cytochrome-c-t and Cell-Transformation--Viral

The inhibitors of apoptosis (IAP) are important regulators of apoptosis. However, little is known about the capacity of Smac mimetics (IAP inhibitor) to overcome virally associated-lymphoma's (VAL) resistance to apoptosis. Here, we explored the pro-apoptotic effect of a novel Smac mimetic, RMT5265.2HCL (RMT) in VAL cells. RMT improved the sensitivity to apoptosis in EBV- and to some extend in HTLV-1- but not in HHV-8-VAL. Furthermore, we identified that RMT promotes caspase 3 and 9 cleavage by inhibiting XIAP and inducing the mitochondrial efflux of Smac and cytochrome C. This investigation further support exploring the use of Smac inhibitors in VAL.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Biomimetics; Cell Line, Tumor; Cell Transformation, Viral; Cytochromes c; Deltaretrovirus Infections; Dipeptides; Epstein-Barr Virus Infections; HEK293 Cells; Herpesvirus 4, Human; Human T-lymphotropic virus 1; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, T-Cell; Mice; Mice, Transgenic; Mitochondria; Mitochondrial Proteins; Models, Biological; Tetrazoles; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

B7-H4 has an inhibitory effect on immune responses via the down-regulation of T cell-mediated immunity, but how the engagement of B7-H4 molecules by counter molecules affects the signaling mechanism of the B7-H4-expressing cells is poorly defined. In this study, we found that B7-H4 expression was enhanced on B cells infected with Epstein-Barr virus (EBV) and that triggering of these molecules induced apoptosis of EBV-transformed B cells. Engagement of B7-H4 initially increased intracellular level of ROS, which then induced the expression of FasL. Engagement of B7-H4 subsequently provoked Fas-mediated and caspase-dependent apoptosis in association with cytochrome c and AIF, and EndoG was released from the mitochondria on EBV-transformed B cells. These results suggest that B7-H4 may be a potential therapeutic target for EBV involved malignancy diseases.

Topics: Apoptosis; Apoptosis Inducing Factor; B-Lymphocytes; B7-1 Antigen; Caspases; Cell Transformation, Viral; Cells, Cultured; Cytochromes c; Fas Ligand Protein; fas Receptor; Herpesvirus 4, Human; Humans; Mitochondria; Signal Transduction; Up-Regulation; V-Set Domain-Containing T-Cell Activation Inhibitor 1

After primary infection of B cells with Epstein-Barr virus (EBV), infected B cells express several viral homologs of human genes that promote activation (LMP1 and CD40) or survival (BHRF and BCL2). EBV-infected B cells also express germinal center phenotype markers, such as CD77, PNA, CD95, and CD38. This transformation of B cells by EBV infection resembles normal B-cell activation and differentiation arising in the germinal center. In the present study, we found that EBV-transformed B cells expressed centrocyte/centroblast marker 1 (CM1), a possible marker of GC B cells and an inducer of their apoptosis. Moreover, ligation of CM1 on EBV-transformed B cells by immobilized anti-CM1 monoclonal antibody induced cell death. The ligation of CM1 immediately increased the generation of intracellular reactive oxygen species (ROS) and disrupted the mitochondrial membrane potential. Pretreatment with N-acetyl cystein (an ROS inhibitor) almost completely blocked this cell death, but Z-VAD-fmk (a caspase inhibitor) did not. We further investigated whether apoptosis-inducing factor (AIF) and endonuclease G (EndoG), which are both related to caspase-independent cell death, would be translocated to the nucleus during the ligation of CM1. We found that AIF and EndoG were released to the cytosplam but not translocated to the nucleus. Moreover, cytochalasin D, a cytoskeleton disruptor, rescued the cells from CM1-mediated cell death and blocked ROS generation. Therefore, it is conceivable that CM1 signaling might provoke cytoskeleton polymerization and trigger ROS generation. Taking these observations together, we conclude that the ligation of CM1 on EBV-transformed B cells can cause cell death via the ROS produced by F-actin polymerization in a caspase-independent manner, although this cell death might be unrelated to AIF and EndoG release from the mitochondria.

