Department of Biological & Biomedical Sciences, Glasgow Caledonian University, City Campus, Glasgow, UK.
FEBS J. 2010 Jan;277(2):441-52. doi: 10.1111/j.1742-4658.2009.07496.x. Epub 2009 Dec 15.
EVI1 is a nuclear zinc finger protein essential to normal development, which participates in acute myeloid leukaemia progression and transforms Rat1 fibroblasts. In this study we show that enforced expression of Evi1 in Rat1 fibroblasts protects from paclitaxel-induced apoptosis, consistent with previously published studies. Surprisingly, however, these cells show increased sensitivity to hydrogen peroxide (H(2)O(2))-induced apoptosis, demonstrated by elevated caspase 3 catalytic activity. This effect is caused by a reduction in carbonic anhydrase III (caIII) production. caIII transcripts are repressed by 92-97% by Evi1 expression, accompanied by a similar reduction in caIII protein. Reporter assays with the rat caIII gene promoter show repressed activity, demonstrating that Evi1 either directly or indirectly modulates transcription of this gene in Rat1 cells. Targeted knockdown of caIII alone, with Dicer-substrate short inhibitory RNAs, also increases the sensitivity of Rat1 fibroblasts to H(2)O(2), which occurs in the absence of any other changes mediated by Evi1 expression. Enforced expression of caIII in Evi1-expressing Rat1 cells reverts the phenotype, restoring H(2)O(2) resistance. Together these data show that Evi1 represses transcription of caIII gene expression, leading to increased sensitivity to H(2)O(2)-induced apoptosis in Rat1 cells and might suggest the basis for the development of a novel therapeutic strategy for the treatment of leukaemias and solid tumours where EVI1 is overexpressed.
EVI1 是一种核锌指蛋白，对正常发育至关重要，它参与急性髓性白血病的进展并转化 Rat1 成纤维细胞。在这项研究中，我们表明在 Rat1 成纤维细胞中强制表达 Evi1 可防止紫杉醇诱导的细胞凋亡，这与先前发表的研究一致。然而，令人惊讶的是，这些细胞对过氧化氢（H₂O₂）诱导的细胞凋亡表现出更高的敏感性，这表现为 caspase 3 催化活性的升高。这种效应是由碳酸酐酶 III（caIII）产生减少引起的。Evi1 表达使 caIII 转录物减少 92-97%，同时 caIII 蛋白也减少。用大鼠 caIII 基因启动子进行的报告基因实验显示活性受到抑制，表明 Evi1 直接或间接调节 Rat1 细胞中该基因的转录。使用 Dicer 底物短发夹 RNA 靶向敲低 caIII 也会增加 Rat1 成纤维细胞对 H₂O₂的敏感性，而这种敏感性在没有 Evi1 表达介导的任何其他变化的情况下发生。在表达 Evi1 的 Rat1 细胞中强制表达 caIII 可恢复表型，恢复对 H₂O₂的抗性。这些数据表明，Evi1 抑制 caIII 基因表达的转录，导致 Rat1 细胞对 H₂O₂诱导的细胞凋亡的敏感性增加，并且可能为治疗 EVI1 过表达的白血病和实体瘤提供一种新的治疗策略提供了基础。
