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

细胞因子依赖性白血病细胞系分化诱导分析及髓系非转录因子鉴定

• 批准号: 07671192
• 负责人: MURATE Takashi
• 金额: $ 1.28万
• 依托单位: NAGOYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671192/
• 关键词: MB-02 CELLS; IL-3 RECEPTOR a TRANSFECTION; EOSINOPHIL; MAJOR BASIC PROTEIN; TRANSCRIPTION FACTOR; PROMOTER REGION; IL-3レセプターα遺伝子導入; IL-3; MB-02 cell; eosinophilic differentiation; transcription factor; サイトカイン依存性細胞株; 分化誘導; 顆粒球系分化; 特異的転写因子

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

使用人白血病细胞系 MB-02 分析了细胞因子对细胞存活、增殖和分化承诺的作用，该细胞的存活和增殖依赖于 GM-CSF。高浓度的 IL-3 不支持增殖，但诱导骨髓分化或凋亡。为了澄清这一点，用IL-3受体a的cDNA转染MB-02细胞，以避免用IL-3培养的MB-02细胞的细胞损失或凋亡。稳定的转染子 MB-02-3R 在 IL-3 的作用下增殖，并且对细胞凋亡具有抵抗力。在低浓度的IL-3下，转染细胞不能增殖但存活，细胞可以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和增殖之间的平衡。使用该分化诱导系统，进行凝胶迁移测定以检测嗜酸性粒细胞谱系特异性诱导核因子的存在。使用嗜酸性粒细胞主要碱性蛋白的 250 个碱基对的 5' 启动子区域，对用低浓度 Il-3 处理的 MB-02-3R 细胞的核蛋白进行分析，以确定是否存在独特的蛋白质，该蛋白质被认为与嗜酸性粒细胞分化有关。根据之前的报道，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（新蛋白质的出现）。然而，DNA 结合蛋白的数量变化仍然是嗜酸性粒细胞分化的可能原因。因此，我们对与MBP启动子区CAAT盒结合的C/EBP5蛋白的变化特别感兴趣。同时，以MBP基因启动子的较长5'端(1.5-2Kb)为探针，继续分析MBP基因表达启动子区域的研究。我们还没有得到结论性的结果来表明哪个部分对于 MBP 基因表达最重要。我们现在正在继续使用与荧光素酶基因连接的 MBP 5 个侧翼区域的缺失突变体进行瞬时转染实验。较少的