Translational State Assay for Human Samples

描述(由申请人提供)：解除对翻译控制的管制是致癌过程中的一个强制性步骤；然而，缺乏检查人类细胞和组织中翻译机制活动的诊断工具。这阻碍了将预后和治疗反应与翻译活动作为癌症类型和阶段的函数联系起来的研究，并阻碍了大规模药物发现努力来调节翻译起始机制，尽管它具有治疗靶点的潜力。为了响应美国国立卫生研究院计划宣布的RFA-RR-06-004，我们在此请求通过R21机制提供支持，以开发一种简单、快速、准确和高通量兼容的分析系统来量化人类细胞和组织的翻译状态。我们的方法是基于成熟的兔网织红细胞自由翻译系统，除了我们替代直接从感兴趣的细胞或组织制备的标准化细胞裂解物。在提取物中加入一个编码两种形式的荧光素酶的双顺反子报告RNA，一种是以帽依赖的方式翻译的，另一种是通过内部核糖体进入位点(IRES)翻译的；并将这两种形式的发光定量为读出起源的细胞或组织固有的帽依赖和帽非依赖的翻译活性。我们建议通过四个特定目标发展该方法：目的1)开发和优化方法以生成和量化原代人类细胞系裂解物中的翻译活性；目的2)使用具有明确作用机制的翻译激动剂和拮抗剂来确定分析的受试者操作特征(ROC)曲线；目的3)制定和优化方法以生成和量化乳腺和肺组织标本的裂解物中的翻译活性；目的4)比较乳腺和肺组织标本裂解物中细胞及其相应起源组织的翻译活性；目的2)确定对照细胞及其相应的疾病组织；以及对照组织和其相应的疾病组织。如果我们成功，这将是发展医学诊断学的第一步，根据组织的翻译属性区分健康和病理组织，将预后和治疗反应与癌症类型和分期的翻译活动联系起来，并为新兴药物发现努力开发翻译修饰物提供强大的工具。 翻译的病理激活，即将mRNA模板转换为蛋白质的过程，是导致癌症的原因；然而，缺乏在人类细胞和组织中量化这一过程的诊断工具。这阻碍了将预后和治疗反应与翻译机制的活动作为癌症类型和阶段的函数联系起来的研究，并阻碍了大规模药物发现努力来调节翻译机制，尽管它有可能成为治疗靶点。在这里，我们建议开发一种简单、快速、准确和高通量兼容的检测系统来量化人类细胞和组织的翻译状态。如果成功，这将是发展医学诊断学的第一步，根据其翻译属性区分健康和病理组织，将预后和治疗反应与癌症类型和分期的翻译活动联系起来，并为新兴药物发现努力开发翻译修饰物提供强有力的工具。

DESCRIPTION (provided by applicant): Deregulation of translational control is an obligatory step in carcinogenesis; however, diagnostic tools to examine the activity of the translational machinery in human cells and tissue are lacking. This has hampered studies to link prognosis and therapeutic response to translational activity as a function of cancer type and stage, and has precluded large scale drug discovery efforts to modulate the translation initiation machinery despite its potential as a therapeutic target. In response to NIH Program Announcement RFA-RR-06-004, here we request support through the R21 mechanism to develop an assay system to quantify the translational state of human cells and tissues that is simple, rapid, accurate and high throughput-compatible. Our approach is based on the well established rabbit reticulocyte cell free translation system, except we substitute standardized cell lysates prepared directly from the cells or tissue of interest. To the extract we add a bicistronic reporter RNA encoding 2 forms of luciferase, one translated in a cap-dependent manner and the other via an internal ribosomal entry site (IRES); and quantify the 2 forms of luminescence as a read out of the intrinsic cap-dependent and cap- independent translational activity of the cell or tissue of origin. We propose to develop this assay through 4 Specific Aims: Aim 1) Develop and optimize protocols to generate and quantify translational activity in lysates from primary human cell lines; Aim 2) Define receiver operating characteristic (ROC) curves for the assay using agonists and antagonists of translation with well-defined mechanisms of action; Aim 3) Develop and optimize protocols to generate and quantify translational activity in lysates from breast and lung tissue specimens; Aim 4) Compare translational activity in lysates from breast and lung tissue specimens between cells and their corresponding tissue of origin; control cells and their corresponding diseased counterparts; control tissue and their corresponding diseased counterparts. If we are successful, this would represent the first step in developing medical diagnostics to differentiate healthy and pathological tissues according to their translational properties, to link prognosis and therapeutic response to translational activity as a function of cancer type and stage and provide a powerful tool for emerging drug discovery efforts to develop translational modifiers. Pathological activation of translation, the process of converting mRNA template into protein, is on the causal pathway to cancer; however, diagnostic tools to quantify this process in human cells and tissue are lacking. This has hampered studies to link prognosis and therapeutic response to the activity of the translational machinery as a function of cancer type and stage, and has precluded large-scale drug discovery efforts to modulate the translational machinery despite its potential as a therapeutic target. Here we propose to develop an assay system to quantify the translational state of human cells and tissues that is simple, rapid, accurate and high throughput-compatible. If successful, this would represent the first step in developing medical diagnostics to differentiate healthy and pathological tissues according to their translational properties, to link prognosis and therapeutic response to translational activity as a function of cancer type and stage and provide a powerful tool for emerging drug discovery efforts to develop translational modifiers.