ANTIMETABOLITE-INDUCED APOPTOSIS IN TENONS CAPSULE FIBROBLASTS

PURPOSE. To determine whether treatment with mitomycin-c and 5-fluorou racil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS. Cultured human subconjunctival Tenon's capsule fibroblasts w ere 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, apopto sis-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 re lease. RESULTS. Morphologic changes characteristic of apoptosis includ ed nuclear and cytoplasmic condensation and occasional nuclear fragmen tation while the plasma membrane remained intact. Apoptosis-specific p rotein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibr oblasts. The amount of apoptosis induced was dose dependent and partia lly inhibited by the addition of fetal calf serum to growth medium imm ediately after treatment. CONCLUSIONS. Mitomycin-C and high-dose 5-flu orouracil 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 populat ion to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.