Drug-Induced Destabilization of Bcl-2 mRNA

描述（由申请人提供）：bcl-2的过表达是b细胞淋巴瘤和某些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）含有几种促进mRNA不稳定的富au元件（AREs）。我们已经观察到，核仁蛋白与bcl-2 mRNA中的re -1结合，可能保护该mRNA免受核酸酶降解。因此，本研究的中心假设是核素通过与3'-UTR中的AREs相互作用稳定bcl-2 mRNA，而ATRA、紫杉醇和冈底酸通过诱导核素下调或失活来破坏bcl-2 mRNA的稳定。在第一个特定目标中提出的研究将评估bcl-2 ARE的1-4对HL-60、MCF-7和来自正常志愿者和B细胞慢性淋巴细胞白血病（CLL）患者的纯化B细胞提取物中bcl-2 mRNA稳定性的影响。接下来将测试紫杉醇和ATRA对细胞提取物中bcl-2 ARE mRNA稳定性的影响。目的2是确定功能性核蛋白-bcl-2 mRNA在完整细胞中是否发生相互作用。重组核蛋白稳定含有不稳定AREs （Specific Aim 1）的bcl-2 mrna的能力将通过转染试验进行测试。我们还将确定亲代HL-60细胞或MCF-7细胞中外源性核蛋白的过度表达是否会导致对ATRA （HL-60）或紫杉醇（HL-60和MCF-7）的抗性，同时bcl-2 mRNA和蛋白增加。进一步的实验将揭示用sirna敲除核仁蛋白表达是否会导致bcl-2 mRNA的不稳定。第三个具体目标是确定核蛋白与bcl-2 are mRNA结合的RNA结合域。最后，Aims 1-3中产生的信息将指导高通量、机器人筛选不同化学文库的发展，以识别特异性抑制bcl-2 mRNA中核蛋白与AREs结合的小分子。

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.