[CANCER RESEARCH 59, 771–775, February 15, 1999] Advances in Brief Mutational Inactivation of the Xeroderma Pigmentosum Group C Gene Confers Predisposition to 2-Acetylaminofluorene-induced Liver and Lung Cancer and to Spontaneous Testicular Cancer in Trp53 / Mice 1 David L. Cheo, Dennis K. Burns, Lisiane B. Meira, Jean Francois Houle, and Errol C. Friedberg 2 Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 Abstract Mice that are genetically engineered to mimic the human hereditary cancer-prone DNA repair-defective disease xeroderma pigmentosum (XP) are highly predisposed to UV radiation-induced skin cancer. It is not clear, however, whether XP mice or humans are predisposed to cancers in other tissues associated with exposure to environmental carcinogens. To test the importance of nucleotide excision repair in protection against chemical carcinogenesis in internal organs, we treated XPC mutant (XPC / ) mice with 2-acetylaminofluorene and NOH-2-acetylaminoflu- orene. We observed a significantly higher incidence of chemically induced liver and lung tumors in XPC / mice compared with normal and het- erozygous littermates. In addition, the progression of liver tumors in XPC / Trp53 / mice is accelerated compared with XPC / Trp53 / animals. Finally, we demonstrate a higher incidence of spontaneous tes- ticular tumors in XPC / Trp53 / double mutant mice compared with XPC / Trp53 / mice. Introduction The hereditary human disease XP 3 is characterized by defective NER of base damage that results from exposure to UV radiation and to a diverse range of chemical carcinogens (1, 2). It is therefore anticipated that this disease would confer a predisposition not only to skin cancer, as has been extensively documented (3, 4), but also to cancers of internal organs typically associated with environmental exposure (5). However, the rarity of the disease coupled with the fact that very few XP patients have been closely monitored for cancer of tissues and organs other than the skin have left this issue in consid- erable doubt (5). Thus, it remains unproved that humans with defec- tive NER are indeed prone to cancer in organs other than those normally exposed to sunlight. Here we report that mice defective in the NER gene XPC, which are known to be highly predisposed to skin cancer after exposure to UVB radiation (6, 7), are also highly predis- posed to liver and lung cancer after treatment with either AAF or its activated derivative, NOH-AAF. We also demonstrate that XPC -/- Trp53 -/- double mutant mice have an increased susceptibility to spontaneous testicular tumors. Materials and Methods XPC mutant mice were generated previously by us (8), and Trp53 mutant mice were purchased from The Jackson Induced Mutant Resource. Mice used in this study were generated from crosses between mice heterozygous for both XPC and Trp53 (as described in Ref. 7) and consisted of all nine possible combinations of mutant and normal alleles of the two genes. Two week-old pups were either mock-treated or treated with a single i.p. injection of AAF (400 nmol/g body weight; Sigma Chemical Co.) or N-OH-AAF (200 nmol/g body weight; CCR, Inc., Chanhassen, MN) dissolved in DMSO and diluted one-tenth in tricaprylin (Sigma), as described (9). All animals were of the same strain background (75% 129/Sv, 25% C57Bl/6) and maintained under standard laboratory conditions until they were sacrificed 14 –16 months after treatment for complete autopsy examination. Histological analysis was routinely per- formed on the lungs, liver, heart, spleen, intestine, and stomach of all animals and on any other organs in which gross pathology was noted at autopsy. Results and Discussion Chemically Induced Liver and Lung Tumors in XPC Trp53 Mutant Mice. All Trp53 -/- animals succumbed to spontaneous tu- mors frequently associated with this genotype, i.e., lymphomas, soft tissue sarcomas, or testicular tumors (10), before completion of the experiment and are not included in the data sets presented here. Gross examination of the liver and lungs of animals treated with AAF or NOH-AAF revealed multiple and frequently confluent hepatic and/or pulmonary nodules of varying size and shape (Fig. 1). We typically observed multiple tumor nodules in affected organs. However, these were often confluent and precluded precise determination of the number of tumors/organ. We estimate that two to five lesions were present in every affected organ on the average. Microscopic exami- nation of these nodules showed that they comprised either premalig- nant or frankly malignant lesions. The former presented as hyperplas- tic nodules in the liver or benign adenomas of the lung, and the latter presented as hepatocellular carcinoma or adenocarcinoma of the lung (Fig. 1). In some cases, foci of malignant change were detected in otherwise benign hyperplastic lesions. These were scored as malig- nant. The histological appearance of the premalignant and malignant lesions of the liver and lungs was very distinctive (Fig. 1). We did not observe lung tumors that were metastases from the liver, or vice versa. Tables 1 and 2 show the proportion of premalignant hyperplastic or adenomatous and malignant lesions in the lungs and/or liver in the various genotypes examined. Results from Trp53 +/+ animals are shown in Table 1 and from Trp53 +/- animals in Table 2. Liver and lung lesions in every affected animal are recorded in the Tables on a case by case basis. Of 17 mock-treated animals representing all six genotypes, none developed neoplastic lesions of any kind, with the exception of a single osteosarcoma in one XPC -/- Trp53 +/- animal (data not shown). Because the liver and lungs represent distinct target organs, either of which can undergo AAF- or NOH-AAF-induced neoplastic change, we documented the fraction of total target organs affected in each genotype with each carcinogen. We also documented the fraction of total animals in which both the liver and lungs were affected and the fraction of total animals in which either target organ was affected in each genotype with each carcinogen. A marked predisposition to chemically induced neoplastic change Received 11/20/98; accepted 1/5/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These studies were supported by research Grant CA44247 (to E. C. F.) and a postdoctoral fellowship from the American Cancer Society (to D. L. C.). 2 To whom requests for reprints should be addressed, at Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX 75235-9072. Phone: (214) 648-4020; Fax: (214) 648-4067; E-mail: friedberg.errol@pathology.swmed.edu. 3 The abbreviations used are: XP, xeroderma pigmentosum; XPC, xeroderma pigmen- tosum group C gene; NER, nucleotide excision repair; AAF, 2-acetylaminofluorene; NOH-AAF, N-hydroxy-2-acetylaminofluorene. 771