Introduction: Malondialdehyde (MDA), a major end product of lipid peroxidation, is commonly used as a marker for lipid peroxidation. Previously we have shown that cyclosporin A (CsA) increased lipid hydroperoxide formation EBV infected B cells. In this report we found that CsA increased MDA formation in EBV-infected and un-infected B cells and this effect of CsA is inhibited by Vit.E. Methods: Human B cells were purified from human spleen and incubated with EBV(B95-8) overnight for infection. To assay MDA formation, un-infected and EBV-infected human B cells were treated with CsA (0-2000ng/ml) or H2O2 (0.1-0.3mM, positive control) or DMSO (0.1% vehicle control) for 10 min. at 37°C. MDA was assayed according to published method (Esterbauer and Cheeseman,1990). Vit.E from 0 to 160μM was added to the cells 15 min before addition of CsA. Results: B cells treated with H2O2 had increased MDA formation from control of 0.15 ± 0.04 to 0.76 ± 0.06 and 0.63 ± 0.15 (arbitrary units, n=3, p<0.05). CsA concentration dependently increased MDA formation in both un-infected and EBV-infected human B cells. In the un-infected group, the control level of MDA was 0.28 ± 0.03, CsA at 125, 250, 500, 1000 and 2000ng/ml increased MDA to 0.43 ± 0.01, 0.73 ± 0.03, 0.94 ± 0.05, 0.65 ± 0.04 and 0.84 ± 0.1 respectively (n=3, P<0.05). In the EBV-infected group, the control level of MDA was 0.34 ± 0.03, CsA at 125, 250, 500, 1000 and 2000ng/ml increased MDA to 0.56 ±0.02, 0.64 ± 0.02, 0.74 ± 0.02, 0.68 ± 0.05 and 0.66 ± 0.018 respectively (n=3, P<0.05). Vit.E had no significant effect on MDA formation in the cells without CsA treatment. However, Vit.E (at 20, 40, 80 and 160μM) dose dependently reduced MDA formation in CsA treated un-infected human B cells from 0.77 ± 0.03 (+CsA, No Vit.E) to 0.64 ± 0.01, 0.45 ±0.006, 0.41 ± 0.01 and 0.25 ± 0.01 (n=3, P<0.05). In EBV-infected cells, Vit.E (at 20, 40, 80 and 160μM) inhibited CsA induced MDA formation from 0.83 ±0.04(+CsA, No Vit.E) to 0.46 ± 0.04, 0.37 ±0.02, 0.24 ± 0.01and 0.32 ± 0.01 respectively (n=3, P<0.05) Conclusions: This report supports our earlier hypothesis that CsA exerts a direct oxidative stress on human B cells. This oxidative stress may lead to promotion of EBV-B cell proliferation and transformation. Vit. E, a dietary antioxidant, can effectively block this effect of CsA.

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [H-3]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units SE) by 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 a physiological concentration of HNE (1 mu m) for 0.5 and 1h and cultured for 2 and 4 weeks showed significantly increased [H-3]-thymidine incorporation. EBV-B cells treated with HNE (I pm) for I It and subsequently cultured for 2 and 4 weeks had a significantly higher (> 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD). Copyright (c) 2005 John Wiley & Sons, Ltd.

Introduction: NFκB, a transcription factor, plays an important role in Epstein-Barr virus (EBV) transformation of human B cells. We hypothesized that cyclosporin A (CsA) induced promotion of EBV-B cell transformation via oxidative activation of NFκB in EBV-B cells. Methods: Human B cells were purified from human spleen and incubated with EBV (B95-8) overnight for infection. Human B cells and EBV-infected human B cells were treated with TNF-α (positive control), with CsA(0-2000ng/ml) and DMSO (0.1%, vehicle control) for 0.5 and 1 h. Nuclear extracts were prepared according to Dignam et al or to BD ClonTech nuclear extraction protocol. NFκB in the nuclear extracts were assayed by EMSA (according to protocol of Promega) or by ELISA (according to protocol of BD ClonTech). When Vit.E was used, Vit.E was added 15 min before addition of other chemicals to the cells. EBV-B cell transformation was assayed by colony counting and 3H-thymidine incorporation. Results: TNF-α (800 units) activated NFκB 153% over control while CsA (500ng/ml, 30 min) activated NFκB 246% as compared to vehicle DMSO control in non-infected human B cells. CsA dose dependently activated NFκB with optimal CsA concentration at 500ng/ml in EBV infected B cells. At this optimal concentration, CsA increased NFκB from control of 0.186 ± 0.01 to 0.349 ± 0.026 (mean OD readings ± SE, n=3, P<0.05). In EBV transformed human B cell line (LCL), CsA (30 min treatment) dose dependently activated NFκB from control of 0.16 ± 0.01 to 0.209 ± 0.02, 0.251 ±0.007, 0.343 ±0.015, 0.221 ±0.026 and 0.225 ±0.003 at CsA concentration of 125, 250, 500, 1000, and 2000ng/ml respectively (n=3, P<0.05). Vit.E (40-160 μM) dose dependently inhibited CsA activation of NFκB in EBV-infected human B cells. Vit.E also inhibited CsA activation of NFκB in LCL cell line. CsA (500ng/ml) activated NFκB from control of 0.372 ±0.033 to 0.653 ±0.026 (in LCL). While Vit.E had no significant effect on NFκB in the control (no CsA, vehicle DMSO only) samples, Vit.E significantly inhibited CsA induced NFκB activation from 0.653 ±0.026(+CsA, No Vit.E) to 0.498 ±0.035, 0.435 ±0.039, 0.389 ±0.022 at 40, 80 and 160 μM respectively(n=3, P<0.05, in LCL). CsA increased EBV-B cell colony formation (colonies/well in 96 well plate) from control of 28.0 ± 2.27, to 49.0 ±2.16, Vit.E at 40 μM reduced CsA promoted EBV- B cell colony formation to 22.5 ±1.76. CsA induced EBV-B cell 3H-thymidine uptake from control of 12, 481±670 to 26,514 ±2732 (CPM/well). Vit.E at 40μM reduced CsA promoted 3H-thymidine uptake to 16,146 ±2088 (n=6, P<0.05). Conclusions: This observation provides evidence that CsA induced oxidative stress activates NFκB and promotes EBV infected B cell transformation. These events could be blocked by antioxidant Vit.E. These findings add to the understanding of the mechanism of CsA promoted EBV related post-transplant lympho-proliferative disorders (PTLD).

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).