MOLECULAR MECHANISMS OF MALIGNANT TRANSFORMATION

恶性转化的分子机制

• 批准号: 6160920
• 负责人: T MIKI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6160920
• 关键词: biological signal transduction; fibroblast growth factor; fluorescent in situ hybridization; gene expression; growth factor receptors; human genetic material tag; human tissue; molecular cloning; molecular oncology; neoplastic transformation; oncogenes

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.

我们设计并利用表达克隆策略来分离 新的癌基因。通过对相关信号通路的分析 通过这种策略分离出癌基因，我们试图澄清 恶性转化的分子机制。在本财年， 我们之前分离的两个癌基因被详细表征。 (1) 已经提出了双受体系统，其中基本相互作用 具有细胞表面蛋白聚糖的成纤维细胞生长因子（bFGF） 碱性 FGF 与 FGF 受体 1 高亲和力结合所需 （FGFR1）。在表达克隆过程中鉴定转化 从骨肉瘤 cDNA 文库中的基因中，我们鉴定出了一种独特的亚型 FGFR2 在单个分子中含有双受体系统。这 受体被硫酸乙酰肝素糖胺聚糖修饰 由替代外显子编码。此外，编码序列的存在 通过另一个替代外显子废除了这种修饰。这个高 亲和受体是增加和持续有丝分裂原所必需的 激活的蛋白激酶活性，三元复合因子独立 基因表达，特别是DNA合成。我们提议写一本小说 FGFR2信号转导的调控机制 糖胺聚糖修饰。 (2) 转化 cDNA 的搜索 大鼠脑 cDNA 表达文库导致同种型的分离 我们之前在骨肉瘤中发现了 ost 癌基因。 除了原始 Ost 同工型中发现的基序外，该 来自大脑的亚型包含一个扩展的 N 末端结构域，即 SH3 域和一个专门表达的附加唯一域 在大脑中。相反，包含 SH3 结构域但不包含 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亚型是由组织产生的 特定的选择性RNA剪接事件。而删除 N- 末端结构域激活了 Ost 的转化活性， SH3 或大脑特定结构域不影响此活动。这 Ost 的转化活性受到共表达的抑制 Rho 家族蛋白的显性失活形式。转录调控 血清反应元件的表达被有效诱导 Ost 的截断形式。截断的ost的表达也谦虚 在 NIH/3T3 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发挥作用。 标题由高效分离新癌基因更改 表达克隆系统。