San José-Eneriz Edurne, Agirre Xabier, Jiménez-Velasco Antonio, Cordeu Lucia, Martín Vanesa, Arqueros Victor, Gárate Leire, Fresquet Vicente, Cervantes Francisco, Martínez-Climent José A, Heiniger Anabel, Torres Antonio, Prósper Felipe, Roman-Gomez Jose
Hematology Department and Area of Cell Therapy, Clinica Universitaria, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.
Eur J Cancer. 2009 Jul;45(10):1877-89. doi: 10.1016/j.ejca.2009.04.005. Epub 2009 May 4.
Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1(+) cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established.
We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2'-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed.
We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression (p<0.05). Expression of BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2'-deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis.
Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM.
促凋亡的 BCL-2 相互作用介质（BIM）的表达最近被认为与伊马替尼诱导的 BCR-ABL1(+) 细胞凋亡有关。然而，慢性粒细胞白血病（CML）中 BIM 调控的机制及其在临床环境中的作用尚未明确。
我们通过 Q-RT-PCR 分析了 100 例新诊断的慢性期 CML 患者中 BIM 的 mRNA 表达，并通过蛋白质印迹分析检测了蛋白质水平。通过亚硫酸氢盐基因组测序和甲基化特异性 PCR（MSP）分析甲基化状态。用伊马替尼和 5-氮杂-2'-脱氧胞苷处理 CML 细胞系，并用两种不同的针对 BIM 的小干扰 RNA（siRNA）转染细胞，然后分析细胞增殖和凋亡情况。
我们发现 36%的患者存在 BIM 表达下调，与 BIM 表达正常的患者相比，这与对伊马替尼缺乏最佳反应显著相关，表现为在 6、12 和 18 个月时细胞遗传学和分子反应降低（p<0.05）。如用 5-氮杂-2'-脱氧胞苷处理 CML 细胞后 BIM 表达恢复所示，BIM 的表达由启动子高甲基化介导。使用 BIM 低表达和正常表达的 CML 细胞系，我们进一步证明伊马替尼诱导的 CML 细胞凋亡需要 BIM 的表达。
我们的数据表明，在一部分 CML 患者中，BIM 的下调由甲基化表观遗传控制且具有不良预后影响，并且伊马替尼与去甲基化剂联合使用可能会改善 BIM 表达降低患者的反应。
