risk in a differentiated cells. Mutat Res 2007;614:16–23. 51–8. Chinese population: a case-control study. BMC Cancer 8. Kraemer KH, Patronas NJ, Schiffmann R, Brooks BP, 23. Wijnhoven SW, Kool HJ, Mullenders LH, et al. Age- 2007;7:81. Tamura D, DiGiovanna JJ. Xeroderma pigmentosum, dependent spontaneous mutagenesis in Xpc mice 40. Hu Z, Wang Y, Wang X, et al. DNA repair gene XPC trichothiodystrophy and Cockayne syndrome: a complex defective in nucleotide excision repair. Oncogene 2000; genotypes/haplotypes and risk of lung cancer in a genotype-phenotype relationship. Neuroscience 2007; 19:5034–7. Chinese population. Int J Cancer 2005;115:478–83. 145:1388–96. 24. Hollander MC, Philburn RT, Patterson AD, et al. 41. Cheo DL, Burns DK, Meira LB, Houle JF, Friedberg 9. de Boer J, Hoeijmakers JH. Nucleotide excision repair Deletion of XPC leads to lung tumors in mice and is EC. Mutational inactivation of the xeroderma pigmen- and human syndromes. Carcinogenesis 2000;21:453–60. associated with early events in human lung carcinogen- tosum group C gene confers predisposition to 2- 10. Bootsma D, Weeda G, Vermeulen W, et al. Nucleotide esis. Proc Natl Acad Sci U S A 2005;102:13200–5. acetylaminofluorene-induced liver and lung cancer and À À excision repair syndromes: molecular basis and clinical 25. de Vries A, van Steeg H. Xpa knockout mice. Semin to spontaneous testicular cancer in Trp53 / mice. symptoms. Philos Trans R Soc Lond BBiolSci 1995;347: Cancer Biol 1996;7:229–40. Cancer Res 1999;59:771–5. 75–81. 26. Cheo DL, Ruven HJ, Meira LB, et al. Characterization 42. Nahari D, McDaniel LD, Task LB, Daniel RL, Velasco- 11. Yasuda T, Sugasawa K, Shimizu Y, Iwai S, Shiomi of defective nucleotide excision repair in XPC mutant Miguel S, Friedberg EC. Mutations in the Trp53 gene of T, Hanaoka F. Nucleosomal structure of undamaged mice. Mutat Res 1997;374:1–9. UV-irradiated Xpc mutant mice suggest a novel Xpc- DNA regions suppresses the non-specific DNA 27. Dolle ME, Giese H, Hopkins CL, Martus HJ, Hausdorff dependent DNA repair process. DNA Repair (Amst) binding of the XPC complex. DNA Repair (Amst) JM, Vijg J. Rapid accumulation of genome rearrange- 2004;3:379–86. 2005;4:389–95. ments in liver but not in brain of old mice. Nat Genet 43. Dolle ME, Busuttil RA, Garcia AM, et al. Increased 12. Hanawalt PC, Ford JM, Lloyd DR. Functional 1997;17:431–4. genomic instability is not a prerequisite for shortened characterization of global genomic DNA repair and its 28. Wijnhoven SW, Kool HJ, Mullenders LH, Slater R, van lifespan in DNA repair deficient mice. Mutat Res 2006; implications for cancer. Mutat Res 2003;544:107–14. Zeeland AA, Vrieling H. DMBA-induced toxic and 596:22–35. 13. Yokoi M, Masutani C, Maekawa T, Sugasawa K, mutagenic responses vary dramatically between NER- 44. Giese H, Dolle ME, Hezel A, van Steeg H, Vijg J. Ohkuma Y, Hanaoka F. The xeroderma pigmentosum deficient Xpa, Xpc and Csb mice. Carcinogenesis 2001; Accelerated accumulation of somatic mutations in mice group C protein complex XPC-HR23Bplays an impor- 22:1099–106. deficient in the nucleotide excision repair gene XPA. tant role in the recruitment of transcription factor IIH 29. Tates AD, van Dam FJ, Natarajan AT, Zwinderman AH, Oncogene 1999;18:1257–60. to damaged DNA. J Biol Chem 2000;275:9870–5. Osanto S. Frequencies of HPRT mutants and micronuclei 45. van der Vliet A and Cross CE. Oxidants, nitrosants, 14. Sugasawa K. The xeroderma pigmentosum group C in lymphocytes of cancer patients under chemotherapy: and the lung. Am J Med 2000;109:398–421. protein complex and ultraviolet-damaged DNA-binding a prospective study. Mutat Res 1994;307:293–306. 46. Hazra TK, Das A, Das S, Choudhury S, Kow YW, Roy protein: functional assays for damage recognition 30. Wijnhoven SW, Sonneveld E, Kool HJ, van Teijlingen R. Oxidative DNA damage repair in mammalian cells: a factors involved in global genome Repair. Methods CM, Vrieling H. Chemical carcinogens induce varying new perspective. DNA Repair (Amst) 2007;6:470–80. Enzymol 2006;408:171–88. patterns of LOH in mouse T-lymphocytes. Carcinogen- 47. Shimizu Y, Iwai S, Hanaoka F, Sugasawa K. Xero- 15. Park CJ, Choi BS. The protein shuffle. Sequential esis 2003;24:139–44. derma pigmentosum group C protein interacts physi- interactions among components of the human nucleo- 31. Dolle ME, Martus HJ, Gossen JA, Boerrigter ME, Vijg cally and functionally with thymine DNA glycosylase. tide excision repair pathway. FEBS J 2006;273:1600–8. J. Evaluation of a plasmid-based transgenic mouse EMBO J 2003;22:164–73. 16. Costa RM, Chigancas V, Galhardo RS, Carvalho H, model for detecting in vivo mutations. Mutagenesis 48. D’Errico M, Parlanti E, Teson M, et al. New functions Menck CF. The eukaryotic nucleotide excision repair 1996;11:111–8. of XPC in the protection of human skin cells from pathway. Biochimie 2003;85:1083–99. 32. Peto R, Pike MC, Day NE, et al. Guidelines for simple, oxidative damage. EMBO J 2006;25:4305–15. 17. Cleaver JE. Cancer in xeroderma pigmentosum and sensitive significance tests for carcinogenic effects in 49. Kassam SN, Rainbow AJ. Deficient base excision related disorders of DNA repair. Nat Rev Cancer 2005;5: long-term animal experiments. IARC Monogr Eval repair of oxidative DNA damage induced by methylene 564–73. Carcinog Risk Chem Hum Suppl 1980;(2 Suppl):311–426. blue plus visible light in xeroderma pigmentosum group 18. Hoogervorst EM, van Oostrom CT, Beems RB, et al. 2- 33. Moon H, Ahn H, Kodell RL. An age-adjusted C fibroblasts. Biochem Biophys Res Commun 2007;359: AAF-induced tumor development in nucleotide excision bootstrap-based Poly-k test. Stat Med 2005;24:1233–44. 1004–9. www.aacrjournals.org 1353 Cancer Res 2008; 68: (5). March 1, 2008

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Mouse Models for Xeroderma Pigmentosum Group A and Group C Show Divergent Cancer Phenotypes

Joost P.M. Melis, Susan W.P. Wijnhoven, Rudolf B. Beems, et al.

Cancer Res 2008;68:1347-1353.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/68/5/1347

Cited articles This article cites 49 articles, 5 of which you can access for free at: http://cancerres.aacrjournals.org/content/68/5/1347.full#ref-list-1

Citing articles This article has been cited by 9 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/68/5/1347.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/68/5/1347. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.