Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, USA.
Am J Pathol. 2011 Feb;178(2):652-61. doi: 10.1016/j.ajpath.2010.10.023.
Carbamoyl phosphate synthetase 1 (CPS1) is a liver-specific, intramitochondrial, rate-limiting enzyme in the urea cycle. A previous study showed that CPS1 is the antigen for hepatocyte paraffin 1 antibody, a commonly used antibody in surgical pathology practice; and CPS1 expression appears to be down-regulated in liver cancer tissue and cell lines. The aim of this study is to understand how the CPS1 gene is regulated in liver carcinogenesis. In this report, we show that human hepatocellular carcinoma (HCC) cells do not express CPS1, whereas cultured human primary hepatocytes express abundant levels. In addition, CPS1 was silenced or down-regulated in liver tumor tissues compared with the matched noncancerous tissues. The expression of CPS1 in HCC cells was restored with a demethylation agent, 5-azacytidine. We show that two CpG dinucleotides, located near the transcription start site, and a CpG-rich region in the first intron were hypermethylated in HCC cells. The hypermethylation of the two CpG dinucleotides was also detected in HCC tumor tissues compared with noncancerous tissues. Further molecular analysis with mutagenesis indicated that the two CpG dinucleotides play a role in promoter activity of the CPS1 gene. In conclusion, our study demonstrates that DNA methylation is a key mechanism of silencing CPS1 expression in human HCC cells, and CPS1 gene hypermethylation of the two CpG dinucleotides is a potential biomarker for HCC.
氨甲酰磷酸合成酶 1（CPS1）是尿素循环中肝脏特异性的线粒体内部限速酶。先前的研究表明，CPS1 是肝石蜡 1 抗体的抗原，肝石蜡 1 抗体是外科病理学实践中常用的抗体；并且 CPS1 的表达似乎在肝癌组织和细胞系中下调。本研究旨在了解 CPS1 基因在肝癌发生中的调控机制。在本报告中，我们表明人肝癌（HCC）细胞不表达 CPS1，而培养的人原代肝细胞则表达丰富的 CPS1。此外，与匹配的非癌组织相比，CPS1 在肝肿瘤组织中被沉默或下调。用去甲基化剂 5-氮杂胞苷可恢复 HCC 细胞中的 CPS1 表达。我们表明，两个位于转录起始位点附近的 CpG 二核苷酸和第一个内含子中的 CpG 丰富区在 HCC 细胞中发生了甲基化。与非癌组织相比，HCC 肿瘤组织中也检测到两个 CpG 二核苷酸的高甲基化。进一步的突变分析表明，这两个 CpG 二核苷酸在 CPS1 基因启动子活性中起作用。总之，我们的研究表明，DNA 甲基化为人类 HCC 细胞中 CPS1 表达沉默的关键机制，并且 CPS1 基因的两个 CpG 二核苷酸的高甲基化是 HCC 的潜在生物标志物。
