MECHANSISM OF CELL GROWTH INHIBITION BY RNASE-L

描述：(申请人摘要)干扰素(IFN)是一类 通过细胞停滞或细胞分裂来限制细胞增殖的细胞因子 对某些人类癌症有很强的抗肿瘤作用。 干扰素诱导其生长抑制的分子机制/细胞 死亡效应在很大程度上仍是未知的。2-5A系统是一种RNA降解 激活依赖2-5A的核糖核酸酶-L的途径是 IF抑制细胞生长的关键调节因子及其作用机制 抗肿瘤活性；最近的研究已经确定了其更广泛的作用 核糖核酸酶-L与细胞凋亡。核糖核酸酶-L在抗肿瘤中的作用 IF的影响，了解其潜在的机制是至关重要的 核糖核酸酶-L介导的生长抑制/细胞死亡及其活性 在细胞中被调节。为了实现这一广泛的目标，RNA底物， 核糖核酸酶-L的下游效应因子和细胞调控因子将被确定。 核糖核酸酶-L表达和活性均可调控的细胞系 在互补差分中使用正、负方向 表达式方法(目标1)和基于函数的方法(目标2)。在目标1中，mRNAs 哪些是未调控的核糖核酸酶-L抑制的细胞和下调的后续 核糖核酸酶-L诱导代表候选核糖核酸酶-L底物和下游 效应器，将通过差异显示分析进行识别。 转导核糖核酸酶-L基因的NIH-3T3细胞对IF的敏感性 在对照细胞无反应的条件下诱导生长抑制； 因此，核糖核酸酶-L活性的特定调节导致了可选择的 表型变化。鉴定在人体内起作用的基因产物 核糖核酸酶-L介导的生长抑制(AIM 2)，一种遗传抑制元件 (GSE)文库，由编码基因抑制的短片段组成 代表核糖核酸酶-L调节剂、底物或下游效应物的产品 将挽救核糖核酸依赖L的成长停滞，提供正面选择 对于表达这些GSE的细胞。在AIMS 1和2中确定的cDNA将是 其特征为核糖核酸酶-L活性的上游或下游介体 测序以确定它们是否代表已知或新的基因(AIMS 3AB)。 内源性核糖核酸酶-L调节子/效应子的表达调控 外源调节剂/效应物对细胞增殖的影响 在正常和癌症细胞系中将被检查以阐明它们在 生长抑制(目标3c，d)。一种体外RNA衰变系统将是 研究特定的核糖核酸酶-L底物是如何被靶向的 退化(目标3E)。核糖核酸酶-L调节剂、底物的鉴定 和效应将促进我们对几个重要的生物学 问题：1)IF诱导生长抑制的机制；2)IF的作用 核糖核酸酶-L在抗癌中的作用；3)核糖核酸稳定性的控制 在基因表达的调控中。

DESCRIPTION: ( applicant's abstract) The interferons (IFNs) are a family of cytokines which function to limit cell proliferation by cytostasis or cell death and exhibit potent antitumor effects against certain human cancers. The molecular mechanisms by which IFNs elicit their growth inhibitory/cell death effects remain largely unknown. The 2-5A system is an RNA degradation pathway which functions through the activation of 2-5A-dependent RNASE-L is a critical mediator of cell growth inhibition by IF and is implicated in its antitumor activity; recent studies have identified a broader role for RNase-L in apoptosis. To determine how RNASE-L functions in the antitumor effects of IF, it is essential to understand the mechanisms underlying RNase-L mediated growth inhibition/cell death and how this activity is regulated in cells. To accomplish this broad objective, RNA substrates, downstream effectors and cell ular regulators of RNASE-L will be identified. Cell lines in which RNASE-L expression and activity can be modulated in both positive and negative directions will be used in complementary differential expression (aim 1) and function based (aim 2) methods. In aim 1, mRNAs which are unregulated in RNase-L inhibited cells and downregulated following RNase-L induction represent candidate RNase-L substrates and downstream effectors and will be identified by differential display analysis. Expression of transfected RNASE-L renders NIH-3T3 cells sensitive to IF induced growth inhibition in conditions where control cells do not respond; thus, the specific modulation of RNase-L activity results in a selectable phenotypic change. To identify gene products which function in RNase-L-mediated growth inhibition (aim 2), a Genetic Suppressor Element (GSE) library, composed of short cDNA fragments encoding inhibition of gene products representing RNase-L regulators, substrates or downstream effectors will rescue RNase-L dependent growth arrest, providing a positive selection for cells expressing these GSEs. cDNAs identified in aims 1 and 2 will be characterized as upstream or downstream mediators of RNASE-L activity and sequenced to determine if they represent known or novel genes (aims 3ab). The regulation of endogenous RNase-L regulator/effector expression and the effects of transfected exogenous regulators/effectors on cell proliferation in normal and cancer cells lines will be examined to elucidate their role in growth inhibition (aims 3c,d). An in vitro RNA decay system will be developed to study how specific RNase-L substrates are targeted for degradation (aim 3e). The identification of RNASE-L regulators, substrates and effects will advance our understanding of several important biological issues: 1) the mechanism of IF induced growth inhibition; 2) the role of RNase-L in the antitumor effects of IF; and 3) the control of RNA stability in the regulation of gene expression.