Activation of the Ha-, Ki-, and N-Ras Genes in Chemically Induced Liver Tumors from CD-I Mice Sujata Mananu' Richard D

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(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 -70Â°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

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 alÃelewasalso 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. over 200 tumors in each treatment group (see "Materials and ras Gene Analysis Using the DNA Transfection Assay. For Methods"). In contrast, only 9 liver tumors were observed in comparative purposes, in addition to the direct tumor DNA vehicle-treated control mice (9 to 12 months old), indicating analysis described above, we also examined the liver tumors that the vast majority of tumors in each of the carcinogen- (from which a sufficient amount of DNA was isolated) using treated groups were induced by the single dose of the chemical. the NIH3T3 cell transfection assay. Note that after transfection, Spontaneous liver tumors were isolated from an aging colony DNA samples from transformed foci of NIH3T3 cells were of male CD-I mice that were approximately 22 months old. At analyzed for ras mutations by the same procedures used for this age, 58% of the surviving mice had liver tumors. Although direct tumor DNA analysis, i.e., sequencing of PCR-amplified the spontaneous tumors were taken from older mice, they were genomic DNA. Thus, only the source of DNA differed between similar to the carcinogen-induced tumors in size range and in the two assays (transfectant DNA versus direct tumor DNA). the proportion of adenomas versus carcinomas (data not Table 2 shows the percentage of spontaneous and carcinogen- shown). induced tumors that gave positive transformed foci in the Thus, the 9- to 12-month-old carcinogen-treated mice and transfection assay and also shows the profile of ras mutations the 22-month-old aging mice provided us with essentially pure that were detected in DNA isolated from the transfected cells. populations of chemically induced tumors and spontaneous As expected, tumors with ras mutations detected by direct tumors, respectively. For this study, 25 to 40 liver tumors from tumor analysis were usually positive in the transfection assay, each treatment group (representing a partial sampling of each and the transfectant DNA generally had the same mutation as tumor group) were examined histologically and assayed for ras the direct tumor DNA (data not shown on an individual tumor gene mutations. basis). Several Ki-ras and N-ras mutations that were detected Frequency and Profile of ras Mutations Detected by Direct by direct tumor DNA analysis were missed by the transfection Tumor DNA Analysis. As shown in Table 1, each group of assay, which is not unexpected given the known limitations of carcinogen-induced liver tumors exhibited a fairly high fre the transfection assay (see "Discussion"). quency of ras gene activation, ranging from 32 to 64% as More notably, transfection detected mutations in a number detected by direct tumor DNA analysis. In contrast, only about of tumors that were negative in the direct tumor DNA analysis 8% of spontaneous liver tumors had activated ras oncogenes. and thereby enhanced the percentage of tumors found to be Furthermore, the three mutations detected in spontaneous liver positive for ras mutation by over 3-fold in spontaneous tumors tumors were all located in Ha-ras codon 61 (CAA to AAA or and AAB-induced tumors, and to a lesser extent in DEN- and CGA), whereas at least one-half of the ras mutations found in N-OH-AAF-induced tumors (compare Table 2 with Table 1). each group of carcinogen-induced tumors were located in other In order to determine the nature of these mutations, we tabu ras gene codons. An example of the DNA sequencing results lated (Table 2) the ras mutation profile for only those tumors

Table 1 Frequency and profile of activating ras mutations in CD-I mouse liver tumors: direct tumor DNA analysis Tumors were assayed for ras mutations by direct sequencing of PCR-amplified tumor DNA. Codons 12-13 and 59-61 of the Ha-, Ki-, and N-ras genes were examined in all samples. DNACarcinogenSpontaneous Profile of ras mutations observed in tumor 6 1 12 13 of (CAA)CGA (CGC)CGC1(1 (GGT)TGT (GGT)TGT3 tumors with rasmutations3/36 CTA14Ki-roj]3 GTT1 (8)" AAB* 12/37(32) N-OH-AAF 19/34(56) 617N-raf 14N-ras 3 DEN 8/25 (32) DM BAFraction 21/33(64)AAA294Ha-ras ' Numbers in parentheses, percentage. *Two AAB-induced tumors had coincident mutations detected in two ras gene codons. 3348

