CHARACTERIZATION OF A NEW GENE IN P53 MUTANT LFS FAMILY

这项研究的长期目标是了解p53生殖系突变对易感性和癌症发展的影响。 肿瘤抑制基因p53在超过一半的人类癌症中发生突变。 特别是在Li-Fraumeni综合征（LFS）中，p53的杂合生殖系突变是使LFS家族成员易于发生癌症的主要遗传缺陷。 野生型p53的功能由参与信号转导途径的许多下游基因介导。 本研究的主要目的是分离、鉴定和表征在所研究的LFS家族成员的肿瘤发生过程中差异表达的候选基因。 为了更好地了解LFS基因型和表型之间的相关性，我们建议招募一对兄弟姐妹从这个LFS家庭包含一个正常的p53（wt/wt）和杂合突变（wt/mt）。 对来自这些兄弟姐妹的非皮肤成纤维细胞（NSF）进行mRNA差异显示方法以分离可能受p53调节的新基因，否则预期这些兄弟姐妹具有非常相似的遗传同一性。 我们建议克隆和表征的全长cDNA的差异表达的基因片段（ZS-3），似乎没有显示出序列或功能同源性的基因在Genbank数据库中。 该3 kb基因片段将通过RACE方法延伸，并将用于筛选正常成纤维细胞cDNA文库。 通过FISH实验将所得基因定位到染色体位置，并确定其组织特异性。 为了评估其在其他LFS家族成员的肿瘤组织中的表达，将用全长ZS-3基因筛选来自所研究的LFS家族的肿瘤样品的子集。 我们还将使用反义寡核苷酸来验证新基因的wt功能，并分析其对具有或不具有p53功能形式的NSFs中细胞增殖的影响。 我们将通过将其重新引入NSF细胞系并分析对细胞增殖的影响来表达该基因。 然后将评估差异表达基因的序列特异性DNA结合特性及其与p53的相互作用。 这些LFS的研究应该描绘LFS的基因型和表型之间的相关性，并有助于对致癌过程的一般理解。

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.