C-FES PROTEIN-TYROSINE KINASE IN MYELOID DIFFERENTIATION

这项提议的目的是确定髓系特异的原核如何- 癌基因蛋白酪氨酸激酶，c-fes可调节肿瘤细胞的分化。 髓系白血病细胞。C-fes原癌基因编码一个93 kDa的蛋白质。 与正常和白血病特异相关的酪氨酸激酶 骨髓单核细胞。C-fes基因在髓系细胞中起着关键作用。 基于我们实验室的研究证明的区别 基因组c-Fes基因对未成熟成髓细胞系K562的转染作用 基因导致克隆变异体稳定表达，具有更多的 成熟粒细胞表型。 这项提议的目的有两个。第一个目标是检查 C-fes基因产物P93c-fes调节其 蛋白酪氨酸激酶活性及其对分化的影响。 这将通过两种方式实现。第一种方法将是 表达c-fes基因及其各种缺失和点突变 杆状病毒表达系统生产重组形式的P93c-Fes 体外研究氨基末端结构域和蛋白的作用 自磷酸化位点在其催化活性的调节中。 其次，将c-fes的cdna及其变异体用于转染法。 研究K562细胞在髓系中的结构与功能关系 差异化。重组P93c-Fes的表达也将提供 作为多克隆生产免疫原的蛋白质的充足来源 用于本提案第二部分的抗体。 第二个目标是评估影响细胞因子的作用 髓系分化，如粒细胞/巨噬细胞集落刺激 巨噬细胞集落刺激因子(GM-CSF)在c-Fes转录调控中的作用 P93c-Fes蛋白酪氨酸激酶活性的表达。这将是 通过利用依赖于脑脊液或反应灵敏的人类 确定c-Fes与粒单核细胞白血病细胞株的关系 在增殖或分化过程中的表达。这个 P93c-Fes在体内的自磷酸化及其激酶的测定 体外活性将通过免疫沉淀法完成 抗P93c-Fes的单特异性多克隆抗体及其亲和力 分别进行层析和非变性聚丙烯酰胺凝胶实验。 将通过核糖核酸酶保护试验进行mRNA水平的分析 或通过竞争性聚合酶链式反应，转录将 可以通过核连续分析来测量。潜在的内源底物 脑脊液依赖或反应性髓系细胞系中的P93c-FES 免疫化学、电泳法和免疫印迹法检测 使用抗磷酸酪氨酸抗体的程序。 从这项研究中获得的信息具有诊断和预后意义。 实用，因为c-fes基因是区分 其他类型白血病的髓样细胞。理解规则中的 C-fes基因也具有治疗意义，因为 这个基因可能表明白血病细胞是否有潜在的 分化，从而降低其致癌潜力。认定 这一监管过程应该有助于差异化的设计- 髓系白血病的特殊治疗方法。

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.