Oxidative stress and LINE-1 reactivation in bladder cancer are epigenetically linked through active chromatin formation.

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.