TELOMERASE INACTIVATION BY RNASE L SIGNALS CELL DEATH

RNase L是IFN系统中普遍存在的核糖核酸内切酶，已被用于选择性和催化切割在信号转导和肿瘤发生中起作用的RNA靶标。嵌合寡核苷酸（odn）是由RNase L的2',5'-四腺苷酸激活剂（2- 5A）共价偶联到反义odn化学合成的。本质上，嵌合体的2- 5A部分结合并激活RNase L，而ODN的反义臂结合RNA靶标，从而指导RNase L特异性降解RNA。本项目的目标是确定2- 5a -反义靶向人类端粒酶RNA （hTR）的杀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不朽中的作用。待验证的假设是，2-5A-anti-hTR的杀瘤作用是由于抑制端粒酶功能，直接或间接引发细胞凋亡反应。设计不同化学形式的2- 5a反义蛋白以提高生物稳定性、杂交亲和力、细胞摄取和RNase L活化能力。我们将探讨2- 5a -端粒酶RNA反义处理肿瘤细胞后导致细胞凋亡的信号通路。将确定caspases、STAT 1和bcl-2家族成员的贡献。2- 5a -反义对基因表达模式、端粒长度和染色体稳定性的影响将被确定。由于人类RNase L可被2- 5a -反义激活，而非小鼠RNase L可被2- 5a -反义激活，因此在RNase L-/-小鼠中表达人类RNase L将产生研究2- 5a -反义对端粒酶RNA抗肿瘤作用的动物模型。IFN对RNase L的诱导作用将在2- 5a反义处理的细胞和小鼠中进行研究。由于2- 5a抗hTR是一种有效的实验性癌症治疗剂，因此该建议有可能对顽固性癌症（如恶性胶质瘤）产生影响。

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.