IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

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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.