IN VITRO SCREENING OF CHEMOPREVENTIVE AGENTS

Bischoff等（Cancer Res 50：7979，1990）发现成纤维细胞 从患有Li-Fraumeni综合征（LFS）的患者获得， p53的种系突变可以发育成永生细胞系。 随后，Shay等人（Mol. cell Biol. 15：425，1995），其培养人乳腺上皮细胞， p53杂合的LFS患者，显示基因组 不稳定，细胞周期失去控制，并可能发展成 永生细胞系永生化人乳腺上皮细胞 表现出剩余的正常p53等位基因，端粒酶， 再激活、端粒稳定化和连续细胞 增殖这项合同的目的是筛选选定的 化学预防剂，使用基于机制的测定， LFS乳房来源，"正常"和自发永生化 上皮细胞通过分析最早获得的性状 利用以下终点标记：（1）自发性 （2）基因组稳定性（3）细胞周期和其他 伴随永生化的分子变化（4） 端粒DNA通过激活端粒酶或替代机制。 (5)锚定独立生长/在血清中生长的能力。关键 要确定的问题是，如果更少的永生事件发生在 含有最低有效浓度的 不会引起急性毒性的化学预防剂。的 永生化频率应与一系列其他 可以客观和定量测量的终点， 在不存在或存在化学预防剂的情况下进行评估。五 代理商将接受测试。

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.