Tanaka K, Imoto I, Inoue J, Kozaki K, Tsuda H, Shimada Y, Aiko S, Yoshizumi Y, Iwai T, Kawano T, Inazawa J
Department of Molecular Cytogenetics, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan.
Oncogene. 2007 Sep 27;26(44):6456-68. doi: 10.1038/sj.onc.1210459. Epub 2007 Apr 16.
Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylation-dependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G(0)-G(1), whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumor-suppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.
表观遗传改变以及由此导致的肿瘤抑制基因失活常常促使各种癌症的发生。为了鉴定可能因食管鳞状细胞癌（ESCC）中异常甲基化而沉默的新候选基因，我们采用一种最近开发的名为基于细菌人工染色体阵列的甲基化CpG岛扩增（BAMCA）的方法分析了ESCC细胞系，并通过BAMCA辅助策略筛选候选基因。在这个项目过程中，我们在一组ESCC细胞系中发现了编码细胞视黄酸结合蛋白1（CRABP1）的基因频繁发生CpG甲基化依赖性沉默。用5-氮杂-2'-脱氧胞苷处理后，基因沉默的ESCC细胞中CRABP1 mRNA的表达得以恢复。具有明确启动子活性的CRABP1 CpG岛的DNA甲基化状态与该基因的表达呈负相关。在原发性ESCC组织中也经常观察到CRABP1的CpG甲基化。在缺乏该蛋白的ESCC细胞中恢复CRABP1表达可通过诱导细胞停滞在G(0)-G(1)期来降低细胞生长，而在表达CRABP1的细胞中敲低该基因则促进细胞生长。在113例原发性ESCC肿瘤中，免疫反应性CRABP1的缺失与癌细胞的去分化以及患者的远处淋巴结转移显著相关。这些结果表明，CRABP1在食管上皮中似乎具有肿瘤抑制功能，其表观遗传沉默可能在食管癌发生过程中起关键作用。其在活检或切除肿瘤中的表达状态可能作为一个指标，用于识别推荐联合治疗模式的ESCC患者。
