Targeting Bcl-x splicing for cancer treatment

靶向 Bcl-x 剪接用于癌症治疗

基本信息

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
项目类别：
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&apos 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

项目摘要

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，癌症的治疗取得了进展，然而，仍然没有有效的方法来治愈它们。已鉴定出许多癌基因和肿瘤抑制基因。这些蛋白质中的一类形成了 Bcl-2 蛋白质家族。最近的研究表明，Bcl-2 家族成员之一 Bcl-x 在外显子 2 中选择性剪接。该外显子的选择性剪接产生抗凋亡（L 形式）或凋亡（S 形式）的亚型，表明 Bcl-x L/S 形式的调节对癌症治疗具有意义。本申请的目标是开发一种基于细胞的测定法，用于鉴定通过使用外显子 2 内的替代剪接位点调节外显子 2 剪接的生物分子/药物，从而使 Bcl-xL 和 Bcl-xS 的比率以及活性Bcl-x 可以被调节。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。该项目的第一个目标是生成用于 Bcl-x 剪接研究的微型基因构建体，并附有报告基因荧光素酶，以便可以通过以下方式测量位于 Bcl-x 前 mRNA 外显子 2 内的剪接位点的替代使用效率：荧光素酶活性。小基因构建体将通过 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细胞系并鉴定生物试剂/小分子，这些生物试剂/小分子可减少 Bcl-xL 的产量，同时在低通量设置 (LTS) 下从小分子文库中增加 Bcl-xS 亚型的产量。这些化合物将在细胞系（例如 PCS 细胞）中测试其对细胞凋亡细胞死亡的活性，PCS 细胞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选择性剪接调节的作用机制将作为长期目标进行研究。这项研究与公共健康的相关性：Bcl-xL 是 Bcl-x 蛋白的大分子形式，在许多肿瘤中含量升高，可保护细胞免受化疗的影响。我们计划在这个项目中建立一个基于细胞的系统来鉴定减少 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。