salicylates and herbimycin

Focal adhesion kinase (FAK) is a signaling molecule associated with cell survival. Previously, we showed that thimerosal, a reactive oxygen species (ROS) generator, can acutely induce FAK tyrosine phosphorylation (within minutes) and chronically induce apoptosis (within days) by redox modulation in HeLa S cells. In the present study, we report that a prolonged oxidative stress by thimerosal induces a remarkable cleavage of FAK, which is accompanied with apoptosis. In fact, the kinetics of FAK cleavage has a good correlation with and actually preceding the apoptosis that was independent of anoikis. The effects were almost completely blocked by the pretreatment with either N-acetyl-l-cysteine (ROS scavenger) or Z-VAD-FMK (pan-caspase inhibitor), suggesting ROS-induced caspase activation as a key mechanism. They could be also reproduced by hydrogen peroxide alone, which appeared to be responsible for thimerosal-mediated oxidative stress-induced apoptosis. Additionally, the down regulation of FAK with antisense oligonucleotide dramatically augmented thimerosal-induced apoptosis. We could observe similar results using human corneal epithelial cells. Taken together, our results show that FAK is a critical cellular target of caspases during oxidative stress (particularly by hydrogen peroxide), resulting in the acceleration of subsequent apoptosis regardless of the anchorage status of cells. From the present results, it is more likely that not cell detachment but the proteolytic cleavage (or inhibition) of FAK is a key modulator as well as a promising indicator of apoptosis in epithelial cells under oxidative stress.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Benzoquinones; Caspase 3; Caspase Inhibitors; Cell Line, Transformed; Chelating Agents; Cornea; Cysteine Proteinase Inhibitors; Egtazic Acid; Epithelial Cells; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; HeLa Cells; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Oxidative Stress; Protein Kinase Inhibitors; Reactive Oxygen Species; Rifabutin; RNA, Small Interfering; Salicylates; Sulfhydryl Compounds; Thimerosal

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Topics: Animals; Antibodies, Monoclonal; Azides; Benzoquinones; Calcium Signaling; CD8 Antigens; Clone Cells; Cytotoxicity, Immunologic; Enzyme Inhibitors; Epitopes; Fas Ligand Protein; fas Receptor; H-2 Antigens; Immunoglobulin Fab Fragments; Interferon-gamma; Lactams, Macrocyclic; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Mast-Cell Sarcoma; Membrane Glycoproteins; Membrane Proteins; Mice; Peptide Fragments; Perforin; Phosphorylation; Photoaffinity Labels; Plasmodium berghei; Pore Forming Cytotoxic Proteins; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Protozoan Proteins; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Salicylates; T-Lymphocytes, Cytotoxic; ZAP-70 Protein-Tyrosine Kinase