Topics: Acetylcysteine; Actins; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Antioxidants; Apoptosis; Apoptosis Inducing Factor; B-Lymphocytes; Caspase Inhibitors; Caspases; Cell Nucleus; Cell Transformation, Viral; Cells, Cultured; Cytochalasin D; Cytochromes c; Endodeoxyribonucleases; Herpesvirus 4, Human; Humans; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylserines; Reactive Oxygen Species

Hepatitis C virus (HCV) core protein has multifunctional activities. We have previously reported that the core protein of HCV immortalizes primary human hepatocytes, which may relate to multistage hepatocarcinogenic events. These immortalized human hepatocytes (IHH) served as a model to study the mechanism of HCV core protein-mediated cell growth regulation. Inhibition of core protein expression in earlier stages after hepatocyte immortalization leads to the induction of apoptosis. Here, we have observed that introduction of antisense core (AS-Core) sequences for inhibition of core protein expression enhanced the expression of E2F1 and p53 levels in early passage IHH. Inhibition of core protein expression also altered the expression level of Bcl-2 family proteins, displaying an increase of the proapoptotic Bax and a decrease in the level of the anti-apoptotic Bcl-xL proteins. These alterations, however, did not result in the release of cytochrome c from the mitochondria. Apaf-1 is frequently deregulated under various pathologic conditions, and examination of AS-Core-expressing apoptotic cells indicated a significant increase in the level of Apaf-1, which coincided with caspase-9 activation. Knockdown of Apaf-1 or the transcriptional regulatory proteins, E2F1 or p53, by small interfering RNA (siRNA) duplexes inhibited the activation of caspase-9 and enhanced cell viability in AS-Core-expressing cells. These findings may contribute to the understanding of the pathophysiology of HCV core protein-mediated hepatocyte growth regulation and disease progression.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Caspases; Cell Cycle Proteins; Cell Transformation, Viral; Cytochromes c; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Gene Expression Regulation, Viral; Hepacivirus; Hepatocytes; Humans; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Antisense; Transcription Factors; Tumor Suppressor Protein p53; Viral Core Proteins

To characterize the UVB radiation-dependent patterns of caspase activation and cell death in SV 40 immortalized corneal epithelial cells.. Cell death in immortalized human corneal epithelial cells (T-HCEC) was induced by exposure to low (50 mJ/cm2) and high (450 mJ/cm2) doses of UVB. Cell death morphology was examined by fluorescence microscopy using the cell death marker propidium iodide (PI). Apoptosis of T-HCEC was analyzed by DNA fragmentation assays, and enzyme activity was measured for caspase 3 and 9 by fluorophotometry. Changes in mitochondrial inner membrane potential were measured by flow cytometry using the fluorescent marker, rhodamine 123. Redistribution of cytochrome c, the upstream trigger of caspase 9, was measured in the cytosol fraction of T-HCEC following irradiation.. PI staining revealed a fragmented staining pattern of the nucleus consistent with apoptosis in detached cells irradiated with low-dose UVB, while cells receiving high dose UVB demonstrated round, well bordered staining of the nucleus. Flow cytometry revealed irreversible mitochondrial damage in the high dose group shown by decreased levels of rhodamine 123 fluorescence. Cells in the low-dose group had intact mitochondrial inner membrane potential, increased cytosolic cytochrome c, and showed a significantly higher rate of DNA fragmentation and caspase activation than the high dose group.. Low dose UVB caused cytochrome c redistribution, caspase activation and apoptosis of corneal epithelial cells, which was not observed at high irradiation levels of UVB.

Topics: Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Line, Transformed; Cell Transformation, Viral; Cytochromes c; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Flow Cytometry; Humans; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Rhodamine 123; Simian virus 40; Ultraviolet Rays

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase Inhibitors; Caspases; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytochromes c; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Etoposide; Fibroblasts; Kinetics; Mitochondria; Nucleic Acid Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Staurosporine; Temperature; Tumor Suppressor Protein p53