IFN-beta induces serine phosphorylation of Stat-1 in Ewing's sarcoma cellsand mediates apoptosis via induction of IRF-1 and activation of caspase-7

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS , were investigated to establish the effects of human recombinant interfero n-alpha 2a and human recombinant interferon-beta on cell proliferation and apoptosis, All four cell lines were much more sensitive to the antiprolifer ative effects of IFN-beta than of IFN-alpha. Analysis of the early signals triggered by IFN-alpha and IFN-beta demonstrated that the two IFNs were sim ilarly effective in inducing tyrosine phosphorylation of the Jak-1 and Tyk- 2 kinases and the transcription factors Stat-1 and Stat-2, Interestingly, a n additional rapid phosphorylation of Stat-1 on serine was observed after I FN-beta treatment, with concomitant activation of p38 mitogen-activated pro tein kinase, In these cells, Stat-1 Ser727 phosphorylation in response to I FN-beta was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN -beta induced the formation of the Interferon Stimulated Gene Factor 3 comp lex more efficiently than IFN-alpha, as well as sustained induction of IRF- 1, which may account for its greater induction of 2'5'oligo(A)synthetase an d greater inhibition of cell proliferation. IFN-beta, but not IFN-alpha, in duced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mu tated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-beta in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activ ation of caspase-7, Ectopic expression of IRF-1 induced apoptosis in all fo ur cell lines which correlated with the activation of caspase-7 and,vith th e downregulation of the Bcl-2 oncoprotein, as observed for IFN-beta-induced apoptosis in parental EW-7 and COH cell lines.