MECHANSISM OF CELL GROWTH INHIBITION BY RNASE-L

描述：（申请人的摘要）干扰素（IFN）是一个家族， 细胞因子通过细胞停滞或细胞生长抑制来限制细胞增殖， 死亡，并对某些人类癌症表现出有效的抗肿瘤作用。 干扰素诱导细胞生长抑制的分子机制 死亡影响仍然很大程度上未知。 2-5A系统是RNA降解系统， 通过激活2- 5A依赖性RNASE-L发挥作用的途径是 一个关键的调解人的细胞生长抑制的IF和牵连， 抗肿瘤活性;最近的研究已经确定了更广泛的作用， RNase-L在细胞凋亡中的作用。 为了确定RNASE-L如何在抗肿瘤细胞中发挥作用， 影响，它是至关重要的，以了解机制的基础 RNase-L介导的生长抑制/细胞死亡以及这种活性是如何影响 在细胞中调节。 为了实现这一广泛的目标，RNA底物， 将鉴定RNASE-L的下游效应物和细胞调节物。 细胞系，其中RNASE-L表达和活性可以在两种细胞中调节 在互补差分中将使用正方向和负方向， 基于表达式（aim 1）和基于函数（aim 2）的方法。 在目标1中，mRNA 在RNase-L抑制细胞中不受调节， RNase-L诱导代表候选RNase-L底物和下游 效应器，并将通过差异显示分析来识别。 转染RNASE-L基因的NIH-3 T3细胞对干扰素的敏感性 在对照细胞不响应的条件下诱导生长抑制; 因此，RNase-L活性的特异性调节导致可选择的 表型变化 为了鉴定基因产物， RNase-L-mediated growth inhibition（aim 2），a Genetic Suppressor Element (GSE)文库，由编码抑制基因的短cDNA片段组成 代表RNase-L调节剂、底物或下游效应物的产品 将拯救RNase-L依赖性生长停滞，提供正选择 表达这些GSE的细胞 目标1和目标2中鉴定的cDNA将被 表征为RNASE-L活性的上游或下游介质， 测序以确定它们是否代表已知或新的基因（aims 3ab）。 内源性RNase-L调节子/效应子表达的调节和RNA酶的表达 转染的外源调节因子/效应因子对细胞增殖的影响 在正常和癌细胞系中的作用，以阐明它们在 生长抑制（目标3c、d）。 体外RNA衰变系统将是 开发用于研究特定RNase-L底物如何靶向 降解（目标3e）。 RNASE-L调节剂、底物的鉴定 和影响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） RNase-L在IF抗肿瘤作用中的作用; 3）RNA稳定性的调控 在基因表达的调节中。

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.