Apoptosis in keratocytes caused by mitomycin C.
OBJECTIVE
The purpose of this study was to quantify the effect of mitomycin C on rabbit keratocytes, with a view to determining its potential in modulating corneal stromal wound healing. In addition, the pathway by which this regulation occurs was investigated.


METHODS
Keratocytes were isolated from New Zealand White rabbits and cultured. Hoechst staining and flow cytometric analyses with annexin V were used to identify the nature of the keratocyte response to mitomycin C. The response of cultured keratocytes to 0.005%, 0.01%, 0.02%, 0.04%, and 0.06% mitomycin C was evaluated with the lactate dehydrogenase (LDH) assay. In addition, after exposure of keratocytes to 0.01% mitomycin C, the LDH assay was performed at different times of 6, 12, and 24 hours. Keratocytes were preincubated with various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) and treated with 0.01% mitomycin C. The LDH assay was performed after 12 hours. Cytochrome c immunostain was performed after exposure to 0.01% mitomycin C.


RESULTS
Hoechst staining revealed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation. Apoptotic changes in cells were detected by flow cytometry. LDH activities increased significantly at concentrations of 0.005% mitomycin C or greater and were time dependent until 24 hours. Treatment with a CPP32-like protease inhibitor caused a decrease in LDH activity, although the results were not statistically significant. Specific inhibitors of caspase-8 and -9 significantly reduced the LDH activity induced by mitomycin C. Cytochrome c immunostaining of keratocytes pretreated with mitomycin C showed strongly positive findings.


CONCLUSIONS
Mitomycin C induced apoptosis, not necrosis, in cultured corneal keratocytes through the caspase pathway-specifically, caspase-8 and -9-related to the mitochondrial pathway.
Kim
Tae-im
TI
Department of Ophthalmology, University of Ulsan, College of Medicine, Asan Medical Center, Seoul, Korea.
Tchah
Hungwon
H
Lee
Seung-ah
SA
Sung
Kyungrim
K
Cho
Beom Jin
BJ
Kook
Michael S
MS
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D011817:Rabbits
D000903:Antibiotics, Antineoplastic; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016685:Mitomycin; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases