Cancer cell telomere dynamics and responses to perturbations

描述（由申请人提供）：为了更好地理解端粒和端粒酶在癌症中的作用，重要的是分析癌细胞中的端粒行为、端粒故障的后果以及端粒酶激活和失活。我们最近开始使用一种新的高时间分辨率光学显微镜系统来探测活细胞中的端粒动态，并在活的人类细胞中发现了新的端粒运动类别。目的1将在短时间（秒）内分析这种实时端粒动力学，量化活细胞中端粒的时间分辨3D运动，以分析癌细胞和正常人细胞中端粒动力学对端粒扰动的响应。我们之前已经设计并测试了突变模板hTers（MT-hTer），其迫使高活性端粒酶（癌细胞的特征）向端粒添加突变序列重复，这诱导端粒的快速去帽，并引发细胞反应，包括凋亡。这些作用是快速的，端粒长度无关，不需要功能性p53或Rb。目的2将确定端粒的机制和参与者，在端粒脱帽后，启动最终以凋亡结束的信号转导反应。敲低癌细胞中的近端端粒酶也能迅速抑制癌细胞的生长，引起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，已知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改变被预测与癌症进展减少有关。目的3将测试端粒酶/端粒的哪些方面在丢失/改变时引起对端粒酶RNA敲低的细胞应答，并且还分析人黑素瘤细胞对端粒酶耗竭的细胞和代谢应答。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的超短端粒（“t-stumps”）。在目标4中，我们将进一步对t残端进行结构分析，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的基础的假设。重要性：许多先前的证据已经指出端粒酶促进肿瘤的维持和生长，并且端粒酶已经被提议作为抗癌治疗的靶点。我们的工作将推进对癌症端粒生物学的基本理解，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将是重要的。

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.