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，分析癌细胞中的端粒行为、端粒功能障碍的后果以及端粒酶的激活和失活非常重要。我们最近开始使用新推出的高时间分辨率光学显微镜系统探索活细胞中的端粒动力学，并在活人类细胞中发现了新型端粒运动。目标 1 将分析短时间（秒）内的实时端粒动态，量化活细胞中端粒的时间分辨 3D 运动，以分析癌性和正常人类细胞中端粒动态对端粒扰动的反应。我们之前设计并测试了突变模板 hTers (MT-hTer)，它迫使高活性端粒酶（癌细胞的特征）在端粒上添加突变序列重复，从而诱导端粒快速脱帽，并引发细胞反应，包括细胞凋亡。这些效应是快速的、与端粒长度无关的并且不需要功能性 p53 或 Rb。目标 2 将确定端粒的机制和参与者，端粒脱帽后启动信号反应，最终导致细胞凋亡。敲低癌细胞中的近端粒酶也能迅速抑制其生长，引发明显的细胞和转录变化。这些和其他最近的结果表明，端粒酶可能在癌症的其他方面发挥作用，这些方面对癌症进展至关重要。端粒酶 RNA 敲低后基因表达谱的独特变化预计与癌症进展减缓有关。目标 3 将测试端粒酶/端粒丢失/改变时的哪些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”）。在目标 4 中，我们将对 t 残端进行进一步的结构分析，确定 t 残端对端粒酶和检查点通路的依赖性，并测试以下假设：t 残端通过以端粒酶特异性方式向细胞发出信号，可能是端粒酶敲低的快速细胞效应。意义：许多先前的证据表明端粒酶促进肿瘤的维持和生长，并且端粒酶已被提议作为抗癌治疗的靶点。我们的工作将增进对癌症端粒生物学的基本了解，这对于开发新的治疗策略以利用癌细胞独特的端粒酶状态非常重要。