Department of Medicine, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island;
Am J Physiol Cell Physiol. 2013 Nov 15;305(10):C1069-79. doi: 10.1152/ajpcell.00080.2013. Epub 2013 Sep 11.
Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.
肿瘤抑制基因的失活可能通过启动子超甲基化在巴雷特食管（BE）向食管腺癌（EA）的进展中发挥重要作用。我们之前已经表明，酸诱导的 p16 基因启动子超甲基化可能依赖于 BAR-T 细胞和 OE33 EA 细胞中 NADPH 氧化酶 NOX5-S 的激活。已知 DNA 甲基转移酶 1（DNMT1）参与在有丝分裂细胞中维持已建立的 DNA 甲基化模式，并可能在癌症的发展中发挥重要作用。因此，我们研究了 DNMT1 是否参与 BAR-T 细胞中酸诱导的 p16 基因启动子超甲基化。我们发现酸显著增加了 p16 基因启动子的甲基化，降低了 p16 mRNA，并增加了细胞增殖，这些效应可能依赖于 BAR-T 细胞中 DNMT1 的激活。DNMT1 在 EA 细胞 FLO 和 OE33 以及 EA 组织中过度表达。酸处理上调了 DNMT1 mRNA 表达并增加了 DNMT1 启动子活性。酸诱导的 DNMT1 mRNA 表达和启动子活性增加显著降低了 NOX5-S 和 NF-κB1 p50 的敲低。相反，NOX5-S、p50 或 p65 的过表达显著增加了 DNMT1 启动子活性。NOX5-S 的敲低显著降低了转染 pNFκB-Luc 的细胞中酸诱导的荧光素酶活性的增加。在 DNMT1 基因启动子中鉴定到 NF-κB 结合元件 GGGGTATCCC。我们得出结论，酸诱导的 p16 基因启动子甲基化增加、p16 mRNA 下调和细胞增殖增加可能依赖于 BAR-T 细胞中 DNMT1 的激活。酸诱导的 DNMT1 表达可能依赖于 NOX5-S 和 NF-κB1 p50 的顺序激活。
