Cancer cell telomere dynamics and responses to perturbations

癌细胞端粒动力学和对扰动的反应

• 批准号: 7913694
• 负责人: ELIZABETH H BLACKBURN
• 金额: $ 29.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7913694
• 关键词: Affect; Alleles; Apoptosis; Apoptotic; Behavior; Binding; Biology; Cancerous; Cell Cycle Stage; Cell Nucleus; Cells; Characteristics; DNA damage checkpoint; Dependence; Gene Expression; Growth; Human; Knock-out; Length; Life; Maintenance; Malignant Neoplasms; Melanoma Cell; Metabolic; Methods; Mitosis; Modeling; Molecular Profiling; Movement; Normal Cell; Nuclear Envelope; Pathway interactions; Play; Property; Proteins; Research Personnel; Resolution; Role; Signal Transduction; Small Interfering RNA; System; TP53 gene; Technology; Telomerase; Telomerase RNA Component; Telomere Shortening; Testing; Time; Work; cancer cell; cancer therapy; design; knock-down; light microscopy; mutant; novel; novel therapeutics; programs; protein complex; response; senescence; sensor; telomere; tumor; tumor progression

DESCRIPTION (provided by applicant): To better understand the role of telomeres and telomerase in cancer, it is important to analyze telomere behavior, the consequences of telomere malfunctioning, and telomerase activation and inactivation in cancer cells. We recently began to probe telomere dynamics in living cells using a newly available, high time-resolution light microscopy system, and found novel classes of telomere movements in live human cells. Aim 1 will analyze such real-time telomere dynamics over short times (seconds), quantifying time- resolved 3D movements of telomeres in live cells, to analyze responses of telomere dynamics to telomere perturbations, in cancerous and normal human cells. We have previously designed and tested mutant- template hTers (MT-hTer) that force the highly active telomerase (characteristic of cancer cells) to add mutant sequence repeats to telomeres, which induced a rapid uncapping of telomeres, and elicited cellular responses, including apoptosis. These effects were rapid, telomere length-independent and do not require functional p53 or Rb. Aim 2 will determine the mechanisms and players at the telomeres that, upon telomere uncapping, initiate the signaling response that ultimately ends in apoptosis. Knocking down the nigh telomerase in cancer cells also quickly inhibited their growth, eliciting distinct cellular and transcriptional changes. These and other recent results have indicated that telomerase likely plays roles in other aspects of cancer known to be central to cancer progression. The distinctive alterations in the gene-expression profiles upon telomerase RNA knockdown were predicted to be associated with diminished cancer progression. Aim 3 will test which aspect(s) of telomerase/telomeres when lost/altered cause the cellular response to telomerase RNA knockdown, and also analyze the cellular and metabolic responses of human melanoma cells to telomerase depletion. By using the single telomere length analysis (STELA) method, we have discovered a novel class of ultra-short telomeres ("t-stumps") in cancer cells. In Aim 4 we will pursue further structural analysis of t-stumps, determine the dependence of t-stumps on telomerase and checkpoint pathways, and test the hypothesis that t-stumps, by signaling cells in a telomerase-'specific fashion, may underlie the rapid cellular effects of telomerase knockdown. Significance: Much previous evidence has pointed to telomerase promoting tumor maintenance and growth, and telomerase has been proposed as a target for anti-cancer therapies. Our work will advance the basic understanding of cancer telomere biology, which will be important to develop novel therapeutic strategies to exploit the unique telomerase status of cancer cells.

描述（由申请人提供）：为了更好地了解端粒和端粒酶在癌症中的作用，分析端粒行为，端粒出现故障的后果以及端粒酶激活和癌细胞中的灭活非常重要。最近，我们开始使用新的，高时间的光学显微镜系统来探测活细胞中的端粒动力学，并在活细胞中发现了新型的端粒运动。 AIM 1将在短时间内（秒）分析此类实时端粒动力学，量化活细胞中端粒的时间分解的3D运动，以分析端粒动力学对端粒扰动的响应，癌细胞和正常的人类细胞。我们先前已经设计和测试了突变模板hters（mt-hter），迫使高度活性的端粒酶（癌细胞的特征）为端粒添加突变序列重复序列，从而诱导端粒的快速解蛋白，并引起细胞反应，包括凋亡，包括凋亡。这些效果是快速的，端粒长度独立的，不需要功能性p53或rb。 AIM 2将确定端粒的机制和参与者，这些机制和参与者在端粒解膜时会启动最终以凋亡结束的信号反应。击倒癌细胞中的附近端粒酶也很快抑制了它们的生长，从而引起了不同的细胞和转录变化。这些和其他最近的结果表明，端粒酶可能在已知的癌症的其他方面起着对癌症进展至关重要的作用。预测端粒酶RNA敲低时基因表达谱的明显变化与癌症进展减少有关。 AIM 3将在丢失/改变时测试端粒酶/端粒的哪个方面会导致细胞对端粒酶RNA敲低的反应，并分析人类黑色素瘤细胞对端粒酶耗竭的细胞和代谢反应。通过使用单个端粒长度分析（Stela）方法，我们在癌细胞中发现了一类新型的超短端粒（“ t-stumps”）。在AIM 4中，我们将对T-T-Tumps进行进一步的结构分析，确定T-支柱对端粒酶和检查点途径的依赖性，并检验以端粒酶 - 特异性方式信号传导细胞来测试T-T-Tumps的假设，这可能是端粒酶敲除的快速细胞影响。意义：以前的许多证据表明，端粒酶促进肿瘤的维持和生长，并且已提出端粒酶作为抗癌疗法的靶标。我们的工作将促进对癌症端粒生物学的基本理解，这对于开发新型治疗策略以利用癌细胞的独特端粒酶状态非常重要。