MOLECULAR MECHANISMS OF MALIGNANT TRANSFORMATION

我们设计并利用了一种表达克隆策略， 新癌基因通过对参与细胞凋亡的信号通路的分析， 通过这种策略分离的癌基因，我们试图阐明 恶性转化的分子机制。在本财政年度， 我们先前分离的两个癌基因被详细表征。（一） 已经提出了一种双受体系统，其中基本的 成纤维细胞生长因子（bFGF）与细胞表面蛋白聚糖， 碱性FGF与FGF受体-1的高亲和力结合所需 （FGFR 1）。在表达克隆过程中， 基因从骨肉瘤cDNA文库，我们确定了一个独特的亚型 FGFR 2在单个分子中含有双重受体系统。这 受体在一个位点被硫酸乙酰肝素糖胺聚糖修饰 由选择性外显子编码此外，编码的序列的存在 通过另一个选择性外显子废除了这种修饰。这种高 亲和力受体是增加和持续促分裂原所必需的， 激活的蛋白激酶活性，三元复合物因子非依赖性 基因表达，尤其是DNA合成。我们提出了一种新 FGFR 2信号转导的调控机制 糖胺聚糖修饰(2)转化cDNA的研究 一个大鼠脑cDNA表达文库导致了一个异构体的分离 我们之前在骨肉瘤中发现的ost癌基因。 除了在原始Ost同种型中发现的基序外， 来自脑的同种型含有延伸的N-末端结构域，即SH 3 结构域和一个额外的独特结构域， 在大脑中。相反，含有SH 3结构域但不含SH 3结构域的同种型可以表达。 脑特异性结构域，在各种组织中普遍表达。 基因组分析表明，这些异构体是由组织产生的- 特异性RNA剪接事件。而删除N- 末端结构域激活了Ost的转化活性， SH 3或脑特异性结构域不影响这种活性。的 Ost的转化活性被共表达的 Rho家族蛋白的显性负性形式。转录调控 由血清反应元件的表达， Ost的截断形式。截断Ost的表达也适度 在NIH/3 T3成纤维细胞中诱导侵袭表型，表明 它可能在细胞骨架组织中起作用。 标题由一种高效的分离新癌基因的方法改为 表达克隆系统。

We have devised and utilized an expression cloning strategy to isolate novel oncogenes. By the analysis of signaling pathways involving the oncogenes isolated by this strategy, we are attempting to clarify the molecular mechanisms of malignant transformation. In this fiscal year, two oncogenes we previously isolated were characterized in detail. (1) A dual receptor system has been proposed where interaction of basic fibroblast growth factor (bFGF) with cell surface proteoglycans is required for high affinity binding of basic FGF to FGF receptor-1 (FGFR1). During a course of expression cloning to identify transforming genes from an osteosarcoma cDNA library, we identified a unique isoform of FGFR2 containing a dual receptor system in a single molecule. This receptor is modified by heparan sulfate glycosaminoglycan at a site encoded by an alternative exon. Moreover, presence of a sequence encoded by another alternative exon abrogated this modification. This high affinity receptor is required for increased and sustained mitogen- activated protein kinase activity, ternary complex factor-independent gene expression, and especially for DNA synthesis. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification. (2) A search of transforming cDNAs from a rat brain cDNA expression library led to the isolation of an isoform of the ost oncogene, which we previously identified in osteosarcomas. In addition to the motifs found in the original Ost isoform, this isoform from brain contained an extended N-terminal domain, an SH3 domain and an additional unique domain which was exclusively expressed in brain. In contrast, an isoform containing the SH3 domain, but not the brain-specific domain, was ubiquitously expressed in various tissues. Genomic analysis suggested that these isoforms were generated by tissue- specific alternative RNA splicing events. Whereas deletion of the N- terminal domain activated the transforming activity of Ost, presence of the SH3 or brain-specific domain did not affect this activity. The transforming activity of Ost was inhibited by the coexpression of dominant negative forms of Rho family proteins. Transcription regulated by the serum responsive element was potently induced by expression of the truncated form of Ost. Expression of truncated Ost also modestly induced an invasive phenotype in NIH/3T3 fibroblasts, suggesting that it may play a role in cytoskeletal organization. Titled changed from Isolation of Novel Oncogenes by an Efficient Expression Cloning System.