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Lung Cancer (2007) 58, 15—20
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Point mutation of K-ras gene in cisplatin-induced lung tumours in A/J mice夽 Akiko Hisamoto, Eisei Kondo, Katsuyuki Kiura ∗, Toshiaki Okada, Shinobu Hosokawa, Junko Mimoto, Nagio Takigawa, Masahiro Tabata, Mitsune Tanimoto Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 7 March 2007; received in revised form 5 April 2007; accepted 13 May 2007
KEYWORDS Second primary cancer; Cisplatin; K-ras mutation; A/J mouse
Summary The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1—2% and 2—10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP. © 2007 Elsevier Ireland Ltd. All rights reserved.
1. Introduction 夽
This work was in part presented at the annual meeting of the American Association for Cancer Research in April 2005 [Abstract #5737] (Anaheim, CA, USA). ∗ Corresponding author. Tel.: +81 86 235 7225; fax: +81 86 232 8226. E-mail address: [email protected] (K. Kiura).
Lung cancer is still a leading cause of cancer death in the developed countries [1]. Approximately 90% of the patients with lung cancer will die of their disease; however, those patients with a specific stage of lung cancer can survive without the disease. The introduction of combined modality therapy (cisplatin-based chemotherapy with concurrent
0169-5002/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2007.05.012
16 radiation therapy) has produced a long-term disease freesurvival for patients with non-small cell lung cancer, as well as small cell lung cancer [2—6]. In fact, concomitant chemoradiotherapy produced disease free-survival in about 30% of the patients with locally advanced non-small cell lung cancer [2—5], however, a substantial portion of the longterm survivors suffer from secondary primary cancers [5]. The second malignancies which occur after the treatment of lung cancer will be an important issue in the development of lung cancer treatment. The risks of secondary lung cancer in patients with even early stage non-small cell lung cancer are estimated to range from 1 to 2% per patient per year [7]. In the locally advanced stage of non-small cell lung cancer after concomitant chemoradiotherapy, the incidence of secondary primary malignancy is 2.3 per 100 patient-years [5], and, surprisingly, the cumulative incidence of second primary cancer at 10 years after concomitant chemoradiotherapy extended to 61% [5]. In addition, the occurrence of second primary cancers has also markedly increased in small cell lung cancer after intensive chemotherapy. For patients with small cell lung cancer, the risk has been reported to increase to >2—10% per patient per year at 10 years after the initial treatment [7]. The development of a second primary cancer will be a critical issue for those patients on the road to being cured. It is essential to explore a new screening system for the second primary cancer, and we still have only one A/J mouse model for the second primary cancer. The strain A mouse lung tumour bioassay has been extensively validated and used to screen chemicals for carcinogenicity [8—10]. Positive responses have been found with a wide variety of carcinogens, including 4-(methyl-nitrosamino)1-(3-pyridyl)-1-butanone (NNK) [11], benzo(a)pyrene (BaP) [12] and urethane [13]. The A/J mouse model tends to have a specific mutation that corresponds to one carcinogen. The tumours induced by NNK [14] or BaP [15] harbour the K-ras mutations at codon 12. Urethane induces the K-ras mutation at codon 61 [16]. Cisplatin is widely used for lung cancer, and at the same time cisplatin is known to be a strong carcinogen in the animal model [17—19]. Cisplatin [17,20], irinotecan (unpublished data) and radiation (unpublished data) could induce tumourigenesis in the A/J mice; however, the specific mutations were not discovered. We previously demonstrated the partial chemopreventive effect of (−)−epigallocatechin gallate (EGCG) in green tea on the cisplatin-induced lung tumourigenesis [20], thereby confirming the complete inhibition of EGCG on the NNK-induced lung tumourigenesis in the A/J mice [20]. At that time we could not elucidate the mechanisms of carcinogenicity by cisplatin. The ras family of oncogenes has three primary members (H-ras, K-ras and N-ras) [21—24]. The ras genes have GTPase activity and they play a role in transducing growth-promoting and survival signals from membranebound receptor tyrosine kinases. The hydrolysis of bound to GTP to GDP abrogates ras signaling, but oncogenic mutations in ras impair GTP hydrolysis, thus, causing persistent signaling [25]. The ras oncogenes acquire their transforming capacity by point mutations that are detected in 20—30% of human primary lung adenocarcinoma. These mutations are found most frequently in codon 12, followed by mutations in codons 13 and 61. Ninety percent of the mutations are
A. Hisamoto et al. found in K-ras in primary lung adenocarcinoma [25]. The status of K-ras mutations, however, has not yet been reported in second primary lung cancers as far as we could determine from a search of the pertinent literature. As a result, no genetic changes in second primary lung cancer have yet been investigated. In this study we tried to detect specific K-ras mutations in the cisplatin-induced tumours in the A/J mouse model in comparison to those in the NNK-induced lung tumourigenesis, in which G to A transition at the second base of codon 12 occurred.
2. Materials and methods 2.1. Animals and chemicals A total of 40 female A/J mice, aged 4 weeks, were purchased from Japan SLC, Shizuoka, Japan. The animals were housed, five per plastic cage, and were given free access to tap water and standard laboratory food (MF; Oriental Yeast, Tokyo, Japan). They were kept in an air-conditioned room with 55 ± 10% humidity, under a daily cycle of alternating 12 h periods of light and darkness, in the Animal Center for Medical Research, Okayama University. Nippon Kayaku Co. (Tokyo Japan) kindly provided cisplatin, while NNK was purchased from Toronto Research Chemicals (Ontario, Canada).
