Analysis of mechanism of transcriptional regulation of retinoic acid-responsive genes during the differentiation of human leukemia cells.

我们先前报道了全反式维甲酸(ATRA)和粒细胞巨噬细胞集落刺激因子(GM-CSF)协同诱导人髓系白血病ML-1细胞向粒细胞分化。为了探讨维甲酸反应基因在分化过程中的转录调控机制，我们用Northern或Western印迹方法检测了维甲酸受体(RARs)、维甲酸X受体(RXRs)及相关辅助因子在经ATRA和/或GM-CSF处理的ML-1细胞中的表达。我们首次发现全反式维甲酸和GM-ε协同诱导C/EBP-CSFmRNA的表达。我们进一步检测了GM-CSF上调RARαmRNA和蛋白的表达，以及两种试剂协同增加类固醇受体辅活化子3(SRC3/AIB 1)的表达，SRC3/AIB 1是包括RARs、mRNA和蛋白质在内的核受体的激活剂。这些变化在其他髓系白血病细胞系(THP-1和KG-1)中也被检测到，它们在ATRA和GM-CSF的反应中表现出与ML-1细胞相似的协同效应。此外，我们发现调控组蛋白精氨酸甲基化的基因(PRMTs和PAD4)的表达发生了变化。为了研究这些变化是否与分化有关，我们检测了SRC3 siRNA对ATRA和GM-CSF处理的ML-1细胞分化的影响。SRC3 siRNA对诱导的SRC3/AIB1基因表达的抑制抑制了两种试剂诱导的NBT还原活性。这些结果提示，ATRA和GM-CSF协同诱导分化与上调SRC3/AIB1基因表达，增加细胞对维甲酸的敏感性有关。

We reported previously that treatment with all-trans retinoic acid (ATRA) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces synergistically differentiation of human myeloblastic leukemia ML-1 cells to granulocytes. To investigate the mechanism of transcriptional regulation of retinoic acid-responsive genes during the differentiation, we examined expression of the responsive genes, retinoic acid receptors (RARs), retinoid X receptors (RXRs) and the cofactors in ML-1 cells treated with ATRA and/or GM-CSF using Northern or Western blot analysis. We first showed that expression of C/EBP ε mRNA was induced synergistically by treatment with ATRA and GM-CSF. We next detected up-regulation of expression of RAR a mRNA and protein by GM-CSF and synergistic increase of expression of steroid receptor coactivator-3 (SRC3/AIB 1), which is known as activator of nuclear receptors including RARs, mRNA and protein by both reagents. These changes were also detected in other myeloid leukemia cell lines (THP-1 and KG-1) that showed a synergistic effect similar to that seen in ML-1 cells in response to ATRA and GM-CSF. Furthermore, we revealed alteration of expression of genes regulating histone arginine methylation (PRMTs and PAD4). To investigate whether these alterations are associated with the differentiation, we examined effect of SRC3 siRNA on the differentiation in ML-1 cells treated with ATRA and GM-CSF. The inhibition of induced SRC3/AIB1 gene expression by SRC3 siRNA suppressed NBT reducing activity induced by treatment with both reagents. These results suggest that the synergistic induction of differentiation by ATRA and GM-CSF is associated with the increase of sensitiveness of cells to retinoic acid via up-regulation of expression of SRC3/AIB1 gene.