PRODUCTION OF ALTERNATIVE FGF RECEPTOR FORMS IN TUMOR

恶性星形细胞瘤在中枢神经系统中占多数 人类中的肿瘤，并与黯淡的预后相关，尽管 综合治疗的应用。据推测，地球的起源 这些肿瘤的发生是良性星形细胞瘤的多步骤进展。 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，数量众多， 到目前为止，人们对细胞遗传学和生化变化知之甚少。 最近的研究表明，星形胶质细胞从一个 良性到恶性的表型伴随着RNA的变化 成纤维细胞生长因子受体1基因的加工。关卡 结果是FGFR-1的高亲和力形式的数量显著增加 表达改变和RNA剪接。两者之间的强相关性 FGFR基因表达改变和星形胶质细胞恶性强调 了解FGFR在正常和恶性病变中作用的重要性 星形胶质细胞生长。这项提议将检验这样一个假设： FGFR RNA剪接的改变导致肿瘤的异常生长 恶性星形细胞。这项建议的具体目标是：1) 开发一个模拟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)使用这种方法 识别FGFR-1前体RNA中的序列元件的模型 对剪接进行调控，3)利用调控序列信息 确定在TRANS中起作用的监管因素，4)确定 反式作用因子表达与转化的关系。 目标1将通过检查现有的星形胶质细胞系来实现 FGFR-1转录本的RNA剪接模式。我们还将表示一个 在这些细胞系中嵌合FGFR-1微型基因以确定其RNA是否 转录本的处理方式类似于内源基因。 目标2将通过引入序列改变、删除、 FGFR-1微型基因的替换和突变，并确定 它们对RNA加工决策的影响。在目标3中，我们将开发一种 用于纯化参与调节的因子(S)的检测系统 FGFR-1 RNA剪接。将使用两种方法：RNA的鉴定- 通过序列特异性相互作用和使用结合蛋白质 从功能上检测RNA剪接的表达克隆。最后，在 目的4我们将使用从目标3获得的cdna序列信息来测量 肿瘤分级标本中反式作用因子的表达水平。一个 对FGFR-1变化机制的进一步认识 恶性星形细胞瘤的RNA处理可能有助于揭示 星形胶质细胞的转化过程并提供新的靶点 抑制它们的生长。

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.