Analysis of Telomerase-Independence Telomere Maintenance

端粒酶非依赖性端粒维持分析

• 批准号: 7405168
• 负责人: DOMINIQUE BROCCOLI
• 金额: $ 24.84万
• 依托单位: MEMORIAL HEALTH UNIVERSITY MED CTR, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405168
• 关键词: alleles; carcinogenesis; cell line; electron microscopy; fluorescent in situ hybridization; genetic recombination; immunoprecipitation; nucleic acid structure; p53 gene /protein; telomerase; telomere; temperature sensitive mutant

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the mechanism of telomerase-independent telomere maintenance. Although telomerase activation is the most common mechanism for maintenance of telomeric DNA, a subset of tumors and cell lines use a telomerase-independent mechanism, termed Alternative Lengthening of Telomeres (ALT), to sustain telomeric DNA. In telomerase-deficient yeast strains, telomere stabilization is achieved through recombination-based mechanisms. Similarly, although the molecular mechanism has yet to be established, the evidence from mammalian systems is consistent with ALT involving recombination. Based on data from yeast studies, it has been suggested that telomere recombination requires loss of the capping activity by which telomeres were originally defined. To test this hypothesis in mammalian cells, the structure of ALT telomeres will be determined using molecular biological methods and electron microscopy. Clarifying the structure of ALT telomeres will provide insight into the molecular mechanism of ALT. Secondly, factors involved in DNA recombination that are essential for telomere maintenance by ALT will be identified using dominant negative and knockdown approaches. These data will characterize the molecular requirements for ALT and will differentiate between possible recombination-based mechanisms. The only genetic alteration in mammalian cells linked to a predisposition to activate ALT is p53 status. Among its other functions, p53 has been shown to be involved in suppression of recombination. Thus, the role of p53 in regulating ALT will be characterized using temperature sensitive alleles of p53. These data will be the first that illustrate how alterations in a factor might predispose cells to ALT. Inhibition of telomerase has become an attractive target for future chemotherapeutic agents. However, ALT tumors would be refractory to these treatments. This proposal will identify structural changes and factors essential for activation of ALT and thus identify new potential targets for chemotherapeutic agents.

描述（由申请人提供）：该提案的目标是表征不依赖于端粒酶的端粒维持机制。尽管端粒酶激活是维持端粒 DNA 的最常见机制，但肿瘤和细胞系的一部分使用不依赖端粒酶的机制（称为端粒替代延长 (ALT)）来维持端粒 DNA。在端粒酶缺陷的酵母菌株中，端粒稳定是通过基于重组的机制实现的。同样，虽然分子机制尚未确定，但来自哺乳动物系统的证据与涉及重组的 ALT 一致。根据酵母研究的数据，有人认为端粒重组需要丧失最初定义端粒的加帽活性。为了在哺乳动物细胞中检验这一假设，将使用分子生物学方法和电子显微镜来确定 ALT 端粒的结构。阐明 ALT 端粒的结构将有助于深入了解 ALT 的分子机制。其次，将使用显性失活和敲除方法来鉴定参与 DNA 重组的因素，这些因素对于 ALT 维持端粒至关重要。这些数据将表征 ALT 的分子需求，并将区分可能的基于重组的机制。哺乳动物细胞中唯一与激活 ALT 倾向相关的基因改变是 p53 状态。除其他功能外，p53 已被证明参与抑制重组。因此，p53 在调节 ALT 中的作用将使用 p53 的温度敏感等位基因来表征。这些数据将首次说明某个因素的改变如何使细胞易患 ALT。端粒酶的抑制已成为未来化疗药物的一个有吸引力的目标。然而，ALT 肿瘤对这些治疗无效。该提案将确定 ALT 激活所必需的结构变化和因素，从而确定化疗药物的新潜在靶点。

项目成果

Doxorubicin resistance in a novel in vitro model of human pleomorphic liposarcoma associated with alternative lengthening of telomeres.

• DOI: 10.1158/1535-7163.mct-09-0705
• 发表时间: 2010-03
• 期刊: Molecular cancer therapeutics
• 影响因子: 5.7
• 作者: Mitchell MA;Johnson JE;Pascarelli K;Beeharry N;Chiourea M;Gagos S;Lev D;von Mehren M;Kipling D;Broccoli D
• 通讯作者: Broccoli D