IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

Bischoff等人(癌症研究50：7979,1990)发现成纤维细胞 来自Li-Fraumeni综合征(LFS)患者，含有 胚系突变的P53，可以发展成永生细胞系。 随后，Shay等人(Mol.细胞生物。 15：425,1995)培养人乳腺上皮细胞 P53杂合子的LFS患者显示基因组 不稳定，失去对细胞周期的控制，并可发展为 永生细胞系。永生化的人乳腺上皮细胞 发现剩余的正常p53等位基因端粒酶缺失 重新激活、端粒稳定和细胞继续 扩散。这份合同的目标是筛选选定的 化学预防药物在体外培养中的机理分析 LFS乳房衍生，“正常”，自然永生 通过对最早获得性特征的分析获得上皮细胞 利用以下终点标记：(1)自发 永生化(2)基因组稳定性(3)细胞周期及其他 伴随永生化的分子变化(4)稳定 端粒DNA通过激活端粒酶或其他机制。 (5)锚定独立生长/在血清中生长能力。钥匙 需要确定的问题是，如果在 含有最低有效浓度的 不会造成急性毒性的化学预防药物。这个 永生频率应与一系列其他 可客观和定量测量的端点，可以 在没有或存在化学预防药物的情况下进行评估。五 代理将接受测试。

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.