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Apoptosis in Keratocytes Caused by Mitomycin C

Authors
 Tae-im Kim  ;  Hungwon Tchah  ;  Michael S. Kook  ;  Beom Jin Cho  ;  Kyungrim Sung  ;  Seung-ah Lee 
Citation
 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Vol.44(5) : 1912-1917, 2003 
Journal Title
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
ISSN
 0146-0404 
Issue Date
2003
MeSH
Animals ; Antibiotics, Antineoplastic/pharmacology* ; Apoptosis/drug effects* ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Caspase Inhibitors ; Caspases/metabolism ; Cell Count ; Cell Division/drug effects ; Cells, Cultured ; Corneal Stroma/drug effects* ; Corneal Stroma/enzymology ; Corneal Stroma/pathology* ; Enzyme Inhibitors/pharmacology ; Fibroblasts/drug effects ; Fibroblasts/enzymology ; Fibroblasts/pathology ; L-Lactate Dehydrogenase/metabolism ; Mitomycin/pharmacology* ; Rabbits
Keywords
12714623
Abstract
PURPOSE:
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.
Files in This Item:
T200306929.pdf Download
DOI
10.1167/iovs.02-0977
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Ophthalmology (안과학교실) > 1. Journal Papers
Yonsei Authors
Kim, Tae-Im(김태임) ORCID logo https://orcid.org/0000-0001-6414-3842
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/114597
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