Screening of Synthetic Lethality with C. elegans Rb

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E2 F的转录因子的功能来阻断细胞周期进程。Rb对于保护细胞免于不适当的细胞分裂的重要性通过癌中Rb途径的几乎普遍失活来证明。 由于该途径在几乎所有癌中是有缺陷的，特异性攻击癌细胞的一种方法是抑制对具有Rb途径缺陷的细胞的存活至关重要但对具有完整Rb途径的那些细胞不是必需的蛋白质。线虫秀丽隐杆线虫有一个单一的Rb家族成员，lin-35 Rb，它可以被灭活，对动物的生长影响相对较小。lin-35 Rb与许多哺乳动物Rb相互作用蛋白的蠕虫同源物具有遗传相互作用，因此可以作为研究体内Rb功能的模型。 我用C。elegans寻找与Rb合成致死的基因，以鉴定与Rb途径平行的新分子途径，这可以定义新的癌症相关途径。为此，我使用了RNA干扰（RNAi）文库。由于lin-35 Rb动物比野生型动物更不健康，因此在lin-35 Rb突变体中观察到的而在野生型动物中未观察到的一些合成表型可能是由两种有害突变的非特异性累加效应引起的。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测定。将在组织特异性试验中检测其通过RNAi失活导致lin-35 Rb蠕虫中与野生型蠕虫中不同表型的所有基因。对于似乎是细胞自主合成致死的并且具有哺乳动物同源物的候选物，将用针对哺乳动物同源物的RNAi处理含有或缺乏Rb的哺乳动物细胞，以评估在蠕虫中观察到的合成致死性是否也发生在哺乳动物中。 其失活与失活的lin-35 Rb一起合成致死的基因产物可能是哺乳动物中的治疗靶点，因为它们的失活可以特异性地杀死肿瘤细胞（其中Rb途径几乎总是失活的）并且使含有功能性Rb的细胞不受伤害。

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.