TELOMERASE INACTIVATION BY RNASE L SIGNALS CELL DEATH

RNA酶L是IFN系统中普遍存在的核糖核酸内切酶，已被利用来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）用共价偶联至反义ODN的RNA酶L的2 '，5'-四腺苷酸激活剂（2- 5A）化学合成。实质上，嵌合体的2- 5A部分结合并激活RNA酶L，而ODN的反义臂结合RNA靶标，从而指导RNA酶L特异性降解RNA。本项目的目的是确定针对人端粒酶RNA（hTR）的2- 5A反义核酸的杀肿瘤活性的分子机制，从而更好地理解端粒酶在癌症和细胞永生中的作用。待检验的假设是2- 5A-抗-hTR的杀肿瘤作用是由于抑制端粒酶功能，其直接或间接触发凋亡反应。将设计不同化学形式的2- 5A-反义以增强生物稳定性、杂交亲和力、细胞摄取和RNase L活化能力。我们将探讨2- 5A反义端粒酶RNA处理肿瘤细胞后导致细胞凋亡的信号事件。将确定半胱天冬酶、STAT 1和bcl-2家族成员的贡献。将确定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。由于2 - 5A反义核酸能激活人的RNase L而不能激活小鼠的RNase L，因此，在RNase L-/-小鼠中表达人的RNase L，可以建立研究端粒酶RNA 2- 5A反义核酸抗肿瘤作用的动物模型。将在2- 5A-反义处理的细胞和小鼠中确定IFN诱导RNA酶L的作用。由于2- 5A-抗hTR是一种有效的癌症实验治疗剂，因此该提议有可能对难治性癌症形式产生影响，例如恶性神经胶质瘤。

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.