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Human Serum Reduces Mitomycin-C Cytotoxicity in Human Tenon’s Fibroblasts

¹From the Center for Vision Research, Westmead Hospital and Westmead Millennium Research Institute, Sydney, NSW, Australia; the ²Hamilton Glaucoma Center, University California San Diego, La Jolla, California; the ³Wound Healing Research Unit, Institute of Ophthalmology Bath Street, London, United Kingdom, and the ⁴Department of Oral Pathology and Oral Medicine, Westmead Hospital, Sydney, NSW, Australia.

PURPOSE. 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.

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				X. Y. Wang, J. G. Crowston, H. Zoellner, and P. R. Healey Interferon-{alpha} and Interferon-{gamma} Sensitize Human Tenon Fibroblasts to Mitomycin-C Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3655 - 3661. [Abstract] [Full Text] [PDF]