2.2. Experimental design Forty mice were divided into two groups for the treatment with NNK (n = 20) or with cisplatin (n = 20). In the NNK treatment group, the mice were treated with a single injection of NNK (100 mg/kg body wt., i.p.) at 7 weeks of age. In the cisplatin-treatment group, the mice were treated with cisplatin (1.62 mg/kg body wt., i.p.) from 7 to 16 weeks of age, once a week for 10 weeks, as previously described [20]. At week 45 (28 weeks after the last cisplatin-treatment), all the mice were killed, and the lungs were removed and fixed for 24 h in 10% buffered formalin and embedded in paraffin. Four-micrometer-thick sections were stained by hematoxylin-eosin (HE). After confirming the histological appearance, DNA was extracted from each tumour using a PixCell laser-capture microscope (LCM) with an infrared diode laser (Arcturus Engineering, Santa Clara, CA). The maximum diameter of each tumour analyzed was determined using an Absolute Coolant Proof Caliper (Mitsutoyo Co., Tokyo, Japan).
2.3. LCM and DNA extraction The slides stained by HE were dissected using a PixCell LCM with an infrared diode laser. All visible tumours were carefully isolated from the lung under dissecting microscopy, in order to avoid contamination with any non-tumour lung tissue, as much as possible. All microdissected samples were transferred to the film on the cap by irradiation with laser, using a spot size of 10 m and a laser power of 45 mW for 2 ms. Tumour DNA was extracted from these cells using the QIAgen DNA Microkit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
K-ras gene in cisplatin-induced tumorigenesis
17
2.4. K-ras mutation analysis of tumour samples DNA was amplified by polymerase chain reaction (PCR). Two sets of primers were used to amplify the first and second exons of the K-ras gene in DNA isolated from the paraffin-embedded lung tumours. First, separate amplification reactions of 40 cycles (94 ◦ C for 2 min, 94 ◦ C for 15 s, 52 ◦ C [exon 1] or 54 ◦ C [exon 2] for 30 s, 68 ◦ C for 1 min), using outer primers (0.1 M) near the 5 and 3 ends of the exons (5 -TTTTTATTGTAAGGCCTGCTGAA-3 and 5 -TCTATCGTAGGGTCGTACTCA-3 for exon 1 and 5 -GGACTCCTACAGGAAACAAGTAGTA-3 and 5 -TCTATAATGGTGAATATCTTCAAAT-3 for exon 2), were used to generate a 90-base pair fragment for exon 1, or a 131-base pair fragment for exon 2. This was followed by 40 cycles of amplification (94 ◦ C for 2 min, 94 ◦ C for 15 s, 52 ◦ C [exon 1] or 54 ◦ C [exon 2] for 30 s, 68 ◦ C for 1 min) with a fresh reaction mix containing 5 l of the first PCR mixture as the source of the DNA template and other sets of inner primers (1.5 l) (5 -ATGACTGAGTATAAACTTGT-3 and 5 -TCGTACTCATCCACAAAGTG-3 for exon 1 and 5 -TACAGGAAACAAGTAGTAATTGATGGAGAA-3 and 5 -ATAATGGTGAATATCTTCAAATGATTTAGT-3 or exon 2) to generate a 58-base pair fragment for exon1, or a 1 1 1-base pair fragment for exon 2. The length and concentration of the PCR products were checked by electrophoresis on 4% agarose gels and then were visualized with ethidium bromide. All PCR products were directly sequenced using the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Co., Foster City, CA) with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). All sequence variants were confirmed by independent PCR amplifications from at least two independent microdissections, and then were sequenced in both directions.
3. Results A total of 42 tumours, classified as adenocarcinoma according to the description of Yang et al. [26], were obtained. Four tumours induced by NNK showed a solid pattern, while 10 tumours demonstrated a papillary pattern. Three tumours induced by cisplatin showed a solid pattern, while 25 tumours demonstrated a papillary pattern (Fig. 1). The mean maximum diameters (range) of NNK-induced tumours and cisplatin-induced tumours were 0.928 mm (0.86—1.66) and 0.576 mm (0.35—2.31), respectively. To adjust for tumour size, the tumours with a maximum diameter of more than 0.85 mm were selected in both groups. As a result, 14 NNKinduced tumours and 19 cisplatin-induced tumours were, thus, analyzed. In the NNK-treatment group, the K-ras codon 12 mutations were found in 10 (71.4%) of 14 tumours (Table 1). Nine mutations consisted of G to A transition at the second position of codon 12 (GGT: glycine to GAT: glutamic acid), and one mutation was G to T transversion at the second position of codon 12 (GGT: glycine to GTT: valine). The frequency of the K-ras mutations in the NNK-induced tumours was almost the same as that described in previous reports [27—30]. In the cisplatin-treatment group, one tumour (3.5%) had a Kras gene mutation in codon 13, and five tumours (17.8%) possessed it in codon 61. These mutations consisted of G to
Fig. 1 Pulmonary tumours induced by NNK and cisplatin week 45 of age (H&E stain). A, the solid growth pattern of adenocarcinoma (original magnification, ×500); B, the papillary growth pattern of adenocarcinoma (original magnification, ×500).
C transversion at the first position of codon 13 (GGC; glycine to CGC; arginine) in one tumour, A to G transition at the second position of codon 61 (CAA; glutamine to CGA; arginine) in two tumours, and A to T transversion at the third position of codon 61 (CAA; glutamine to CAT; histidine) in three tumours. We could not find any difference in the size and histological appearance between the tumours with the K-ras codon 61 mutation and those with the K-ras codon 13
Table 1
Mutation pattern in the K-ras gene in the A/J mice Cisplatin-treatment group (n = 28)
NNK-treatment group (n = 14)
Codon 12
0 (0%)
10 tumours (71.4%) GGT → GTT one tumour GGT → GAT 9 tumours
Codon 13
One tumour (3.5%) GGC → CGC one tumour
0 (0%)
Codon 61
Five tumours (17.8%) CAA → CGA two tumours CAA → CAT three tumours
0 (0%)
18
Fig. 2 Sequencing of the K-ras gene. A: Nine NNK-induced lung tumours had the point mutation in codon 12 (GGT to GAT). B: One cisplatin-induced tumour had the point mutation in codon 13 (GGC to CGC). C: Two cisplatin-induced tumours had the point mutation in codon 61 (CAA to CGA).
