Cancer cell telomere dynamics and responses to perturbations

描述(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、端粒故障的后果以及癌细胞中端粒酶的激活和失活是很重要的。我们最近开始使用一种新的、高时间分辨率的光学显微镜系统来探测活细胞中的端粒动力学，并在活的人类细胞中发现了新型的端粒运动。目的1将分析这种短时间(秒)的实时端粒动力学，量化活细胞中端粒的时间分辨3D运动，分析端粒动力学对端粒扰动的响应，在癌细胞和正常人类细胞中。我们之前已经设计并测试了突变模板hTer(MT-hTer)，它迫使高活性的端粒酶(癌细胞的特征)在端粒上添加突变序列重复序列，从而诱导端粒快速去封顶，并引发包括凋亡在内的细胞反应。这些作用迅速，端粒长度无关，不需要功能性的P53或Rb。目标2将确定端粒的机制和角色，在端粒去封顶时，启动信号反应，最终导致细胞凋亡。击倒癌细胞中的NIGH端粒酶也会迅速抑制它们的生长，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，这些方面是已知的癌症进展的核心。端粒酶RNA基因敲除后基因表达谱的独特变化被预测与减缓癌症进展有关。目的3将检测端粒酶/端粒的哪个方面(S)丢失/改变时引起细胞对端粒酶RNA敲除的反应，并分析人黑色素瘤细胞对端粒酶缺失的细胞和代谢反应。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的超短端粒(“t-stump”)。在目标4中，我们将对t-残基进行进一步的结构分析，确定t-残基对端粒酶和检查点通路的依赖性，并检验这一假说，即t-残基通过以端粒酶特异性的方式向细胞发出信号，可能是端粒酶敲除的快速细胞效应的基础。意义：许多先前的证据表明端粒酶促进肿瘤的维持和生长，并且端粒酶被认为是抗癌治疗的靶点。我们的工作将促进对癌症端粒生物学的基本了解，这将对开发利用癌细胞独特的端粒酶状态的新的治疗策略将是重要的。

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.