4-Hydroxynonenal mediates cyclosporine promoted EBV-induced B cell transformation

Abstract

Introduction. We had previously shown that cyclosporine A (CsA) directly promoted Epstein--Barr virus (EBV) infected human B cell (EBV-B cell) immortalization via oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end product of lipid peroxidation. We hypothesized that HNE mediates a direct oxidative stress promoting the effect of CsA on EBV-B cell transformation. Methods. Human B cells were purified from human spleen and incubated with EBV (B95-8) overnight for infection. To assay HNE production, EBV-B cells were treated with 0, 500, and 1000 ng/ml of CsA or with HNE (positive control) or DMSO (0.1%, vehicle control) for 10 min at 37°C. HNE--Protein adducts in the cell extract was assayed by slot-blot using polyclonal HNE antibody. For 3H-thymidine uptake and LMP1 assay, EBV-B cells were treated with HNE (1 μM) for 0, 0.5, 1, and 4 h at 37°C and cultured for 2 and 4 weeks. Cells were then assayed for expression of EBV oncogene LMP1 by using PE-conjugated anti-LMP1 antibody in flow cytometry and for 3H-thymidine uptake. Results. CsA at 500 and 1000 ng/ml significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units ± SE) of 251.3 ± 7.5 to 361.3 ± 9.7 and 342.7 ± 10.7, respectively ( P < 0.05, n = 3). EBV-B cells treated with physiological concentration of HNE (1 μM) for 0.5 h and 1 h and cultured for 2 weeks had significantly increased 3H-thymidine incorporation (cpm ± SE) over a control of 839.2 ± 54 to 1192 ± 41.6 and 1998 ± 24.8, respectively ( P < 0.02, n = 6). EBV-B cells treated with HNE (1 μM) for 0.5 and 1 h and cultured for 4 weeks also had significantly increased 3H-thymidine uptake over a control of 1035 ± 206 to 2022 ± 293 and 1941 ± 229, respectively ( P < 0.03, n = 6). HNE (1 μM) treated for 4 h did not have a significant effect on cell proliferation at 2 or 4 weeks. EBV-B cells treated with HNE (1 μM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher (>2.0 times ) LMP1 positive cell population over the control. Conclusion. This observation provides evidence for further understanding of CsA-induced oxidative stress and direct promotion of EBV related post-transplant lymphoproliferative disorder (PTLD).