DNA Interstrand Crosslinking Agents and Human Ocular Fibroblasts: Differential Sensitivity to Mitomycin-C and cis-Diaminedichloroplatinum(II)

Abstract

Although clinical application of mitomycin-C (MMC) as an adjunct to glaucoma filtration and pterygium ablation surgeries is based on its inhibitory effect on the local fibroblast proliferation, the extent to which fibroblasts of Tenon's capsule and adjacent tissues (ocular fibroblasts) are susceptible to MMC still remains to be investigated. In this context, we substantially compared the sensitivity of ocular fibroblasts to MMC with normal fibroblasts of other tissue origins and with Fanconi's anemia (FA) fibroblasts known to be supersensitive to MMC due to presumable genetic susceptibility. Since MMC kills cells mainly by forming DNA interstrand crosslinks, cis -diaminedichloroplatinum (CDDP), another crosslinking agent, was also used to evaluate the cytotoxic effect of crosslinks on ocular and other fibroblasts. Following the treatment with various concentrations of MMC or CDDP for 1 hr, fibroblasts were incubated for 14 days until colonies developed. Based on the colony number reflecting the cell number which survived, the regression lines of survival rates were plotted in a semilogarithmic fashion. D 0 and D 10, doses which reduced survival rate to 37% and 10%, respectively, were interpolated and compared among the three cell groups. Ocular fibroblasts were about 2·5 times more sensitive than other normal fibroblasts, but 4-5 times more resistant than FA fibroblasts, to MCC, since the mean D 0 of ocular, other normal, and FA fibroblasts was 0·063, 0·171 and 0·015 μg ml -1, respectively, and the mean D 10 of these was 0·154, 0·401 and 0·029 μg ml -1, respectively. On the other hand, ocular fibroblasts exhibited an isosensitivity to CDDP compared with other normal fibroblasts. FA fibroblasts showed a supersensitivity to CDDP as well. DNA synthesis assay analysed by immunocytochemistry using bromodeoxyuridine (BrdU) and anti-BrdU antibody showed a similar hypersensitivity to MMC but a normosensitivity to CDDP of ocular fibroblasts. These results may indicate that intracellular activation of MMC to the reactive reduced form progresses more effectively in ocular fibroblasts than in fibroblasts originating in other tissues. The hypersensitivity to MMC of ocular fibroblasts is not attributed to genetic susceptibility because of the normosensitivity to CDDP, CDDP may be a promising agent as an alternative to MMC in the ophthalmic fields.