REGULATION AND ONCOGENIC FUNCTION OF GROWTH FACTOR FGF4

生长因子FGF4的调控及致癌功能

• 批准号: 2414306
• 负责人: JAMES C LANG
• 金额: $ 10.48万
• 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/2414306
• 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.

我们最近从患者的 DNAS 中分离出一个转化基因 使用 NIH3T3 细胞灶形成测定法治疗慢性粒细胞白血病。 负责转化的序列已被克隆并 特点。 该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，不是 在体细胞组织中表达。 FGF4基因的编码序列 （位于 11 号染色体上）已与来自 15 号染色体上的一个未识别基因。此和中 FGF4 的转化 先前描述的研究是表达失调的结果，而不是 而非编码序列的突变。 因此，有价值的是 研究FGF4表达的调控机制。 矛盾的是， NIH3T3 细胞中内源性 FGF4 的表达受到抑制，但 正常基因的外源添加拷贝被表达并且能够 诱导转变。 拟议研究的目标有三个。 首先我们希望进一步深入研究并探讨其机制 FGF4表达的局部控制。 具体来说，迄今为止发起人 已被证明在所有测试的细胞系中均无活性，除非与 激活 3' 增强子序列。 功能启动子结构域的研究已 因而被证明是困难的。 使用瞬态报告分析系统 （荧光素酶）比之前使用的系统（CAT）更敏感 通过其他人的研究，我们已经确定了 5' 内的活性启动子结构域 FGF4 的侧翼序列，并显示启动子在两种情况下均具有活性 允许 FGF4 表达的胚胎癌细胞 (F9) 海拉细胞却不是。 我们希望进一步开展这些研究并 表征启动子和增强子内的功能域 序列并研究这些序列如何相互作用来控制 FGF4 的局部表达。 其次，根据以往的数据证明 携带 FGF4 基因的粘粒克隆正在转化，而 内源基因沉默，我们推测FGF4表达的可能性 也可以从远处进行调控。 我们将对此进行调查 通过将含有 FGF4基因。 因此，这些克隆将含有更多的 与之前描述的克隆相比，FGF4 基因侧翼的序列。 如果 YAC 的转移产生正常细胞集落而不是转化细胞集落 形态学上，可以推断推定的抑制基因座已被 共同转移，然后将进一步表征该基因座。 第三， 对于响应 FGF4 而被激活的基因知之甚少 NIH3T3 细胞的诱导转化。 我们将尝试识别并 利用最新的基于 PCR 的方法来表征这些基因 它允许从差异中识别和克隆 cDNA 表达的基因。