Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.

McMahon, Gerald; Hanson, Linda O.; Lee, Jung-Ja; Wogan, Gerald N.

doi:10.1073/pnas.83.24.9418

Cited by 87 publications

(41 citation statements)

References 36 publications

(30 reference statements)

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“…and N. Crawford, unpublished results). Other oncogenes, notably ras and raf, have been detected in some rat and mouse hepatomas induced with chemical carcinogens (25)(26)(27), but Reynolds et al (28) detected virtually no transforming genes in spontaneous hepatomas of F344 rats. One consistent finding, though, has been an elevation of c-myc transcripts in hepatocellular carcinomas (3)(4)(5)(6)(7)(8).…”

Section: Discussionmentioning

confidence: 99%

c-myc gene amplification during hepatocarcinogenesis by a choline-devoid diet.

Chandar

Lombardi

Locker

1989

Proc. Natl. Acad. Sci. U.S.A.

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Liver tumors arise in rats fed a choline-devoid diet without added carcinogens. We found amplification of the c-myc gene in 13/13 of these tumors. The amplification ranged from 2-to 70-fold and was accompanied by an increase in c-myc gene expression. Amplification of c-myc was larger in tumors of rats fed a choline-devoid diet followed by a choline-supplemented diet than in tumors from animals fed a choline-devoid diet exclusively. In the former animals, low levels of c-myc gene amplification were also detected in nontumorous regions of tumor-bearing livers. The choline-devoid diet provides an in vivo experimental model for the induction of gene amplification in the rat liver. In this setting, amplification of the c-myc gene may be an early and critical event in carcinogenesis.There is evidence that the c-myc gene is involved in the control of normal cell division and in the process of cell transformation (1). In the liver, increased c-myc expression is seen during liver regeneration (2) and in hepatocellular carcinomas (3-8).Rats fed a choline-devoid (CD) diet, without exposure to chemical carcinogens, develop hepatocellular carcinomas after 14-16 months (9-11). The mechanism of this carcinogenesis is unknown, but major consequences of the diet include cell damage and a marked increase in hepatocyte turnover (12). DNA adducts were not detected in the livers of these animals, ruling out the possibility that contamination of the diet or the rats' environment with chemical carcinogens was responsible for the liver pathology and tumor induction (13). The CD diet also causes a general reduction in hepatocyte DNA methylation (14,15). During a study of ras genes, we noticed changes in endogenous viral sequences in tumor DNA and surmised that the changes may be characteristic of CD diet hepatocarcinogenesis (16). As liver cell death and regeneration persist during CD diet feeding, we undertook studies of c-myc expression and gene structure.In an earlier study, groups of rats were fed a continuous diet, either CD or choline-supplemented (CS), for 14-16 months, with tumor incidences of 26% and 0%, respectively (11,17). In later studies, groups were fed sequential diets CD diet for 3, 6, 9, and 12 months followed by CS diet for a combined total of 16 months; respective tumor incidences were 13%, 27%, 33%, and 73% (17). All tumors were well to moderately well differentiated hepatocellular carcinomas of trabecular, adenomatous, or mixed type. In this paper we report our analysis of c-myc gene structure and expression in these livers and tumors. MATERIALS AND METHODSExperimental Animals, Diets, and Tumors. All studies were performed on male Fisher 344 rats weighing 90-100 g when fed CD or CS diets, as described (11,16,17 Nucleic Acid Purification and Hybridization Methods. DNA and total RNA were purified from tumors and livers as described (16). Southern blotting and hybridization were also carried out as described (20). DNA was labeled by the random primer method (21) to a specific activity of 4 X 10' dpm/pxg and hy...

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Section: Discussionmentioning

confidence: 99%

c-myc gene amplification during hepatocarcinogenesis by a choline-devoid diet.

Chandar

Lombardi

Locker

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…The treatment protocol used to induce tumors was essentially the same as in the previous experiment, except that carcinogen dosing was initiated at 36 rather than 21 days of age. As expected, this change in protocol resulted in a longer latency period for tumor development and, consequently, reduced the incidence of end-stage hepatocellular carcinomas at the time the experiment was terminated (68 weeks after termination of dosing), compared with similar studies involving treatment of younger animals (22). All liver tissues obtained from animals killed 63 weeks after termination of dosing and beyond showed morphological changes characteristic of malignant neoplasms (21).…”

Section: Discussionmentioning

confidence: 84%

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

Soman

Wogan

1993

Proc. Natl. Acad. Sci. U.S.A.

