thapsigargin and Burkitt-Lymphoma

Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinβ (C/EBPβ) in a variety of tumor cell lines. C/EBPβ activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPβ inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPβ knock-down. Therefore, C/EBPβ mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.

Topics: Activating Transcription Factor 4; Activating Transcription Factor 6; Antineoplastic Agents; Boronic Acids; Bortezomib; Burkitt Lymphoma; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; HEK293 Cells; Herpesvirus 4, Human; Humans; Pyrazines; Regulatory Factor X Transcription Factors; Response Elements; Thapsigargin; Trans-Activators; Transcription Factor CHOP; Transcription Factors; Unfolded Protein Response; Virus Activation; X-Box Binding Protein 1

Permeability transition, and a subsequent drop in mitochondrial membrane potential (DeltaPsi(m)), have been suggested to be mechanisms by which cytochrome c is released from the mitochondria into the cytosol during apoptosis. Furthermore, a drop in DeltaPsi(m) has been suggested to be an obligate early step in the apoptotic pathway. Didemnin B, a branched cyclic peptolide described to have immunosuppressive, anti-tumour, and anti-viral properties, induces rapid apoptosis in a range of mammalian cell lines. Induction of apoptosis by didemnin B in cultured human pro-myeloid HL-60 cells is the fastest and most complete ever described with all cells being apoptotic after 3 h of treatment. By utilizing the system of didemnin B-induced apoptosis in HL-60 cells, and the potent inhibitors of mitochondrial permeability transition, cyclosporin A and bongkrekic acid, we show that permeability transition as determined by changes in DeltaPsi(m) and mitochondrial Ca2+ fluxing, is not a requirement for apoptosis or cytochrome c release. In this system, changes in mitochondrial membrane potential and cytochrome c release are shown to be dependent on caspase activation, and to occur concurrently with the release of caspase-9 from mitochondria, genomic DNA fragmentation and apoptotic body formation.

Topics: Apoptosis; Bongkrekic Acid; Burkitt Lymphoma; Calcium Signaling; Caspase 9; Caspases; Cyclosporine; Cytochrome c Group; Depsipeptides; DNA Fragmentation; Enzyme Activation; HL-60 Cells; Humans; Intracellular Membranes; Ion Channels; Melanoma; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Peptides, Cyclic; Permeability; Thapsigargin; Tumor Cells, Cultured

The present study has used a panel of 23 monoclonal antibodies to IgM and 4 to IgD in order to probe parameters influencing sIg-dependent apoptosis in an IgM/IgD-expressing Burkitt lymphoma line. No direct correlation was observed between the capacity of the different anti-mu to drive cells into apoptosis and either their domain specificity or their affinity for sIgM. There was, however, a direct correlation between the functional outcome and the ability of the monoclonal antibodies to elicit a rise in intracellular Ca2+. For apoptosis to occur, the Ca2+ response had to attain a threshold value of approximately 100 nM. A direct role for Ca2+ in the delivery of the apoptotic signal was demonstrated using thapsigargin to raise intracellular Ca2+ levels. Antigen receptor ligation was linked to Ca2+ increases by tyrosine kinases as revealed by direct analysis of protein tyrosine phosphorylation and the effects of selective protein tyrosine kinase-inhibiting tyrphostins. These findings reveal a central role for the antigen receptor-generated Ca2+ signal in driving apoptosis in human B lymphoma cells and stresses the need to use a panel of reagents when probing function with presumed ligand-mimetic monoclonal antibodies.

Topics: Antibodies, Monoclonal; Apoptosis; B-Lymphocytes; Burkitt Lymphoma; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Epitope Mapping; Humans; Immunoglobulin D; Immunoglobulin M; Intracellular Fluid; Kinetics; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Signal Transduction; Thapsigargin; Tumor Cells, Cultured