Apoptosis in keratocytes caused by mitomycin C

Tae-im Kim, Hungwon Tchah, Seung ah Lee, Kyungrim Sung, Beom Jin Cho, Michael S. Kook

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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 immuno-stain 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.

Original languageEnglish
Pages (from-to)1912-1917
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number5
DOIs
Publication statusPublished - 2003 May 1

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Mitomycin
Apoptosis
L-Lactate Dehydrogenase
Caspase 9
Caspase 8
Caspase Inhibitors
Cytochromes c
Protease Inhibitors
Corneal Keratocytes
Staining and Labeling
Rabbits
Annexin A5
Caspases
Wound Healing
Flow Cytometry
Cytoplasm
Necrosis
Coloring Agents

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Kim, T., Tchah, H., Lee, S. A., Sung, K., Cho, B. J., & Kook, M. S. (2003). Apoptosis in keratocytes caused by mitomycin C. Investigative Ophthalmology and Visual Science, 44(5), 1912-1917. https://doi.org/10.1167/iovs.02-0977
Kim, Tae-im ; Tchah, Hungwon ; Lee, Seung ah ; Sung, Kyungrim ; Cho, Beom Jin ; Kook, Michael S. / Apoptosis in keratocytes caused by mitomycin C. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 5. pp. 1912-1917.
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title = "Apoptosis in keratocytes caused by mitomycin C",
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 immuno-stain 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.",
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Kim, T, Tchah, H, Lee, SA, Sung, K, Cho, BJ & Kook, MS 2003, 'Apoptosis in keratocytes caused by mitomycin C', Investigative Ophthalmology and Visual Science, vol. 44, no. 5, pp. 1912-1917. https://doi.org/10.1167/iovs.02-0977

Apoptosis in keratocytes caused by mitomycin C. / Kim, Tae-im; Tchah, Hungwon; Lee, Seung ah; Sung, Kyungrim; Cho, Beom Jin; Kook, Michael S.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 5, 01.05.2003, p. 1912-1917.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Apoptosis in keratocytes caused by mitomycin C

AU - Kim, Tae-im

AU - Tchah, Hungwon

AU - Lee, Seung ah

AU - Sung, Kyungrim

AU - Cho, Beom Jin

AU - Kook, Michael S.

PY - 2003/5/1

Y1 - 2003/5/1

N2 - 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 immuno-stain 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.

AB - 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 immuno-stain 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.

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