Background: We have previously shown that oxidative stress induced by H2O2 or cyclosporin A (CsA) can promote Epstein-Barr virus (EBV) transformation of human B cells as analyzed by colony formation, cell number, and by 3H-thymidine incorporation. In this report, we used EBV oncogene LMP1 as a marker to analyze H2O2 or CsA promotion of EBV transformation of human B cells and to test whether antioxidant vitamin E could inhibit H2O2 or CsA promoted LMP1 expression in the EBV-infected cells.
Materials and methods: Human splenocytes were prepared by centrifugation and plating technique to provide a greater than 80% pure preparation of B cells and were used for EBV infection. The EBV infected cells were treated with H2O2 (0.1 mM, 10 min), or with CsA (500 ng/ml) with or with out vitamin E (40 microM). The cells were cultured for up to 4 weeks. Samples were taken every week and were stained with phycoerythrin-conjugated mouse anti-LMP1 monoclonal antibody to assay LMP1 positive population by flow cytometry.
Results: In EBV-infected cells, the LMP1-positive cell population reached 14% after 4 weeks of culture. CsA or H2O2 treatment promoted LMP1 positive population to 43% and 41% after 4 weeks of culture. Vitamin E (40 microM) completely inhibited LMP1 expression in EBV-infected cells and in CsA- or H2O2-treated cells.
Conclusion: In agreement with our previous observation, CsA or H2O2 can promote EBV transformation of human B cells. This oxidative stress induced promotion of EBV transformation can be blocked by antioxidant Vitamin E. This finding may have future therapeutic implications for post-transplant lymphoproliferative disorder.