Interferon-alpha and interferon-gamma sensitize human tenon fibroblasts to mitomycin-C.
OBJECTIVE
To investigate the effect of interferon (IFN)-alpha and IFN-gamma pretreatment on mitomycin C (MMC)-induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-alpha and IFN-gamma modulate the susceptibility of HTFs to MMC.


METHODS
HTFs were pretreated with IFN-alpha and IFN-gamma for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining.


RESULTS
MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-alpha nor IFN-gamma alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-alpha and IFN-gamma had a synergistic effect. IFN-alpha and IFN-gamma pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells.


CONCLUSIONS
IFN-alpha and IFN-gamma enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3-dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.
Wang
Xiao Yang
XY
Center for Vision Research, University of Sydney, Westmead, Hospital Sydney, NSW, Australia. xy_wang@wmi.usyd.edu.au
Crowston
Jonathan G
JG
Zoellner
Hans
H
Healey
Paul R
PR
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004347:Drug Interactions; D053222:Fas Ligand Protein / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D016685:Mitomycin / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects; D019014:fas Receptor / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D000998:Antiviral Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D053222:Fas Ligand Protein; D016898:Interferon-alpha; D019014:fas Receptor; D016685:Mitomycin; D007371:Interferon-gamma; D020169:Caspases