Fas- and interferon gamma-induced apoptosis in Chang conjunctival cells: Further investigations

PURPOSE. Previously interferon (IFN)gamma-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were dem onstrated. The aim of this study was to further investigate the mechanisms of IFN gamma-, Fas-, and cycloheximide (CHX)-induced programmed cell death, with special attention to the role of transcriptional factors NF-kappa B a nd STAT1. METHODS. In a human conjunctival cell line (Chang conjunctival cells) apopt osis was induced with 500 ng/ml anti-Pas antibody (anti-Fas ab) alone (24 o r 48 hours) or, as previously reported, with 300 U/ml of human recombinant: IFN gamma alone (48 hours). To study the role of IFN gamma on Fas-induced apoptosis, cells were treated first with IFN gamma at 30 U/ml during 24 hou rs (nontoxic dose), and then anti-Fas ab was applied for 24 hours. Moreover , to study thr influence of CHX on Fas- and IFN gamma-induced apoptosis, ce lls were treated for 24 hours with 300 U/ml IFN gamma together with a nonto xic concentration (1 mu g/ml) of CHX, or with 500 ng/ml anti-Fas ab togethe r with 1 mu g/ml CHX (24 hours). After treatment, cell viability (neutral r ed assay), mitochondrial membrane potential (rhodamine 123 assay), chromati n condensation (Hoechst 33342 assay), and the index Hoechst/neutral red wer e studied by cold light microplate cytometry, The apoptotic process tvas so ught fur by contrast phase microscopy and DAPI staining and was confirmed b y immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 wer e investigated by Western blot analysis. NF-kappa B and STAT DNA-binding ac tivities 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 gamma pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell d eath. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by art index Hoechst/neutral red greater than one. hll the se modifications were preceded by a decrease in mitochondrial membrane pote ntial. EMSA revealed that NF-kappa B was activated after IFN gamma and anti -Fas alo treatments and inhibited after CHX treatment. STAT1 was strongly a ctivated after IFN gamma treatment and only in a minor degree after anti-Fa s ab treatment. STAT1-binding activity persisted after CHX treatment. CONCLUSIONS. The relative resistance of Chang cells toward Fas-induced apop tosis could be related to the activation of NF-kappa B. IFN gamma-induced p rogrammed cell death preferentially involves the activation of STAT1 that c ounterbalances NF-kappa B antiapoptotic effects. In fact, Fas-induced apopt osis was potentiated by IFN gamma or CHX treatments. These results suggest that NF-kappa B activation could maintain cell viability as well as partici pate in IFN gamma-induced inflammatory modifications, whereas STAT1 activat ion could provide, in this model, a proapoptotic signal.