HEMATOPOIETIC SPECIFIC GENES IN STEM CELL DEVELOPMENT

该续期资助的长期目标是了解RhoGTPases及其调控因子在造血、造血细胞功能和造血恶性肿瘤中的作用。RhoGTPases （Rho/Rac/CDC42）是一个重要的蛋白家族。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、基因转录、细胞分裂和分化。GTPase作用的一个关键分子机制是它在非活性gtp结合状态和活性gtp结合状态之间的循环。被外部细胞信号激活的gtpase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因子之一。作为gtp解离的有效抑制剂，RhoGDIs在gtp酶的水平和位置调节中起着关键作用。先前拨款的目的是a)破坏RhoGDIbeta（一种在造血细胞中优先表达的GDI），并研究其对胚胎干细胞分化的影响；b)寻找其他GDI相互作用蛋白；c)探讨rhogdi可能的抑瘤作用。来自RhoGDIbeta-/- ES的吞噬细胞被发现在其超氧化物生产方面存在缺陷。RhoGDIbeta- /-动物现已生成。一种推定的rhogdi相互作用蛋白已被确定，它与一种功能未知的新型丝氨酸/苏氨酸磷酸酶相同。RhoGDI相互作用蛋白的基因已被确定，它与一种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RhoGTPase，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RhoGTPase突变的第一个例子，并提出了是否可能有其他例子的问题。本更新提案的具体目的是：目的1)对RhoGDIbeta-/-动物进行表型分析，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挑战的影响；破坏无处不在的RhoGDIalpha基因并使动物表型化；计划利用这两个突变体获得双gdi基因敲除的动物；目的2)破译与rhogdi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gdi如何通过癌基因影响细胞分裂和转化，检测RhoH是否可以转化因子依赖的造血细胞，并在数据库中寻找更多RhoGTPase及其调控因子参与人类疾病的证据。这项更新提案的直接目的将继续告诉我们RhoGTPase途径如何在癌变和转移的生化步骤中收敛。期望和长期目标是建立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，并设计出能够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.