Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Division of Urology, Buriram Hospital, Buriram Province 31000, Thailand.
Free Radic Biol Med. 2019 Apr;134:419-428. doi: 10.1016/j.freeradbiomed.2019.01.031. Epub 2019 Jan 29.
Oxidative stress and reactivation of long interspersed element-1 (LINE-1) are coincidently observed in bladder cancer (BlCa), but the mechanistic connection between these two oncogenic phenomena is unknown. Previously, we reported increases in oxidative stress and LINE-1 protein (ORF1p) expression in human BlCa tissues. In this study, we measured 5-methylcytosine (5mC), 8-hydroxydeoxyguanosine (8-OHdG), 8-oxoguanosine DNA glycosylase-1 (OGG1), H3K9me3 and HP1α in bladder tissues obtained from BlCa patients. Reactivation of LINE-1 by reactive oxygen species (ROS) through chromatin remodeling was investigated in seven BlCa cell lines. We found that 5mC was decreased, but 8-OHdG, H3K9me3 and HP1α levels were increased in BlCa tissues relative to the adjacent non-cancerous tissues. OGG1, H3K9me3 and HP1α expression in BlCa tissues were positively correlated with 8-OHdG levels. Following HO treatment, LINE-1 transcript expression was increased in VM-CUB-1 and TCCSUP, whereas AluYa5 and AluYb8 transcripts were increased in BFTC905 cells. Basal expression of LINE-1 ORF1p varied among BlCa cell lines from none to very high. HO treatment clearly increased expression of ORF1p in VM-CUB-1, TCCSUP and BFTC905. Chromatin immunoprecipitation experiments revealed that 5'-LINE-1 promoters became further enriched in H3K4me3 and H3K18ac in VM-CUB-1 and BFTC905 cells treated with HO. In contrast, 5'-LINE-1 promoters became more enriched in H3K9me3 and H3K27me3 in UM-UC-3 treated with HO. In summary, decreased 5mC, but increased 8-OHdG, H3K9me3 and HP1α expression were demonstrated in human BlCa tissues, indicating global DNA hypomethylation, increased oxidative stress and altered histone methylation in BlCa. Chromatin structures were profoundly changed in BlCa cells exposed to ROS, but expression of LINE-1 transcript and protein were at most modestly increased. ROS enhanced expression of full-length LINE-1 elements only in cell lines with pre-existing activation, which was paralleled by increased formation of active chromatin at LINE-1 promoter loci.
氧化应激和长散在元件-1（LINE-1）的重新激活在膀胱癌（BlCa）中同时观察到，但这两种致癌现象之间的机制联系尚不清楚。此前，我们报道了人类膀胱癌组织中氧化应激和 LINE-1 蛋白（ORF1p）表达的增加。在这项研究中，我们测量了从膀胱癌患者获得的膀胱组织中的 5-甲基胞嘧啶（5mC）、8-羟基脱氧鸟苷（8-OHdG）、8-氧鸟嘌呤 DNA 糖基化酶-1（OGG1）、H3K9me3 和 HP1α。通过染色质重塑，研究了活性氧（ROS）对 LINE-1 的重新激活。我们发现，与相邻非癌组织相比，BlCa 组织中的 5mC 减少，但 8-OHdG、H3K9me3 和 HP1α 水平升高。BlCa 组织中 OGG1、H3K9me3 和 HP1α 的表达与 8-OHdG 水平呈正相关。HO 处理后，VM-CUB-1 和 TCCSUP 中的 LINE-1 转录物表达增加，而 BFTC905 细胞中的 AluYa5 和 AluYb8 转录物增加。不同膀胱癌细胞系中 LINE-1 ORF1p 的基础表达从无到高不等。HO 处理明显增加了 VM-CUB-1、TCCSUP 和 BFTC905 中 ORF1p 的表达。染色质免疫沉淀实验显示，在 VM-CUB-1 和 BFTC905 细胞中，HO 处理后 5'-LINE-1 启动子进一步富含 H3K4me3 和 H3K18ac。相比之下，在 HO 处理的 UM-UC-3 中，5'-LINE-1 启动子富含 H3K9me3 和 H3K27me3。总之，在人类膀胱癌组织中，5mC 减少，8-OHdG、H3K9me3 和 HP1α 表达增加，表明膀胱癌中存在全基因组 DNA 低甲基化、氧化应激增加和组蛋白甲基化改变。ROS 暴露的膀胱癌细胞中的染色质结构发生了深刻变化，但 LINE-1 转录物和蛋白质的表达最多只是适度增加。ROS 仅在具有预先存在激活的细胞系中增强全长 LINE-1 元件的表达，这与 LINE-1 启动子位点活性染色质的形成增加相平行。
