Zhao, X; Bausano, B; Pike, BR; Newcomb-Fernandez, JK; Wang, KK; Shohami, E; Ringger, NC; Deford, SM; Anderson, DK; Hayes, RL
121-31. [Journal of neuroscience research]
Primary septo-hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF-alpha; 0.3-500 ng/ml) to examine proteolysis of the cytoskeletal protein alpha-spectrin (240 kDa) to a signature 145 kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3. The effects of TNF-alpha incubation on morphology and cell viability were assayed by fluorescein diacetate-propidium iodide (FDA-PI) staining, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alterations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF-alpha produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF-alpha yielded maximal, 3-fold, increase in LDH release and was associated with caspase-specific 120-kDa fragment but not calpain-specific 145-kDa fragment as early as 3.5 hr after injury. Incubation with the pan-caspase inhibitor, carbobenzosy- Asp-CH(2)-OC (O)-2-6-dichlorobenzene (Z-D-DCB, 50-140 microM) significantly reduced LDH release produced by TNF-alpha. Apoptotic-associated oligonucleosomal-sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF-alpha. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF-alpha may induce caspase-3 activation but not calpain activation in septo-hippocampal cultures and that this activation of caspase-3 at least partially contributes to TNF-alpha-induced apoptosis.
Animals; Apoptosis/drug effects; Aspartic Acid/analogs & derivatives/pharmacology; Calpain/analysis; Caspase 3; Caspase Inhibitors; Caspases/metabolism; Cells, Cultured/drug effects; Cycloheximide/pharmacology; Cysteine Proteinase Inhibitors/pharmacology; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation/drug effects; Hippocampus/cytology/drug effects; Nerve Tissue Proteins/antagonists & inhibitors/metabolism; Neurons/drug effects/metabolism; Protein Synthesis Inhibitors/pharmacology; Septum Pellucidum/cytology/drug effects; Spectrin/metabolism; Tumor Necrosis Factor-alpha/pharmacology; 12634-43-4; 30KYC7MIAI; 98600C0908; EC 3.4.22.-