TNF-alpha 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-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.
Zhao
X
X
The Vivian L. Smith Center for Neurologic Research, Department of Neurosurgery, The University of Texas Health Science Center, Houston, Texas, USA.
Bausano
B
B
Pike
B R
BR
Newcomb-Fernandez
J K
JK
Wang
K K
KK
Shohami
E
E
Ringger
N C
NC
DeFord
S M
SM
Anderson
D K
DK
Hayes
R L
RL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002154:Calpain / Q000032:analysis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D012688:Septum Pellucidum / Q000166:cytology / Q000187:drug effects*; D013049:Spectrin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009419:Nerve Tissue Proteins; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D013049:Spectrin; D001224:Aspartic Acid; D003513:Cycloheximide; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases