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Involvement of TLCK‐sensitive serine protease in colchicine‐induced cell death of sympathetic neurons in culture
Author(s) -
Mitsui Chizu,
Sakai Kazuhisa,
Ninomiya Takafumi,
Koike Tatsuro
Publication year - 2001
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.10008
Subject(s) - dna fragmentation , apoptosis , caspase , programmed cell death , microbiology and biotechnology , serine protease , fragmentation (computing) , biology , colchicine , protease , biochemistry , enzyme , ecology , genetics
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 ‐α‐tosyl‐ L ‐lysine chloromethyl ketone (TLCK), but not N ‐α‐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. J. Neurosci. Res. 66:601–611, 2001. © 2001 Wiley‐Liss, Inc.