A. Normal B. CAA â€”¿AAA C. CAA - CGA D. CAA - CTA

ACGT A CG T AC G T A CG T Fig. 1. Representative DNA sequences of the codon 61 region of the Ha-ras gene. All .=' sequences shown are of the sense strand and were obtained by direct sequencing of PCR- amplified liver tumor (or normal liver tissue) DNA. A, wild-type sequence derived from nor mal liver tissue DNA; B, Câ€”Â»Atransversionin codon 61 detected in a spontaneous tumor; C, A4, transition in codon 61 detected in a Câ€”-A spontaneous tumor; D, Aâ€”Â»Ttransversionin codon 61 detected in a DMBA-induced tumor. *-â€¢¿

that were negative in the direct tumor DNA assay but positive type of ras mutation found in a tumor, as exemplified by the in the transfection assay. Remarkably, all of the ras mutations DMBA-induced liver tumors. DMBA is thought to have a detected in these transfectants were Ha-ras codon 61 mutations strong preference for chemically interacting with adenine (20- characteristic of spontaneous tumors, regardless of whether 22), and the predominant mutation detected in mouse skin and they were from spontaneous or induced tumors. mammary tumors induced by DMBA is an A to T transversion Silent ras Gene Mutations in Carcinogen-induced Liver Tu in the second base of Ha-ras codon 61 (3, 4). A small fraction mors. Using direct tumor DNA analysis, we detected silent raÃ of the DMBA-induced liver tumors in our study had the A to gene mutations (mutations that do not change the amino acid T transversion in Ha-ras codon 61, which was specific for sequence of the encoded ras protein) in 4 DEN-induced tumors DMBA since it was not found in the other groups of chemically and in 2 N-OH-AAF-induced tumors (data not shown). In 3 of induced or spontaneous tumors. However, the majority of the 4 DEN-induced tumors and in both N-OH-AAF-induced DMBA-induced liver tumors had a G to C transversion in Ki- tumors, the silent mutation was an A to C transversion in the ras codon 13, which has not been seen in DMBA-induced skin third base of Ha-ras codon 12. The fourth DEN-induced tumor or mammary tumors. had a silent T to C transition in the third base of Ki-ras codon The tissue specificity in the type of ras mutations detected in 12. Four of the 6 silent mutations were accompanied by a DMBA-induced tumors could be due to DMBA actually caus coincident activating mutation within the same tumor. The ing different types of mutations in liver versus skin or mammary silent mutations were not detected in the transfection assay (not cells or to the different ras mutations induced by DMBA having shown) and thus appear to be nontransforming, as expected. different transforming potencies in each tissue-type. In support These results suggest that the third base of codon 12 in Ha- of the latter possibility, Nakazawa et al. (23) demonstrated that and Ki-ras may be hypermutable in CD-I mouse liver cells. DMBA efficiently induced Ha-ras (A to T) transversions in Histopatholgy: Correlation of Specific Carcinogen-induced BALB/c 3T3 cells, but transformed foci that selectively grew Mutations with Tumor Malignancy. Comparison of the muta- out of the DMBA-treated cultures did not carry this mutation. tional patterns observed between adenomas and carcinomas The Ha-ras codon 61 (A to T) transversion may be weakly induced by each carcinogen revealed an interesting correlation in the case of DMBA-induced tumors (data not shown). The Ki-ras codon 13 (G to C) mutation was found in 7 adenomas A. Normal B. GCC - GCG and 10 carcinomas induced by DMBA. However, the Ha-ras codon 61 (A to T) mutation was in 4 adenomas but no carci nomas (data not shown). This mutation was not detected in any ACGT A C GT of the other carcinogen-induced or spontaneous CD-I mouse liver tumors (Tables 1 and 2). These results suggest that CD-I mouse liver tumors with the A to T transversion may have a low probability of progressing into carcinomas relative to liver tumors carrying the Ki-ras codon 13 mutation.

