C-FES PROTEIN-TYROSINE KINASE IN MYELOID DIFFERENTIATION

C-FES 蛋白酪氨酸激酶在骨髓分化中的作用

• 批准号: 3198719
• 负责人: ROBERT I. GLAZER
• 金额: $ 21.7万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198719
• 关键词: Baculoviridae; RNase protection assay; affinity chromatography; antibody formation; cell differentiation; colony stimulating factor; complementary DNA; cytokine; gel electrophoresis; gene expression; genetic transcription; immunochemistry; immunoprecipitation; messenger RNA; molecular cloning; monoclonal antibody; myelogenous leukemia; neoplasm /cancer genetics; nucleic acid sequence; polymerase chain reaction; protein tyrosine kinase; protooncogene; tissue /cell culture; transfection; western blottings

The purpose of this proposal is to determine how a myeloid-specific proto- oncogene protein-tyrosine kinase, c-fes can regulate the differentiation of myeloid leukemia cells. The c-fes proto-oncogene encodes a 93 kDa protein- tyrosine kinase that is specifically associated with normal and leukemic myelomonocytic cells. The c-fes gene plays a pivotal role in myeloid differentiation based on studies in our laboratory demonstrating that transfection of immature myeloblast cell line K562 with the genomic c-fes gene results in the stable expression of clonal variants possessing a more mature granulocytic phenotype. The aims of this proposal are two-fold. The first objective is to examine the manner by which the c-fes gene product, P93c-fes, regulates its protein-tyrosine kinase activity and how this influences differentiation. This will be accomplished in two ways. The first approach will be to express the c-fes cDNA and its various deletions and point mutations in a baculovirus expression system to produce recombinant forms of P93c-fes to investigate in vitro, the role of the amino-terminal domain and autophosphorylation sites in the regulation of its catalytic activity. Secondly, the c-fes cDNA and its variants will be utilized for transfection of K562 cells to study its structure-function relationships in myeloid differentiation. The expression of recombinant P93c-fes will also provide an ample source of protein as an immunogen for the production of polyclonal antibodies for use in the second part of this proposal. The second objective is to assess the role of cytokines which influence myeloid differentiation, such as granulocyte/macrophage colony-stimulating factor (GM-CSF), in the regulation of transcription of c-fes and in the expression of the protein-tyrosine kinase activity of P93c-fes. This will be accomplished by utilizing CSF-dependent or -responsive human myelomonocytic leukemia cell lines to determine the relationship of c-fes expression to the proliferative or differentiative processes. The autophosphorylation of P93c-fes in vivo, and measurement of its kinase activity in vitro will be accomplished by immunoprecipitation with monospecific polyclonal antibodies to P93c-fes, and by affinity chromatography and a non-denaturing polyacrylamide gel assay, respectively. The analysis of mRNA levels will be carried out by RNase protection assay or by competitive polymerase chain reaction assay, and transcription will be measured by nuclear run-on assays. Potential endogenous substrates of P93c-fes in CSF-dependent or -responsive myeloid cell lines will be investigated by immunochemical, electrophoretic and immunoblotting procedures using anti-phosphotyrosine antibodies. The information derived from this study has diagnostic and prognostic utility since the c-fes gene is a specific marker which distinguishes myeloid from other types of leukemias. Understanding the regulation of the c-fes gene has therapeutic implications as well, since the activity of this gene may indicate whether a leukemic cell has the potential for differentiating and thereby reducing its oncogenic potential. Cognizance of this regulatory process should aid in the design of differentiation- specific therapies for myeloid leukemias.

该提议的目的是确定髓样特异性的原始 癌基因蛋白 - 酪氨酸激酶，C-FES可以调节 髓样白血病细胞。 C-FES原始癌基因编码93 kDa蛋白 与正常和白血病特别相关的酪氨酸激酶 脊髓细胞细胞。 C-FES基因在髓样中起关键作用 基于我们实验室的研究的分化表明 与基因组C-FES的未成熟骨髓细胞细胞系K562转染 基因导致具有更多的克隆变体的稳定表达 成熟的粒细胞表型。 该提议的目的是两个方面。 第一个目标是检查 C-FES基因产物P93C-FES调节其的方式 蛋白质酪氨酸激酶活性及其如何影响分化。 这将通过两种方式完成。 第一种方法是 在A中表达C-FES cDNA及其各种缺失和点突变 杆状病毒表达系统以产生重组形式的p93c-fes 在体外调查，氨基末端结构域的作用和 自磷酸化位点的催化活性调节。 其次，C-FES cDNA及其变体将用于转染 K562细胞研究其在髓样的结构功能关系 分化。 重组P93C-FES的表达也将提供 充分的蛋白质来源作为多克隆生产的免疫原 该提案第二部分中使用的抗体。 第二个目标是评估影响影响的细胞因子的作用 髓样分化，例如粒细胞/巨噬细胞刺激 因子（GM-CSF），在调节C-FES和在 p93c-FES蛋白酪氨酸激酶活性的表达。 这会 可以通过利用CSF依赖或反应性人来完成 脊髓细胞性白血病细胞系以确定C-FES的关系 表达对增殖或分化过程。 这 体内p93c-FES的自磷酸化及其激酶的测量 体外活动将通过免疫沉淀与 单特异性多克隆抗体抗P93C-FES，并且按亲和力 色谱和非降解聚丙烯酰胺凝胶测定法。 mRNA水平的分析将通过RNase保护分析进行 或通过竞争性聚合酶链反应测定法，转录将 可以通过核跑步测定法测量。 潜在的内源性基材 CSF依赖性或反应性髓样细胞系中的p93c-fes将是 通过免疫化学，电泳和免疫印迹研究 使用抗磷酸酪氨酸抗体的程序。 从这项研究中得出的信息具有诊断和预后 效用是因为C-FES基因是区分的特定标记 其他类型的白血病。 了解调节 C-FES基因也具有治疗意义，因为 该基因可能表明白血病细胞是否具有 区分并降低其致癌潜力。 认知 在这个监管过程中，应有助于设计分化 - 髓样白血病的特定疗法。