A. Hisamoto et al. however, we also demonstrated that the K-ras codon 12 mutations did not exist in the cisplatin-induced tumours. The mechanisms of carcinogenicity by NNK were investigated in detail. NNK is known to be activated in the lung, and activated NNK produces methylating and pyridyloxobutylating agents [27,31,32], which subsequently form methylated DNA adducts, such as O6 -methylguanine and 7-methylguanine, in the bronchial and bronchiolar epithelium [33,34]. O6 -methylguanine induces G to A transitions, and such transitions have been frequently observed in the mutated K-ras genes at the second base of codon 12 found in NNK-induced tumours [27,35]. The mechanisms of carcinogenesis by cisplatin, however, have not yet been investigated. We could not find any specific mutations in the cisplatin-induced tumours, in contrast to the specific K-ras codon 12 mutation found in the NNKinduced tumours; however, cisplatin might be associated with the K-ras codon 61 mutation. The K-ras codon 61 mutation was also found in the tumours induced by the high doses of NNK, urethane and vinyl carbamate [14,36,37], although we could not find any K-ras codon 61 mutations in the NNKinduced tumours in this experiment. It is unclear why the point mutations occur predominantly at the second base of codon 61 in the K-ras gene. The NNK-induced lung tumours in the A/J mice are commonly used as an animal model for cancer chemoprevention studies, because tumours can be induced at a high incidence in a relatively short period of time, and tumours with either a papillary or solid growth pattern can be induced [27,32,38]. The relative frequency of carcinomas also appears to increase almost linearly from 34 to 50 weeks [27]. The majority of tumours were adenocarcinoma, since we obtained the lung tumours from the A/J mice at 45 weeks of age. The characteristics of the cisplatin-induced tumours have not yet been investigated. The histologic appearances in the tumour induced by cisplatin or NNK were very similar, although the NNK-induced tumours were of relatively larger size than the cisplatin-induced tumours. In conclusion, the K-ras codon 12 mutations do not play a key role in cisplatin-induced tumourigenesis in the A/J mice. The mechanisms of carcinogenicity by cisplatin are different from those by NNK or BaP. Other genetic changes or K-ras mutations at locations other than codon 12 might therefore be associated with the multistage carcinogenic process.
Conflict of interest mutation. We could not detect the K-ras codon 12 mutations in the cisplatin-induced tumours (Fig. 2).
None.
4. Discussion
Acknowledgements
We found the K-ras codons 13 and 61 mutations in the cisplatin-induced tumours, but the incidence of these mutations was low; only one tumour (3.5%) had the K-ras codon 13 mutation, while 5 (17.8%) tumours had the K-ras codon 61 mutations. Accordingly, the activation of K-ras mutation is not likely the main pathway for lung oncogenesis induced by cisplatin in the A/J mice. In a sharp contrast, we confirmed the presence of the K-ras codon 12 mutations in the NNK-induced tumours as previously reported;
We thank Drs. Daizo Kishino, Ken Sato (Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences) and Yukinari Isomoto (Central Research Laboratory, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences) for their excellent technical assistance and helpful suggestions and Dr. Brian Quinn, Kyushu University for his critical reading of this manuscript. This work was in part supported by a
K-ras gene in cisplatin-induced tumorigenesis Japan Society for the Promotion of Science grant-in-aid for Scientific Research (C) (no. 16590740).
References [1] Schottenfeld D. Epidemiology of lung cancer. In: Pass HI, Mitchell JB, Jhonson DH, Turrusi AT, editors. Lung cancer: principles and practice. Philadelphia: Lipponcot-Raven; 1996. [2] Rigas JR, Lara PN. Current perspectives on treatment strategies for locally advanced, unresectable stage III non-small cell lung cancer. Lung Cancer 2005;50:S17—24. [3] Segawa Y, Ueoka H, Kiura K, Kamei H, Tabata M, Sakae K, et al. A phase II study of cisplatin and 5-fluorouracil with concurrent hyperfractionated thoracic radiation for locally advanced nonsmall cell lung cancer: a preliminary report from the Okayama Lung Cancer Study Group. Br J Cancer 2000;82(1):104—11. [4] Kiura K, Ueoka H, Segawa Y, Tabata M, Kamei H, Takigawa N, et al. Phase I/II study of docetaxel and cisplatin with concurrent thoracic radiation therapy for locally advanced non-small cell lung cancer. Br J Cancer 2003;89(5):795—802. [5] Takigawa N, Kiura K, Segawa Y, Watanabe Y, Kamei H, Moritaka T, et al. Second primary cancer in survivors following concurrent chemoradiation for locally advanced non-small cell lung cancer. Br J Cancer 2006;95(9):1142—4. [6] Turrisi 3rd AT, Kim K, Blum R, Sause WT, Livingston RB, Komaki R, et al. Twice-daily compared with once-daily thoracic radiotherapy in limited small cell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med 1999;340(4):265—71. [7] Johnson BE. Second lung cancers in patients after treatment for an initial lung cancer. J Natl Cancer Inst 1998;90:1335—45. [8] Shimkin MB, Stoner GD. Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res 1975;21:1—58. [9] Theiss JC, Stoner GD, Shimkin MB, Weisburger EK. Test for carcinogenicity of organic contaminants of United States drinking waters by pulmonary tumor response in strain A mice. Cancer Res 1997;37(8 Pt 1):2717—20. [10] Theiss JC, Stoner GD, Shimkin MB. Murine pulmonary adenoma bioassay of potentially effective agents against slow-growing solid tumors. Cancer Res 1977;37(1):305—9. [11] Hecht SS, Abbaspour A, Hoffman D. A study of tobacco carcinogenesis. XLII. Bioassay in A/J mice of some structural analogues of tobacco-specific nitrosamines. Cancer Lett 1988;42(1—2):141—5. [12] Hecht SS, Isaacs S, Trushin N. Lung tumor induction in A/J mice by the tobacco smoke carcinogens 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone and benzo[a]pyrene: a potentially useful model for evaluation of chemopreventive agents. Carcinogenesis 1994;15(12):2721—5. [13] Witschi H, Williamson D, Lock S. Enhancement of urethan tumorigenesis in mouse lung by butylated hydroxytoluene. J Natl Cancer Inst 1977;58(2):301—5. [14] Belinsky SA, Devereux TR, Maronpot RR, Stoner GD, Anderson MW. Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines. Cancer Res 1989;49(19):5305—11. [15] Manjanatha MG, Li EE, Fu PP, Heflich RH. H- and K-ras mutational profiles in chemically induced liver tumors from B6C3F1 and CD-1 mice. J Toxicol Environ Health 1996;47(2):195—208. [16] Nuzum EO, Malkinson AM, Beer DG. Specific Ki-ras codon 61 mutations may determine the development of urethan-induced mouse lung adenomas or adenocarcinomas. Mol Carcinog 1990;3(5):287—95. [17] Leopold WR, Miller EC, Miller JA. Carcinogenicity of antitumor cis-platinum (II) coordination complexes in the mouse and rat. Cancer Res 1979;39:913—8.
