METABOLIC DETERMINANTS OF HEMATOPOIETIC CELL DEVELOPMENT

虽然现在对促进存活的特定细胞因子知道得很多 以及造血干细胞和祖细胞的生长，相对较少 知道控制自我更新损失的监管机制 这些细胞的能力、分化和终末成熟 血细胞发育正常。在低等真核生物中，例如酿酒酵母和 发育开关是由代谢变化触发的 是由营养缺乏引起的，对这些生物的研究表明 鸟嘌呤核苷酸(如GTP)的枯竭是一个关键 发育开始时的代谢事件。对两者的平行研究 人白血病细胞株和纯化的、正常的CD34+骨髓细胞也 在鸟嘌呤核糖核酸代谢和 控制造血细胞发育。为了理解如何 细胞内鸟苷酸池的调节是一种代谢信号 造血细胞的发展，我们建议在两个中心进行研究 目标。 我们的第一个目标是了解IMP-脱氢酶(IMPDH)是如何 控制De的中间嘌呤代谢高度保守的酶 新鸟嘌呤核苷酸合成，在生长和生长过程中受到调节 造血细胞的发育。使用依赖IL-3的小鼠 造血细胞系。32D(Cl3)和免疫亲和纯化的CD34+人 骨髓细胞，我们将表征GTP合成、鸟苷酸池和 低氧条件下IMPDH基因表达、蛋白和活性的水平 IL-3刺激生长，G-CSF诱导髓系成熟和细胞凋亡 在停止使用生长因子之后。我们还将研究细胞因子 IMPDH亚型的依赖翻译后修饰，检查 细胞内pH变化和IL-3信号变化对IMPDH的影响 表达和活性，并确定IMPDH如何过度表达和低于表达 基因导入会影响细胞的成熟。我们的第二个 目的是确定鸟苷的调节机制。 血池会影响造血细胞的发育。我们将研究 抑制IMPDH对GTP依赖、ras介导的信号转导的影响 通过检测GTP结合的ras和IL-3刺激的信号转导通路 英国皇家空军。由于蛋白质合成受到GTP的高度调控，我们还将 确定鸟粪的耗尽和再利用是否影响周转和 短命原癌基因蛋白(例如，c-myb)的供应 造血细胞的生长发育。最后，我们将定义 鸟苷耗竭和再耗竭对血管内皮细胞表达的影响 转录控制基因PU.1和C/EBF与髓系细胞有关 发育，以及髓系特异性基因(例如，CD11b和 乳铁蛋白)。这些关于造血细胞调控的基础研究 发展可能导致与体外繁殖相关的新见解 以及用于临床目的的这些细胞的基因操作。

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.