Figure 8. Mild oxidative stress reduces transcription from the EBV BamHI-Cp in a lymphoblastoid cell-line.

(A) Paraquat treatment reduces the level of BamHI-Cp transcripts in treated NOLA-1 cells. Total RNA was extracted from control or cells exposed to 150 µM of paraquat for 48 or 72, reverse-transcribed with random hexamers. cDNA was used for real time PCR performed as described in the Methods section to evaluate changes in Cp transcripts relative to the endogenous control GAPDH transcripts. The results are presented relative to the BamHI-Cp transcript level observed in control cells, which was assigned a value of 1. Significant decreases in the level of BamHI-Cp transcripts were observed in cells with paraquat for 48 and 72 hours, such that transcript level was reduced to approximately 60% after 48 hours of treatment, and approximately 40% after 72 hours of treatment. Cell-cycle analyses conducted with several treated samples indicates that paraquat treatment does not increase the sub-G1 population. However, treated cells were consistently observed to accumulate in G0/G1. Treatment caused a small increase in the number of necrotic cells doubly-positive for annexin V and PI staining. Small increases were also seen for cells positive for only annexin V or only PI. (B) Paraquat treatment decreases expression of EBNA2 and LMP1, as evaluated by immunoblot. 2×10⁵ control or cells treated with 150 µM paraquat (P150) for 72 hours were examined as described in Methods for expression of EBNA1, EBNA2 and LMP1. Band intensity was compared to that observed in control cells, and is expressed as percent of control along with standard deviation from two experiments. Expression was normalized to the level of β-actin protein detected in each sample. (C) Paraquat treatment does not alter the expression or localization of EBNA1. Control or treated cells were transferred to slides by cytospin, prior to fixation and permeabilization. EBNA1 expression was evaluated using rabbit polyclonal Ab 2638 as described in the methods, and detected using a TexasRed conjugated secondary anti-rabbit Ab. Cells were counterstained with Hoechst 33342. The absence of paraquat exposure, or length of treatment with 150 µm paraquat is indicated above each panel. Detection of EBNA1-specific signal, Hoechst signal and the merge is also indicated. The scale bar represents a distance of 10 µM.