CHARACTERIZATION OF TELOMERASE AND ITS INHIBITORS

癌细胞的一般特征之一是基因组不稳定。 尽管目前还不清楚是什么导致了这种不稳定，但有一种假设 引起越来越多的关注的是，自由染色体的末端，要么来自 染色体断裂或由于端粒序列的丢失而导致的 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 也许，随着每次细胞分裂，端粒序列逐渐丢失 因为末端复制问题。肿瘤细胞确实缩短了 端粒，但它们也具有极高的酶水平 端粒酶可以克服末端复制问题，而正常细胞可以 不。因此，端粒酶是新型抗癌药物的一个有吸引力的靶点。 因为对肿瘤细胞具有预期的选择性。此外，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，我们建议对端粒酶及其抑制物进行大量的表征 更详细地，为了选择最有效的代理并定义他们的 生物效应。具体地说，我们提出：L)分离、提纯和 鉴定几种人类肿瘤类型的端粒酶(S)。端粒酶来自 选定的乳腺癌、肺癌和结肠癌样本将被分离并 具有生物化学特性，以及端粒酶的良好特性 HeLa人肿瘤细胞株将得到纯化。此外，我们还将设计 新的、更快、更灵敏的端粒酶活性分析。2)至 阐明人肿瘤端粒酶的作用机制和特异性 被选定的药物的抑制作用。代理商将包括：(A) 核苷/核苷酸类似物，(B)非核苷逆转录酶 抑制剂，以及(C)反义分子。它们能够抑制孤立的 端粒酶将被测定。与其他核苷酸加工方法的比较 逆转录酶、末端转移酶和DNA等酶 聚合酶将有助于描述特定的结构要求 端粒酶抑制。3)比较药物的形成和修复- 诱导的端粒损伤与块状DNA相比。特别是，我们将 研究DNA反应物对结构和功能的作用 端粒DNA。助剂将包括：(A)一种AT特定的烷基化辅助剂 凹槽粘结剂，(B)GC特异性DNA插层剂，和(C)一个 烯二炔链断裂剂。 从这些研究中，我们希望了解到生物化学和生物化学 端粒酶抑制的后果，原型结构提供 特异性抑制的最好前景，以及肿瘤细胞如何应对 端粒的损伤。总体而言，这项工作将为我们揭示 对端粒/端粒酶系统的干扰可能提供新的和 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.