Translational State Assay for Human Samples

描述（由申请人提供）：解除对转化控制的管制是致癌的必要步骤；然而，检测人类细胞和组织中翻译机制活性的诊断工具是缺乏的。这阻碍了将预后和治疗反应与翻译活性作为癌症类型和分期的功能联系起来的研究，并且阻碍了调节翻译起始机制的大规模药物发现工作，尽管它具有作为治疗靶点的潜力。根据美国国立卫生研究院项目公告RFA-RR-06-004，在此，我们请求通过R21机制支持开发一种简单、快速、准确和高通量兼容的测定系统来量化人类细胞和组织的转化状态。我们的方法是基于完善的兔网织细胞自由翻译系统，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，我们添加了一个双链报告RNA，编码两种形式的荧光素酶，一种以帽依赖的方式翻译，另一种通过内部核糖体进入位点（IRES）翻译；并将两种形式的发光量化为源自细胞或组织的固有帽依赖和帽独立的翻译活性的读数。我们建议通过4个特定目标来发展这种检测：目标1)开发和优化方案，以产生和量化来自原代人细胞系的裂解物的翻译活性；目标2)定义具有明确作用机制的翻译激动剂和拮抗剂的受试者工作特征（ROC）曲线；目标3)开发和优化方案，以产生和量化乳腺和肺组织标本裂解物的翻译活性；目的4)比较乳腺和肺组织标本中细胞及其相应的原组织的裂解物的翻译活性；对照细胞及其相应的病变细胞；控制组织和相应的病变组织。如果我们取得成功，这将是发展医学诊断的第一步，根据其翻译特性区分健康和病理组织，将预后和治疗反应与翻译活性联系起来，作为癌症类型和阶段的功能，并为开发翻译修饰剂的新兴药物发现工作提供强大的工具。

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.