19 [18] Hennings H. Enhanced malignant conversion of benign mouse skin tumors by cisplatin. J Natl Cancer Inst 1990;82:836—40. [19] Reed E, Gupta-Burt S, Litterst CL, Poirier MC. Characterization of the DNA damage recognized by an antiserum elicited against cis-diamminedichlorplatinum (II)-modified DNA. Carcinogenesis 1990;11:2117—21. [20] Mimoto J, Kiura K, Matsuo K, Yoshino T, Takata I, Ueoka H, et al. (−)−Epigallocatechin gallate can prevent cisplatin-induced lung tumorigenesis in A/J mice. Carcinogenesis 2000;21: 915—9. [21] Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 1982;297(5866):474—8. [22] McCoy MS, Toole JJ, Cunningham JM, Chang EH, Lowy DR, Weinberg RA. Characterization of a human colon/lung carcinoma oncogene. Nature 1983;302(5903):79—81. [23] Der CJ, Krontiris TG, Cooper GM. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl Acad Sci USA 1982;79(11):3637—40. [24] Shimizu K, Goldfarb M, Suard Y, Perucho M, Li Y, Kamata T, et al. Three human transforming genes are related to the viral ras oncogenes. Proc Natl Acad Sci USA 1983;80(8):2112—6. [25] Onn A, Vaporciyan AA, Chang JY, Komaki R, Roth JA, Herbst RS. The thorax cancer of the lung. In: Kufe DW, Bast Jr RC, Hait WH, Hong WK, Pollock RE, Weichselbaum RR, Holland JF, Frei III E, editors. Cancer 7 medicine. London: BC Decker Inc.; 2006. p. 1179—224. [26] Yang G, Wang ZY, Kim S, Liao J, Seril DN, Chen X, et al. Characterization of early pulmonary hyperproliferation and tumor progression and their inhibition by black tea in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis model with A/J mice. Cancer Res 1997;57:1889—94. [27] Belinsky SA, Devereux TR, Forly JF, Maronpot RR, Anderson MW. Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonone in the A/J mouse. Cancer Res 1992;52:3164—73. [28] Chen B, Liu L, Castonguay A, Maronpot RR, Anderson MW, You M. Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone induced mouse lung tumors. Carcinogenesis 1993;14:1603—8. [29] Matzinger SA, Crist KA, Stoner GD, Anderson MW, Pereire MA, Steele VE, et al. K-ras mutations in lung tumors from A/J and A/JxTSG-p53 F1 mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and phenethyl isothiocyanate. Carcinogenesis 1995;16:2487—92. [30] Kawano R, Takeshima Y, Inai K. Effects of K-ras gene mutations in the development of lung lesions induced by 4-(N-methyln-nitorosamino)-1-(3-pyridyl)-1-butanone in A/J mice. Jpn J Cancer Res 1996;87:44—50. [31] Nettesheim P. Cells of primaly pulmonary neoplasm in mice. Exp Lung Res 1991;17:215—7. [32] Foley JF, Anderson MW, Stoner GD, Gaul BW, Hardisty JF, Maronpot RR. Proliferative lesions of the mouse lung: progression studies in strain A mice. Exp Lung Res 1991;17:157—68. [33] Belinsky SA, White CM, Trushin N, Hecht SS. Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fisher rat. Cacinogenesis 1989;10:2269—74. [34] Van BJ, Feron VJ, Leeman WR, Wilmer JW, Vermeulen E, Den EL, et al. Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis 1994;15:2023—9. [35] Ronai ZA, Gradia S, Peterson LA, Hecht SS. G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene
20 isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and related DNA methylating and pyridyloxobutylating agents. Carcinogenesis 1993;14:2419—22. [36] You M, Candrian U, Maronpot R, Stoner G, Anderson M. Activation of K-ras protooncogene in spontaneously occurring and chemically-induced lung tumors of strain A mouse. Proc Natl Acad Sci USA 1989;86:3070—4.
A. Hisamoto et al. [37] Ohmori H, Abe T, Hirano H, Murakami T, Katoh T, Gotoh S, et al. Comparison of Ki-ras gene mutation among simultaneously occurring multiple urethane-induced lung tumors in individual mice. Carcinogenesis 1992;13:851—5. [38] Hecht SS, Morse MA, Amin S, Stoner GD, Jordan KG, Choi CI, et al. Rapid single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonone and the effect of diet. Carcinogenesis 1989;10:1901—4.
