HEMATOPOIETIC SPECIFIC GENES IN STEM CELL DEVELOPMENT

这项更新资助的长期目标是了解RhoGTPases及其调节剂在造血，造血细胞功能和造血恶性肿瘤中的作用。RhoGTP酶（Rho/Rac/CDC 42）已经成为具有核心重要性的蛋白质家族。它们共同调节多种基本细胞功能，包括细胞骨架组织、膜运输、运动性、基因转录、细胞分裂和分化。一个关键的分子机制，其依赖于一个GTP酶的操作是它的循环之间的一个非活性的GDP结合的活性GTP结合状态。由外部细胞信号激活的GTP酶必须被引导到适当的细胞内位置，在那里它们短暂地起作用，然后再次切换到非活性状态。人们对这些信号是如何传输和协调的知之甚少。GDP-解离抑制剂是分子开关的三个关键调节剂之一。RhoGDIs作为GDP解离的强有力抑制剂，在调节GTP酶的水平和定位中起关键作用。先前资助的目的是a）破坏RhoGDI β，一种优先在造血细胞中表达的GDI，并研究对ES细胞分化的影响; B）寻找其他GDI相互作用蛋白;和c）研究RhoGDI的可能的肿瘤抑制作用。发现来源于RhoGDIbeta-/- ES的吞噬细胞在其超氧化物产生中具有缺陷。RhoGDIbeta- /-动物现已生成。一个假定的RhoGDI相互作用蛋白已被确定，是一种新的丝氨酸/苏氨酸磷酸酶的未知功能相同。RhoGDI相互作用蛋白的基因已被鉴定为与功能未知的新型丝氨酸/苏氨酸磷酸酶相同。Rho H基因是一种新的造血特异性RhoGT 3基因，已被发现参与淋巴瘤和骨髓瘤的非随机易位。这是人类疾病中RhoGT 3突变的第一个例子，并提出了是否可能有其他例子的问题。本更新提案中的具体目标是：目标1）表型RhoGDI β-/-动物，比较野生型与突变型动物组织中的超氧化物产生，并测试这种差异对致癌攻击的影响;破坏普遍存在的RhoGDI α基因和动物表型;计划使用两种突变体来衍生具有双GDI敲除的动物;目的2）研究与RhoGDIs相互作用的丝氨酸/苏氨酸磷酸酶的功能; 3）进一步研究GDIs如何影响细胞分裂和癌基因转化，以测试RhoH是否可以转化因子依赖性造血细胞，并开始在数据库中搜索RhoGTdR及其调节剂参与人类疾病的更多证据。这项更新提案的直接目标将继续告知我们RhoGT 3途径如何汇聚在致癌和转移的生化步骤上。期望和长期目标是建立在这些信息的基础上，并确定促进癌细胞生长和消散的特定步骤和分子，并设计出阻止这种病理过程的药物。

The long term goal of this renewal grant is to understand the role of RhoGTPases and their regulators in hematopoiesis, hematopoietic cell function and hematopoietic malignancies. RhoGTPases (Rho/Rac/CDC42) have emerged as a family of proteins of central importance. Together, they regulate a diverse array of fundamental cellular functions including cytoskeletal organization, membrane-trafficking, motility, gene transcription, cell division and differentiation. A key molecular mechanism upon which rests the operation of a GTPase is its cycling between an inactive GDP-bound an active GTP-bound state. GTPases, activated by external cellular cues, must be directed to proper intracellular locations where they act transiently followed by a switching to an inactive state again. Little is known about how these signals are transmitted and coordinated. GDP-Dissociation Inhibitors are one of the three key regulators of the molecular switch. As powerful inhibitors of the dissociation of GDP, RhoGDIs play critical role in modulating levels and location of GTPases. The objectives of the previous grant were a) to disrupt RhoGDIbeta, a GDI which is preferentially expressed in hematopoietic cells, and to study the consequences on ES cell differentiation; b) to search for other GDI interacting proteins; and c) to investigate the possible tumor suppressor effect of RhoGDIs. Phagocytes derived from RhoGDIbeta-/- ES were found to have a defect in their superoxide production. RhoGDIbeta- /- animals have now been generated. A putative RhoGDI-interacting protein has been identified that is identical to a novel serine/ threonine phosphatase of unknown function. The gene for RhoGDI- interacting protein has been identified that is identical to a novel serine/threonine phosphatase of unknown function. The gene for Rho H, a new hematopoietic specific RhoGTPase, has been found to be involved in non-random translocations in lymphoma and myeloma. This is the first example of a RhoGTPase mutation in human disease and raises the question whether there may be other examples. The specific aims in this renewal proposal are: Aim 1) to phenotype RhoGDIbeta-/- animals, to compare superoxide production in tissues of wildtype versus mutant animals and to test for the effect of such differences on oncogenic challenge; to disrupt the ubiquitous RhoGDIalpha gene and phenotype the animals; to plan to use the two mutants to derive animals with dual-GDI knockout; Aim 2) to decipher the function of the novel serine/threonine phosphatases that was found to interact with RhoGDIs; 3) to further investigate how GDIs affect cell division and transformation by oncogenes, to test whether RhoH can transform factor dependent hematopoietic cells, and to initiate a search in data base for more evidence of involvement of RhoGTPase and their regulators in human diseases. The immediate aims of this renewal proposal will continue to inform us about how RhoGTPase pathways converge upon the biochemical steps of carcinogenesis and metastasis. The expectations and long term goals are to build upon this information and identify specific steps and molecules that promote growth and dissipation of cancer cells and to design drugs that will stop this pathological process.