ELAV-LIKE PROTEINS IN NEUROBLASTOMA

N-myc的扩增和非调控表达起着至关重要的作用 在确定神经母细胞瘤临床行为中的作用。 同样，在许多NB细胞系中，已显示出致瘤性 与N-myc的表达水平直接相关。差异化 在不同表型中观察到N-myc的表达水平 NB细胞系BBLW(W-N和W-S)的亚克隆似乎 这在很大程度上是由于N-myc mRNA周转率的细胞差异所致。 与c-myc和c-fos不同，人们对N-myc mRNA知之甚少 新陈代谢。其他不稳定的mRNAs的更替似乎是 RNA结合蛋白与蛋白质相互作用的结果 3‘非翻译区(3’UTR)富Au元素。RNA结合 用W-N和W-S细胞进行的研究表明 一种40 kDa蛋白(P40)与高密度脂蛋白结合的差异活性 N-myc 3‘非编码区内富含AU序列的特异性。 免疫沉淀实验表明，p40是 ELAV样RNA结合蛋白家族。因此，它是， 可能是ELAV样蛋白调节N-myc mRNA周转， 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 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 N-myc 3‘非编码区富含AU序列的功能表征 它绑定了p40。N-myc 3‘非编码区嵌合mRNAs I 取代稳定的mRNAs的3‘非编码区的序列将被表达 在NB细胞中。含突变N-的mRNAs的衰变动力学 MYC 3‘非编码区结合位点将与未突变的进行比较 也将使用嵌合体来研究mrna、mrna周转 通过井间结构域交换产生的转录本 表征c-fos 3‘UTR不稳定元件(已知 与ELAV样蛋白相互作用)和N-myc 3‘非编码区 结合部位。第二个具体目的是研究N-myc mRNA 使用体外系统在存在和不存在的情况下进行衰变 竞争对手N-myc RNA片段。第三个具体目标是 ELAV样蛋白在N-myc基因表达中的作用 体内周转和Nb表型。ELAV样蛋白 外源基因转移将改变NB细胞的表达 或通过添加反义ELAV mRNA或寡聚体。vt.N- MYC基因半衰期与肿瘤细胞表型和生长 将在NB细胞中检测增强或下调的潜力- 调节ELAV样蛋白的表达。此外，类ELAV 将通过以下方法检测原发NB肿瘤中的蛋白表达 Western印迹分析，以及两者之间的关系 将分析其在体内的表达和临床行为。一个 更好地了解N-myc mRNA的降解可能会导致 N-myc表达与NB表型的新策略 是可以改变的。因此，这些研究最终可能会提供 开发有效的治疗方法所需的线索 N-myc和N-myc高表达的儿童NB肿瘤 在临床上具有攻击性。

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.