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Role of tumor suppressor genes in transplacental lung carcinogenesis
L A Rollins1, S Leone-Kabler, M G O'Sullivan
1Department of Cancer Biology, Bowman Gray School of Medicine, Comprehensive Cancer Center of Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Abstract:
Most human cancers involve multiple genetic changes, including activation 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 retinoblastoma (Rb) regulatory axis. As part of an ongoing project to determine how in utero exposure to chemical carcinogens affects the molecular pathogenesis of murine lung tumors, the p53 and p16Cdkn2a genes were analyzed by using paraffin-embedded lung tissues from mice treated transplacentally with 3-methylcholanthrene. Single-strand conformation polymorphism analysis of exons 5-8 of the p53 gene, as well as their flanking introns, demonstrated an absence of mutations at this gene 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 exhibited a CAC-->TAC transition at nt 301 (His74-->Tyr74), and tumor 36-1 exhibited a GGG-->GAG transition at nucleotide 350 (Gly90-->Glu90). Northern blot analysis of 14 of the larger tumors showed a marked decrease in the levels of Rb RNA expression. Immunohistochemical analysis revealed a spectrum of pRb expression, with the smaller adenomas showing moderate numbers of nuclei with heterogeneous staining for pRb in contrast 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 inactivation of tumor suppressor genes was a late event in murine lung tumor pathogenesis. The identification of both mutations at the Cdkn2a gene locus and reduced levels of Rb expression combined with previous studies demonstrating a high incidence of mutated Kras2 alleles in these tumors implies that alterations of the Rb regulatory axis, in combination with mutation of Kras2, may be the preferred pathway for the pathogenesis of pulmonary tumors in transplacentally exposed mice.
Insights
Maternal exposure to carcinogens during pregnancy can lead to lung tumors in mice. These tumors often show Kras2 mutations and alterations in the Rb pathway, suggesting a key role in cancer development.
Area of Science:
- Molecular biology
- Cancer research
- Toxicology
Background:
- Human cancers often result from multiple genetic alterations, including oncogene activation (e.g., Kras2) and tumor suppressor gene inactivation (e.g., p53, Rb pathway).
- Understanding the impact of in utero exposure to chemical carcinogens on lung tumor development is crucial for identifying carcinogenic mechanisms.
Purpose of the Study:
- To investigate the molecular pathogenesis of murine lung tumors induced by transplacental exposure to 3-methylcholanthrene.
- To analyze mutations in the p53 and p16Cdkn2a genes and assess the expression of the retinoblastoma (Rb) protein in these tumors.
Main Methods:
- Paraffin-embedded lung tissues from mice treated transplacentally with 3-methylcholanthrene were analyzed.
- Single-strand conformation polymorphism (SSCP) and sequence analyses were used to detect mutations in p53 and Cdkn2a genes.
- Northern blot and immunohistochemical analyses were performed to evaluate Rb RNA and pRb protein expression.
Main Results:
- No mutations were found in the p53 gene; however, a genetic polymorphism was identified in intron 4 of the DBA/2 strain.
- Mutations in exon 2 of the Cdkn2a gene were detected in 7% of the tumors, including specific point mutations (His74Tyr74, Gly90Glu90).
- A marked decrease in Rb RNA expression and reduced nuclear pRb expression were observed, particularly in larger tumors.
Conclusions:
- Inactivation of tumor suppressor genes appears to be a late event in murine lung tumor development.
- Mutations in Cdkn2a and reduced Rb expression, coupled with Kras2 mutations, suggest the Rb regulatory axis plays a critical role in pulmonary tumor pathogenesis in transplacentally exposed mice.