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

研究计划包括研究融合中的三种不同突变， 基因P210 BCR/ABL，费城染色体的分子标志 (Ph慢性粒细胞性白血病（CML）。 这些突变涉及到 BCR/ABL最近显示出潜在的重要作用， 在几个体外系统中引起转化。 该三个突变 包括：（1）缺失SH 2结构域，一个保守的调节区， c-Abl和许多其他蛋白质，在细胞间的信号转导中起重要作用。 酪氨酸磷酸化的蛋白质和增殖介质， （2）BCR（Y177 F）区域中的点突变体，显示为 Ras与BCR/ABL在细胞转化中的重要联系;（3） 删除c-Abl羧基末端的一个区域，该区域最近 已显示介导Abl与F-肌动蛋白细胞骨架的相互作用。 关于这些地区是否在世界上重要， 造血细胞在组织培养和体内的转化。 的 实现这些目标的基本研究设计包括两个阶段， 第一个涉及在组织培养中研究这些突变，第二个涉及在组织培养中研究这些突变， 将使用体外获得的见解来建立体内相关性 通过研究它们在小鼠骨髓移植实验中的作用。 在 第一部分，将这些突变体构建体引入小鼠中， 造血因子依赖性细胞系Ba/F3（淋巴样）和FDCP-1 （髓样）分别通过逆转录病毒基因转移和电穿孔。 结果将通过这些P210 BCR/ABL突变体在其 将这些细胞转化为不依赖生长因子的能力， 野生型P210。 在第二阶段，这些突变体将被引入到 小鼠骨髓细胞经Rat-1成纤维细胞逆转录病毒包装细胞 是林书 感染后，骨髓细胞将被用来重建 同系致死性照射受者的发病率和白血病 将结果与平行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，也许有一天受益患者患有 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.