IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

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Bischoff et al (Cancer Res 50:7979, 1990) found that fibroblasts obtained from patients with the Li-Fraumeni syndrome (LFS) containing germline mutations in p53, can develop into immortal cell lines. Subsequently, it was demonstrated by Shay et al (Mol. Cell Biol. 15:425, 1995) that cultured human breast epithelial cells from patients with LFS which are heterozygous for p53, show genomic instability, loss of cell cycle control, and can develop into immortal cell lines. The immortalized human breast epithelial cells exhibit loss of the remaining normal p53 allele, telomerase reactivation, telomere stabilization, and continued cell proliferation. The objective of this contract is to screen selected chemopreventive agents using mechanism-based assays in cultures of LFS breast-derived, "normal" and spontaneously immortalized epithelial cells by analysis of the earliest acquired traits utilizing the following endpoint markers: (1) Spontaneous immortalization (2) Genomic stability (3) Cell cycle and other molecular changes that accompany immortalization (4) Stabilization of telomeric DNA via activation of telomerase or alternative mechanisms. (5) Anchorage independent growth/ability to grow in serum. The key question to determine is if fewer immortalization events occur in the cultures containing the lowest efficacious concentrations of chemopreventive agents that do not cause acute toxicity. 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. Five agents will be tested.