PRODUCTION OF ALTERNATIVE FGF RECEPTOR FORMS IN TUMOR

恶性星形细胞瘤占中枢神经系统的大部分 人类的肿瘤，并与一个令人沮丧的预后， 多模式治疗的应用。据推测， 这些肿瘤的发生是从良性星形细胞瘤的多步进展 到间变性星形细胞瘤（AA），最后到多形性胶质母细胞瘤 （GBM）。在这种从良性到恶性的转变中， 到目前为止，人们对细胞遗传学和生物化学的变化知之甚少。 最近研究表明，星形胶质细胞从星形胶质细胞分化为星形胶质细胞， 良性表型向恶性表型转变伴随着RNA的变化， 成纤维细胞生长因子受体1（FGFR-1）基因的加工。水平 结果，FGFR-1的高亲和力形式的表达显著升高， 改变表达和RNA剪接。之间的强相关性 FGFR基因表达改变和星形胶质细胞恶性肿瘤的关系强调了 了解FGFR在正常和恶性肿瘤中作用的重要性 星形胶质细胞生长。这一提议将检验以下假设： FGFR RNA剪接的改变有助于细胞的异常生长， 恶性星形胶质细胞本提案的具体目标是：（1） 开发模拟FGFR-1 RNA加工途径的模型系统 在正常脑胶质细胞和胶质母细胞瘤中观察到，2）使用这种 该模型用于鉴定FGFR-1前体RNA内的序列元件， 作用于调节剪接，3）使用调节序列信息， 确定反式作用的调节因素，4）以确定 反式作用因子表达与转化的关系。 目的1将通过检查可用的星形胶质细胞系来实现， FGFR-1转录物的RNA剪接模式。我们还将表达一个 在这些细胞系中嵌合FGFR-1小基因，以确定其RNA 转录本的加工方式与内源基因类似。 目的2将通过引入序列改变，缺失， FGFR-1小基因中的取代和突变，并确定 它们对RNA加工决策的影响。在目标3中，我们将开发一个 用于纯化参与调节的因子的测定系统 FGFR-1 RNA剪接。将使用两种方法：鉴定RNA- 通过序列特异性相互作用的结合蛋白及其用途 表达克隆，其功能性地检测RNA剪接。最后在 目的4我们将使用目的3中的cDNA序列信息来测量 分级肿瘤样品中反式作用因子表达水平。一 进一步了解FGFR-1变化的机制 恶性星形细胞瘤中的RNA加工可能有助于阐明 星形胶质细胞的转化过程，并提供新的目标， 抑制他们的成长。

Malignant astrocytomas comprise the majority of central nervous system tumors in humans and are associated with a dismal prognosis despite the application of multimodality therapy. It is hypothesized that the genesis of these tumors occurs as a multistep progression from benign astrocytoma to anaplastic astrocytoma (AA), and finally, to glioblastoma multiforme (GBM). In this transformation from benign to malignant, numerous, and as of now, poorly understood cytogenetic and biochemical changes take place. Recently it was demonstrated that the progression of astrocytes from a benign to a malignant phenotype is accompanied by a change in the RNA processing of fibroblast growth factor receptor 1 (FGFR-1) gene. The level of a high affinity form of the FGFR-1 is dramatically elevated as a result altered expression and RNA splicing. The strong correlation between altered FGFR gene expression and astrocyte malignancy underscores the importance of understanding the role of FGFR in normal and malignant astrocyte cell growth. This proposal will test the hypothesis that alterations in FGFR RNA splicing contribute to abnormal growth of malignant astrocytes. The specific objectives of this proposal are 1) to develop a model system which mimics the FGFR-1 RNA processing pathways observed in normal brain glial cells and glioblastoma, 2) to use this model to identify sequence elements within the FGFR-1 precursor RNA which act to regulate splicing, 3) to use the regulatory sequence information to identify regulatory factors acting in trans, 4) to determine the relationship between trans-acting factor expression and transformation. Aim 1 will be accomplished by examining available astrocyte cell lines for the RNA splicing patterns of FGFR-1 transcripts. We will also express a chimeric FGFR-1 minigene in these cell lines to determine if its RNA transcripts are processed in a manner analogous to the endogenous gene. Aim 2 will be accomplished by introducing sequence alterations, deletions, substitutions and mutations, into the FGFR-1 minigene and determining their effect on RNA processing decisions. In Aim 3 we will develop an assay systems to be used to purify the factor(s) involved in regulation of FGFR-1 RNA splicing. Two approaches will be used: identification of RNA- binding proteins through sequence-specific interaction and use of expression cloning which functionally detects RNA splicing. Finally, in Aim 4 we will use cDNA sequence information derived from Aim 3 to measure the level of trans-acting factor expression in graded tumor samples. A further understanding of the mechanisms underlying the changes in FGFR-1 RNA processing in malignant astrocytomas may shed light on the transformation process in astrocytes and provide new targets for suppressing their growth.