Function-based selection of target genes in tumor cells

基于功能的肿瘤细胞靶基因选择

• 批准号: 6786073
• 负责人: IGOR B RONINSON
• 金额: $ 51.65万
• 依托单位: ORDWAY RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786073
• 关键词: RNA interference; biomaterial development /preparation; biotechnology; bromodeoxyuridine; cell growth regulation; cell line; complementary DNA; functional /structural genomics; genetic library; genetic regulatory element; genetic transduction; growth inhibitors; neoplasm /cancer genetics; neoplastic cell; neoplastic growth; nucleic acid sequence; small interfering RNA; telomerase; transfection /expression vector

DESCRIPTION (provided by applicant): This proposal uses a new methodology to identify human genes that are required for tumor cell growth. Such genes, which provide potential targets for cancer treatment, will be identified through expression selection of genetic suppressor elements (GSEs). GSEs are biologically active sense- or antisense-oriented cDNA fragments that inhibit the function of the gene from which they are derived. Genes that are essential for cell proliferation are expected to give rise to GSEs that inhibit cell growth. Such GSEs can be isolated by bromodeoxyuridine (BrdU) suicide selection from a normalized (reduced-redundance) library of human cDNA fragments in an inducible retroviral vector. In preliminary studies, selection for growth-inhibitory GSEs has been carried out in breast carcinoma cells, yielding growth-inhibitory GSEs from about 60 genes. Many of the genes identified by GSE selection are known oncogenes or positive regulators of cell growth, while other genes have no known function or had not been previously implicated in cell proliferation. This analysis will now be extended to several other types of tumor and normal cells. A normalized cDNA fragment library in an inducible retroviral vector will be generated from a mixture of RNA preparations from multiple human tumor cell lines. This library will be transduced into recipient cell lines derived from several major types of human cancer and into telomerase-immortalized lines of normal human cells. Prior to transduction, the recipient cell lines will be derivatized to provide for high efficiency of retroviral infection and for the ability to regulate gene expression from retroviral vectors. The transduced cells will be subjected to BrdU suicide selection for growth-inhibitory GSEs, and GSE-enriched population of cDNA fragments will be recovered from the selected cells. Genes enriched by GSE selection will be identified by sequencing, and representative GSEs from each gene will be tested by several functional assays. The role of GSE-cognate genes in cell proliferation will be confirmed via siRNA inhibition. Genes identified through GSE selection will be prioritized as potential targets by comparing the ability of their cognate GSEs to inhibit cell growth in different types of tumor and normal cells and by analyzing the ability of the GSEs to induce tumor cell death through mitotic catastrophe. This analysis will provide a database of potential new targets for the development of anticancer drugs.

描述（由申请人提供）： 该提案使用了一种新的方法来识别肿瘤细胞生长所需的人类基因。这些基因为癌症治疗提供了潜在的靶点，将通过遗传抑制元件（GSE）的表达选择来鉴定。GSE是具有生物活性的正义或反义cDNA片段，其抑制它们所来源的基因的功能。预期细胞增殖所必需的基因产生抑制细胞生长的GSE。这样的GSE可以通过溴脱氧尿苷（BrdU）自杀选择从诱导型逆转录病毒载体中的人cDNA片段的标准化（还原型）文库中分离。在初步研究中，已经在乳腺癌细胞中进行了生长抑制GSE的选择，从大约60个基因中产生了生长抑制GSE。通过GSE选择鉴定的许多基因是已知的癌基因或细胞生长的正调节因子，而其他基因没有已知的功能或先前没有涉及细胞增殖。这种分析现在将扩展到其他几种类型的肿瘤和正常细胞。诱导型逆转录病毒载体中的标准化cDNA片段文库将由来自多种人肿瘤细胞系的RNA制备物的混合物产生。该文库将被转导入来源于几种主要类型的人类癌症的受体细胞系和正常人类细胞的端粒酶永生化细胞系。在转导之前，受体细胞系将被衍生化以提供高效率的逆转录病毒感染和调节来自逆转录病毒载体的基因表达的能力。将对转导的细胞进行BrdU自杀选择以获得生长抑制性GSE，并从所选细胞中回收GSE富集的cDNA片段群体。将通过测序鉴定通过GSE选择富集的基因，并通过几种功能测定法检测每个基因的代表性GSE。GSE同源基因在细胞增殖中的作用将通过siRNA抑制来证实。通过比较其同源GSE抑制不同类型肿瘤和正常细胞中细胞生长的能力，并通过分析GSE通过有丝分裂灾难诱导肿瘤细胞死亡的能力，将通过GSE选择鉴定的基因优先作为潜在靶标。这项分析将为抗癌药物的开发提供一个潜在的新靶点数据库。