ELAV-LIKE PROTEINS IN NEUROBLASTOMA

N-myc的扩增和表达失调在肿瘤的发生发展中起着关键作用。 在决定神经母细胞瘤（NB）的临床行为中的作用。 类似地，在许多NB细胞系中，致瘤性已被证明是 与N-myc表达水平直接相关。 差动 N-myc表达水平在表型不同的 NB细胞系BBL-W（W-N和W-S）的亚克隆似乎 这在很大程度上是由于N-myc mRNA周转的细胞差异。 与c-myc和c-fos不同，N-myc mRNA的研究很少 新陈代谢. 其他不稳定的mRNA的周转似乎是 RNA结合蛋白与 3'非翻译区（3' UTR）富含AU的元件。 RNA结合 用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内富含AU序列的特异性。 免疫沉淀实验表明，p40是一个成员， ELAV样RNA结合蛋白家族。 因此， ELAV样蛋白可能调节N-myc mRNA的更新， 从而调节N-myc表达的稳态水平 和NC表型。这项研究的重点将是了解 NB中N-myc mRNA降解的细胞控制，和 研究ELAV样蛋白在确定NB中的作用 表型。 这项建议的第一个具体目标是 功能性表征N-myc 3 'UTR AU富集序列 与p40结合 嵌合mRNA I，其N-myc 3' UTR 替换稳定mRNA的3 'UTR的序列将被表达 NB细胞。 含有突变的N- myc 3 'UTR结合位点将与未突变的 mRNA，mRNA周转也将使用嵌合的 转录本是由阱之间的结构域交换产生的， 特征性的c-fos 3 'UTR不稳定性元件（其是已知的 与ELAV样蛋白相互作用）和N-myc 3 'UTR 结合位点。 第二个具体目标是研究N-myc mRNA 在存在和不存在以下物质的情况下使用体外系统进行衰变 竞争者N-myc RNA片段。 第三个具体目标是 描述ELAV样蛋白在N-myc mRNA中的作用 转换和NB表型。 ELAV样蛋白 通过外源基因转移将改变NB细胞中的表达 或通过加入反义ELAV mRNA或寡聚体。 不，不 myc mRNA半衰期与肿瘤细胞表型和生长 将在NB细胞中检查增强或降低的潜力， 调节ELAV样蛋白表达。 此外， 蛋白质表达将在原发性NB肿瘤中通过 蛋白质印迹分析，以及 将分析在体内的表达和临床行为。 一 更好地理解N-myc mRNA的降解可能会导致 N-myc表达和NB表型的新策略 是可以改变的 因此，最终，这些研究可能会提供 开发有效治疗的必要线索， 患有NB肿瘤的儿童表达高水平的N-myc， 具有临床攻击性。

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.