DISCUSSION 12 Consistent with earlier studies in mice, our results demon strate that populations of chemically induced CD-I mouse liver C 13 tumors can be distinguished from spontaneous liver tumors by ras gene analysis. In contrast to B6C3F, mice, spontaneous liver tumors in CD-I mice have a relatively low frequency of Fig. 2. Representative DNA sequences of the codon 12-13 region of the Ki- ras mutations, and chemically induced CD-I mouse liver tu ras gene. The sequences shown are of the antisense strand and were obtained by mors can be distinguished by quantitative as well as qualitative direct sequencing of PCR-amplified liver tumor (or normal liver tissue) DNA. A, wild-type sequence derived from normal liver tissue DNA; B, Câ€”Â»Gtransversion differences in ras gene mutations. in antisense strand of codon 13 (a CGCâ€”Â»CGCtransversionin the sense strand) Both chemical- and tissue-specific factors can influence the detected in a DMBA-induced tumor. 3349

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. ACTIVATED raj GENES IN MURINE LIVER TUMORS A.Normal B.ACC - AAC (12)C.ACC - ACA (13) D.ACC - ACA (12) ACC-Â»ACA (13) Fig. 3. Representative DNA sequences of the codon 12-13 region of the N-ras gene. The sequences shown are of the antisense strand A C GT A C GT ACGT ACGT and were obtained by direct sequencing of PCR-amplified liver tumor (or normal liver tissue) DNA. A, wild-type sequence derived from normal liver tissue DNA; B, Câ€”Â»Atrans- â€¢¿C. version in antisense strand of codon 12 (a GGTâ€”Â»GTTtransversion in (he sense strand) detected in a N-OH-AAF-induced tumor; C, Câ€”Â»Atransversionin antisense strand of codon 13 (a GGTâ€”Â»TGTtransversion in the sense C â€”¿Â»A strand) detected in a N-OH-AAF-induced tu !>. mor: D, two coincident VMM mutations de 13 tected in an AAB-induced tumor, Câ€”Â»Atrans- versions in the antisense strand of both codons 12 and 13 (corresponding to GGTâ€”Â»TGT transversions in the sense strands of both co dons 12 and 13).

Table 2 Analysis afras mutations in NIH3T3 cell transfectants Tumor DNA was transfected into NIH3T3 cells, and DNA samples isolated from transformed foci were examined for raÃmutations by PCR amplification and sequencing. Codons 12-13 and 59-61 of the Ha-, Ki-, and N-ros genes were examined in all samples. Profile of activating ras mutations detected in transformed foci Ha-roj 61 Ki-rail3 N-r

Profile of mutations detected in transfectant DNA but missed in the direct tumor analysis Spontaneous AAB N-OH-AAF 6 DEN 3 11 DMBA622 4 Â°Thesefoci did not contain point mutations in codons 12, 13, or 61 of Ha-, Ki-, or N-ras. ' Numbers in parentheses, percentage. ' Two AAB-induced tumors had coincident mutations detected in two ras gene codons. transforming in CD-I mouse liver tumors, since all 4 of the tion found in spontaneous liver tumors, and thus tumors in DMBA-induced liver tumors carrying this transversion were duced by these carcinogens could not clearly be distinguished adenomas, whereas 10 of 17 DMBA-induced liver tumors car from spontaneous tumors. In this study, we found that in rying the Ki-ras codon 13 (G to C) transversion were carcino addition to the Ha-ras codon 61 (C to A) transversions, N-OH- mas. Interestingly, Brown et al. (24) noted that even in skin, AAF- and DEN-induced CD-I mouse liver tumors also contain tumors with the A to T transversion have a low probability of G to T transversions in the N-ras gene (codons 12 or 13). These progressing into carcinomas. results show that N-OH-AAF- and DEN-induced mouse liver Mutations at guanine residues by DMBA are not unprece tumors can be distinguished from spontaneous liver tumors and dented, since ras mutations detected in primary bladder epithe represent the first demonstration of N-ras mutations in mouse lial cells treated with DMBA in vitro were invariably G to A liver tumors (AAB also caused N-ras mutations), illuminating transitions in Ki-ras codon 12 (24). In addition, DMBA is a role for the N-ras gene activation in mouse liver known to cause guanosine adducts as well as adenosine adducts carcinogenesis. (3, 4, 22). Also, other polycyclic aromatic hydrocarbons are The primary assay we used to detect ras mutations involved known to interact extensively with guanine residues (20, 24). a direct analysis of tumor DNA, which is the most straightfor Similarly, in our study, we found that a high percentage of the ward and consistent method for detecting ras mutations. For ras mutations detected in AAB and N-OH-AAF-induced CD-I comparative purposes, we also used the NIH3T3 cell transfec mouse liver tumors were the same Ki-ras codon 13 (G to C) tion assay as a means of detecting ras mutations. We found transversion found in DMBA-induced liver tumors. that transfection missed several Ki-ras and N-ras mutations Previously, N-OH-AAF- (14) and DEN-induced (16) liver that were readily detected in direct tumor DNA samples. Trans tumors in B6C3F, mice were shown to primarily contain C to fection has several well-known inherent limitations that can A transversions in Ha-ras codon 61, which is the major muta cause it to miss ras mutations. Transfection is sensitive to 3350

