Zhuang Wen-Yue, Li Zheng-Yi, Zhao Yun, Cen Jian-Nong, Zhuang Wen-Zhuo, Chen Zi-Xing
Medical Ecsomatics College of Beihua University, Jilin 132013, Jilin Province, China.
Department of Laboratorial Examination, Jilin Medical College, Jilin 132013, Jilin Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1394-8. doi: 10.7534/j.issn.1009-2137.2013.06.006.
This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.
本研究旨在探讨AML1-ETO融合蛋白对白血病细胞中抗凋亡基因BCL-2的影响，并探讨其在白血病发生中的作用。通过流式细胞术检测U937-WT、U937-Mock和U937-A/E1-4细胞的凋亡水平。通过蛋白质免疫印迹法检测裂解的caspase-3蛋白表达。采用定量PCR评估AML1-ETO表达细胞或U937非表达细胞以及有或无t(8;21)的AML患者白血病细胞中BCL-2基因的表达。采用基于染色质免疫沉淀(ChIP)的PCR研究AML1-ETO阳性白血病细胞系中AML1-ETO与BCL-2启动子之间的直接相互作用。结果表明，在U937-A/E细胞中而非U937-WT或U937-Mock细胞中，凋亡细胞在统计学上显著增加，且AML1-ETO表达也显著增强了caspase-3的激活。与未转染的U937相比，表达AML1-ETO的细胞亚克隆显示出显著低水平的BCL-2 mRNA。在含有AML1-ETO的急性髓系白血病原代骨髓细胞中，与其他缺乏这种易位的急性髓系白血病相比，BCL-2 mRNA水平明显较低。转染细胞中的富集区域位于BCL-2启动子内。结论是BCL-2是AML1-ETO的直接靶基因。AML1-ETO可下调BCL-2的表达。
