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Induces Apoptosis and Expression of Inflammation-Related Proteins in Chang Conjunctival Cells
1 From the Services d’Ophthalmologie et 3 d’Immunohématologie, Hôpital Ambroise Paré, AP-HP, Université René Descartes Paris V, Boulogne, France; and 2 Laboratoire de Biologie Cellulaire, Institut National de la Santé et de la Recherche Médicale U327, Faculté de Médecine Xavier Bichat, Université Paris VII, France.
Abstract
PURPOSE. The purpose of this study was to investigate the effect of
interferon (IFN)
on cell viability, cell growth, and apoptosis and
on expression of apoptotic and inflammation-related proteins in
epithelial conjunctival cells in vitro. Some aspects of transduction
pathways of IFN-induced alterations were also investigated,
especially the role of protein kinase C (PKC) and IFN
transcriptional factor STAT1.
METHODS. A human conjunctival cell line was treated with different
concentrations (30 and 300 U/ml) of human recombinant IFN. After 24,
48, and 72 hours of treatment, cell viability and relative cell number
were studied with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium
bromide (MTT) and crystal violet colorimetric assays. The apoptotic
process was sought by phase-contrast microscopy,
4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and
transmission electron microscopy and was confirmed by DNA
electrophoresis and immunoblotting of poly(ADP-ribose) polymerase
(PARP). The cell cycle and expression of apoptotic proteins Fas, bax,
and p53; of inflammation-related proteins HLA-DR and intercellular
adhesion molecule (ICAM)-1; and of IFN signal-transducing factor
STAT1 were evaluated by flow cytometry and/or western blot analysis. To
investigate PKC-related transduction pathways, two PKC modulators,
12-O-tetradecanoyl-phorbol-13-acetate (TPA) and staurosporine,
were applied for 3 hours, followed by IFN treatment for 72 hours.
Moreover, the effects of PKC depletion were studied after a 24-hour
application of TPA, also followed by IFN treatment for 72 hours.
Then, Fas, ICAM-1, and HLA-DR expressions were studied by flow
cytometry.
RESULTS. IFN at 30 U/ml induced no change in cell cycle and in cell
viability. Cell viability significantly decreased after 48 hours of
treatment with 300 U/ml IFN, associated with cell cycle alterations
(decrease in number of cells in the S–M phase), apoptotic chromatin
condensation and fragmentation, ladder pattern on DNA electrophoresis
assay, and cleavage of PARP. Moreover, IFN-treated cells
overexpressed plasma membrane Fas, HLA-DR, and ICAM-1 in a dose- and
time-dependent manner, and STAT1 in both nuclear and cytosolic cell
fractions. Only 300 U/ml IFN-treated cells overexpressed bax,
whereas Bcl-2 and p53 proteins were not modified. HLA-DR and Fas were
upregulated after addition of staurosporine or after PKC-depleting
treatment and repressed with TPA. Staurosporine, PKC depletion, and TPA
all enhanced ICAM-1 expression.
CONCLUSIONS. In our model, IFN induced expression of inflammatory molecules
and apoptotic mediators, cell growth arrest, and apoptosis of Chang
conjunctival cells. Moreover, our results suggest that activation of
PKC is not involved in some IFN cellular effects that possibly imply
the upregulation and nuclear translocation of STAT1. IFN-induced
apoptosis could explain in part the recently reported coexistence of
inflammation and programmed cell death in ocular surface inflammatory
disorders such as Sjögren’s syndrome.
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