IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

Li-Fraumeni患者的培养人成纤维细胞 含有胚系p53突变的综合征(LFS)迅速发展 染色体不稳定，他们退出了经历的生长危机 通过正常的人类成纤维细胞，发育成永生细胞系。 该项目涉及筛选选定的化学预防措施。 在LFS成纤维细胞培养中使用基于机制的分析的药物 利用以下方法分析最早获得的性状 终点标记：(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学变化 伴随着永生化(4)端粒DNA通过 端粒酶的激活或替代机制和(5)锚定 自主成长。两个独立的LFS成纤维细胞 胚系p53突变--一种没有胚系p53的LFS成纤维细胞 突变和正常对照成纤维细胞在培养中生长， 并对观察到的肿瘤进展参数进行分析 在永生化过程中自发地在LFS培养中。DNA与细胞 正在从每个成纤维细胞中提取蛋白质提取物 每隔5PD进行一次应变。每种菌株的10种独立培养物正在进行 在没有和存在化学预防的情况下并行进行 探员。永生频率应与 一系列其他客观和定量可测量的终点 这可以在没有或存在化学预防的情况下进行评估 探员们。

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.