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journal homepage: www.elsevier.com/locate/lungcan
Point mutation of K-ras gene in cisplatin-induced lung tumours in A/J mice夽 Akiko Hisamoto, Eisei Kondo, Katsuyuki Kiura ∗, Toshiaki Okada, Shinobu Hosokawa, Junko Mimoto, Nagio Takigawa, Masahiro Tabata, Mitsune Tanimoto Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 7 March 2007; received in revised form 5 April 2007; accepted 13 May 2007
KEYWORDS Second primary cancer; Cisplatin; K-ras mutation; A/J mouse
Summary The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1—2% and 2—10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP. © 2007 Elsevier Ireland Ltd. All rights reserved.
1. Introduction 夽
This work was in part presented at the annual meeting of the American Association for Cancer Research in April 2005 [Abstract #5737] (Anaheim, CA, USA). ∗ Corresponding author. Tel.: +81 86 235 7225; fax: +81 86 232 8226. E-mail address: [email protected] (K. Kiura).
Lung cancer is still a leading cause of cancer death in the developed countries [1]. Approximately 90% of the patients with lung cancer will die of their disease; however, those patients with a specific stage of lung cancer can survive without the disease. The introduction of combined modality therapy (cisplatin-based chemotherapy with concurrent
0169-5002/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2007.05.012
16 radiation therapy) has produced a long-term disease freesurvival for patients with non-small cell lung cancer, as well as small cell lung cancer [2—6]. In fact, concomitant chemoradiotherapy produced disease free-survival in about 30% of the patients with locally advanced non-small cell lung cancer [2—5], however, a substantial portion of the longterm survivors suffer from secondary primary cancers [5]. The second malignancies which occur after the treatment of lung cancer will be an important issue in the development of lung cancer treatment. The risks of secondary lung cancer in patients with even early stage non-small cell lung cancer are estimated to range from 1 to 2% per patient per year [7]. In the locally advanced stage of non-small cell lung cancer after concomitant chemoradiotherapy, the incidence of secondary primary malignancy is 2.3 per 100 patient-years [5], and, surprisingly, the cumulative incidence of second primary cancer at 10 years after concomitant chemoradiotherapy extended to 61% [5]. In addition, the occurrence of second primary cancers has also markedly increased in small cell lung cancer after intensive chemotherapy. For patients with small cell lung cancer, the risk has been reported to increase to >2—10% per patient per year at 10 years after the initial treatment [7]. The development of a second primary cancer will be a critical issue for those patients on the road to being cured. It is essential to explore a new screening system for the second primary cancer, and we still have only one A/J mouse model for the second primary cancer. The strain A mouse lung tumour bioassay has been extensively validated and used to screen chemicals for carcinogenicity [8—10]. Positive responses have been found with a wide variety of carcinogens, including 4-(methyl-nitrosamino)1-(3-pyridyl)-1-butanone (NNK) [11], benzo(a)pyrene (BaP) [12] and urethane [13]. The A/J mouse model tends to have a specific mutation that corresponds to one carcinogen. The tumours induced by NNK [14] or BaP [15] harbour the K-ras mutations at codon 12. Urethane induces the K-ras mutation at codon 61 [16]. Cisplatin is widely used for lung cancer, and at the same time cisplatin is known to be a strong carcinogen in the animal model [17—19]. Cisplatin [17,20], irinotecan (unpublished data) and radiation (unpublished data) could induce tumourigenesis in the A/J mice; however, the specific mutations were not discovered. We previously demonstrated the partial chemopreventive effect of (−)−epigallocatechin gallate (EGCG) in green tea on the cisplatin-induced lung tumourigenesis [20], thereby confirming the complete inhibition of EGCG on the NNK-induced lung tumourigenesis in the A/J mice [20]. At that time we could not elucidate the mechanisms of carcinogenicity by cisplatin. The ras family of oncogenes has three primary members (H-ras, K-ras and N-ras) [21—24]. The ras genes have GTPase activity and they play a role in transducing growth-promoting and survival signals from membranebound receptor tyrosine kinases. The hydrolysis of bound to GTP to GDP abrogates ras signaling, but oncogenic mutations in ras impair GTP hydrolysis, thus, causing persistent signaling [25]. The ras oncogenes acquire their transforming capacity by point mutations that are detected in 20—30% of human primary lung adenocarcinoma. These mutations are found most frequently in codon 12, followed by mutations in codons 13 and 61. Ninety percent of the mutations are
A. Hisamoto et al. found in K-ras in primary lung adenocarcinoma [25]. The status of K-ras mutations, however, has not yet been reported in second primary lung cancers as far as we could determine from a search of the pertinent literature. As a result, no genetic changes in second primary lung cancer have yet been investigated. In this study we tried to detect specific K-ras mutations in the cisplatin-induced tumours in the A/J mouse model in comparison to those in the NNK-induced lung tumourigenesis, in which G to A transition at the second base of codon 12 occurred.
2. Materials and methods 2.1. Animals and chemicals A total of 40 female A/J mice, aged 4 weeks, were purchased from Japan SLC, Shizuoka, Japan. The animals were housed, five per plastic cage, and were given free access to tap water and standard laboratory food (MF; Oriental Yeast, Tokyo, Japan). They were kept in an air-conditioned room with 55 ± 10% humidity, under a daily cycle of alternating 12 h periods of light and darkness, in the Animal Center for Medical Research, Okayama University. Nippon Kayaku Co. (Tokyo Japan) kindly provided cisplatin, while NNK was purchased from Toronto Research Chemicals (Ontario, Canada).
