CHARACTERIZATION OF TELOMERASE AND ITS INHIBITORS

癌细胞的一般特征之一是基因组不稳定性。 虽然目前还不清楚是什么导致了这种不稳定性，但有一种假说认为， 越来越受到关注的是，游离染色体末端，无论是从 染色体断裂或端粒序列的丢失， 末端，容易发生非法重组事件。因此，端粒 为染色体提供稳定性。然而，似乎有一个 可能是随着每次细胞分裂端粒序列逐渐丢失 因为末端复制的问题。肿瘤细胞确实缩短了 端粒，但他们也拥有大大提高水平的酶 端粒酶来克服末端复制问题，而正常细胞可以 没有因此，端粒酶是一个有吸引力的新的抗癌药物的目标 这是因为预期的对肿瘤细胞的选择性。而且我们 已经有初步证据表明，抑制端粒酶可以杀死 癌细胞 因此，我们建议在很大程度上表征端粒酶及其抑制剂， 更详细，以选择最有效的代理商，并定义其 生物效应。具体而言，我们建议：1）分离，纯化， 表征来自几种人类肿瘤类型的端粒酶。端粒酶从 将分离选定的乳腺癌、肺癌和结肠癌标本， 生物化学特征，以及来自良好表征的 将纯化HeLa人肿瘤细胞系。此外，我们将设计 新的，更快，更灵敏的端粒酶活性检测。2)到 阐明人肿瘤端粒酶的作用机制和特异性 选择性抑制剂。代理人将包括： 核苷/核苷酸类似物，（B）非核苷逆转录酶 抑制剂和（c）反义分子。它们抑制孤立的 将测定端粒酶。与其他核苷酸加工的比较 酶如逆转录酶、末端转移酶和DNA 聚合酶将有助于描述特定的结构要求， 抑制端粒酶。3)比较药物- 诱导端粒损伤而不是大量DNA损伤。特别是要 检查DNA反应剂对结构和功能的作用 端粒DNA试剂将包括：（a）AT特异性烷基化次要试剂， 沟结合剂，（B）GC特异性DNA嵌入剂，和（c） 烯二炔断链剂。 从这些研究中，我们希望了解生物化学和生物学 端粒酶抑制的后果，原型结构提供了 特异性抑制的最佳前景，以及肿瘤细胞如何科普 端粒的损伤 总的来说，这项工作将揭示新的光如何 干扰端粒/端粒酶系统可以提供一种新的， 人类癌症的选择性治疗策略。

One of the general characteristics of cancer cells is genomic instability. Though it is still unclear what causes this instability, a hypothesis gaining increasing attention is that free chromosome ends, either from chromosome breakage or from loss of the telomere sequences which cap the ends, are prone to illegitimate recombination events. Thus, telomeres provide stability to the chromosomes. However, there appears to be a gradual loss of telomere sequences with each cell division, perhaps because of the end-replication problem. Tumor cells do have shortened telomeres, but they also possess greatly elevated levels of the enzyme telomerase to overcome the end-replication problem, while normal cells do not. Thus, telomerase is an attractive target for new anti-cancer agents because of the expected selectivity for neoplastic cells. Furthermore, we already have preliminary evidence that inhibiting telomerase can kill cancer cells. We therefore propose to characterize telomerase and its inhibitors in much more detail, in order to select the most effective agents and define their biological effects. Specifically, we propose: l) To isolate, purify, and characterize telomerase(s) from several human tumor types. Telomerase from selected breast, lung, and colon cancer specimens will be isolated and characterized biochemically, and telomerase from the well-characterized HeLa human tumor cell line will be purified. In addition, we will design new, faster, and more sensitive telomerase activity assays. 2) To elucidate the mechanism and specificity of human tumor telomerase inhibition by selected agents. Agents will include: (a) nucleoside/nucleotide analogs, (b) nonnucleoside reverse transcriptase inhibitors, and (c) antisense molecules. Their ability to inhibit isolated telomerase will be determined. Comparison with other nucleotide processing enzymes such as reverse transcriptase, terminal transferase, and DNA polymerases will help to delineate structural requirements for specific inhibition of telomerase. 3) To compare the formation and repair of drug- induced lesions in telomeres versus bulk DNA. In particular, we will examine the actions of DNA-reactive agents on the structure and function of telomeric DNA. Agents will include: (a) an AT-specific alkylating minor groove binder, (b) a GC-specific DNA intercalating agent, and (c) an enediyne strand scission agent. From these studies we hope to learn the biochemical and biological consequences of telomerase inhibition, which prototype structures offer the best promise of specific inhibition, and how tumor cells cope with lesions in the telomeres. Overall, this work will shed new light on how interference with the telomere/telomerase system could provide a new and selective therapeutic strategy for human cancer.