Targeting Bcl-x splicing for cancer treatment

靶向 Bcl-x 剪接用于癌症治疗

• 批准号: 7282095
• 负责人: JIANHUA ZHOU
• 金额: $ 7.89万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282095
• 关键词: Alternative Splicing; Alzheimer' s Disease; Apoptosis; Apoptotic; BCL-Xs protein; Biochemical Genetics; Biological; Biological Assay; Cell Death; Cell Line; Cell Nucleus; Cell physiology; Cells; Cessation of life; Characteristics; Class; Collaborations; Complex; DNA; DNA Damage; Degenerative Disorder; Development; Exclusion; Exons; Family member; Future; Genes; Goals; Heterogeneous Nuclear RNA; Homeostasis; L Forms; Lead; Letters; Libraries; Luc Gene; Luciferases; Malignant Neoplasms; Measures; Messenger RNA; Modification; Monitor; Mutation; Necrosis; Neurodegenerative Disorders; Numbers; Oncogenes; Organism; Parkinson Disease; Pattern; Pharmaceutical Preparations; Phosphorylation; Physical condensation; Production; Protein Family; Protein Isoforms; Proteins; Public Health; RNA Splicing; Reagent; Regulation; Regulator Genes; Reporter; Reporter Genes; Research; Reverse Transcriptase Polymerase Chain Reaction; Screening procedure; Signal Pathway; Site; Spices; Spinal Muscular Atrophy; System; Testing; Tumor Suppressor Proteins; Universities; base; bcl-x protein; cancer therapy; carcinogenesis; chemotherapy; design and construction; high throughput screening; human disease; inhibitor/antagonist; mRNA Precursor; novel; receptor; small molecule; small molecule libraries; stable cell line; tau Proteins; tumor

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）：在过去的二十年中，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它们。许多癌基因以及肿瘤抑制基因已被确定。这些蛋白质中的一类形成Bcl-2蛋白质家族。最近的研究表明，Bcl-2家族成员之一，Bcl-x是在外显子2选择性剪接。该外显子的选择性剪接产生抗凋亡（L型）或凋亡（S型）的同种型，表明Bcl-x的L/S型的调节对癌症治疗具有意义。本申请的目的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内的选择性剪接位点调节外显子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Bcl-xL的抗凋亡形式，从而治疗癌症。该项目的第一个目的是产生用于Bcl-x剪接研究的微型基因构建体，其具有附接的报告基因荧光素酶，使得可以通过荧光素酶活性来测量位于Bcl-x前体mRNA的外显子2内的剪接位点的选择性使用的效率。将通过RT-PCR验证微基因构建体，然后建立稳定的细胞系。该项目的第二个目的是优化稳定的细胞系，并从小分子文库中鉴定减少Bcl-xL产生同时在低通量环境（LTS）中增加Bcl-xS同种型产生的生物试剂/小分子。将测试化合物对细胞系如PCS细胞中的凋亡性细胞死亡的活性，PCS细胞是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长期目标进行研究。这项研究与公共卫生的相关性：Bcl-xL是Bcl-x蛋白的一种大形式，在许多肿瘤中升高，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，用于识别减少Bcl-xL的药物。这些化合物可能与其他化学疗法组合用于癌症治疗。