MOLECULAR BASIS FOR CYTOKINE INDUCED MYELOID CELL DIFFERENTIATION

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，以及激活成熟细胞，在髓系分化过程中发挥关键的调节作用。粒细胞集落刺激因子(G-CSF)和粒细胞集落刺激因子(GM-CSF)是指导髓系分化的重要细胞因子。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通路的组件。两个髓系细胞系可以作为这些信号事件的模型：1)EML是一种多能干细胞因子依赖细胞系，具有显性的阴性维甲酸受体α。尽管EML细胞表达GM-CSF受体，但未诱导的EML细胞不会对GM-CSF产生反应。用IL-3/ATRA诱导细胞分化，并对GM-CSF有反应。癌基因EVI-1在这些细胞中的过度表达阻止了GM-CSF反应性的获得。2)32个DCl3细胞在G-CSF刺激下增殖分化。将衍生品系32D.C10(缺乏G-CSFR)与携带Y764F突变的受体一起转染后，细胞无法对G-CSF做出反应而增殖，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来分析早期髓系分化所需的分子事件。到目前为止，介导G-或GM-CSF诱导的髓系分化的信号通路仍未确定。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是一组调控基因中的一个，其中许多基因尚未被识别，这些基因代表了起始于细胞膜的分化诱导信号的核靶标。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.确定在EML诱导分化的哪个时间点，GM-CSF信号通路被启动，探讨该通路对GM-CSF传导的增殖信号的反应机制，并表征EVI-1过表达诱导的通路的阻断。2.确定G-CSF信号的“分化臂”上调的一组基因。3.通过在EML和32D中过表达，确定AIMS 1和AIMS 2中发现的下游基因的功能作用。转导的EML细胞的表型将被检测是否有能力绕过早期启动细胞因子反应的需要；转导的32D细胞将被检测以分离对G-CSF的增殖和分化反应。

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.