REGULATION AND ONCOGENIC FUNCTION OF GROWTH FACTOR FGF4

生长因子FGF4的调控及致癌功能

• 批准号: 2104795
• 负责人: JAMES C LANG
• 金额: $ 9.33万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2104795
• 关键词: 3T3 cells; DNA binding protein; DNA footprinting; artificial chromosomes; carcinoma; cell differentiation; cell transformation; embryo neoplasm; fibroblast growth factor; gene expression; gene induction /repression; genetic enhancer element; genetic promoter element; genetic regulation; human genetic material tag; molecular cloning; nucleic acid sequence; oncogenes; polymerase chain reaction; pulsed field gel electrophoresis

We have recently isolated a transforming gene from the DNAS of a patient with chronic myeloid leukemia using the NIH3T3 cell focus formation assay. The sequences responsible for transformation have been cloned and characterized. The transforming gene has been identified as fibroblast growth factor 4 (FGF4). Expression of FGF4 is restricted to undifferentiated embryonic stem and embryonal carcinoma cells and is not expressed in somatic tissue. The coding sequences of the FGF4 gene (located on chromosome 11) have been fused to RNA processing signals from an unidentified gene on chromosome 15. Transformation by FGF4 in this and previously described studies is the result of deregulated expression rather than mutation of the coding sequences. It is therefore of value to investigate the mechanism of regulation of FGF4 expression. Paradoxically, expression of endogenous FGF4 is suppressed in NIH3T3 cells yet an exogenously added copy of the normal gene is expressed and capable of inducing transformation. The goals of the proposed study are threefold. Firstly we wish to further previous studies and investigate the mechanism of local control of FGF4 expression. Specifically, to date the promoter has been shown to be inactive in all cell lines tested unless linked to an activating 3' enhancer sequence. Study of functional promoter domains has thus proven difficult. Using a transient reporter assay system (luciferase) which is more sensitive than the system (CAT) used previously by others, we have identified active promoter domains within the 5' flanking sequence of FGF4 and have shown the promoter to be active in both embryonal carcinoma cells (F9) which are permissive for FGF4 expression and HeLa cells which are not. We wish to further these studies and characterize functional domains both within the promoter and the enhancer sequence and to investigate how such sequences might interact to control local expression of FGF4. Secondly, based on previous data demonstrating that cosmid clones harboring the FGF4 gene are transforming while the endogenous gene is silent, we suggest the possibility that FGF4 expression may also be regulated from a distant locus. We will investigate this possibility by the transfer into NIH3T3 cells of YAC clones containing the FGF4 gene. These clones will therefore contain substantially greater sequence flanking the FGF4 gene than clones previously described. If transfer of YACs produce cell colonies of normal rather than transformed morphology, it may be inferred that a putative suppressor locus has been co-transferred and this locus will then be further characterized. Thirdly, little is known about the genes which are activated in response to FGF4 induced transformation of NIH3T3 cells. We will attempt to identify and characterize these genes by utilizing a recent and novel PCR based method which allows the identification and cloning of cDNA from differentially expressed genes.

我们最近从一名患者的DNA中分离出了一种转化基因 使用NIH3T3细胞病灶形成试验检测慢性粒细胞白血病。 负责转化的序列已经被克隆并 特色化的。转化基因已被鉴定为成纤维细胞。 生长因子4(FGF4)。FGF4的表达仅限于 未分化的胚胎干细胞和胚胎癌细胞 在体细胞组织中表达。成纤维细胞生长因子4基因的编码序列 (位于11号染色体)被融合到RNA处理信号中 15号染色体上的一个未知基因。FGF4在这个和 之前描述的研究是放松表达管制的结果 而不是编码序列的突变。因此，它是有价值的 探讨FGF4表达的调控机制。矛盾的是， 内源性FGF4在NIH3T3细胞中的表达受到抑制 外源添加的正常基因拷贝被表达并能够 诱导转化。这项拟议研究的目标有三个。 首先，我们希望对以前的研究进行进一步的研究，并探讨其机制 局部调控FGF4的表达。具体地说，与发起人约会 已经被证明在所有测试的细胞系中都是不活跃的，除非与 激活3‘增强子序列。HAS功能启动子结构域的研究 因此，这被证明是困难的。使用瞬时报告分析系统 (荧光素酶)，比以前使用的系统(CAT)更敏感 另外，我们已经在5‘端发现了活性启动子结构域 FGF4的侧翼序列，并表明启动子在两个 允许FGF4表达的胚胎癌细胞(F9)和 不是的HeLa细胞。我们希望进一步开展这些研究，并 鉴定启动子和增强子中的功能结构域 并研究这些序列如何相互作用来控制 FGF4的局部表达。其次，基于前人的数据论证 含有FGF4基因的粘粒克隆正在转化，而 内源基因沉默，提示FGF4可能表达 也可能受一个遥远的基因位点的调控。我们将对此进行调查 将含有该基因的YAC克隆转入NIH3T3细胞的可能性 FGF4基因。因此，这些克隆将包含更大的 序列侧翼的FGF4基因比前面描述的克隆。如果 YAC的转移产生正常的细胞克隆，而不是转化的细胞克隆 形态，可以推断一个可能的抑制基因已经被 共同转移，然后这一轨迹将进一步表征。第三， 对FGF4激活的基因知之甚少 诱导NIH3T3细胞转化。我们将尝试确定和 利用一种新的基于聚合酶链式反应的方法来鉴定这些基因 这使得鉴定和克隆差异表达的基因成为可能。 表达的基因。