The long-term goal of the proposed study is to understand the influence of germline mutations of p53 in predisposition and cancer development. The tumor suppression gene, p53, is mutated in more than half of all human cancers. In a Li-Fraumeni Syndrome (LFS) in particular, a heterozygous germline mutation of p53 is the primary genetic defect that predisposes LFS family members to cancer development. The functions of a wt p53 are mediated by a number of downstream genes involved in the signal transduction pathway. The main aim of this proposal is to isolate, identify and characterize candidate genes that are differentially expressed during the process of tumorigenesis in members of LFS family under study. To better understand the correlation between genotype and phenotype in LFS, we propose to recruit a pair of siblings from this LFS family containing a normal p53 (wt/wt) and a heterozygous mutation (wt/mt). To non- skin fibroblasts (NSFs) from theses siblings who otherwise would be expected to have very similar genetic identity, will be subjected to the mRNA differential display methodology to isolate novel genes that might be regulated by p53. We propose to clone and characterize the full-length cDNA of a differentially expressed gene fragment (ZS-3) that seems to show no sequence or functional homology to genes in the Genbank database. This 3 kb gene fragment will be extended by the RACE method and will be used to screen a normal fibroblast cDNA library. The resulting gene will be mapped to a chromosome location by the FISH experiment and its tissue-specificity determined. To assess its expression in tumor tissues of other LFS family members, a subset of tumor samples from the LFS family under study will be screened with the full-length ZS-3 gene. We will also inactivate the wt function of the new gene with antisense oligonucleotides and analyze its effects on cell proliferation in NSFs with or without the functional form of p53. We will express the gene by reintroducing it into NSF cell lines and analyzing the resulting effects on cell proliferation. The differentially expressed gene will then be evaluated for its sequence-specific DNA binding properties and its interaction with p53. These studies of LFS should delineate the correlation between genotype and phenotype in LFS and contribute to the general understanding of the processes leading to carcinogenesis.
