THE ANALYSIS OF DIFFERENTIATION INDUCTION OF CYTOKINE DEPENDENT LEUKEMIA CELLL LINE AND THE IDENTIFICATION OF MYELOID LINEAGE APESIFIC TRANSCRIPTION FACTORS

以GM-CSF依赖的人白血病细胞株MB-02为研究对象，分析了细胞因子对细胞存活、增殖和分化的影响。高浓度的IL-3不支持细胞增殖，但可诱导髓系分化或凋亡。为了阐明这一点，将IL-3受体α基因导入MB-02细胞，观察与IL-3共同培养的MB-02细胞的细胞丢失或凋亡。稳定的转染体MB-02-3R可在IL-3刺激下增殖，并能抵抗细胞凋亡。在低浓度的IL-3作用下，转基因细胞不能增殖而存活，可分化为嗜酸性粒细胞。这些结果表明，IL-2介导的信号系统决定了分化与增殖之间的平衡。利用该分化诱导体系，用凝胶移位实验检测嗜酸性粒细胞系特异性诱导核因子的存在。使用250base…对低浓度IL-3处理的MB-02-3R细胞的嗜酸性粒细胞主要碱性蛋白、核蛋白的多对5‘端启动子区进行分析，以寻找与嗜酸性粒细胞分化相关的独特蛋白。根据以往的报道，GATA1、GATA2、GATA3、NF-E2和PU-1被认为是嗜酸性粒细胞承诺因子的候选因子。然而，我们的实验表明，在我们的MB-02-3R细胞分化系统中，低剂量IL-3处理没有观察到新的移位条带(新蛋白的出现)。然而，DNA结合蛋白的数量变化仍然是导致嗜酸性粒细胞分化的可能原因。因此，我们特别关注与MBP启动子区域CAAT盒结合的C/EBP5蛋白的变化。同时，以MBP基因启动子较长的5‘端(1.5-2kb)为探针，继续对MBP基因表达的启动子区域进行分析。我们还没有得到对MBP基因表达最重要的部分的确凿结果。我们现在继续利用与荧光素酶基因相连的5个MBP侧翼区的缺失突变体进行瞬时转染实验。较少

The role of cytokine on cell survival, proliferation and differentiation commitment was analyzed with a human leukemia cell line, MB-02 whose survival and proliferation was GM-CSF dependent. High concentration of IL-3 did not support proliferation but induced either myeloid differentiation or apoptosis. To clarify this point, MB-02 cells were transfected with cDNA for IL-3 receptor a to circum vent cell loss or apoptosis of MB-02 cells cultured with IL-3. A stable transfectant, MB-02-3R,proliferated in response to IL-3 and was resistant to apoptosis. In low concentration of IL-3, in which the transfectant cells could not proliferate but survived, the cells could differentiate into eosinophils. These results suggest that signaling system mediated by IL-2 determines the balance between differentiation and proliferation. Using this differentiation induction system, gel-shift assay was performed to detect the presence of eosinophil lineage specific induction nuclear factors. Using 250 base … More pair of 5' promoter area of eosinophil major basic protein, nuclear protein of MB-02-3R cells treated with low concentration of Il-3 was analyzed for the presence of unique protein which was thought to be involved with eosinophilic differentiation. According to previous reports, GATA1, GATA2, GATA3, NF-E2 and PU-1 were supposed to be the candidate of eosinophil commitment factor of factors. However, our experiments showed that new shifted bands (appearance of new proteins) were not observed with low dose IL-3 treatment in our MB-02-3R cell differentiation system. However, quantitative changes of DNA binding proteins remain as the possible cause eosinophilic differentiation. So, we are especially interested with the change of C/EBP5 protein which binds to CAAT box of MBP promoter region. Simultaneously, the research to analyze the promoter region of MBP gene expression are continued using longer 5' side of MBP gene promoter (1.5-2Kb) as the probes. We did not get yet the conclusive results showing what part is the most important for MBP gene expression. We are now continuing the transient transfection experiments using the deletion mutants of 5, flanking region of MBP connected with luciferase genes. Less