title: Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts.
abstract: OBJECTIVE
  To examine the effect of mitomycin-C on the expression of apoptosis genes in human Tenon capsule fibroblasts and to evaluate whether death receptor signaling modulates mitomycin-C cytotoxicity.
  
  
  METHODS
  Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor expression was determined by flow cytometry in control and mitomycin-C-treated Tenon fibroblasts. Fibroblast death was quantified using a lactate dehydrogenase release assay. The effect of Fas and TNF-receptor signaling was evaluated using Fas-specific antibodies and soluble TNF-alpha.
  
  
  RESULTS
  Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01-1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50-500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C-induced apoptosis.
  
  
  CONCLUSIONS
  Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.
authors.affiliation: Wound Healing Research Unit, Institute of Ophthalmology, Bath Street, London EC1V 9EL, United Kingdom. jcrowston@lineone.net
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Crowston
authors.forename: Lydia H
authors.initials: LH
authors.lastname: Chang
authors.forename: Peter H
authors.initials: PH
authors.lastname: Constable
authors.forename: Julie T
authors.initials: JT
authors.lastname: Daniels
authors.forename: Arne N
authors.initials: AN
authors.lastname: Akbar
authors.forename: Peng T
authors.initials: PT
authors.lastname: Khaw
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000235:genetics*; D002478:Cells, Cultured; D003238:Connective Tissue / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018124:Receptors, Tumor Necrosis Factor; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor; D016685:Mitomycin; D007770:L-Lactate Dehydrogenase