[CANCER RESEARCH 49, 1103-1109, March 1, 1989] Infrequent Activation of K-ras, ll-ras, and Other Oncogenes in Hepatocellular Neoplasms Initiated by Methyl(acetoxymethyl)nitrosamine, a Methylating Agent, and Promoted by Phénobarbitalin F344 Rats Masahiro Watatani,1 Alan O. Perantoni, Carl D. Reed, Takayuki Enomoto, Martin L. Wenk, and Jerry M. Rice2 Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 {M. W., A. O. P., C. D. R., T. E., J. M. R.J, and Microbiological Associates, Inc., Bethesda, Maryland 20816 [M. L. W.] ABSTRACT Fischer 344/Ncr rats of both sexes were subjected to partial hepatec- tomy and then initiated 21-24 h later by a single injection of methyl(acetoxymethyl)nitrosamine at 0.1 mmol/kg body weight via the portal vein. Beginning 3 weeks later, development of hepatocellular neoplasms in initiated rats was promoted by feeding 0.05% phénobarbital (PB) in the diet. Not only intrahepatic lesions but also a variety of extrahepatic tumors were induced. High-molecular-weight DNAs were prepared from 67 samples of grossly normal liver containing multiple preneoplastic foci/areas of microscopic dimensions, 137 hepatocellular adenomas (nodules), 93 hepatocellular carcinomas (HCC), 10 cholan- giomas, and 25 extrahepatic tumors in 95 rats and tested for transforming activity in the NIH 3T3 transfection assay. DNA preparations from 7 of 93 HCCs, 2 of 10 cholangiomas, 2 of 137 nodules, 1 histiocytic sarcoma, and 1 thyroid carcinoma were positive in the transfection assay. Southern blot analysis showed that NIH 3T3 transformants induced by DNA from 5 HCCs, 1 hepatocellular adenoma, 1 cholangioma, 1 histiocytic sarcoma, and 1 thyroid carcinoma contained an activated K-rav gene of rat origin. Rat-derived 11-ra.s was identified in transformants from 2 additional HCCs and rat c-ra/from 1 hepatocellular adenoma. The transforming gene from one cholangioma showed no sequence homology to the ras genes, neu, or c-ra/. Immunoprecipitation analysis of ras M, 21,000 protein in 11 transformants indicated that, based upon protein electro- phoretic mobilities, activation of the ras genes consistently resulted from mutations in codon 12 of these genes. Selective oligonucleotide analysis revealed that a G —¿ » A transition in the second base of codon 12 of K-ras was present in the 9 K-roj-positive transformants and also in DNAs prepared from the original tumors. In contrast, oligonucleotide hybridi zation experiments with DNAs from 35 hepatocellular tumors that were negative in transfection assays revealed the presence of mutant K-ras in 1 of 15 HCCs; no mutation could be detected in 20 transfection-negative adenomas. The infrequency of detection of a specific oncogene, more frequent detection of oncogenes in malignant tumors, and failure to observe activated oncogenes in preneoplastic lesions suggest that activa tion of ras oncogenes may occur as a late and infrequent event in the evolution of some rat hepatocellular neoplasms and that mutation of a specific ras locus is not an obligatory early event in the genesis of these neoplasms. INTRODUCTION Carcinogenesis in many tissues is a multistep process involv ing successive stages of initiation, promotion, and progression of lesions from preneoplastic foci to benign and malignant neoplasms. This process has been particularly well documented for chemically induced HCC3 in rats (1, 2). Histologically, a variety of cellular lesions have been demonstrated, beginning Received 9/1/88; revised 11/28/88; accepted 12/5/88. 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 Present address: Second Department of Surgery, Osaka University Medical School, 1-1-50. Fukushima. Fukushima-ku, Osaka 553, Japan. ! To whom requests for reprints should be addressed. ' The abbreviations used: HCC, hepatocellular carcinoma; DMN-OAc, methyl(acetoxymethyl)nitrosamine; PB. phénobarbital;MNU, /V-nitrosomethyl- urea; DMBA, 7,12-dimethylbenz[a]anthracene; LGL, large granular lymphocyte; SSC, standard saline citrate: SDS. sodium dodecyl sulfate; p21, M, 21,000 protein. with focal areas of preneoplastic cells, some of which progress to nodules (adenomas) and finally to HCC (3). Many structur ally diverse carcinogens can initiate this sequence, and of these, DMN-OAc, an ester of the presumed carcinogenic metabolite of dimethylnitrosamine (4, 5), is among the most shortlived. Given as a single injection via the portal vein at appropriate intervals after partial hepatectomy, this agent is perhaps the most effective known initiator of phenobarbital-promotable hepatocellular tumors among the P-450-independent alkylating agents (6). Recent studies of carcinogen-induced rodent tumors have indicated that the activation of cellular oncogenes plays an important role in the initiation of certain tumors. By gene transfer assay, it has been demonstrated that cellular oncogenes that are members of the ras gene family are activated at high frequency in some carcinogen-induced animal tumors, among them some that result from single exposures to alkylating agents (7-9). However, specific classes of carcinogens and specific types of neoplasms are not invariably associated with activation of a specific member of the ras gene family (10). Moreover, there is little information on the stage of tumor development at which cellular oncogenes become activated. Studies of rat mammary tumors induced by MNU or of mouse skin tumors initiated by DMBA suggest that activation of the Harvey ras (H-ras) gene occurs at or close to the time of initiation (11, 12). These conclusions are based upon the single- point mutations observed in codons 12 or 61 of the H-ras gene in these tumors and the ability of both MNU and DMBA to act directly upon and mutate the cellular genome. More re cently, a point mutation in the 61st codon of the H-ras gene was demonstrated in mouse hepatomas induced by three struc turally different carcinogens, suggesting that H-ras activation is an early event in mouse hepatocarcinogenesis as well (13). It is of considerable importance to elucidate at which stage of tumor development particular cellular oncogenes become acti vated. The treatment protocol described in our present study has the advantage of eliciting a well-defined sequence of initia tion, promotion, and progression using a single exposure to DMN-OAc with subsequent PB promotion. Thus, we are able to evaluate rat liver tissues for the presence of activated onco genes in hepatocellular lesions at all stages of progression to HCC. MATERIALS AND METHODS Chemicals. DMN-OAc (M, 132) was synthesized as described previ ously (4). The carcinogen was dissolved in phosphate-buffered saline at pH 7.0 at a concentration of 2.64 mg/ml within 24 h of use and stored at —¿ 20°C until needed. Induction of Tumors. Ninety-five F344/NCr rats of each sex, at 4 weeks of age, were obtained from the Animal Production Area of the NCI-Frederick Cancer Research Facility (Frederick, MD). When they weighed 100 g, rats were subjected to a two-thirds partial hepatectomy under ether anesthesia (6). Twenty-one to 24 h after partial hepatec- 1103 on July 12, 2021. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from