2.2. Experimental design Forty mice were divided into two groups for the treatment with NNK (n = 20) or with cisplatin (n = 20). In the NNK treatment group, the mice were treated with a single injection of NNK (100 mg/kg body wt., i.p.) at 7 weeks of age. In the cisplatin-treatment group, the mice were treated with cisplatin (1.62 mg/kg body wt., i.p.) from 7 to 16 weeks of age, once a week for 10 weeks, as previously described [20]. At week 45 (28 weeks after the last cisplatin-treatment), all the mice were killed, and the lungs were removed and fixed for 24 h in 10% buffered formalin and embedded in paraffin. Four-micrometer-thick sections were stained by hematoxylin-eosin (HE). After confirming the histological appearance, DNA was extracted from each tumour using a PixCell laser-capture microscope (LCM) with an infrared diode laser (Arcturus Engineering, Santa Clara, CA). The maximum diameter of each tumour analyzed was determined using an Absolute Coolant Proof Caliper (Mitsutoyo Co., Tokyo, Japan).
2.3. LCM and DNA extraction The slides stained by HE were dissected using a PixCell LCM with an infrared diode laser. All visible tumours were carefully isolated from the lung under dissecting microscopy, in order to avoid contamination with any non-tumour lung tissue, as much as possible. All microdissected samples were transferred to the film on the cap by irradiation with laser, using a spot size of 10 m and a laser power of 45 mW for 2 ms. Tumour DNA was extracted from these cells using the QIAgen DNA Microkit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
K-ras gene in cisplatin-induced tumorigenesis
17
2.4. K-ras mutation analysis of tumour samples DNA was amplified by polymerase chain reaction (PCR). Two sets of primers were used to amplify the first and second exons of the K-ras gene in DNA isolated from the paraffin-embedded lung tumours. First, separate amplification reactions of 40 cycles (94 ◦ C for 2 min, 94 ◦ C for 15 s, 52 ◦ C [exon 1] or 54 ◦ C [exon 2] for 30 s, 68 ◦ C for 1 min), using outer primers (0.1 M) near the 5 and 3 ends of the exons (5 -TTTTTATTGTAAGGCCTGCTGAA-3 and 5 -TCTATCGTAGGGTCGTACTCA-3 for exon 1 and 5 -GGACTCCTACAGGAAACAAGTAGTA-3 and 5 -TCTATAATGGTGAATATCTTCAAAT-3 for exon 2), were used to generate a 90-base pair fragment for exon 1, or a 131-base pair fragment for exon 2. This was followed by 40 cycles of amplification (94 ◦ C for 2 min, 94 ◦ C for 15 s, 52 ◦ C [exon 1] or 54 ◦ C [exon 2] for 30 s, 68 ◦ C for 1 min) with a fresh reaction mix containing 5 l of the first PCR mixture as the source of the DNA template and other sets of inner primers (1.5 l) (5 -ATGACTGAGTATAAACTTGT-3 and 5 -TCGTACTCATCCACAAAGTG-3 for exon 1 and 5 -TACAGGAAACAAGTAGTAATTGATGGAGAA-3 and 5 -ATAATGGTGAATATCTTCAAATGATTTAGT-3 or exon 2) to generate a 58-base pair fragment for exon1, or a 1 1 1-base pair fragment for exon 2. The length and concentration of the PCR products were checked by electrophoresis on 4% agarose gels and then were visualized with ethidium bromide. All PCR products were directly sequenced using the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Co., Foster City, CA) with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). All sequence variants were confirmed by independent PCR amplifications from at least two independent microdissections, and then were sequenced in both directions.
3. Results A total of 42 tumours, classified as adenocarcinoma according to the description of Yang et al. [26], were obtained. Four tumours induced by NNK showed a solid pattern, while 10 tumours demonstrated a papillary pattern. Three tumours induced by cisplatin showed a solid pattern, while 25 tumours demonstrated a papillary pattern (Fig. 1). The mean maximum diameters (range) of NNK-induced tumours and cisplatin-induced tumours were 0.928 mm (0.86—1.66) and 0.576 mm (0.35—2.31), respectively. To adjust for tumour size, the tumours with a maximum diameter of more than 0.85 mm were selected in both groups. As a result, 14 NNKinduced tumours and 19 cisplatin-induced tumours were, thus, analyzed. In the NNK-treatment group, the K-ras codon 12 mutations were found in 10 (71.4%) of 14 tumours (Table 1). Nine mutations consisted of G to A transition at the second position of codon 12 (GGT: glycine to GAT: glutamic acid), and one mutation was G to T transversion at the second position of codon 12 (GGT: glycine to GTT: valine). The frequency of the K-ras mutations in the NNK-induced tumours was almost the same as that described in previous reports [27—30]. In the cisplatin-treatment group, one tumour (3.5%) had a Kras gene mutation in codon 13, and five tumours (17.8%) possessed it in codon 61. These mutations consisted of G to
Fig. 1 Pulmonary tumours induced by NNK and cisplatin week 45 of age (H&E stain). A, the solid growth pattern of adenocarcinoma (original magnification, ×500); B, the papillary growth pattern of adenocarcinoma (original magnification, ×500).
C transversion at the first position of codon 13 (GGC; glycine to CGC; arginine) in one tumour, A to G transition at the second position of codon 61 (CAA; glutamine to CGA; arginine) in two tumours, and A to T transversion at the third position of codon 61 (CAA; glutamine to CAT; histidine) in three tumours. We could not find any difference in the size and histological appearance between the tumours with the K-ras codon 61 mutation and those with the K-ras codon 13
Table 1
Mutation pattern in the K-ras gene in the A/J mice Cisplatin-treatment group (n = 28)
NNK-treatment group (n = 14)
Codon 12
0 (0%)
10 tumours (71.4%) GGT → GTT one tumour GGT → GAT 9 tumours
Codon 13
One tumour (3.5%) GGC → CGC one tumour
0 (0%)
Codon 61
Five tumours (17.8%) CAA → CGA two tumours CAA → CAT three tumours
0 (0%)
18
Fig. 2 Sequencing of the K-ras gene. A: Nine NNK-induced lung tumours had the point mutation in codon 12 (GGT to GAT). B: One cisplatin-induced tumour had the point mutation in codon 13 (GGC to CGC). C: Two cisplatin-induced tumours had the point mutation in codon 61 (CAA to CGA).
