Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.
OBJECTIVE
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.
Kim
Tae-im
TI
Corneal Dystrophy Research Institute, Department of Ophthalmology, Yonsei University, College of Medicine, Seoul, Korea.
Choi
Seung-il
SI
Lee
Hyung Keun
HK
Cho
Young Jae
YJ
Kim
Eung Kweon
EK
D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000473:pathology*; D003317:Corneal Dystrophies, Hereditary / Q000473:pathology*; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000473:pathology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D016685:Mitomycin; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D051028:bcl-2-Associated X Protein; D000099222:betaIG-H3 Protein