Drug-Induced Destabilization of Bcl-2 mRNA

药物诱导的 Bcl-2 mRNA 不稳定

• 批准号: 7208037
• 负责人: DANIEL James FERNANDES
• 金额: $ 21.87万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7208037
• 关键词: 3' Untranslated Regions; Acids; Affect; Antineoplastic Agents; Apoptosis; Applications Grants; B-Cell Lymphomas; B-Lymphocytes; Be++ element; Beryllium; Binding; Binding Sites; Biological Assay; Cell Extracts; Cell Line; Cells; Chronic Lymphocytic Leukemia; Development; Down-Regulation; Drug resistance; Elements; Endoribonucleases; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Genetic Transcription; Growth; HL-60 Cells; HL60; Human; Human Volunteers; Indium; MCF7 cell; Malignant Neoplasms; Maps; Messenger RNA; Okadaic Acid; Paclitaxel; Pancreatic ribonuclease; Patients; Pharmaceutical Preparations; Phosphorylation; Protein Binding; Protein Overexpression; Proteins; Proteolysis; RNA Recognition Motif; RNA Sequences; Recombinants; Reporting; Resistance; Ribonucleases; Ribosomal RNA; Robotics; Role; Screening procedure; Structure; Testing; Time; Transfection; Tretinoin; Work; base; cancer cell; deletion analysis; design; high throughput screening; inhibitor/antagonist; leukemia; mRNA Stability; mRNA Transcript Degradation; novel strategies; nuclease; nucleolin; research study; small molecule; small molecule libraries; volunteer

DESCRIPTION (provided by applicant): Over expression of bcl-2 is an important component in the development of B-cell lymphomas and some B-cell leukemia as well as in the emergence of cellular resistance to certain anticancer drugs. The 3'-untranslated region (3'-UTR) of bcl-2 mRNA contains several AU-rich elements (AREs) that promote mRNA destabilization. We have observed that the protein, nucleolin, binds to ARE-1 in bcl-2 mRNA, potentially protecting this mRNA from nuclease degradation. Accordingly, the central hypothesis of this proposal is that nucleolin stabilizes bcl-2 mRNA through interaction with AREs in the 3'-UTR, and that ATRA, taxol and okadiac acid destabilize bcl-2 mRNA by inducing down regulation or inactivation of nucleolin. The studies proposed in the first specific aim will evaluate the effects of bcl-2 ARE's 1-4 on bcl-2 mRNA stability in extracts of HL-60, MCF-7 and purified B cells from normal volunteers and patients with B-cell chronic lymphocytic leukemia (CLL). This will be followed by testing the effects of taxol and ATRA on bcl-2 ARE mRNA stability in cell extracts. Aim 2 is to determine whether functional nucleolin-bcl-2 mRNA interactions occur in intact cells. The ability of recombinant nucleolin to stabilize the bcl-2 mRNAs containing destabilizing AREs (Specific Aim 1) will be tested using transfection assays. We will also determine if exogenous over expression of nucleolin in parental HL-60 cells or MCF-7 cells can confer resistance to ATRA (HL-60) or taxol (HL-60 and MCF-7) concomitant with increased bcl-2 mRNA and protein. Additional experiments will reveal if knockdown of nucleolin expression with siRNAs leads to destabilization of bcl-2 mRNA. The third specific aim is to identify the RNA binding domains of nucleolin that are involved in binding to bcl-2 ARE mRNA. Finally, the information generated in Aims 1-3 will guide the development of high-throughput, robotic screening of diverse chemical libraries to identify small molecules that specifically inhibit nucleolin binding to AREs in bcl-2 mRNA.

描述（由申请人提供）：bcl-2的过度表达是B细胞淋巴瘤和一些B细胞白血病的发展以及细胞对某些抗癌药物产生耐药性的重要组成部分。 bcl-2 mRNA 的 3'-非翻译区 (3'-UTR) 包含多个富含 AU 的元件 (ARE)，可促进 mRNA 不稳定。我们观察到核仁蛋白与 bcl-2 mRNA 中的 ARE-1 结合，可能保护该 mRNA 免受核酸酶降解。因此，该提议的中心假设是核仁素通过与 3'-UTR 中的 ARE 相互作用来稳定 bcl-2 mRNA，而 ATRA、紫杉醇和冈田酸通过诱导核仁素的下调或失活来不稳定 bcl-2 mRNA。第一个具体目标中提出的研究将评估 bcl-2 ARE 1-4 对正常志愿者和 B 细胞慢性病患者的 HL-60、MCF-7 和纯化 B 细胞提取物中 bcl-2 mRNA 稳定性的影响。淋巴细胞白血病（CLL）。随后将测试紫杉醇和 ATRA 对细胞提取物中 bcl-2 ARE mRNA 稳定性的影响。目标 2 是确定完整细胞中是否发生功能性核仁素-bcl-2 mRNA 相互作用。将使用转染测定来测试重组核仁素稳定含有不稳定 ARE 的 bcl-2 mRNA 的能力（具体目标 1）。我们还将确定亲本 HL-60 细胞或 MCF-7 细胞中核仁素的外源过度表达是否可以赋予对 ATRA (HL-60) 或紫杉醇 (HL-60 和 MCF-7) 的抗性，同时增加 bcl-2 mRNA 和蛋白质。其他实验将揭示用 siRNA 敲低核仁素表达是否会导致 bcl-2 mRNA 不稳定。第三个具体目标是鉴定参与与 bcl-2 ARE mRNA 结合的核仁素的 RNA 结合域。最后，目标 1-3 中生成的信息将指导开发高通量、机器人筛选不同化学文库，以识别特异性抑制核仁素与 bcl-2 mRNA 中 ARE 结合的小分子。