Previous studies from this and other laboratories have shown that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis of paraffin-embedded lung tissue for Ki-ras-2 mutations indicated that 79% of the lesions examined contained point mutations in codons 12 and 13 of the Ki-ras-2 gene locus, the majority of which (84%) were G → T transversions. The mutational spectrum was dependent on the tumor stage, as both the incidence of mutation and type of mutation produced correlated with malignant progression of the tumor. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas, and 100% of the adenocarcinomas. In the tumors with mutations, GLY12 → CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas, and 14% of the adenocarcinomas. GLY12 → VAL12 transversions were not observed in hyperplasias and occurred in 42% of the ade- nomas and 57% of the adenocarcinomas. The remaining ASP12 and ARG13 mutations occurred only in adenomas (17%) and adenocarcinomas (29%). The tumors were also analyzed for alterations in the structure or function of the tumor suppressor genes Rb, p53, and Cdkn2a. No mutations were observed in exons 5-8 of the p53 gene. SSCP analysis demonstrated that 2 of 15 lung tumors contained shifted bands at the Cdkn2a gene locus. Sequence analysis has identified these as mutations in exon 2, with a CAC → TAC transition at base 301 (HIS74 → TYR74) in tumor 23-1 and GGG → GAG transition at base 350 (GLY90 → GLU90) in tumor 36-1. Northern blot analysis of the larger tumors revealed that 14 of 14 of these large lung tumors exhibited markedly decreased expression of Rb gene transcripts. These results were confirmed by immunohistochemistry. The larger tumors, which exhibited features of adenocarcinomas, showed a marked reduction or almost complete absence of nuclear pRb staining compared with smaller adenomas and normal lung tissue. The results suggest that Ki-ras-2 mutations are an early and frequent event in lung tumorigenesis, and that the type of mutation produced by environmental chemicals can influence the carcinogenic potential of the tumor. The results obtained with the Cdkn2a and Rb genes suggest that alterations in the Rb regulatory axis may play a key role in the pathogenesis of the pulmonary tumors and appear to occur later in the neoplastic process. It appears from these experiments that the combination of mutated Ki-ras-2 and alterations in the Rb regulatory gene locus, which are frequent alterations in human lung tumors, may be the preferred pathway for lung tumor pathogenesis in mice exposed transplacentally to environmental carcinogens.
Bibliographical noteFunding Information:
Received 10 February 1998; accepted 10 February 1998. Dr. Miller thanks Gary Stoner of the Ohio State University and Alvin Malkinson of the University of Colorado Health Sciences Center for the invitation to speak at the Second International Mouse Lung Tumorigenesis Symposium. The authors acknowledge the National Institutes of Health in supporting this work, in particular Grants R01 ES06501 (to MSM) from the National Institute of Environmental Health Sciences and Cancer Center Support Grant P30 CAI2197 from the National Cancer Institute, which provided support for the Analytical Imaging Core Facility, the DNA Synthesis Core Laboratory, and the DNA Sequencing and Gene Analysis Facility. The present address of Lisa L. Wessner is Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 191 11, USA. The present address of Manxia Fan is Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Address correspondence to Dr. Mark Steven Miller, Department of Cancer Biology, N'ake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA, E-mail: firstname.lastname@example.org
- Lung cancer
- Tumor suppression genes