Mechanism of growth inhibitory effect of Mitomycin-C on cultured human retinal pigment epithelial cells: apoptosis and cell cycle arrest.
OBJECTIVE
To investigate the therapeutic potential of Mitomycin-C (MMC) in the management of proliferative vitreoretinopathy, the antiproliferative effect of MMC on cultured human retinal pigment epithelial (RPE) cells were investigated in vitro.


METHODS
Drug sensitivities of cultured human RPE cells to MMC were determined using the tetrazolium dye assay. In order to detect the presence of apoptosis, DNA fragmentation was assessed by DAPI staining, and TdT-dUTP nick-end labeling (TUNEL) assay. The relative amount of DNA fragmentation was quantified by flow cytometric analysis. To analyze the cell cycle response of RPE cells to MMC, flow cytometric analysis of propidium iodide stained nuclei was performed. The levels of proteins related to DNA damage in the RPE cells were then determined by Western blot analysis.


RESULTS
MMC inhibited cell proliferation in a dose-dependent manner. The majority of RPE cells following treatment with 10 microg/ml of MMC exhibited fragmented nuclei as observed by DAPI staining and TUNEL assay. Cell cycle analysis demonstrated an accumulation of cells arrested in S and G2/M phase following treatment with 1 microg/ml of MMC. At 10 microg/ml of MMC, a dramatic increase of the cell population in the sub G1 peak, which can be considered a marker of cell death by apoptosis, was observed by flow cytometry. Western blot analysis of p53 and p21 revealed a gradual increase in the level of these proteins when RPE cells were exposed to increasing concentrations of MMC.


CONCLUSIONS
This study demonstrated that the response of RPE cells to MMC was bi-directional: 1) partial arrest of the cell cycle at S, G2/M phase, and 2) induction of apoptotic cell death.
Kang
S G
SG
Department of Ophthalmology, Eulji University School of Medicine, Seoul, Korea.
Chung
H
H
Yoo
Y D
YD
Lee
J G
JG
Choi
Y I
YI
Yu
Y S
YS
D000293:Adolescent; D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D020287:In Situ Nick-End Labeling; D007211:Indoles / Q000378:metabolism; D008875:Middle Aged; D016685:Mitomycin / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005456:Fluorescent Dyes; D007211:Indoles; D016159:Tumor Suppressor Protein p53; C007293:DAPI; D016685:Mitomycin; D004247:DNA