Screening of Synthetic Lethality with C. elegans Rb

线虫 Rb 的综合致死率筛选

• 批准号: 7245150
• 负责人: MICHAEL E HURWITZ
• 金额: $ 13.61万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7245150
• 关键词: Screening; Synthetic; Lethality; C.; elegans

DESCRIPTION (provided by applicant): The tumor suppressor Rb is a phosphoprotein that blocks cell cycle progression by modulating the functions of E2F-containing transcription factors. The importance of Rb for protecting the cell from inappropriate cell division is demonstrated by the almost universal inactivation of the Rb pathway in carcinomas. Since this pathway is defective in almost all carcinomas, one approach to specifically attack cancer cells is to inactivate proteins essential for the survival of cells with Rb pathway defects but not essential for those with an intact Rb pathway. The nematode Caenorhabditis elegans has a single Rb family member, lin-35 Rb, which can be inactivated with relatively minor effects on the growth of the animal. lin-35 Rb has been shown to interact genetically with the worm homologs of many mammalian Rb-interacting proteins and therefore can serve as a model to study Rb function in vivo. I am using C. elegans to search for genes that are synthetically lethal with Rb to identify new molecular pathways parallel to the Rb pathway which could define new cancer-related pathways. To do so, I am using an RNA interference (RNAi) library. Because Iin-35 Rb animals are less healthy than wild-type animals, it is possible that some of the synthetic phenotypes seen in lin-35 Rb mutants but not in wild-type animals are caused by the non-specific additive effects of two harmful mutations. To address the issue of cell-autonomous synthetic lethality (i.e., whether the synthetic effects of two mutations occur within a single cell), I am developing tissue-specific assays for synthetic lethality. All genes whose inactivation by RNAi results in a different phenotype in lin-35 Rb worms from that in wild-type worms will be tested in a tissue-specific assay. For candidates that appear to be cell-autonomously synthetically lethal and that have mammalian homologs, mammalian cells either containing or lacking Rb will be treated with RNAi to the mammalian homologs to assess whether the synthetic lethality seen in worms also occurs in mammals. Gene products whose inactivation is synthetically lethal with inactivated lin-35 Rb may be therapeutic targets in mammals since their inactivation may specifically kill tumor cells (in which the Rb pathway is almost always inactivated) and leave cells containing functional Rb unharmed.

描述（由申请人提供）：肿瘤抑制剂RB是一种磷酸蛋白，通过调节含E2F的转录因子的功能来阻断细胞周期的进展。 RB对保护细胞免受不适当细胞分裂的重要性证明了癌中RB途径的普遍失活。 由于该途径在几乎所有癌中都是有缺陷的，因此一种特异性攻击癌细胞的一种方法是使蛋白质灭活对于具有RB途径缺陷的细胞生存所必需的蛋白质，但对于具有完整RB途径的细胞的生存而言。线虫秀丽隐杆线虫有一个单一的RB家族成员LIN-35 RB，可能会使动物生长的影响相对较小。 LIN-35 RB已显示与许多哺乳动物RB相互作用蛋白的蠕虫同源物相互作用，因此可以作为研究体内RB功能的模型。 我正在使用秀丽隐杆线虫来搜索与RB合成致命的基因，以识别与RB途径平行的新分子途径，该途径可以定义新的癌症相关途径。为此，我正在使用RNA干扰（RNAi）库。由于IIN-35 RB动物不如野生型动物健康，因此在LIN-35 RB突变体中看到的某些合成表型可能是由两个有害突变的非特异性加性作用引起的。为了解决细胞自主的合成致死性问题（即，在单个细胞中是否发生了两个突变的合成作用），我正在开发组织特异性的测定法以进行合成致死性。 RNAi失活的所有基因在LIN-35 RB蠕虫中导致与野生型蠕虫中的表型不同，将在组织特异性测定中进行测试。对于似乎是单个自主性合成且具有哺乳动物同源物的候选者，将用RNAi治疗含有或缺乏RB的哺乳动物细胞与哺乳动物同源物治疗，以评估哺乳动物中蠕虫中是否也发生合成致死性。 灭活与失活的LIN-35 RB合成致死的基因产物可能是哺乳动物的治疗靶标，因为它们的失活可能特异性杀死肿瘤细胞（其中几乎总是灭活了RB途径），并使含有功能性RB的细胞保持不受限制。