Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.
OBJECTIVE
To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts.


METHODS
Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling.


RESULTS
Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts.


CONCLUSIONS
Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.
Crowston
Jonathan G
JG
Center for Vision Research, Westmead Hospital and Westmead Millennium Research Institute, Sydney, NSW, Australia. jcrowston@eyecenter.ucsd.edu
Wang
Xiao Y
XY
Khaw
Peng T
PT
Zoellner
Hans
H
Healey
Paul R
PR
D017304:Annexin A5 / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002452:Cell Count; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D005205:Fascia / Q000166:cytology*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016685:Mitomycin / Q000633:toxicity*; D044967:Serum / Q000502:physiology*; D013778:Tetrazolium Salts; D013844:Thiazoles
D017304:Annexin A5; D000903:Antibiotics, Antineoplastic; D006133:Growth Substances; D013778:Tetrazolium Salts; D013844:Thiazoles; D016685:Mitomycin; D004247:DNA; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue