The goal of this project is to identify genetic alterations in tumor suppressor genes and proto-oncogenes that play a role in mouse carcinogenesis. Allelic losses of specific chromosomal regions, detected by restriction fragment length polymorphism (RFLP) analyses, suggest that tumor suppressor gene inactivation occurs in most common human cancers. We have extended RFLP analyses to F1 mouse tumor DNA in order to map potential tumor suppressor genes in mice. DNA from a panel of butadiene-induced lung adenocarcinomas, lymphomas, and hepatomas of B6C3F1 mice (NTP bioassay) have been examined for allelic losses. RFLP analyses revealed that one copy of chromosome 4 was deleted in five of eight lung tumors as well as one of ten lymphomas; one allele of the retinoblastoma gene was also deleted in a lung tumor. Lung tumor DNAs were examined by PCR and direct sequencing for point mutations in the P53 tumor suppressor gene, which is frequently mutated in a wide variety of human tumors. A G->T transition in a P53 mutation hotspot was observed in one tumor. RFLP analysis and a novel PCR assay for a CA repeat near the P53 gene revealed allelic losses in DNA of another lung tumor and three lymphomas. These tumors are likely to contain mutant P53 genes which we will characterize by single-strand conformation polymorphism analyses and direct sequencing. In order to generate additional tumors for RFLP studies, we have constructed a series of 9 transgenic mouse lines containing mutated mouse P53 gene constructs in collaboration with M. Eddy and G. Goulding (LRDT). These lines are currently being characterized for transgene expression and tumor formation (lung adenocarcinomas, osteosarcomas, and lymphomas). In addition we have obtained transgenic lines that develop mammary carcinomas (MMTV-ras, MMTV-neu, and MMTV-myc), osteosarcomas (polyoma early region) and lung carcinomas (Abl-ras) from outside investigators for analysis of allelic losses and P53 mutations.
