CHARACTERIZATION OF A NEW GENE IN P53 MUTANT LFS FAMILY

这项拟议研究的长期目标是了解p53胚系突变在易感性和癌症发展中的影响。肿瘤抑制基因p53在超过一半的人类癌症中发生突变。尤其是在Li-Fraumeni综合征(LFS)中，P53的杂合胚系突变是使LFS家族成员易患癌症的主要遗传缺陷。Wt p53的功能是由参与信号转导途径的多个下游基因介导的。这一建议的主要目的是分离、鉴定和鉴定在研究中的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的基因型和表型之间的相关性，我们建议从这个LFS家族中招募一对包含正常P53(wt/wt)和杂合突变(wt/mt)的兄弟姐妹。来自这些兄弟姐妹的非皮肤成纤维细胞(NSF)将受到mRNA差异显示方法学的影响，以分离可能受P53调控的新基因。我们建议克隆和鉴定一个差异表达基因片段(ZS-3)的全长cDNA，该基因片段似乎没有序列或功能上与Genbank数据库中的基因同源性。这一3kb的基因片段将通过RACE方法进行延伸，并用于筛选正常成纤维细胞的cDNA文库。由此产生的基因将通过FISH实验被定位到染色体位置，并确定其组织特异性。为了评估它在LFS家族其他成员的肿瘤组织中的表达，将用全长ZS-3基因对正在研究的LFS家族的一部分肿瘤样本进行筛选。我们还将用反义寡核苷酸使新基因的wt功能失活，并分析其对具有或不具有P53功能形式的神经干细胞增殖的影响。我们将通过将该基因重新导入NSF细胞系并分析其对细胞增殖的影响来表达该基因。然后将评估差异表达的基因的序列特异性DNA结合特性以及它与P53的相互作用。这些对LFS的研究应该描绘出LFS的基因型和表型之间的相关性，并有助于对导致癌症发生的过程的总体理解。

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.