METABOLIC DETERMINANTS OF HEMATOPOIETIC CELL DEVELOPMENT

虽然现在对促进存活的特定细胞因子有很多了解， 造血干细胞和祖细胞的生长， 已知有控制自我更新丧失的调节机制 能力，分化，并在这些细胞的终末成熟， 正常的血细胞发育 在低等真核生物中，如S.酿酒酵母和 发育开关由代谢变化触发 对这些生物的研究表明， 鸟嘌呤核糖核苷酸（如GTP）的消耗是一个关键 在发育开始时发生代谢事件。 两者的平行研究 人白血病细胞系和纯化的正常CD 34+骨髓细胞也已经 建立了鸟嘌呤核苷酸代谢和 控制造血细胞发育。 理解如何 细胞内鸟苷酸池的调节作为代谢信号， 造血细胞的发展，我们提出了两个中心的研究 目标. 我们的第一个目标是了解IMP-脱氢酶（IMPDH）， 一种高度保守的中间嘌呤代谢酶，它控制鹿 新鸟嘌呤核苷酸合成，在生长过程中受到调节， 造血细胞的发育。 使用IL-3依赖性，鼠 造血细胞系 32 D（cl 3）和免疫亲和纯化的CD 34+人 骨髓细胞，我们将表征GTP合成，鸟苷酸池， IMPDH基因表达、蛋白质和活性的水平， IL-3刺激生长、G-CSF诱导骨髓成熟和凋亡 在生长因子退出后。 我们还将研究细胞因子 IMPDH亚型的依赖性翻译后修饰，检查 细胞内pH变化和改变的IL-3信号传导对IMPDH的影响 表达和活性，并确定如何IMPDH过表达和表达不足， 诱导的基因转染影响细胞成熟。 我们的第二 目的是确定鸟苷酸的调节机制， 池影响造血细胞发育。 我们会研究 IMPDH抑制对GTP依赖性ras介导的信号传导的影响 通过测量GTP结合的ras和IL-3刺激的 RAF. 由于蛋白质合成是高度GTP调节的，我们还将 确定鸟苷酸消耗和补充是否影响营业额， 短寿命原癌基因蛋白的供应（例如，c-myb）影响 造血细胞生长和发育。 最后，我们将定义 鸟苷酸缺失和补充对 转录控制基因PU.1和C/EBF与骨髓细胞相关 发育，以及骨髓特异性基因（例如，cd 11b和 乳铁蛋白）。 这些造血细胞调控的基础研究 发展可能会导致新的见解有关的体外繁殖 并对这些细胞进行基因操作以用于临床目的。

While much is now known about specific cytokines that promote the survival and growth of hematopoietic stem cells and progenitors, relatively little is known about regulatory mechanisms that control the loss of self-renewal capacity, differentiation, and terminal maturation of these cells during normal blood cell development. In lower eukaryotes, e.g. S. cerevisiae and Dictyostelium, developmental switches are triggered by metabolic changes caused by nutrient deprivation, and studies of these organisms have shown that depletion of guanine ribonucleotides (e.g. GTP) is a critical metabolic event int he onset of development. Parallel studies of both human leukemia cell lines and purified, normal CD34+ marrow cells have also established a link between guanine ribonucleotide metabolism and the control of hematopoietic cell development. To understand how the regulation of intracellular guanylate pools serve as a metabolic signal for the development of hematopoietic cells, we propose studies with two central objectives. Our first objective is to understand how IMP-dehydrogenase (IMPDH), a highly conserved enzyme of intermediary purine metabolism which controls de novo guanine ribonucleotide synthesis, is regulated during the growth and development of hematopoietic cells. Using both the Il-3 dependent,murine hematopoietic cell line. 32D(cl3) and immuno-affinity purified CD34+ human marrow cells, we will characterize GTP synthesis, guanylate pools, and levels of IMPDH gene expression, protein, and activity under conditions of Il-3 stimulated growth, G-CSF induced myeloid maturation, and apoptosis following growth factor withdrawal. We will also investigate cytokine dependent post-translational modifications of IMPDH isoforms, examine the effects of intracellular pH changes and altered Il-3 signalling on IMPDH expression and activity, and determine how IMPDH over- and under-expression induced by gene transfection affect cellular maturation. Our second objective is to define mechanisms by which the regulation of guanylate pools influences hematopoietic cell development. We will examine the effects of IMPDH inhibition on GTP-dependent, ras mediated signalling pathways by measuring GTP-bound ras and IL-3 stimulated phosphorylation of raf. Because protein synthesis is highly GTP-regulated, we will also determine whether guanylate depletion and repletion affect the turnover and supply of short-lived proto oncogene proteins (e.g., c-myb) that influence hematopoietic cell growth and development. Finally, we will define the effects of guanylate depletion and repletion on the expression of transcription control genes, PU.1 and C/EBF, implicated in myeloid cell development, as well as myeloid specific genes (e.g., CD11b and lactoferrin). These basic studies of the control of hematopoietic cell development may lead to new insights relevant to the in vitro propagation and genetic manipulation of these cells for clinical purposes.