A. Hisamoto et al. however, we also demonstrated that the K-ras codon 12 mutations did not exist in the cisplatin-induced tumours. The mechanisms of carcinogenicity by NNK were investigated in detail. NNK is known to be activated in the lung, and activated NNK produces methylating and pyridyloxobutylating agents [27,31,32], which subsequently form methylated DNA adducts, such as O6 -methylguanine and 7-methylguanine, in the bronchial and bronchiolar epithelium [33,34]. O6 -methylguanine induces G to A transitions, and such transitions have been frequently observed in the mutated K-ras genes at the second base of codon 12 found in NNK-induced tumours [27,35]. The mechanisms of carcinogenesis by cisplatin, however, have not yet been investigated. We could not find any specific mutations in the cisplatin-induced tumours, in contrast to the specific K-ras codon 12 mutation found in the NNKinduced tumours; however, cisplatin might be associated with the K-ras codon 61 mutation. The K-ras codon 61 mutation was also found in the tumours induced by the high doses of NNK, urethane and vinyl carbamate [14,36,37], although we could not find any K-ras codon 61 mutations in the NNKinduced tumours in this experiment. It is unclear why the point mutations occur predominantly at the second base of codon 61 in the K-ras gene. The NNK-induced lung tumours in the A/J mice are commonly used as an animal model for cancer chemoprevention studies, because tumours can be induced at a high incidence in a relatively short period of time, and tumours with either a papillary or solid growth pattern can be induced [27,32,38]. The relative frequency of carcinomas also appears to increase almost linearly from 34 to 50 weeks [27]. The majority of tumours were adenocarcinoma, since we obtained the lung tumours from the A/J mice at 45 weeks of age. The characteristics of the cisplatin-induced tumours have not yet been investigated. The histologic appearances in the tumour induced by cisplatin or NNK were very similar, although the NNK-induced tumours were of relatively larger size than the cisplatin-induced tumours. In conclusion, the K-ras codon 12 mutations do not play a key role in cisplatin-induced tumourigenesis in the A/J mice. The mechanisms of carcinogenicity by cisplatin are different from those by NNK or BaP. Other genetic changes or K-ras mutations at locations other than codon 12 might therefore be associated with the multistage carcinogenic process.
Conflict of interest mutation. We could not detect the K-ras codon 12 mutations in the cisplatin-induced tumours (Fig. 2).
None.
4. Discussion
Acknowledgements
We found the K-ras codons 13 and 61 mutations in the cisplatin-induced tumours, but the incidence of these mutations was low; only one tumour (3.5%) had the K-ras codon 13 mutation, while 5 (17.8%) tumours had the K-ras codon 61 mutations. Accordingly, the activation of K-ras mutation is not likely the main pathway for lung oncogenesis induced by cisplatin in the A/J mice. In a sharp contrast, we confirmed the presence of the K-ras codon 12 mutations in the NNK-induced tumours as previously reported;
We thank Drs. Daizo Kishino, Ken Sato (Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences) and Yukinari Isomoto (Central Research Laboratory, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences) for their excellent technical assistance and helpful suggestions and Dr. Brian Quinn, Kyushu University for his critical reading of this manuscript. This work was in part supported by a
K-ras gene in cisplatin-induced tumorigenesis Japan Society for the Promotion of Science grant-in-aid for Scientific Research (C) (no. 16590740).
References [1] Schottenfeld D. Epidemiology of lung cancer. In: Pass HI, Mitchell JB, Jhonson DH, Turrusi AT, editors. Lung cancer: principles and practice. Philadelphia: Lipponcot-Raven; 1996. [2] Rigas JR, Lara PN. Current perspectives on treatment strategies for locally advanced, unresectable stage III non-small cell lung cancer. Lung Cancer 2005;50:S17—24. [3] Segawa Y, Ueoka H, Kiura K, Kamei H, Tabata M, Sakae K, et al. A phase II study of cisplatin and 5-fluorouracil with concurrent hyperfractionated thoracic radiation for locally advanced nonsmall cell lung cancer: a preliminary report from the Okayama Lung Cancer Study Group. Br J Cancer 2000;82(1):104—11. [4] Kiura K, Ueoka H, Segawa Y, Tabata M, Kamei H, Takigawa N, et al. Phase I/II study of docetaxel and cisplatin with concurrent thoracic radiation therapy for locally advanced non-small cell lung cancer. Br J Cancer 2003;89(5):795—802. [5] Takigawa N, Kiura K, Segawa Y, Watanabe Y, Kamei H, Moritaka T, et al. Second primary cancer in survivors following concurrent chemoradiation for locally advanced non-small cell lung cancer. Br J Cancer 2006;95(9):1142—4. [6] Turrisi 3rd AT, Kim K, Blum R, Sause WT, Livingston RB, Komaki R, et al. Twice-daily compared with once-daily thoracic radiotherapy in limited small cell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med 1999;340(4):265—71. [7] Johnson BE. Second lung cancers in patients after treatment for an initial lung cancer. J Natl Cancer Inst 1998;90:1335—45. [8] Shimkin MB, Stoner GD. Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res 1975;21:1—58. [9] Theiss JC, Stoner GD, Shimkin MB, Weisburger EK. Test for carcinogenicity of organic contaminants of United States drinking waters by pulmonary tumor response in strain A mice. Cancer Res 1997;37(8 Pt 1):2717—20. [10] Theiss JC, Stoner GD, Shimkin MB. Murine pulmonary adenoma bioassay of potentially effective agents against slow-growing solid tumors. Cancer Res 1977;37(1):305—9. [11] Hecht SS, Abbaspour A, Hoffman D. A study of tobacco carcinogenesis. XLII. Bioassay in A/J mice of some structural analogues of tobacco-specific nitrosamines. Cancer Lett 1988;42(1—2):141—5. [12] Hecht SS, Isaacs S, Trushin N. Lung tumor induction in A/J mice by the tobacco smoke carcinogens 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone and benzo[a]pyrene: a potentially useful model for evaluation of chemopreventive agents. Carcinogenesis 1994;15(12):2721—5. [13] Witschi H, Williamson D, Lock S. Enhancement of urethan tumorigenesis in mouse lung by butylated hydroxytoluene. J Natl Cancer Inst 1977;58(2):301—5. [14] Belinsky SA, Devereux TR, Maronpot RR, Stoner GD, Anderson MW. Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines. Cancer Res 1989;49(19):5305—11. [15] Manjanatha MG, Li EE, Fu PP, Heflich RH. H- and K-ras mutational profiles in chemically induced liver tumors from B6C3F1 and CD-1 mice. J Toxicol Environ Health 1996;47(2):195—208. [16] Nuzum EO, Malkinson AM, Beer DG. Specific Ki-ras codon 61 mutations may determine the development of urethan-induced mouse lung adenomas or adenocarcinomas. Mol Carcinog 1990;3(5):287—95. [17] Leopold WR, Miller EC, Miller JA. Carcinogenicity of antitumor cis-platinum (II) coordination complexes in the mouse and rat. Cancer Res 1979;39:913—8.
