Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3.
Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.
Miyashita
T
T
Department of Genetics, National Children's Medical Research Center, Tokyo, Japan. tmiyashita@nch.go.jp
Nagao
K
K
Krajewski
S
S
Salvesen
G S
GS
Reed
J C
JC
Inoue
T
T
Yamada
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053178:Caspase 6; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D015448:Leukemia, B-Cell; D009842:Oligopeptides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D005938:Glucocorticoids; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003907:Dexamethasone; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases