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-Induced Apoptosis in Chang Conjunctival Cells: Further Investigations
1 From the Services d’Ophthalmologie et 2 d’Immunohématologie, Hôpital Ambroise Paré, AP-HP, Université René Descartes Paris V, Boulogne, France; 3 Laboratoire de Biologie Cellulaire, INSERM U327, Faculté de Médecine Xavier Bichat, Université Paris VII, Paris, France; and 4 Service de Pharmacotoxicologie, Centre Hospitalier National d’Ophtalmologie XV-XX, Paris, France.
PURPOSE. Previously interferon (IFN)
-induced apoptosis and expression of
inflammation-related proteins in a human conjunctival cell line were
demonstrated. The aim of this study was to further investigate the
mechanisms of IFN-, Fas-, and cycloheximide (CHX)-induced programmed
cell death, with special attention to the role of transcriptional
factors NF-
B and STAT1.
METHODS. In a human conjunctival cell line (Chang conjunctival cells)
apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab)
alone (24 or 48 hours) or, as previously reported, with 300 U/ml of
human recombinant IFN alone (48 hours). To study the role of IFN
on Fas-induced apoptosis, cells were treated first with IFN at 30
U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied
for 24 hours. Moreover, to study the influence of CHX on Fas- and
IFN-induced apoptosis, cells were treated for 24 hours with 300 U/ml
IFN together with a nontoxic concentration (1 µg/ml) of CHX, or
with 500 ng/ml anti-Fas ab together with 1 µg/ml CHX (24 hours).
After treatment, cell viability (neutral red assay), mitochondrial
membrane potential (rhodamine 123 assay), chromatin condensation
(Hoechst 33342 assay), and the index Hoechst/neutral red were studied
by cold light microplate cytometry. The apoptotic process was sought
for by contrast phase microscopy and DAPI staining and was confirmed by
immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8
were investigated by Western blot analysis. NF-B and STAT
DNA-binding activities were studied by electrophoretic mobility shift
assays (EMSA).
RESULTS. After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20%
and 30%, respectively, of apoptotic cells were observed. When anti-Fas
sera were applied after IFN pretreatment or together with CHX, 50%
to 80% of cells demonstrated morphologic characteristics of programmed
cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by
caspase-8 activation, and by an index Hoechst/neutral red greater than
one. All these modifications were preceded by a decrease in
mitochondrial membrane potential. EMSA revealed that NF-B was
activated after IFN and anti-Fas ab treatments and inhibited after
CHX treatment. STAT1 was strongly activated after IFN treatment and
only in a minor degree after anti-Fas ab treatment. STAT1-binding
activity persisted after CHX treatment.
CONCLUSIONS. The relative resistance of Chang cells toward Fas-induced
apoptosis could be related to the activation of NF-B. IFN-induced
programmed cell death preferentially involves the activation of STAT1
that counterbalances NF-B antiapoptotic effects. In fact,
Fas-induced apoptosis was potentiated by IFN or CHX treatments.
These results suggest that NF-B activation could maintain cell
viability as well as participate in IFN-induced inflammatory
modifications, whereas STAT1 activation could provide, in this model, a
proapoptotic signal.
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