Responses to perturbing telomeres in human cells

描述(申请人提供)：细胞核糖核蛋白端粒酶通过复制固有的RNA模板在染色体末端合成端粒DNA。这项拟议研究的长期目标是了解最近发现的端粒和端粒酶在细胞增殖中的具体作用，并研究如何利用颠覆这些作用来影响癌细胞和衰老。在大多数癌细胞中，端粒酶高度活跃。我们最近报道，即使包含七种不同突变模板(但不是对照野生型模板)的人端粒酶RNA基因构建物的低阈值表达水平也足以降低人类癌细胞的存活率，增加细胞凋亡率和减缓肿瘤生长，我们感兴趣的是将这种突变模板端粒酶RNA表达作为抗肿瘤策略的潜在可行性。前三个特定目标解决了正常细胞和癌细胞对突变模板端粒酶RNA的反应机制。它们是：#1：测试七个特定的模板突变的端粒酶RNA是否在体外组装成具有合成突变重复序列能力的核心酶；#2：确定这种突变模板端粒酶RNA在培养的人类细胞中的表达是否导致突变的端粒DNA序列被整合到端粒中，并测试人类端粒蛋白的结合是否受到突变的影响。测试一种特定的突变模板端粒酶RNA的作用机制的特定模型；#3：使用改进的载体系统传递突变模板端粒酶RNA基因，并对细胞生长和凋亡进行短期检测，以分析哪些DNA损伤检查点和凋亡途径参与了对突变模板端粒酶RNA表达的反应。确定与人类端粒功能、DNA损伤和其他反应途径有关的特定基因的参与。最近积累的证据表明，端粒酶通过不需要端粒延长的机制(S)促进细胞增殖。特定目的#4检验特定假设，即端粒酶在体内具有端粒保护和促进细胞增殖的功能，端粒DNA的合成与端粒DNA的合成是分不开的。我们的目标是更全面地理解(S)端粒酶激活在癌症和衰老中所起的作用。

DESCRIPTION (provided by applicant): The cellular ribonucleoprotein telomerase synthesizes telomeric DNA at the ends of chromosomes by copying an intrinsic RNA template. The long term goals of the proposed research are to understand specific roles in cell proliferation that have been recently uncovered for telomeres and telomerase, and investigate how subverting these roles can be used to impact on cancer cells and aging. In most cancer cells, telomerase is highly active. We recently reported that even a low threshold level of expression of human telomerase RNA gene constructs containing seven different mutant templates (but not the control wild-type template) was sufficient to decrease cellular viability, increase apoptosis and slow tumor growth of human cancer cells, We are interested in the potential feasibility of using this mutant-template telomerase RNA expression as an anti-neoplastic strategy for human cancers. The first three Specific Aims address the mechanism of the response to mutant-template telomerase RNA in normal and cancer cells. They are: #1: Test whether the seven specific template-mutated telomerase RNAs assemble into core enzyme with the ability to synthesize mutant repeats in vitro; #2: Determine whether expression of such mutant-template telomerase RNA in cultured human cells causes mutant telomeric DNA sequences to be incorporated into the telomeres, and test whether the binding of human telomeric proteins is affected by the mutations. Test specific models for the mechanism of action of one particular mutant-template telomerase RNA; #3: Use improved vector systems for delivery of mutant-template telomerase RNA genes, and short term assays for cell growth and apoptosis, to analyze which DNA damage checkpoint and apoptotic pathways are involved in the responses to mutant template telomerase RNA expression. Determine the involvement of specific genes implicated in human telomere functions, DNA damage and other response pathways. Accumulating recent evidence suggests that telornerase promotes cell proliferation by mechanism(s) that do not require telomere elongation. Specific Aim #4 tests the specific hypothesis that telomerase has such functions, in telomere protection and promoting cell proliferation, that are separable its synthesis of telomeric DNA in vivo. The goal is a fuller understanding of the role(s) in cancer and aging played by telomerase activation.