IN VITRO AND IN VIVO STUDIES OF P210 BCR/ABL MUTATIONS

研究计划包括研究融合过程中的三个不同的突变。 基因P210 bcr/abl，费城染色体的分子标志 慢性粒细胞白血病(CML)。这些突变涉及的区域 BCR/ABL最近被证明在如何 在几个体外系统中引起转化。三个突变 包括：(1)SH2结构域的缺失，这是一个保守的调节区 C-Abl和其他许多蛋白质，在信号转导中起重要作用 酪氨酸上磷酸化的蛋白质及其增殖和调节因子 分化；(2)bcr(Y177F)区域中的一个点突变 RAS与bcr/abl在细胞转化中的重要联系； C-Abl羧基末端的一个区域的缺失 研究表明，Abl与F-肌动蛋白细胞骨架的相互作用具有调节作用。 关于这些区域是否在 造血细胞在组织培养和体内的转化。这个 实现这些目标的基本拱门设计包括两个阶段， 第一个涉及研究组织培养中的这些突变，第二个涉及研究这些突变 将利用在体外获得的洞察力来建立体内相关性 通过研究它们在小鼠骨髓移植实验中的作用。在……里面 第一部分，这些突变结构将被引入小鼠体内 造血因子依赖的细胞连接蛋白BA/F3(淋巴样)和FDCP-1 (髓系)分别通过逆转录病毒基因转移和电穿孔。 结果将通过这些P210BCR/ABL突变体在其 将这些细胞转化为生长因子独立的能力 野生型P210。在第二阶段，这些突变体将被引入到 大鼠成纤维细胞逆转录病毒包装细胞对小鼠骨髓细胞的影响 林。感染后，骨髓细胞将被用来重建 同基因致死照射受者及其发病率和白血病 结果将与平行的P210对照进行比较。这些 研究将为BCR/ABL的发病机制提供重要的见解。 相关的恶性肿瘤，也许有一天会让患者受益 这些疾病。

The research plan involves studying three distinct mutations in the fusion gene P210 BCR/ABL, the molecular hallmark of the Philadelphia Chromosome (Ph') of Chronic Myelogenous Leukemia (CML). These mutations involve areas of BCR/ABL recently shown to have potentially important roles in how it causes transformation in several in vitro systems. The three mutations include: (1) deletion of the SH2 domain, a conserved regulatory region in c-Abl and many other proteins, important in signal transduction between proteins phosphorylated on tyrosine and mediators of proliferation and differentiation; (2) a point mutant in a region of BCR (Y177F) shown to be an important link between Ras and BCR/ABL in cellular transformation; (3) deletion of a region in the carboxy-terminus of c-Abl that has recently been shown to mediate the interaction of Abl with the F-actin cytoskeleton. Much remains to be learned about whether these regions are important in transformation of hematopoietic cells in tissue culture and in vivo. The basic res arch design to accomplish these goals consists of two phases, the first involves studying these mutations in tissue culture, and the second will be to use the insight gained in vitro to establish in vivo relevance by studying their effects in murine bone marrow transplant experiments. In the first part, these mutant constructs will be introduced into the murine hematopoietic factor-dependent cell liens Ba/F3 (lymphoid), and FDCP-1 (myeloid) by retroviral gene transfer and electroporation, respectively. Outcome will be assessed by how these P210BCR/ABL mutants compare in their ability to transform these cells to growth factor independence compared to wild type P210. In the second phase these mutants will be introduced into murine bone marrow cells by a Rat-1 fibroblast retroviral packaging cell lin. After infection, the marrow cells will be used to reconstitute syngeneic lethally irradiated recipients, and the incidence and leukemic phenotype which results will be compared to parallel P210 controls. These studies will provide important insight into the pathogenesis of BCR/ABL- related malignancies, and perhaps someday benefit patients suffering from these diseases.