Drug-Induced Destabilization of Bcl-2 mRNA

描述（由申请人提供）：bcl-2的过度表达是B细胞淋巴瘤和某些B细胞白血病发展以及细胞对某些抗癌药物耐药性出现的重要组成部分。bcl-2 mRNA的3 '-非翻译区（3'-UTR）含有几个富含AU的元件（战神），其促进mRNA的不稳定。我们已经观察到，蛋白质，核仁素，结合到bcl-2 mRNA中的ARE-1，潜在地保护该mRNA免受核酸酶降解。因此，该建议的中心假设是核仁素通过与3 '-UTR中的战神相互作用来稳定bcl-2 mRNA，而ATRA、紫杉醇和okadiac酸通过诱导核仁素的下调或失活来使bcl-2 mRNA不稳定。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细胞慢性淋巴细胞白血病（CLL）患者的HL-60、MCF-7和纯化的B细胞提取物中bcl-2 mRNA稳定性的影响。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。目的2是确定完整细胞中是否存在功能性核仁素-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去稳定战神（特异性Aim 1）的bcl-2 mRNA的能力。我们还将确定是否在亲代HL-60细胞或MCF-7细胞中外源性过表达核仁素可以赋予对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。另外的实验将揭示用siRNA敲低核仁素表达是否导致bcl-2 mRNA的不稳定。第三个具体目标是鉴定核仁素的RNA结合结构域，其参与与bcl-2 ARE mRNA的结合。最后，目标1-3中产生的信息将指导开发高通量的机器人筛选不同的化学文库，以鉴定特异性抑制核仁素与bcl-2 mRNA中战神结合的小分子。

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.