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Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFBl)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with aflele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G-C-to-A-T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G-C-to-T-A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G-C-to-T-A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepatocellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.Aflatoxins have been implicated by epidemiologic studies as a causative factor for hepatocellular carcinoma in humans (1). Metabolic studies of aflatoxin B1 (AFB1) have shown that its active form, AFB1 8,9-epoxide, is highly mutagenic and carcinogenic for the liver in rats and other experimental animals, with mutagenicity correlating with carcinogenicity (2, 3). AFB1 binds exclusively to guanine residues of DNA, both in vivo and in vitro (4). The major DNA adduct formed by the binding of AFB1 at the N7 position of guanine is highly unstable and depurinates readily or is converted into the more stable, open-ring formamidopyrimidine adduct (1). The presence of DNA adducts in the genome may contribute to genetic alterations in loci involved in neoplastic development. Previous work by our group and others on AFB1-induced liver carcinogenesis in rodents has demonstrated that activation of cellular ras (c-ras) genes by specific base substitutions occurs at a high frequency in tumor DNA (5, 6).In human tumors as well as in animal tumor models, c-ras genes have been shown to be mutated, the frequency of mutation varying widely among different tumor types. Accumulating evidence suggests that in many human neoplasms, RAS oncogenes may play a role in the early stages of carcinogenesis (7-10). Presently, little information is available regarding the frequency of c-ras oncogene activation during the early stages of liver tumorigenesis in humans or in experimental animal models. Previous studies of AFB1-induced hepatocellular carcinomas in Fischer 344 rats showed activating mutations in codon 12 ofthe c-Ki-ras gene, consisting of single-nucleotide changes (6). To develop a more complete understanding of the role of ras oncogenes in live...

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“…Thus, examination of 4 activated fibrosarcomas induced in rats by 1,8-dinitropyrene (1, revealed that 1 tumor contained K-ras, while the other 3 contained activated genes that were unrelated to ras (70,71). In addition, McMahon et al (72) have provided evidence for transforming gene activation in a high proportion of hepatocellular carcinomas induced in Fischer rats by aflatoxin B1. Activated K-ras was detected in 2 carcinomas, while evidence that 8 of the 11 tumors contained genes that were unrelated to ras was also provided.…”

Section: Uncharacterized Transforming Genesmentioning

confidence: 99%

The role of non-ras transforming genes in chemical carcinogenesis.

Cooper

1991

Environ Health Perspect

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DNA transfection experiments using the NIH 3T3 mouse fibroblast cell line have demonstrated that chemically induced tumors and chemically transformed cell lines frequently contain dominant transforming genes. Although many of the genes detected using the NIH 3T3 transfection-transformation assay are activated versions of H-ras, K-ras, and N-ras, in some experimental systems activated forms of genes such as met and neu that are unrelated to ras have been observed. The activated met gene was originally detected in a human cell line that had been transformed by exposure to N-methyl-N'-nitro-N-nitrosoguanidine. Subsequent studies demonstrated that the met proto-oncogene encodes a novel growth factor receptor and that gene activation involves the production of a chimeric gene in which the regions of met encoding the extracellular and transmembrane domains of the receptor are replaced by the 5'-region of an unrelated gene called trp. The activated neu gene was detected in tumors of the nervous system that arose in mice following transplacental exposure to N-ethyl-N-nitrosourea. The neu gene also encodes a novel growth factor receptor but, in contrast to met, its activation involves a single T:A----A:T point mutation in the region of the neu gene encoding the receptor transmembrane domain. The presence of genetic alterations in chemically induced malignancies has also been assessed in cytogenetic studies and by Southern analysis of DNA from neoplastic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.

Cited by 87 publications

References 36 publications

c-myc gene amplification during hepatocarcinogenesis by a choline-devoid diet.

c-myc gene amplification during hepatocarcinogenesis by a choline-devoid diet.

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

The role of non-ras transforming genes in chemical carcinogenesis.

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