AML1-ETO融合蛋白对BCL-2基因表达的影响

本研究旨在观察AML1-ETO在白血病细胞中对于抗凋亡基因日乳-2表达的影响，探讨其在白血病发生中的作用。应用流式细胞术检测急性单核细胞白血病细胞株U937-WT、U937-Mock和经AML1-ETO基因转染的U937-A／E1-4的细胞凋亡率；使用免疫印迹法检测cleavedcaspase-3蛋白的表达；荧光实时定量PCR检测转染细胞和对照组细胞以及AMLM2患者白血病细胞BCL-2mRNA的表达；染色质免疫沉淀技术研究转染细胞中AML1-ETO与BCL-2基因启动子之间直接的相互作用情况。结果表明：AML1-ETO转染细胞的凋亡率明显增加，且检测到cleavedcaspase-3蛋白的表达；转染了AML1—ETO 的U937细胞系和具有AML1—ETO融合基因的AML-M2患者中，BCL-2的mRNA表达水平显著下调；转染细胞沉淀富集的AML1-ETO直接结合的DNA中含有BCL-2基因的启动子序列。结论：BCL-2是AML-ETO的直接靶基因，AML1-ETO能下调BCL-2的表达。

Effect of AMLI-ETO Fusion Protein on the Expression of BCL-2

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 West- ern 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 ceils, 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 en- riched 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.