salubrinal and Epstein-Barr-Virus-Infections

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Topics: Carcinogens; Cell Line; Cinnamates; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epstein-Barr Virus Infections; Eukaryotic Initiation Factor-2; Gene Expression Profiling; Gene Expression Regulation, Viral; Genes, Immediate-Early; Germinal Center; Herpesvirus 4, Human; Humans; Immediate-Early Proteins; Lymphocytes; Lymphoma; Membrane Glycoproteins; Plasma Cells; Tetradecanoylphorbol Acetate; Thapsigargin; Thiourea; Trans-Activators; Viral Matrix Proteins; Virus Replication

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

Topics: Acetylcysteine; Animals; Anthracenes; Antibodies, Monoclonal; Apoptosis; B-Lymphocytes; Callithrix; CD27 Ligand; Cell Line, Transformed; Chelating Agents; Cinnamates; Egtazic Acid; Endoplasmic Reticulum; Epstein-Barr Virus Infections; Free Radical Scavengers; Herpesvirus 4, Human; MAP Kinase Kinase 4; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Signal Transduction; Thiourea; Unfolded Protein Response