title: Cyclosporin A up-regulates and activates protein kinase C-zeta in EBV-infected and EBV-transformed human B-cells.
abstract: 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.
  
  
  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 microM) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H(2)O(2) (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 microM) in H(2)O(2) (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 PKCalpha 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Surgery, University of Kentucky, College of Medicine, Lexington, KY 40536, USA.
authors.forename: Changguo
authors.initials: C
authors.lastname: Chen
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Johnston
authors.forename: Hoonbae
authors.initials: H
authors.lastname: Jeon
authors.forename: Roberto
authors.initials: R
authors.lastname: Gedaly
authors.forename: Patrick
authors.initials: P
authors.lastname: McHugh
authors.forename: Dinesh
authors.initials: D
authors.lastname: Ranjan
mesh_terms: D001402:B-Lymphocytes / Q000187:drug effects* / Q000821:virology; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D020031:Epstein-Barr Virus Infections / Q000378:metabolism*; D004854:Herpesvirus 4, Human; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D018384:Oxidative Stress; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation; D000099221:Protein Kinase C zeta
chemical_list: D016572:Cyclosporine; D007166:Immunosuppressive Agents; D020033:Protein Isoforms; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta