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Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts
J G Crowston1, A N Akbar, P H Constable
1Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom.
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.
Insights
Mitomycin-C and 5-fluorouracil induce apoptosis in cultured fibroblasts. Fetal calf serum partially inhibits mitomycin-C-induced apoptosis, suggesting a novel target for regulating scarring.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Subconjunctival fibroblasts play a key role in ocular scarring.
- Understanding the mechanisms regulating fibroblast proliferation and death is crucial for developing anti-scarring therapies.
Purpose of the Study:
- To investigate if mitomycin-C and 5-fluorouracil induce apoptotic cell death in cultured subconjunctival fibroblasts.
- To explore factors influencing drug-induced apoptosis in these cells.
Main Methods:
- Human Tenon's capsule fibroblasts were treated with varying concentrations of mitomycin-C or 5-fluorouracil.
- Apoptosis was assessed using morphological analysis, apoptosis-specific protein expression, DNA fragmentation (TUNEL assay), and lactate dehydrogenase release.
Main Results:
- Mitomycin-C and high-dose 5-fluorouracil induced characteristic apoptotic changes in fibroblasts.
- Apoptosis was dose-dependent and observed 48 hours post-treatment with mitomycin-C.
- Fetal calf serum partially inhibited mitomycin-C-induced apoptosis.
Conclusions:
- Mitomycin-C and 5-fluorouracil are potent inducers of apoptosis in cultured Tenon's fibroblasts.
- Exogenous factors, such as fetal calf serum, modulate fibroblast susceptibility to apoptosis.
- Targeting fibroblast apoptosis presents a novel strategy for controlling ocular scarring.