TELOMERASE INACTIVATION BY RNASE L SIGNALS CELL DEATH

核糖核酸酶L是干扰素系统中普遍存在的一种内切核糖核酸酶，它被用来选择性和催化切割在信号转导和肿瘤发生中发挥作用的核糖核酸靶标。嵌合寡核苷酸是由核糖核酸酶L的2‘，5’-四腺苷激活剂(2-5A)共价偶联到反义寡核苷酸上而合成的。本质上，嵌合体的2-5A部分结合并激活核糖核酸酶L，而寡核苷酸的反义臂结合核糖核酸靶标，从而指导核糖核酸酶L特异性地降解核糖核酸。本项目的目标是确定针对人端粒酶RNA(Htr)的2-5A反义抗肿瘤活性的分子机制，从而更好地了解端粒酶在癌症和细胞永生化中的作用。需要检验的假设是，2-5A-anti-HTR的杀瘤作用是由于抑制了端粒酶功能，而端粒酶功能直接或间接地触发了细胞凋亡反应。将设计不同化学形式的2-5A反义核酸，以增强生物稳定性、杂交亲和力、细胞摄取和核糖核酸酶L激活能力。我们将探讨在端粒酶RNA反义2-5A处理的肿瘤细胞中导致细胞凋亡的信号事件。将确定caspase、STAT1和BCL-2家族成员的贡献。2-5A反义对整体基因表达模式、端粒长度和染色体稳定性的影响将被确定。由于2-5A反义核糖核酸酶激活人而不是小鼠核糖核酸酶L，因此在核糖核酸酶L-/-小鼠中表达人核糖核酸酶L可以建立研究2-5A-反义端粒酶核糖核酸抗肿瘤作用的动物模型。干扰素对核糖核酸酶L的诱导作用将在2-5A反义处理的细胞和小鼠中确定。由于2-5A-抗HTR是一种有效的癌症实验性治疗剂，这一提议有可能对难治性癌症产生影响，如恶性胶质瘤。

RNase L, a ubiquitous endoribonuclease in the IFN system, has been harnessed for the selective and catalytic cleavage of RNA targets that function in signal transduction and oncogenesis. Chimeric oligonucleotides (ODNs) are chemically-synthesized with a 2',5'-tetraadenylate activator (2- 5A) of RNase L covalently coupled to antisense ODNs. In essence, the 2- 5A portion of the chimera binds and activates RNase L while the antisense arm of the ODN binds the RNA target thus directing RNase L to degrade the RNA specifically. The goal of this project is to determine the molecular mechanism for the tumoricidal activity of 2-5A-antisense directed against human telomerase RNA (hTR) and thus better understand the role of telomerase in cancer and cell immortality. The hypothesis to be tested is that the tumoricidal effect of 2-5A-anti-hTR is due to inhibition of telomerase function which directly or indirectly triggers an apoptotic response. Different chemical forms of 2-5A-antisense will be designed to enhance biostability, hybridization affinity, cellular uptake and RNase L activation ability. We will probe the signaling events which lead to apoptosis in tumor cells treated with 2-5A-antisense to telomerase RNA. The contributions of caspases, STAT 1, and bcl-2 family members will be determined. Effects of 2-5A-antisense on global gene expression patterns, telomere length and chromosome stability will be determined. Because human but not murine RNase L is activated by 2-5A-antisense, an animal model for studying the anti-tumor effects of 2-5A-antisense against telomerase RNA will be generated by expressing human RNase L in RNase L-/- mice. The effects of IFN induction of RNase L will be determined in 2-5A-antisense treated cells and mice. Because 2-5A-anti- hTR is a potent experimental therapeutic agent for cancer, this proposal has the potential to make an impact on intractable forms of cancer, such as malignant glioma.