Targeting Bcl-x splicing for cancer treatment

靶向 Bcl-x 剪接用于癌症治疗

• 批准号: 7151742
• 负责人: JIANHUA ZHOU
• 金额: $ 8.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7151742
• 关键词: apoptosis; cell line; genes; neoplasm /cancer

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.

描述(由申请人提供)：在过去的二十年里，癌症的治疗已经取得了进展，然而，仍然没有有效的方法来治愈他们。许多癌基因以及肿瘤抑制基因已经被发现。这些蛋白中的一类形成了一个Bcl-2蛋白家族。最近的研究表明，Bc l-2家族成员之一Bc l-x在外显子2上交替剪接。该外显子的选择性剪接可产生抗凋亡(L形式)或凋亡(S形式)的异构体，提示对L/S形式的Bc l-x的调控可能对癌症的治疗有意义。本应用的目的是建立一种基于细胞的分析方法，通过利用外显子2中的选择性剪接位点来鉴定调节外显子2剪接的生物分子/药物，从而调节Bclxl/Bclxs的比例和Bclxs的活性。这些分子可能被用来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生成用于Bcl-x剪接研究的微型基因构建物，并附上报告基因荧光素酶，以便通过荧光素酶活性来衡量位于Bcl-x Pre-mRNA外显子2内的剪接位点的替代使用效率。构建的微小基因将通过RT-PCR进行验证，从而建立稳定的细胞系。该项目的第二个目标是优化稳定的细胞系，并鉴定生物试剂/小分子，这些生物试剂/小分子在低通量环境下(LTS)从小分子文库中减少BclxL的产生，同时增加Bclxs异构体的产生。这些化合物将在PCS细胞等细胞系中测试其对凋亡细胞死亡的活性，PCS细胞系是一种表达异常高水平的抗凋亡Bcl-xL的细胞系。这些化合物对外显子2选择性剪接调控的作用机制将作为一个长期目标进行研究。这项研究与公共健康的相关性：bclxl是一种在许多肿瘤中升高的大形式的bcl-x蛋白，它保护细胞免受化疗的影响。在这个项目中，我们计划建立一个基于细胞的系统，用于识别降低Bc l-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。