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.