期刊
JOURNAL OF SURGICAL RESEARCH
卷 153, 期 1, 页码 156-161出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jss.2008.03.017
关键词
protein kinase-zeta; cyclosporin A; oxidative stress; human B-cells; Epstein-Barr virus; cell signaling
类别
Background. Protein Kinase C (PKC) is a family of enzymes that plays a key role in cell signaling pathways leading to cellular activation and proliferation. Conventional PKC (cPKC) is dependent on calcium for activation. We have proposed that cyclosporin A (CsA), despite being a calcineurin inhibitor, will activate PKC in B cells, thus promoting Epstein-Barr virus (EBV)-induced transformation. Here we show that CsA promoted atypical PKC isoform PKC-zeta in B cells. Materials and methods. Western-blot was used to assay PKC-zeta protein level in EBV-B cells. Confocal microscopy was used to assay PKC-zeta translocation from cytosol to cell membrane, a known process of PKC activation. Results. CsA (500 ng/mL) time dependently increased PKC-zeta from control of 7055 units to 7145, 10,805, 10,914, and 12,705 units, respectively, after 15 min, 1 h, 12 h, and 24 h of incubation in EBV-transformed human B-cell line (LCL). CsA increased PKC-zeta expression was inhibited 50% by Vit.E (40 mu M) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H2O2 (0.1 mM) treatment, PKC-zeta protein level in LCL cells increased 124%, 257%, 349%, and 359% after 15 min, 1 h, 12 h, and 24 h of culture compared with control. Addition of Vit.E (40 mu M) in H2O2 (0.1 mM) treatment and then with Vit.E in the culture decreased PKC-zeta level in LCL cells 26%,20%,41%, and 60% after 15 min, 1 h, 12 h, and 24 h of culture. In confocal microscopy in Jurkat T cell line, phorbol 12-myristate 13-acetate (PMA) activated cPKC isoform PKC alpha after 30 min treatment and activated PKC-zeta after 60 min treatment. CsA inhibited PMA activation of PKC-alpha but not PKC-zeta. CsA alone did not activate PKC-alpha or PKC-zeta in Jurkat T cells. In LCL and in EBV-infected human B-cells, PMA stimulated PKC-alpha activation after 30 min treatment and stimulated PKC-zeta activation after 60 min treatment. CsA inhibited PMA activation of PKC-alpha but not PKC-zeta. In addition, CsA activated PKC-zeta in the EBV-transformed and EBV-infected human B cells. Conclusion. These experiments show that CsA-induced oxidative stress caused PKC-zeta up-regulation in LCL cells, and show the differential effect of CsA in the PKC signaling pathways in T cells versus B cells. CsA-induced PKC-zeta activation may be an important signaling step in EBV-induced post-transplant lymphoproliferative disorders. (C) 2009 Elsevier Inc. All rights reserved.
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