Kumagai Takashi, Akagi Tadayuki, Desmond Julian C, Kawamata Norihiko, Gery Sigal, Imai Yasufumi, Song Jee Hoon, Gui Dorina, Said Jonathan, Koeffler H Phillip
Division of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA.
Int J Cancer. 2009 Feb 15;124(4):827-33. doi: 10.1002/ijc.23994.
Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.
分子靶向治疗是一种治疗胰腺癌的有前景的方法。肿瘤抑制基因的沉默可通过启动子区域的组蛋白去乙酰化和/或DNA甲基化发生。在此，我们鉴定了胰腺癌细胞中表观遗传沉默的基因。胰腺癌细胞系PANC-1细胞分别用或不用5-氮杂-2'-脱氧胞苷（一种DNA甲基转移酶抑制剂）和辛二酰苯胺异羟肟酸（SAHA，一种组蛋白去乙酰化酶抑制剂）处理，然后从这些细胞中分离mRNA。寡核苷酸微阵列分析显示，在PANC-1细胞中用5-氮杂-2'-脱氧胞苷和SAHA处理后，包括泛素羧基末端水解酶L1（UCHL1）、CCAAT增强子结合蛋白α（C/EBPα）、金属蛋白酶组织抑制因子2（TIMP2）和干扰素调节因子7（IRF7）在内的30个基因上调。这4个基因的诱导在几种胰腺癌细胞系中通过实时PCR得到验证。有趣的是，在6个胰腺癌细胞系中的6个中，C/EBPα的表达显著恢复。染色质免疫沉淀分析显示，在用SAHA处理的PANC-1细胞中，C/EBPα启动子区域的组蛋白H3被乙酰化；亚硫酸氢盐测序显示其启动子区域在几种胰腺癌细胞系中存在甲基化。强制表达C/EBPα显著抑制PANC-1细胞的克隆增殖。免疫共沉淀分析显示C/EBPα与E2F1相互作用；并且这种相互作用导致E2F1转录活性受到抑制。免疫组织化学分析显示，C/EBPα在胰腺腺癌细胞中定位于细胞质，而在正常胰腺细胞中主要定位于细胞核。我们的数据表明，C/EBPα的异常沉默以及不适当的细胞质定位导致其功能失调，提示C/EBPα是胰腺癌细胞中一个新的候选肿瘤抑制基因。
