Sequential cleavage of poly(ADP-ribose)polymerase and appearance of a small Bax-immunoreactive protein are blocked by Bcl-X(L) and caspase inhibitors during staurosporine-induced dopaminergic neuronal apoptosis.
To assess the role of Bcl-X(L) and its splice derivative, Bcl-X(S), in staurosporine-induced cell death, we used a dopaminergic cell line, MN9D, transfected with bcl-xL (MN9D/Bcl-X(L)), bcl-xS (MN9D/Bcl-X(S)), or control vector (MN9D/Neo). Only 8.6% of MN9D/Neo cells survived after 24 h of 1 microM staurosporine treatment. Caspase activity was implicated because a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), attenuated staurosporine-induced cell death. Bcl-X(L) rescued MN9D cells from death (89.4% viable cells), whereas Bcl-X(S) had little or no effect. Bcl-X(L) prevented morphologically apoptotic changes as well as cleavage of poly(ADP-ribose)polymerase (PARP) induced by staurosporine. It is interesting that a small Bax-immunoreactive protein appeared 4-8 h after PARP cleavage in MN9D/Neo cells. The appearance of the small Bax-immunoreactive protein, however, may be cell type-specific as it was not observed in PC12 cells after staurosporine treatment. The sequential cleavage of PARP and the appearance of the small Bax-immunoreactive protein in MN9D cells were blocked either by Z-VAD-fmk or by Bcl-X(L). Thus, our present study suggests that Bcl-X(L) but not Bcl-X(S) prevents staurosporine-induced apoptosis by inhibiting the caspase activation that may be directly or indirectly responsible for the appearance of the small Bax-immunoreactive protein in some types of neurons.
Kim
J E
JE
Department of Biology, College of Science, Yonsei University, Seoul, Korea.
Oh
J H
JH
Choi
W S
WS
Chang
I I
II
Sohn
S
S
Krajewski
S
S
Reed
J C
JC
O'Malley
K L
KL
Oh
Y J
YJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D061945:Caspase Inhibitors; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D061945:Caspase Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D019311:Staurosporine; D004298:Dopamine