MOLECULAR BASIS FOR CYTOKINE INDUCED MYELOID CELL DIFFERENTIATION

细胞因子诱导骨髓细胞分化的分子基础

• 批准号: 6336673
• 负责人: NANCY BERLINER
• 金额: $ 30.98万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336673
• 关键词: cell differentiation; cell line; cell proliferation; colony stimulating factor; genetic mapping; hematopoiesis; myeloid stem cell; regulatory gene; transfection

Hematopoietic cytokines play a key regulatory role in myeloid differentiation by stimulating the proliferation of certain precursors, inducing the differentiation of others, and activating mature cells. G-CSF and GM-CSF are the most important cytokines directing myeloid differentiation. Two key steps in the maturation of these cells are the acquisition of cell surface receptors and of the components for the downstream signaling pathways. Two myeloid cell lines can serve as models for these signaling events: 1) EML is a multipotent stem cell factor- dependent cell line immortalized with a dominant negative retinoic acid receptor alpha. Although they express the GM-CSF receptor, uninduced EML cells will not respond to GM-CSF. Induction with IL-3/ATRA induces the cells to differentiate and to become GM-CSF responsive. Over- expression of the oncogene EVI-1 in these cells blocks this acquisition of GM-CSF responsiveness. 2) 32 Dcl3 cells proliferate and differentiate in response to G-CSF. Transfection of a derivative line, 32D.C10 (which lacks G-CSFR), with a receptor carrying a Y764F mutation renders the cells unable to proliferate in response to G-CSF, although they will differentiate with this induction. We propose to use these cell lines as models for analyzing the molecular events required for early myeloid differentiation. To date, signaling pathways that mediate G- or GM-CSF-induced myeloid differentiation remain to be identified. Nonetheless, it is clear that differentiation-specific signaling results in the activation of key regulatory genes, such as C/ebpalpha. We hypothesize that C/ebpalpha is ne of a set of regulatory genes, many of which have yet to be identified, that represent nuclear targets for differentiation-inducing signals that initiate at the membrane. We hypothesize that these genes provide a molecular point of entry into understanding the process of myeloid differentiation. We propose: 1. To determine at which point in the induction of differentiation by EML the GM-CSF signaling pathway becomes primed, to investigate the mechanism by which the pathway becomes responsive to the proliferative signal transduced by GM-CSF, and to characterize the block in the pathway induced by EVI-1 over-expression. 2. To identify the set of genes that are up-regulated by the "differentiation arm" of the G-CSF signaling. 3. To establish the functional role of downstream genes identified in Aims 1 and 2 by over-expression in EML and 32D. The phenotype of transduced EML cells will be examined for the ability to bypass the need for early priming of cytokine response; transfected 32D cells will be assayed for separation of proliferative and differentiative responses to G-CSF.

造血细胞因子通过刺激某些前体细胞的增殖、诱导其他前体细胞的分化和激活成熟细胞，在髓系分化中发挥关键的调节作用。G-CSF和GM-CSF是指导髓系分化的最重要的细胞因子。这些细胞成熟的两个关键步骤是获得细胞表面受体和下游信号传导途径的组分。两种骨髓细胞系可用作这些信号传导事件的模型：1）EML是用显性负性视黄酸受体α永生化的多能干细胞因子依赖性细胞系。尽管它们表达GM-CSF受体，但未诱导的EML细胞将不对GM-CSF应答。用IL-3/ATRA诱导诱导细胞分化并变得对GM-CSF有反应。癌基因EVI-1在这些细胞中的过表达阻断了GM-CSF应答性的获得。2)32 Dcl 3细胞响应于G-CSF而增殖和分化。用携带Y 764 F突变的受体转染衍生细胞系32D.C10（其缺乏G-CSFR）使得细胞不能响应于G-CSF而增殖，尽管它们将在这种诱导下分化。我们建议使用这些细胞系作为模型，用于分析早期髓样分化所需的分子事件。迄今为止，介导G-或GM-CSF诱导的髓样分化的信号通路仍有待确定。尽管如此，很明显，分化特异性信号导致关键调控基因的激活，如C/ebpalpha。我们假设C/ebpalpha是一组调控基因之一，其中许多基因尚未被鉴定，这些基因代表了细胞膜上启动的分化诱导信号的核靶。我们假设这些基因提供了一个了解髓系分化过程的分子切入点。我们建议：1.为了确定在EML诱导分化的哪个点GM-CSF信号传导途径开始启动，研究该途径对GM-CSF转导的增殖信号产生应答的机制，并表征EVI-1过表达诱导的途径阻断。2.鉴定由G-CSF信号传导的“分化臂”上调的基因组。3.通过在EML和32 D中过表达，确定目标1和2中鉴定的下游基因的功能作用。将检查转导的EML细胞的表型绕过细胞因子应答的早期引发需要的能力;将测定转染的32 D细胞对G-CSF的增殖和分化应答的分离。