IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

从Li-Fraumeni患者中培养的人成纤维细胞 LFS综合征（LFS）包含生殖系p53突变， 染色体不稳定，他们退出的增长经历的危机 由正常的人类成纤维细胞，发展成永生细胞系。 该项目涉及筛选选定的化学预防 在LFS成纤维细胞培养物中使用基于机制的测定， 分析最早获得的性状，利用以下 终点标志物：（1）自发永生化（2）基因组 稳定性（3）细胞周期控制和其他分子遗传变化 （4）端粒DNA的稳定， 端粒酶的激活或替代机制和（5）锚定 独立成长。两个独立的LFS成纤维细胞， 生殖系p53突变，无生殖系p53的LFS成纤维细胞 突变，和正常对照成纤维细胞在培养物中生长， 并测定观察到的肿瘤进展的参数 在永生化过程中自发地在LFS培养物中。DNA和细胞 从每个成纤维细胞中制备蛋白质提取物 每隔5 PD进行一次应变。每种菌株的十种独立培养物 在不存在和存在化学预防剂的情况下平行进行 剂永生化频率应与 一系列其他客观和定量可测量的终点 可以在不存在或存在化学预防剂的情况下进行评估 剂.

Cultured human fibroblasts from patients with the Li-Fraumeni syndrome (LFS) containing germline p53 mutations, rapidly develop chromosomal instability, and they exit the growth crisis experienced by normal human fibroblasts, developing into immortal cell lines. This project involves the screening of selected chemopreventive agents using mechanism-based assays in cultures of LFS fibroblasts by analysis of the earliest acquired traits utilizing the following endpoint markers: (1) spontaneous immortalization (2) genomic stability (3) cell cycle control and other molecular genetic changes that accompany immortalization (4) stabilization of telomeric DNA via activation of telomerase or alternative mechanisms and (5) anchorage independent growth. Two independent LFS fibroblasts cells with germline p53 mutations, an LFS fibroblast without a germline p53 mutation, and a normal control fibroblast are being grown in culture, and assayed for parameters of tumor progression that are observed spontaneously in LFS cultures during immortalization. DNA and cell protein extracts are being prepared from each fibroblastic cell strain every 5PD. Ten independent cultures of each strain are being carried in parallel in the absence and presence of a chemopreventive agent. The immortalization frequency shall be correlated with a series of other objectively and quantitatively measurable endpoints that can be assessed in the absence or presence of chemopreventive agents.