HEMATOPOIETIC SPECIFIC GENES IN STEM CELL DEVELOPMENT

这项更新拨款的长期目标是了解RhoGTP酶及其调节因子在造血、造血细胞功能和血液系统恶性肿瘤中的作用。RhoGTP酶(Rho/RAC/CDC42)是一个重要的蛋白质家族。它们共同调节一系列不同的基本细胞功能，包括细胞骨架组织、膜运输、运动、基因转录、细胞分裂和分化。依赖于GTP酶运行的一个关键分子机制是它在GDP结合的非活性状态和GTP结合的活性状态之间的循环。被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，在那里它们短暂地发挥作用，然后再次切换到不活跃状态。关于这些信号是如何传输和协调的，人们知之甚少。GDP解离抑制剂是分子开关的三个关键调节因子之一。作为GDP解离的强效抑制剂，RhoGDIs在调节GTP酶的水平和位置方面起着至关重要的作用。上一次拨款的目的是a)阻断在造血细胞中优先表达的GDI RhoGDIbeta，并研究其对ES细胞分化的影响；b)寻找其他GDI相互作用的蛋白；以及c)研究RhoGDIs可能的肿瘤抑制作用。来自RhoGDIbeta-/-ES的吞噬细胞被发现在其超氧化物产生方面存在缺陷。RhoGDIbeta--现在已经产生了动物。已鉴定出一个与RhoGDI相互作用的蛋白，该蛋白与一种新的丝氨酸/苏氨酸磷酸酶相同，但功能未知。RhoGDI相互作用蛋白的基因已被鉴定为与一种新的功能未知的丝氨酸/苏氨酸磷酸酶相同。Rho H基因是一种新的造血特异性RhoGTP酶，已被发现与淋巴瘤和骨髓瘤的非随机易位有关。这是人类疾病中RhoGTPase突变的第一个例子，并引发了是否可能有其他例子的问题。这一更新建议的具体目标是：1)对RhoGDIbeta/-动物进行表型，比较野生型和突变型动物组织中超氧化物的产生，并测试这种差异对致癌挑战的影响；扰乱普遍存在的RhoGDIpha基因并对动物进行表型；计划使用这两个突变体来衍生具有双GDI基因敲除的动物；目标2)破译与RhoGDIs相互作用的新型丝氨酸/苏氨酸磷酸酶的功能；3)进一步研究GDIs如何通过癌基因影响细胞分裂和转化，测试RhoH是否能转化因子依赖的造血细胞，并在数据库中搜索RhoGTPase及其调控因子参与人类疾病的更多证据。这项更新提案的直接目的将继续告诉我们RhoGTP酶通路如何汇聚到癌症发生和转移的生化步骤上。期望和长期目标是在这些信息的基础上，确定促进癌细胞生长和消散的具体步骤和分子，并设计阻止这一病理过程的药物。

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.