ROS mediates EBV reactivation by MNNG.

<p>(A) MNNG increased the intracellular ROS levels in TW01 or NA cells and the ROS scavengers reduced MNNG-induced ROS production. Cells were pretreated with ROS scavengers including NAC (0.5 or 1mM), catalase (500 or 1000 unit/ml) and reduced glutathione (0.5 or 1mM) for 1 h and then mock treated or treated with MNNG (1μg/ml) for 24 h. Cells were stained with the ROS-sensitive probe dihydroethidium (DHE) for 1 h and the fluorescence levels were measured by flow cytometry. (B) ROS scavengers reduced the expression of Rta, Zta and EA-D proteins induced by MNNG in NA, (C) HA and (D) C666-1 cells. After pretreatment with ROS scavengers for 1 h and MNNG (1μg/ml) treatment for another 72 h, cells were harvested and subjected to immunoblotting using antibodies against the lytic EBV proteins, Rta, Zta, EA-D and cellular β-actin as an internal control. (E) The activity of Rta promoter was induced by MNNG and the effect was inhibited by ROS scavengers. Luciferase activities were measured in TW01 cells transfected with the reporter plasmids of Rp or Zp for 24 h. The cells were pretreatment with ROS scavengers for 1 h, following by treating with MNNG (1μg/ml) for additional 24 h and harvested for luciferase activity assay. (F) Repeated treatment with a low dose concentration of MNNG increased the ROS level. TW01 and NA cells were repeatly treated with MNNG (0.1μg/ml) once a day for 5 days and pretreated with NAC (1mM), catalase (1000 unit/ml) or reduced glutathione (1mM) for 1 h on each occasion. Cells were then stained with the ROS-sensitive probe dihydroethidium (DHE) for 1 h and their fluorescence levels were measured by flow cytometry. (G) ROS scavengers inhibited EBV reactivation by repeated treatment with a low dose MNNG (0.1μg/ml). The treatment procedure was the same as described in (F). Cells were harvested and subjected to immunoblotting. *: <i>p</i><0.05, **: <i>p</i><0.01, compared to mock treatment of the same cell line or the same promoter; +: <i>p</i><0.05, ++: <i>p</i><0.01, compared to MNNG treatment or MNNG treatment 5 times of the same cell line or the same promoter.