The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid [HL60]. Homo sapiens

The product of the ecotropic virus integration site 1 (EVI1) gene, whose overexpression is associated with a poor prognosis in myeloid leukemias and some epithelial tumors, regulates gene transcription both through direct DNA binding and through modulation of the activity of other sequence specific transcription factors. Previous results from our laboratory have shown that EVI1 influenced transcription regulation in response to the myeloid differentiation inducing agent, all-trans retinoic acid (ATRA), in a dual manner: it enhanced ATRA induced transcription of the RARb gene, but repressed the ATRA induction of the EVI1 gene itself. In the present study, we asked whether EVI1 would modulate the ATRA regulation of a larger number of genes, as well as biological responses to this agent, in human myeloid cells. U937 and HL-60 cells ectopically expressing EVI1 through retroviral transduction were subjected to microarray based gene expression analysis, and to assays measuring cellular proliferation, differentiation, and apoptosis. These experiments showed that EVI1 modulated the ATRA response of several dozens of genes, and in fact reinforced it in the vast majority of cases. A particularly strong synergy between EVI1 and ATRA was observed for GDF15, which codes for a member of the TGF-b superfamily of cytokines. In line with the gene expression results, EVI1 enhanced cell cycle arrest, differentiation, and apoptosis in response to ATRA, and knockdown of GDF15 counteracted some of these effects. Overall design: HL60 cells transduced with an EVI1 expression vector or empty vector as a control were treated with all-trans retinoic acid (ATRA) or an equivalent amount of DMSO (solvent) for 24 h prior to gene expression microarray analysis. 3 biological replicates were performed.

嗜性病毒整合位点1（ecotropic virus integration site 1, EVI1）基因的编码产物，其过表达与髓系白血病及部分上皮肿瘤的不良预后密切相关，可通过直接结合DNA以及调控其他序列特异性转录因子的活性两种方式调控基因转录。本实验室既往研究表明，在髓系分化诱导剂全反式维甲酸（all-trans retinoic acid, ATRA）的刺激下，EVI1以双重方式参与转录调控：一方面可增强ATRA诱导的视黄酸受体β（RARβ）基因转录，另一方面却抑制ATRA对EVI1基因自身的诱导表达。
本研究旨在探究EVI1是否会在人类髓系细胞中调控更多基因对ATRA的应答，以及该基因对ATRA相关生物学过程的影响。本研究通过逆转录病毒转导技术在U937与HL-60细胞中外源过表达EVI1，随后对两组细胞开展基于基因芯片的基因表达分析，并检测细胞增殖、分化与凋亡水平。
实验结果显示，EVI1可调控数十个基因对ATRA的应答反应，且在绝大多数情况下会增强该应答。其中，EVI1与ATRA之间的协同效应在生长分化因子15（GDF15）的表达调控中尤为显著——GDF15编码转化生长因子β（TGF-β）细胞因子超家族的一员。与基因表达分析结果一致，EVI1可增强ATRA诱导的细胞周期阻滞、分化与凋亡；而敲低GDF15则会抵消部分上述效应。
总体实验设计：将携带EVI1表达载体的HL60细胞及空载载体对照细胞，分别用全反式维甲酸（ATRA）或等体积溶剂二甲基亚砜（DMSO）处理24小时，随后进行基因表达芯片分析。本实验共设置3次生物学重复。