ELAV-LIKE PROTEINS IN NEUROBLASTOMA

神经母细胞瘤中的 ELAV 样蛋白

• 批准号: 2370768
• 负责人: SUSAN L. COHN
• 金额: $ 20.75万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2370768
• 关键词: RNA binding protein; cell line; chemical binding; fusion gene; gene expression; genetic mapping; genetic promoter element; lethal genes; neoplastic cell; neuroblastoma; northern blottings; nucleic acid sequence; oncogenes; phenotype; polymerase chain reaction; protein structure function; ribonucleoproteins; site directed mutagenesis; transfection; transfection /expression vector; western blottings

N-myc amplification and deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma (NB). Similarly in many NB cell lines, tumorigenicity has been shown to directly correlate with N-myc expression levels. The differential levels of N-myc expression observed in the phenotypically distinct subclones of the NB cell line BBL-W (W-N and W-S), appears to be largely due to cellular disparities in N-myc mRNA turnover. Unlike c-myc and c-fos, little is known about N-myc mRNA metabolism. The turnover of other unstable mRNAs appears to be a consequence of interactions between RNA-binding proteins and 3' untranslated region (3'UTR) AU-rich elements. RNA binding studies performed with W-N and W-S cells demonstrate differential activity of a 40-kDa protein (p40) that binds with high specificity to AU-rich sequences within the N-myc 3'UTR. Immunoprecipitation experiments indicate that p40 is a member of the ELAV-like RNA-binding protein family. It is, therefore, possible that ELAV-like proteins regulate N-myc mRNA turnover, and thereby, modulate the steady-state levels of N-myc expression and NC phenotype. The focus of this study will be to understand the cellular control of N-myc mRNA degradation in NB, and examine the role ELAV-like proteins play in determining NB phenotype. The first specific aim of this proposal is to functionally characterize the N-myc 3'UTR AU-rich sequences that bind p40. Chimeric mRNAs I which N-myc 3' UTR sequences replace the 3'UTR of stable mRNAs will be expressed in NB cells. The decay kinetics ofmRNAs containing mutated N- myc 3'UTR binding sits will be compared to that of non-mutated mRNA, mRNA turnover will also be studied using chimeric transcripts generated by domain interchange between the well characterized c-fos 3'UTR instability elements (which are known to interact with ELAV-like proteins) and the N-myc 3'UTR binding sites. The second specific aim is to study N-myc mRNA decay using in vitro systems in the presence and absence of competitor N-myc RNA fragments. The third specific aim is to characterize the role of ELAV-like proteins in N-myc mRNA turnover and NB phenotype in vivo. ELAV-like protein expression will be altered in NB cells by exogenous gene transfer or by the addition of anti-sense ELAV mRNA or oligomers. N- myc mRNA half-life and tumor cell phenotype and growth potential will be examined in NB cells with enhanced or down- regulated ELAV-like protein expression. In addition, ELAV-like protein expression will be examined in primary NB tumors by Western blot analysis, and the relationship between level of expression and clinical behavior in vivo will be analyzed. A better understanding of N-myc mRNA degradation may lead to novel strategies by which N-myc expression and NB phenotype can be altered. Thus, ultimately, these studies may provide the clues necessary for the development of effective therapy for children with NB tumors that express high levels of N-myc and are clinically aggressive.

N-myc扩增和放松调节表达在至关重要 在确定神经母细胞瘤（NB）的临床行为方面的作用。 同样，在许多NB细胞系中，肿瘤性已被证明 与N-MYC表达水平直接相关。 差异 在表型上观察到的N-MYC表达水平 NB细胞系BBL-W（W-N和W-S）的子克隆似乎 很大程度上是由于N-MYC mRNA转换中的细胞差异。 与C-Myc和C-Fos不同，关于N-MYC mRNA知之甚少 代谢。 其他不稳定mRNA的营业额似乎是 RNA结合蛋白与 3'未翻译区域（3'UTR）AU富含元素。 RNA结合 W-N和W-S细胞进行的研究表明 40 kDa蛋白（p40）的差异活性，该蛋白与高 N-MYC 3'UTR中Au富序列的特异性。 免疫沉淀实验表明p40是 类似Elav的RNA结合蛋白家族。 因此，这是 elav样蛋白可能调节n-myc mRNA周转率， 因此，调节N-MYC表达的稳态水平 和NC表型。这项研究的重点是了解 NB中N-MYC mRNA降解的细胞控制，以及 检查elav样蛋白在确定NB中的作用 表型。 该提议的第一个具体目的是 在功能上表征N-Myc 3'utr Au富序列 结合p40。 嵌合mRNA i哪个n-myc 3'utr 序列替换稳定mRNA的3'UTR将被表示 在NB细胞中。 含有突变的n-的mrNA的衰减动力学 Myc 3'utr绑定将被比较 mRNA，也将使用嵌合研究mRNA更新 井之间由域互换产生的成绩单 表征C-FOS 3'UTR不稳定性元素（已知 与类似elav的蛋白质相互作用）和n-myc 3'utr 绑定位点。 第二个具体目的是研究N-MYC mRNA 在存在和不存在的情况下，使用体外系统衰减 竞争者N-MYC RNA片段。 第三个具体目的是 表征伊拉夫样蛋白在N-myc mRNA中的作用 营业额和体内NB表型。 伊拉夫样蛋白 外源基因转移将在NB细胞中改变表达 或通过添加抗沉思的ELAV mRNA或低聚物。 n- MYC mRNA半衰期和肿瘤细胞表型和生长 在具有增强或下降的NB细胞中将检查潜力 调节的Elav样蛋白表达。 另外，类似于Elav 将通过原发性NB肿瘤检查蛋白质表达 Western印迹分析以及水平之间的关系 将分析体内表达和临床行为。 一个 更好地了解N-MYC mRNA降解可能会导致 N-MYC表达和NB表型的新型策略 可以改变。 因此，最终，这些研究可能提供 开发有效治疗所必需的线索 表达高水平N-MYC和的NB肿瘤儿童 在临床上是侵略性的。