We investigated the molecular basis of the synergistic induction by interferon-␥ (IFN-␥)/tumor necrosis factor-␣ (TNF-␣) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-␥ and/or TNF-␣ action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between ؊73 and ؊36, which is the minimal element inducible by LPS or TNF-␣; 2) an element located between ؊181 and ؊73, which appeared to regulate the response to IFN-␥ and TNF-␣ negatively; and 3) a distal element upstream of ؊224, which was inducible by IFN-␥ alone. LPS signaling was found to involve NFB activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-␥ and TNF-␣, in monocytic cells, involved cooperation between the IRF-1 and NFB p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-␥.

Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzymelinked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a functionblocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of ␤-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases.Invasion and metastasis of tumor cells is a multiple process that depends on uncontrolled interactions between adjacent cells and/or cells and their extracellular environment (1, 2). These interactions are mediated directly by specific adhesion receptors and indirectly by extracellular proteinases that mediate degradation of the extracellular matrix (ECM).

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the eects of human recombinant interferon-a2a and human recombinant interferon-b on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative eects of IFN-b than of IFN-a. Analysis of the early signals triggered by IFN-a and IFN-b demonstrated that the two IFNs were similarly eective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-b treatment, with concomitant activation of p38 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-b was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN-b induced the formation of the Interferon Stimulated Gene Factor 3 complex more eciently than IFN-a, as well as sustained induction of IRF-1, which may account for its greater induction of 2'5'oligo(A)synthetase and greater inhibition of cell proliferation. IFN-b, but not IFN-a, induced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mutated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-b in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-b-induced apoptosis in parental EW-7 and COH cell lines. Oncogene (2000) 19, 3372 ± 3383.