Drug-Induced Destabilization of Bcl-2 mRNA

描述(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某些抗癌药物出现细胞耐药的重要组成部分。BCL-2mRNA的3‘-非翻译区(3’-UTR)含有几个富含AU的元件(ARs)，它们促进了mRNA的失稳。我们已经观察到，核仁蛋白与bcl2mRNA中的ARE-1结合，潜在地保护这种mRNA免受核酸酶的降解。因此，这一建议的中心假设是，核仁通过与3‘-UTR中的ARES相互作用来稳定bCL-2mRNA，而ATRA、紫杉醇和己二酸通过诱导核仁下调或失活来破坏bCL-2mRNA的稳定。第一个特定目的的研究将评估bcl2ARE‘s1-4对正常人和B细胞慢性淋巴细胞白血病(CLL)患者的HL-60、MCF-7和纯化的B细胞提取液中bcl-2mRNA稳定性的影响。接下来将测试紫杉醇和全反式维甲酸对细胞提取物中bcl2are mRNA稳定性的影响。目的2是确定在完整的细胞中是否存在功能性核仁素-bcl2mRNA的相互作用。重组核仁素稳定含有不稳定的ARES的bcl2mRNAs的能力将通过转染实验进行测试。我们还将确定在亲代HL-60细胞或MCF-7细胞中外源性核仁素的过度表达是否会导致对ATRA(HL-60)或紫杉醇(HL-60和MCF-7)的耐药性，并伴随bcl2基因和蛋白的增加。进一步的实验将揭示，如果用siRNAs抑制核仁素的表达，是否会导致bcl2mRNA的不稳定。第三个特定的目的是确定核仁的RNA结合域，这些结构域参与与bcl-2are mRNA的结合。最后，在AIMS 1-3中产生的信息将指导高通量、机器人筛选各种化学文库的发展，以识别特定地抑制核仁素与bcl-2mRNA中的ARE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.