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Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

Authors
 Tae-im Kim  ;  Seung-il Choi  ;  Hyung Keun Lee  ;  Young Jae Cho  ;  Eung Kweon Kim 
Citation
 MOLECULAR VISION, Vol.14 : 1222-1228, 2008 
Journal Title
MOLECULAR VISION
Issue Date
2008
MeSH
Apoptosis/drug effects* ; Cell Survival/drug effects ; Cells, Cultured ; Cornea/pathology* ; Corneal Dystrophies, Hereditary/pathology* ; Dose-Response Relationship, Drug ; Extracellular Matrix Proteins/genetics ; Extracellular Matrix Proteins/metabolism ; Fibroblasts/drug effects* ; Fibroblasts/pathology* ; Gene Expression Regulation/drug effects ; Humans ; Mitomycin/pharmacology* ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Time Factors ; Transforming Growth Factor beta/genetics ; Transforming Growth Factor beta/metabolism ; bcl-2-Associated X Protein/genetics ; bcl-2-Associated X Protein/metabolism
Keywords
Apoptosis/drug effects* ; Cell Survival/drug effects ; Cells, Cultured ; Cornea/pathology* ; Corneal Dystrophies, Hereditary/pathology* ; Dose-Response Relationship, Drug ; Extracellular Matrix Proteins/genetics ; Extracellular Matrix Proteins/metabolism ; Fibroblasts/drug effects* ; Fibroblasts/pathology* ; Gene Expression Regulation/drug effects ; Humans ; Mitomycin/pharmacology* ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Time Factors ; Transforming Growth Factor beta/genetics ; Transforming Growth Factor beta/metabolism ; bcl-2-Associated X Protein/genetics ; bcl-2-Associated X Protein/metabolism
Abstract
PURPOSE: The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts.

METHODS: Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting.

RESULTS: MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types.

CONCLUSIONS: MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.
Files in This Item:
T200800305.pdf Download
DOI
18615204
Appears in Collections:
1. College of Medicine (의과대학) > Research Institute (부설연구소) > 1. Journal Papers
1. College of Medicine (의과대학) > Dept. of Ophthalmology (안과학교실) > 1. Journal Papers
Yonsei Authors
Kim, Eung Kweon(김응권) ORCID logo https://orcid.org/0000-0002-1453-8042
Kim, Tae-Im(김태임) ORCID logo https://orcid.org/0000-0001-6414-3842
Lee, Hyung Keun(이형근) ORCID logo https://orcid.org/0000-0002-1123-2136
Cho, Young Jae(조영재)
Choi, Seung Il(최승일) ORCID logo https://orcid.org/0000-0001-7168-8795
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/106452
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