Point mutation of K-ras gene in cisplatin-induced lung tumors in A/J mice

5737

Risks of secondary lung cancer in patients with non-small and small cell lung cancer are estimated to be 1-2% and 2-10% per patient per year, respectively. Cisplatin is widely used in the treatment of lung cancer and is also known as a carcinogen in experimental animals. We have reported that (-)-epigallocatechin gallate or celecoxib partially inhibits cisplatin-induced lung tumors in A/J mice (Carcinogenesis. 21(5): 915, 2000; Proc AACR 44 (#4920): 981, 2003). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butamina (NNK), a tobacco-specific nitrosamine, induces lung tumors in A/J mice in which G to A transition at the second base of codon12 were frequently detected. Urethane induces K-ras gene mutation, particularly at codon 61. Although cisplatin also induces lung tumors in A/J mice, K-ras mutation in tumors has not been investigated. In this study, we investigated the K-ras mutation of lung tumors in A/J mice. A total of 30 female A/J mice were treated with cisplatin (1.62mg/kg body wt, i.p.) once a week for 10 weeks (from aged 6 weeks). The mice were killed at 18 weeks after the cisplatin treatment. The lungs were removed and fixed in formalin for 12-24h followed by paraffin embedding. 7-micrometer thick sections were cut from a paraffin block, and then deparaffinized. Tumor lesions were picked up using laser capture microdissection method. DNA was amplified by nested PCR method. The purified DNA was directly sequenced codons12, 13, and 61.For a control, the point mutation of codon12 was detected in 10 (71.4%) of 14 lung tumors induced by NNK, which included 4 tumors with papillary pattern, 10 tumors with solid pattern. Mean tumor diameter (range) was 0.928mm (0.86-1.66). Among 19 tumors induced by cisplatin, no point mutation at codon12, 1 (5.3%) at codon13 (GGC;glycine→CGC;arginine), 3 (15.8%) at codon61 (2 tumors: CAA;glutamic acid→CGA;arginine, CAA;glutamic acid→CAT;histidine 1 tumor) were detected. Cisplatin-induced 19 tumors analyzed included 1 tumor with papillary pattern and 18 tumors with solid pattern. Mean tumor diameter (range) was 0.760mm (0.35-1.67). No specific K-ras mutations in the lung tumors induced by cisplatin were detected.