Screening of Synthetic Lethality with C. elegans Rb

线虫 Rb 的综合致死率筛选

• 批准号: 6718572
• 负责人: MICHAEL E HURWITZ
• 金额: $ 13.53万
• 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/6718572
• 关键词: Caenorhabditis elegans; RNA interference; cell differentiation; cell line; cell proliferation; gene deletion mutation; gene expression; genetic library; genetic regulation; genetic screening; helminth genetics; molecular oncology; neoplasm /cancer genetics; retinoblastoma protein; tissue mosaicism

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）：肿瘤抑制因子 Rb 是一种磷蛋白，通过调节含有 E2F 的转录因子的功能来阻断细胞周期进程。 Rb 通路在癌症中几乎普遍失活，证明了 Rb 对于保护细胞免遭不适当细胞分裂的重要性。 由于该通路在几乎所有癌症中都存在缺陷，因此特异性攻击癌细胞的一种方法是灭活对具有 Rb 通路缺陷的细胞生存必需的蛋白质，但对于具有完整 Rb 通路的细胞来说则不是必需的。线虫秀丽隐杆线虫有一个 Rb 家族成员 lin-35 Rb，它可以被灭活，对动物生长的影响相对较小。 lin-35 Rb 已被证明与许多哺乳动物 Rb 相互作用蛋白的蠕虫同源物存在遗传相互作用，因此可以作为研究 Rb 体内功能的模型。 我正在使用秀丽隐杆线虫来寻找与 Rb 合成致死的基因，以识别与 Rb 途径平行的新分子途径，从而定义新的癌症相关途径。为此，我使用 RNA 干扰 (RNAi) 库。由于 Iin-35 Rb 动物的健康状况不如野生型动物，因此在 lin-35 Rb 突变体中看到但在野生型动物中没有看到的一些合成表型可能是由两种有害物质的非特异性累加效应引起的。突变。为了解决细胞自主合成致死率的问题（即两个突变的合成效应是否发生在单个细胞内），我正在开发针对合成致死率的组织特异性测定。所有因 RNAi 失活导致 lin-35 Rb 线虫与野生型线虫表型不同的基因都将在组织特异性测定中进行测试。对于看起来具有细胞自主合成致死性并且具有哺乳动物同系物的候选物，将用哺乳动物同系物的RNAi处理含有或缺乏Rb的哺乳动物细胞，以评估在线虫中看到的合成致死性是否也发生在哺乳动物中。 失活的 lin-35 Rb 失活具有综合致死性的基因产物可能是哺乳动物的治疗靶点，因为它们的失活可以特异性杀死肿瘤细胞（其中 Rb 途径几乎总是失活），而含有功能性 Rb 的细胞不会受到伤害。