(CANCER RESEARCH 52. 3347-3352. June 15. 1992] Activation of the Ha-, Ki-, and N-ras Genes in Chemically Induced Liver Tumors from CD-I Mice Sujata Mananu' Richard D. Storer, Srinivasa Prahalada, Karen R. Leander, Andrew R. Kraynak, Brian J. Ledwith, Matthew J. van Zwieten, Matthews O. Bradley, and Warren W. Nichols Department of Safety Assessment, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486 ABSTRACT although the frequency of these mutations varies among mouse strains (6, 10-13). Previous studies in B6C3F,2 mice demon We compared the profilo of ras gene mutations in spontaneous CD-I strated that liver tumors induced by some genotoxic carcinogens mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene(DMBA), (6, 14, 15) and nongenotoxic carcinogens (11) had different 4-aminoazobenzene, Ar-hydroxy-2-acetylaminofluorene, or /V-nitrosodi- frequencies or profiles of ras gene point mutations than were ethylamine. By direct sequencing of polymerase chain reaction-amplified seen in spontaneous tumors. However, B6C3F, mouse liver tumor DNA, the carcinogen-induced tumors were found to have much tumors induced by benzidine (11), N-OH-AAF (14), and DEN higher frequencies of ras gene activation than spontaneous tumors. Fur (16) were reported to have a similar pattern of Ha-ras codon thermore, each carcinogen caused specific types of ras mutations not 61 mutations to that seen in spontaneous liver tumors. detected in spontaneous tumors, including several novel mutations not Determining the general applicability of ras gene analysis for previously associated with either the carcinogen or mouse hepatocarcin- distinguishing chemically induced tumors from spontaneous ogenesis. For example, the model compound DMBA is known to cause tumors requires further evaluation, including studies of other predominantly A to T tranversions in Ha-rai codon 61 in mouse skin and carcinogens and other mouse strains. In this study, we examined mammary tumors, consistent with the ability of DMBA to form bulky ras gene activation in liver tumors from Crl:CD-l(ICR)BR mice adducts with adenosine. Our results demonstrate that the predominant (referred to as CD-I mice), a strain used for carcinogenicity mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-rflj codon 13 (DMBA is also known to form guanosine adducts), studies in several laboratories including our own (8). We com illustrating the influence of both chemical- and tissue-specific factors in pared spontaneous CD-I mouse liver tumors with liver tumors determining the type of ras gene mutations in a tumor. 4-Aminoazoben- induced by a single dose of either DMBA, N-OH-AAF, DEN, zene and /V-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon or AAB. 13 mutation. In addition, we found that .V-nitrosodiethylamine, 4-ami noazobenzene, and Ar-hydroxy-2-acetylaminofluorene all caused G to T MATERIALS AND METHODS transversions in the N-ra; gene (codons 12 or 13). This is the first demonstration of Vru.v mutations in mouse liver tumors, establishing a Induction of CD-I Mouse Liver Tumors. DEN was purchased from role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison Sigma Chemical Co., St. Louis, MO. DMBA and AAB were purchased of the nix mutations detected in the direct tumor analysis with those from Aldrich Chemical Co., Milwaukee, WI. N-OH-AAF was pur detected after NIH3T3 cell transaction indicates that spontaneous ras chased from CCR, Inc., Chanhassen, MN. Each carcinogen was dis mutations (in Ha-ras codon 61) are often present in only a small fraction solved in 10% dimethyl sulfoxide in trioctanoin. of the tumor cells, raising the possibility that they may sometimes occur Twelve-day-old male CD-I mice (Charles River Breeding Laborato as a late event in CD-I mouse hepatocarcinogenesis. ries, Wilmington, MA), 41-42 animals/treatment group, were given a single i.p. injection of either DMBA (20 nmol/g body weight), AAB (400 nmol/g), N-OH-AAF (200 nmol/g), DEN (20 nmol/g), or the INTRODUCTION vehicle alone. Liver tumors were harvested from surviving mice 9 or 12 months after injection of the carcinogens (29 of 36 N-OH-AAF-treated Characterization of the genetic lesions caused by carcinogens mice had a total of 355 tumors; 23 of 31 DMBA-treated mice had a in tumor DNA enhances our understanding of the mechanisms total of 226 tumors; 26 of 37 AAB-treated mice had a total of 256 involved in chemical carcinogenesis, while helping to delineate tumors; 30 of 35 DEN-treated mice had a total of 256 tumors; 3 of 38 the multiple steps involved in carcinogenesis in different tissues. vehicle-treated mice had a total of only 9 tumors). Spontaneous liver One of the most highly characterized