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. ACTIVATED ras GENES IN MURINE LIVER TUMORS variability in the quality of DNA, since an intact ras gene is ras oncogene may be a particularly effective complement (for needed to transform NIH3T3 cells. In addition, the large size promoting growth of the tumor) to an initiating lesion caused of the Ki-ras gene causes it to be more sensitive than Ha- or N- directly by AAB. Alternatively, AAB-initiated tumors may be ras to factors that reduce transfection efficiency (26). Further hypermutable or more prone to spontaneous Ha-ros codon 61 more, some raÃmutations do not cause a pronounced morpho mutations later during tumor development. logical transformation of NIH3T3 cells and can be missed in In conclusion, our results demonstrate that in CD-I mouse the transfection assay (27). Thus, in addition to the additional liver tumors, genotoxic carcinogens cause an increased fre steps, transfection adds significant inconsistencies to ras gene quency and different profile of ras mutations than that found analysis and therefore direct tumor DNA analysis is generally in spontaneous tumors. All of the ros mutations that are unique the method of choice for analyzing ras gene mutations. to chemically induced tumors are present in the majority of Our results do illustrate a case when transfection is advan cells within the tumors, consistent with a putative role in the tageous. Transfection was able to detect ras mutations in many initiation of the tumors by the carcinogen. However, the Ha tumors that were negative in the direct tumor DNA analysis. ras codon 61 mutations characteristic of spontaneous tumors Since sequencing of the direct tumor DNA preparations from are often present in only a small fraction of cells, suggesting these tumors showed only the normal ras alÃele,the mutant ras that they can occur later in tumor development, independent alÃelesmust have been present in only a small fraction of the of any initiating carcinogen. cells in the tumor sample. The transfectant DNA must be enriched (relative to the direct tumor DNA) for the mutated ACKNOWLEDGMENTS ra.v alÃele,since the same PCR and sequencing methods were used in both assays. The ability to select for activated ras alÃeles We thank Roger W. Wiseman for assistance in the selection of (capable of transforming NIH3T3 cells) is central to the trans carcinogen dose levels and David H. Billman for conduct of the iunior fection assay, and others have demonstrated that transfection igenicity studies. can detect mutations that were present in too few of the cells of a tumor to be detected in the direct tumor DNA (28, 29). REFERENCES Most significantly, all of the mutations that were detected in 1. Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779-827, 1987. transfectant DNA but not in direct tumor DNA, whether they 2. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res., 49: 4682- were found in chemically induced or spontaneous tumors, are 4689, 1989. Ha-ros codon 61 mutations (CAA to AAA or CG A). Since 3. Balmain, A., and Brown, K. Oncogene activation in chemical carcinogenesis. Adv. Cancer Res., 51: 147-182, 1988. these mutations are identical to the mutations characteristic of 4. Sukumar, S. An experimental analysis of cancer: role of ras oncogenes in spontaneous tumors and since they are present in only a small multistep carcinogenesis. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. Activation of the Ha-, Ki-, and N-ras Genes in Chemically Induced Liver Tumors from CD-1 Mice

Sujata Manam, Richard D. Storer, Srinivasa Prahalada, et al.

Cancer Res 1992;52:3347-3352.

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