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

点击翻译按钮获取中文摘要

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