Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.

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Abstract

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.