ROLE OF TUMOR-SUPPRESSOR GENES IN TRANSPLACENTAL LUNG CARCINOGENESIS

Most human cancers involve multiple genetic changes, including activat ion of oncogenes such as Ki-ras-2 (Kras2) and inactivation of any one of a number of tumor suppressor genes such as p53 and members of the r etinoblastoma (Rb) regulatory axis. As part of an ongoing project to d etermine how in utero exposure to chemical carcinogens affects the mol ecular pathogenesis of murine lung tumors, the p53 and p16(Cdkn2a) gen es were analyzed by using paraffin-embedded lung tissues from mice tre ated transplacentally with 3-methylcholanthrene. Single-strand conform ation polymorphism analysis of exons 5-8 of the p53 gene, as well as t heir flanking introns, demonstrated an absence of mutations at this ge ne locus. However, a genetic polymorphism was identified at nt 708 in intron 4 of the DBA/2 strain of mice 5 bp downstream of a 3' branching -point splice signal. Analysis of exons 1 and 2 of the Cdkn2a gene by single-strand conformation polymorphism and sequence analyses revealed mutations in exon 2 in 7% of the tumors examined. Tumor 23-1 exhibite d a CAC-->TAC transition at nt 301 (His(74)-->Tyr(74)) and tumor 36-1 exhibited a GGG-->GAG transition at nucleotide 350 (Gly(90)-Glu90). No rthern blot analysis of 14 of the larger tumors showed a marked decrea se in the levels of Rb RNA expression. Immunohistochemical analysis re vealed a spectrum of pRb expression, with the smaller adenomas showing moderate numbers of nuclei with heterogeneous staining for pRb in con trast with a highly reduced or near-complete absence of expression in the nuclei of larger tumors with features of adenocarcinomas. The low incidence of mutations at tumor suppressor loci suggested that inactiv ation of tumor suppressor genes was a late event in murine lung tumor pathogenesis. The identification of both mutations at the CdknZa gene locus and reduced levels of Rb expression combined with previous studi es demonstrating a high incidence of mutated Kras2 alleles in these tu mors implies that alterations of the Rb regulatory axis, in combinatio n with mutation of Kras2, may be the preferred pathway for the pathoge nesis of pulmonary tumors in transplacentally exposed mice. (C) 1998 W iley-Liss, Inc.