19 [18] Hennings H. Enhanced malignant conversion of benign mouse skin tumors by cisplatin. J Natl Cancer Inst 1990;82:836—40. [19] Reed E, Gupta-Burt S, Litterst CL, Poirier MC. Characterization of the DNA damage recognized by an antiserum elicited against cis-diamminedichlorplatinum (II)-modified DNA. Carcinogenesis 1990;11:2117—21. [20] Mimoto J, Kiura K, Matsuo K, Yoshino T, Takata I, Ueoka H, et al. (−)−Epigallocatechin gallate can prevent cisplatin-induced lung tumorigenesis in A/J mice. Carcinogenesis 2000;21: 915—9. [21] Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 1982;297(5866):474—8. [22] McCoy MS, Toole JJ, Cunningham JM, Chang EH, Lowy DR, Weinberg RA. Characterization of a human colon/lung carcinoma oncogene. Nature 1983;302(5903):79—81. [23] Der CJ, Krontiris TG, Cooper GM. Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl Acad Sci USA 1982;79(11):3637—40. [24] Shimizu K, Goldfarb M, Suard Y, Perucho M, Li Y, Kamata T, et al. Three human transforming genes are related to the viral ras oncogenes. Proc Natl Acad Sci USA 1983;80(8):2112—6. [25] Onn A, Vaporciyan AA, Chang JY, Komaki R, Roth JA, Herbst RS. The thorax cancer of the lung. In: Kufe DW, Bast Jr RC, Hait WH, Hong WK, Pollock RE, Weichselbaum RR, Holland JF, Frei III E, editors. Cancer 7 medicine. London: BC Decker Inc.; 2006. p. 1179—224. [26] Yang G, Wang ZY, Kim S, Liao J, Seril DN, Chen X, et al. Characterization of early pulmonary hyperproliferation and tumor progression and their inhibition by black tea in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis model with A/J mice. Cancer Res 1997;57:1889—94. [27] Belinsky SA, Devereux TR, Forly JF, Maronpot RR, Anderson MW. Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonone in the A/J mouse. Cancer Res 1992;52:3164—73. [28] Chen B, Liu L, Castonguay A, Maronpot RR, Anderson MW, You M. Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone induced mouse lung tumors. Carcinogenesis 1993;14:1603—8. [29] Matzinger SA, Crist KA, Stoner GD, Anderson MW, Pereire MA, Steele VE, et al. K-ras mutations in lung tumors from A/J and A/JxTSG-p53 F1 mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and phenethyl isothiocyanate. Carcinogenesis 1995;16:2487—92. [30] Kawano R, Takeshima Y, Inai K. Effects of K-ras gene mutations in the development of lung lesions induced by 4-(N-methyln-nitorosamino)-1-(3-pyridyl)-1-butanone in A/J mice. Jpn J Cancer Res 1996;87:44—50. [31] Nettesheim P. Cells of primaly pulmonary neoplasm in mice. Exp Lung Res 1991;17:215—7. [32] Foley JF, Anderson MW, Stoner GD, Gaul BW, Hardisty JF, Maronpot RR. Proliferative lesions of the mouse lung: progression studies in strain A mice. Exp Lung Res 1991;17:157—68. [33] Belinsky SA, White CM, Trushin N, Hecht SS. Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fisher rat. Cacinogenesis 1989;10:2269—74. [34] Van BJ, Feron VJ, Leeman WR, Wilmer JW, Vermeulen E, Den EL, et al. Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis 1994;15:2023—9. [35] Ronai ZA, Gradia S, Peterson LA, Hecht SS. G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene
20 isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and related DNA methylating and pyridyloxobutylating agents. Carcinogenesis 1993;14:2419—22. [36] You M, Candrian U, Maronpot R, Stoner G, Anderson M. Activation of K-ras protooncogene in spontaneously occurring and chemically-induced lung tumors of strain A mouse. Proc Natl Acad Sci USA 1989;86:3070—4.
A. Hisamoto et al. [37] Ohmori H, Abe T, Hirano H, Murakami T, Katoh T, Gotoh S, et al. Comparison of Ki-ras gene mutation among simultaneously occurring multiple urethane-induced lung tumors in individual mice. Carcinogenesis 1992;13:851—5. [38] Hecht SS, Morse MA, Amin S, Stoner GD, Jordan KG, Choi CI, et al. Rapid single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonone and the effect of diet. Carcinogenesis 1989;10:1901—4.