Purpose. To investigate the therapeutic potential of Mitomycin-C (MMC) in t
he management of proliferative vitreoretinopathy, the antiproliferative eff
ect of MMC on cultured human retinal pigment epithelial (RPE) cells were in
vestigated in vitro.
Methods. Drug sensitivities of cultured human RPE cells to MMC were determi
ned using the tetrazolium dye assay. In order to detect the presence of apo
ptosis, DNA fragmentation was assessed by DAPI staining, and TdT-dUTP nick-
end labeling (TUNEL) assay. The relative amount of DNA fragmentation was qu
antified by flow cytometric analysis. To analyze the cell cycle response of
RPE cells to MMC, flow cytometric analysis of propidium iodide stained nuc
lei 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 m
ajority of RPE cells following treatment with 10 mug/ml of MMC exhibited fr
agmented nuclei as observed by DAPI staining and TUNEL assay. Cell cycle an
alysis demonstrated an accumulation of cells arrested in S and G2/M phase f
ollowing treatment with 1 mug/ml of MMC. At 10 mug/ml of MMC, a dramatic in
crease 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 t
hese proteins when RPE cells were exposed to increasing concentrations of M
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, a
nd 2) induction of apoptotic cell death.