Tada Yasuhiro, Brena Romulo Martin, Hackanson Björn, Morrison Carl, Otterson Gregory A, Plass Christoph
Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics, The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
J Natl Cancer Inst. 2006 Mar 15;98(6):396-406. doi: 10.1093/jnci/djj093.
Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer.
We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBPalpha promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBPalpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBPalpha promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays.
DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPalpha promoter were associated with low or absent C/EBPalpha expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBPalpha promoter was detected in C/EBPalpha-nonexpressing cell lines; USF binding was detected in C/EBPalpha-expressing cell lines; however, in C/EBPalpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment.
DNA hypermethylation of the upstream C/EBPalpha promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBPalpha expression in human lung cancer.
在包括急性髓性白血病和肺癌在内的多种人类恶性肿瘤中，均可见肿瘤抑制因子CCAAT/增强子结合蛋白α（C/EBPα）表达缺失。我们推测C/EBPα启动子的DNA甲基化和组蛋白乙酰化可能会调节肺癌中C/EBPα的表达。
我们通过Northern印迹、免疫印迹和免疫组织化学分析了15种人肺癌细胞系和122例人肺原发性肿瘤中C/EBPα的表达。使用亚硫酸氢盐测序、联合亚硫酸氢盐限制性分析、甲基化特异性聚合酶链反应和Southern印迹评估C/EBPα启动子甲基化。我们通过染色质免疫沉淀检查了C/EBPα启动子处组蛋白H3和H4的乙酰化状态。通过染色质免疫沉淀和电泳迁移率变动分析，在未处理或用组蛋白脱乙酰酶抑制剂曲古抑菌素A和去甲基化剂5-氮杂-2'-脱氧胞苷（5-aza-dC）处理的细胞系中，检测甲基-CpG结合蛋白MeCP2和MBD2以及上游刺激因子（USF）与C/EBPα启动子的结合。
在15种肺癌细胞系中的12种以及120例原发性肺肿瘤中的81例中，C/EBPα启动子上游区域（-1422至-896）的DNA甲基化和组蛋白乙酰化与C/EBPα低表达或无表达相关。在不表达C/EBPα的细胞系中检测到MeCP2和MBD与C/EBPα启动子上游结合；在表达C/EBPα的细胞系中检测到USF结合；然而，在不表达C/EBPα的细胞系中，仅在曲古抑菌素A和5-aza-dC处理后才检测到USF结合。
如先前报道，并非核心启动子区域，而是C/EBPα启动子上游区域的DNA高甲基化在人类肺癌中C/EBPα表达的调节中起关键作用。