lesions associated with tumors were isolated from an aging colony of male CD-I mice that carcinogenesis is the activation of the ras genes by point mu were approximately 22 months (94 to 98 weeks) old, when survival was tation (1-7). Several genotoxic carcinogens have been shown to about 54% and approximately 58% of the surviving mice had tumors induce particular patterns of ras gene point mutations, which (282 tumors in 125 mice). Note that number of tumors actually refers in certain cases can be correlated to the specificity of the to grossly visible nodules; Histological examination was performed on carcinogen for interacting with particular bases in DNA (re only those tumors that were taken for analysis. viewed in Refs. 3 and 4). Only those tumors greater than 3 mm in diameter were taken for DNA isolation. A section of each tumor with adjacent normal tissue The specificity of certain carcinogens for inducing particular was examined histologically. A portion of the tumor was carefully patterns of ras gene point mutations has been used to help trimmed of surrounding normal tissue and rapidly frozen in liquid distinguish chemically induced liver tumors from spontaneous nitrogen. Tumor samples were stored at -70°C until they were proc liver tumors, which arise at high and variable incidence in essed for DNA extraction. In addition to the liver tumors, normal liver several of the mouse strains used for long-term carcinogenicity (or kidney) tissue from each mouse was also frozen for use as a negative studies (8, 9). In spontaneous mouse liver tumors, ras gene control for â„¢¿.\mutationanalysis. point mutations are exclusively found in Ha-ras codon 61, DNA Isolation. High molecular weight DNA was purified from pulverized frozen tissue by lysis in 4 M guanidinium isothiocyanate Received 10/23/91; accepted 4/3/92. solution followed by CsCl gradient centrifugation, treatment with pro- 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 2The abbreviations used are: B6C3F, mice. C57BL/6 x C3HF, mice; N-OH- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. AAF, A'-hydro?iy-2-acetylaminofluorene; DEN. iV-nitrosodiethylamine: DMBA, 1To whom requests for reprints should be addressed, at WP45-321, Merck 7.12-dimethylbenz(a)anthracene: AAB. 4-aminoazobcnzene; CD-1 mice. Crl:CD- Sharp and Dohme, West Point, PA 19486. 1(ICR)BR mice; PCR, polymerase chain reaction. 3347 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. ACTIVATED ras GENES IN MURINE LIVER TUMORS teinase K and RNase A, phenol-chloroform extraction, and ethanol for each type of point mutation detected in the CD-I mouse precipitation, essentially as described by others (17). liver tumors is shown in Figs. 1, 2, and 3 (for Ha-, Ki-, and N- ras Mutation Analysis. Direct preparations of tumor DNA were ras mutations, respectively). In all tumors that had a ras gene analyzed for ras mutations by PCR amplification followed by Sanger dideoxy-DNA sequencing using 5'-end-labeled primers. The sequences mutation, at least 50% of the normal ras alíelewasalso present, of the oligonucleotide primers used for amplification and sequencing suggesting that most or all of these tumors were heterozygous and the procedures used for amplification, sequencing, and 5'-end for their mutation. labeling are described in detail elsewhere (18). For comparative pur DMBA-induced tumors contained predominantly Ki-ras co poses, tumors were also analyzed by transfection of the tumor DNA don 13 (G to C) transversions. In addition, four DMBA-induced into NIH3T3 cells, essentially as described previously (19). After trans tumors had an A to T transversion in Ha-ras codon 61. Neither fection, DNA isolated from the NIH3T3 cell transformants was assayed of these mutations was detected in the spontaneous CD-I liver for ras mutations by the same procedures used for direct tumor DNA. tumors (Table 1). Similarly, AAB-induced tumors had predom In both the direct tumor DNA analysis and the transfection assay, inantly Ki-ras codon 13 mutations, as well as several N-ras normal tissue DNA (from the same mouse as each tumor) was used as mutations (G to T transversions in codon 12 or 13). Several N- a negative control. OH-AAF- and DEN-induced tumors had the CAA to AAA Ha ras codon 61 mutation that was found in spontaneous tumors RESULTS (Table 1). In addition, DEN caused G to T transversions in Induction of CD-I Mouse Liver Tumors. Each group of car codon 12 of N-ras, and AAF caused G to C transversions in cinogen-treated mice exhibited a high incidence of liver tumors, Ki-ras codon 13 and G to T transversions in N-ras codons 12 ranging from 70 to 86% of the surviving mice, with a total of and 13.