Targeting Bcl-x splicing for cancer treatment

描述（由申请人提供）：在过去的二十年中，癌症的治疗取得了进展，然而，仍然没有有效的治疗方法。许多致癌基因和肿瘤抑制基因已经被发现。其中一类蛋白质形成了Bcl-2蛋白家族。最近的研究表明，Bcl-2家族成员之一Bcl-x在外显子2上被选择性拼接。该外显子的选择性剪接产生抗凋亡（L型）或凋亡（S型）异构体，表明调节L/S型Bcl-x对癌症治疗具有重要意义。本申请的目标是开发一种基于细胞的检测方法，用于鉴定通过使用外显子2内的替代剪接位点来调节外显子2剪接的生物分子/药物，从而调节Bcl-xL和Bcl-xS的比例以及Bcl-x的活性。这些分子可能用于减少肿瘤中抗凋亡形式的Bcl-xL，从而治疗癌症。该项目的第一个目标是为Bcl-x剪接研究生成带有报告基因荧光素酶的迷你基因构建体，以便通过荧光素酶活性来测量Bcl-x前体mrna外显子2内剪接位点的替代使用效率。构建的迷你基因将通过RT-PCR进行验证，然后建立稳定的细胞系。该项目的第二个目标是优化稳定的细胞系，并从小分子文库中确定生物试剂/小分子，这些试剂/小分子可以减少Bcl-xL的产量，同时在低通量设置（LTS）下增加Bcl-xS异构体的产量。这些化合物将在细胞系（如PCS细胞）中测试对凋亡细胞死亡的活性，PCS细胞是一种表达异常高水平抗凋亡Bcl-xL的细胞系。这些化合物调控外显子2选择性剪接的作用机制将作为一个长期目标进行研究。本研究与公众健康的相关性：Bcl-xL，在许多肿瘤中升高的Bcl-x蛋白的大形式，保护细胞免受化疗。我们计划在这个项目中建立一个基于细胞的系统来鉴定降低Bcl-xL的药物。这些化合物可能与其他化疗药物联合用于癌症治疗。

DESCRIPTION (provided by applicant): Progress has been made during the last two decades on treatment of cancers, however, there is still no effective approach for their cure. Many oncogenes as well as tumor suppressors have been identified. One class of these proteins forms a Bcl-2 protein family. Recent studies have shown that one of the Bcl-2 family member, Bcl-x is alternatively spliced in exon2. Alternative splicing of this exon generates isoforms either anti-apoptotic (L form) or apoptotic (S form), suggesting that regulation of L/S forms of Bcl-x has implication for cancer therapy. The goal of this application is to develop a cell-based assay for identification of biological molecules/drugs that modulate exon 2 splicing through usage of an alternative splicing site within exon 2 so that the ratio of Bcl-xL and Bcl-xS, and activities of Bcl-x can be regulated. These molecules are potentially used to reduce anti-apoptotic form of Bcl-xL in tumors, therefore treating cancers. The first aim of this project is to generate mini-gene constructs for Bcl-x splicing studies with a reporter gene luciferase attached so that efficiency of alternative usage of a splicing site residing within exon 2 of Bcl-x pre-mRNA can be measured by luciferase activity. The mini-gene constructs will be validated by RT-PCR and then stable cell lines will be established. The second aim of this project is to optimize stable cell lines and to identify biological reagents/small molecules that reduce production of Bcl-xL while increase the production of the Bcl-xS isoform in a low throughput setting (LTS) from libraries of small molecules. The compounds will be tested for activity on apoptotic cell death in cell lines such as PCS cells, a cell line that expresses an abnormal high level of anti-apoptotic Bcl-xL. Mechanisms of action of these compounds on regulation of exon2 alternative splicing will be investigated as a long-term goal. Relevance of this research to public health: Bcl-xL, the large form of Bcl-x protein that is elevated in many tumors, protects cells from chemotherapy. We plan in this project to establish a cell-based system for identification of drugs that reduce Bcl-xL. These compounds may be potentially used for cancer treatment with combination of other chemotherapies.