Temporospatial sequence of cellular events associated with etoposide-induced neuronal cell death: role of antiapoptotic protein Bcl-X(L).
Etoposide-induced death comprises such nuclear events as the formation of topoisomerase II-DNA cleavable complex and cytosolic events including caspase activation. By first establishing the temporospatial death sequence triggered by etoposide in a neuronal cell line, MN9D overexpressing Bcl-X(L) (MN9D/Bcl-X(L)) or control vector (MN9D/Neo), we examined whether formation of this complex is primarily responsible for cell death and at which strategic points and how Bcl-X(L) blocks etoposide-induced neuronal death. Etoposide induced death that was dependent on caspase, cycloheximide, and calpain in MN9D/Neo cells. Etoposide also induced death in enucleated MN9D/Neo cells, although this was less severe. The level of topoisomerase II-DNA cleavable complex reached at a maximum of 2 hr after etoposide treatment was identical in MN9D/Neo and MN9D/Bcl-X(L) cells. In MN9D/Neo cells, cytochrome c release into the cytosol and caspase activation occurred as early as 2 hr and 3-6 hr after etoposide treatment, respectively. Etoposide-induced DNA laddering potentially via caspase appeared as early as 12 hr after drug treatment, followed by nuclear swelling in MN9D/Neo cells (>18-20 hr). Subsequently, nuclear condensation started by 24-28 hr and became apparent thereafter. All of these events except for nuclear swelling were substantially blocked in MN9D/Bcl-X(L). At the later stage of cell death (<32-36 hr), a specific cleavage of Bax and fodrin appeared that was completely blocked by calpain inhibitor or by Bcl-X(L). Taken together, our data suggest that Bcl-X(L) prevents etoposide-induced neuronal death by exerting its anticaspase and anticalpain effect on cellular events after the formation of topoisomerase II-DNA cleavable complex that may not be a major contributor to cell death.
Kim
J E
JE
Department of Biology, Yonsei University College of Science, 134 Shinchondong Seodaemoonku, Seoul 120-749, Korea.
Han
B S
BS
Choi
W S
WS
Eom
D S
DS
Lee
E H
EH
Oh
T H
TH
Markelonis
G J
GJ
Saido
T C
TC
Lee
G E
GE
Chung
I K
IK
Oh
Y J
YJ
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002490:Central Nervous System / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000037:antagonists & inhibitors*; D006801:Humans; D008840:Microfilament Proteins / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D059005:Topoisomerase II Inhibitors; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D013049:Spectrin
D002154:Calpain; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D020169:Caspases; D003574:Cytochrome c Group; D004247:DNA; D004250:DNA Topoisomerases, Type II; D004791:Enzyme Inhibitors; D005047:Etoposide; D008840:Microfilament Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D059005:Topoisomerase II Inhibitors; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D013049:Spectrin; C494826:BAX protein, human; C494811:BCL2L1 protein, human