Cytochrome c release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells.
The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.
Gorman
A M
AM
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Bonfoco
E
E
Zhivotovsky
B
B
Orrenius
S
S
Ceccatelli
S
S
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003078:Colchicine / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D009474:Neurons / Q000166:cytology* / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D009842:Oligopeptides; C108637:aspartyl-glutamyl-valyl-aspartal; C106566:benzyloxycarbonyl-aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003078:Colchicine