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: 6686567
• 负责人: IGOR B RONINSON
• 金额: $ 47.95万
• 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/6686567
• 关键词: biomaterial development /preparation; biotechnology; bromodeoxyuridine; cell growth regulation; cell line; cell proliferation; complementary DNA; functional /structural genomics; genetic library; genetic regulatory element; genetic transduction; growth inhibitors; neoplasm /cancer genetics; neoplastic cell; neoplastic growth; nucleic acid sequence; 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）： 该提案使用一种新的方法来识别肿瘤细胞生长所需的人类基因。这些为癌症治疗提供潜在靶标的基因将通过遗传抑制元件（GSE）的表达选择来鉴定。 GSE 是具有生物活性的有义或反义 cDNA 片段，可抑制其来源基因的功能。细胞增殖所必需的基因预计会产生抑制细胞生长的 GSE。此类 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 片段标准化（减少冗余）文库中分离出来。在初步研究中，已经在乳腺癌细胞中进行了生长抑制性 GSE 的选择，从大约 60 个基因中产生了生长抑制性 GSE。通过 GSE 选择鉴定出的许多基因是已知的癌基因或细胞生长的正调节因子，而其他基因则没有已知的功能或之前未涉及细胞增殖。该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 片段文库将从多种人类肿瘤细胞系的 RNA 制剂混合物中产生。该文库将被转导到源自几种主要人类癌症类型的受体细胞系中，以及正常人类细胞的端粒酶永生化系中。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以及调节逆转录病毒载体的基因表达的能力。转导的细胞将受到生长抑制性 GSE 的 BrdU 自杀选择，并且将从所选细胞中回收富含 GSE 的 cDNA 片段群体。通过 GSE 选择富集的基因将通过测序进行鉴定，并且来自每个基因的代表性 GSE 将通过多种功能测定进行测试。 GSE 同源基因在细胞增殖中的作用将通过 siRNA 抑制得到证实。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，并分析 GSE 通过有丝分裂灾难诱导肿瘤细胞死亡的能力，通过 GSE 选择鉴定的基因将优先作为潜在靶标。该分析将为抗癌药物的开发提供潜在新靶标的数据库。