TNF-α stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures

Primary septo-hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF-α; 0.3-500 ng/ml) to examine proteolysis of the cytoskeletal protein α-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-α 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-α produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF-α yielded maximal, 3-fold, increase in LDH release and was associated with caspase-specific 120-kDa fragment but not calpainspecific 145-kDa fragment as early as 3.5 hr after injury. Incubation with the pan-caspase inhibitor, carbobenzosy-Asp-CH₂-OC (O)-2-6-dichlorobenzene (Z-D-DCB, 50-140 μM) significantly reduced LDH release produced by TNF-α Apoptotic-associated oligonucleosomal-sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF-α. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF-α 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-α-induced apoptosis.