Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with
colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that
caspase-3 was indeed activated, but its
peptide inhibitor (
Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death.
z-VAD-fmk, the general inhibitor of
caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-
L-lysine chloromethyl
ketone (
TLCK), but not N-alpha-tosyl-
L-phenylalanine chloromethyl
ketone (
TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both
z-VAD-fmk and
TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both
caspase-dependent and -independent pathways.
TLCK-sensitive
serine protease is also likely to act upstream of
caspase-3 in a
caspase-dependent pathway. Electron microscopic observations demonstrated that
z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring
necrosis. More importantly,
TLCK effectively blocked this abundant vesicular formation along with suppressing
chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that
TLCK-sensitive
serine protease plays multiple roles in
caspase-dependent and -independent pathways of
colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.