Screening of Synthetic Lethality with C. elegans Rb

描述(申请人提供)：肿瘤抑制因子RB是一种磷酸蛋白，通过调节含有E2F的转录因子的功能来阻止细胞周期进展。在癌症中，Rb通路几乎普遍失活，证明了Rb对于保护细胞免受不适当细胞分裂的重要性。 由于这一途径在几乎所有癌症中都是缺陷的，因此特异性攻击癌细胞的一种方法是使Rb途径缺陷的细胞生存所必需的蛋白质失活，但对于那些Rb途径完整的细胞来说不是必不可少的。线虫秀丽线虫只有一个RB家族成员，LIN-35RB，可以被灭活，对动物的生长影响相对较小。LIN-35Rb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基因上的相互作用，因此可以作为研究Rb在体内功能的模型。 我正在使用线虫来搜索与Rb合成致命的基因，以确定与Rb途径平行的新分子途径，这可能定义新的癌症相关途径。为此，我使用的是RNA干扰(RNAi)库。由于Iin-35Rb动物的健康状况不如野生型动物，因此在LIN-35Rb突变体中发现的一些合成表型可能是由两个有害突变的非特异性相加效应引起的，而野生型动物中没有。为了解决细胞自主合成致死性的问题(即，两个突变的合成效应是否发生在一个细胞内)，我正在开发合成致死性的组织特异性分析。所有被RNAi灭活导致LIN-35RB蠕虫与野生型蠕虫不同表型的基因都将在组织特异性测试中进行测试。对于那些似乎是细胞自主合成致死的候选细胞和具有哺乳动物同源基因的候选细胞，无论是否含有Rb的哺乳动物细胞都将被针对哺乳动物同源基因的RNAi处理，以评估在蠕虫中看到的合成致死是否也发生在哺乳动物中。 与灭活的Lin-35Rb一起失活的基因产物可能成为哺乳动物的治疗靶点，因为它们的失活可能特异性地杀死肿瘤细胞(其中Rb途径几乎总是失活的)，而使含有功能性Rb的细胞不受损害。

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.