We report here the isolation and characterization of a novel tumor necrosis factor-␣ (TNF-␣)-inducible gene, SCC-S2. Based on the nucleotide sequence, the SCC-S2 open reading frame contains a sequence in the amino terminus that shows a significant homology to death effector domain II of cell death regulatory protein, Fasassociated death domain-like interleukin-1␤-converting enzyme-inhibitory protein (FLIP). Unlike FLIP, the SCC-S2 open reading frame contains only one death effector domain and lacks the carboxyl-terminal caspaselike homology domain, raising the possibility that SCC-S2 may be a novel member of the FLIP family. SCC-S2 mRNA expression is found in most normal tissues and malignant cells. The steady state level of SCC-S2 mRNA is significantly induced by TNF-␣ in different tumor cells (TNF-␣ at 20 ng/ml for 3 h: A549, ϳ2-9-fold; SKOV-3, ϳ3-fold; PCI-04A, ϳ3-6-fold). TNF-␣ treatment (100 ng/ml, 4 h) of HeLa cells transiently transfected with FLAG epitope-tagged SCC-S2 cDNA or expression vector alone led to an increase in the number of apoptotic cells as compared with the untreated counterpart. Interestingly, however, SCC-S2 transfectants revealed a significant decrease in the number of apoptotic cells as compared with the vector transfectants (p < 0.001). These data implicate a role of SCC-S2 as a negative mediator of apoptosis in certain cell types.Increasing evidence suggests that apoptosis requires activation of members of the interleukin-1␤-converting enzyme-like family of cysteine proteases, also known as caspases. The caspase activation appears to be triggered by some members of the TNFR 1 superfamily, including TNFR1 (p55/CD120a) and TNFR2 (p75/CD120b), and Fas/Apo-1 (CD95). TNF binds to TNFR1, and FasL binds to Fas. TNFR1 and Fas, also known as death receptors, are characterized by the presence of a cytoplasmic sequence motif called the death domain, which interacts with the death domain of the adaptor molecules FADD and TNFR-associated death domain, recruiting them to the membrane. TNFR-associated death domain interacts with FADD, and FADD, in turn, associates with an apical caspase, FLICE (caspase 8/MACH/Mch5), through death effector domains (DEDs) present at the carboxyl terminus of FADD and the amino terminus of FLICE, resulting in the assembly of a receptor-associated death-inducing signaling complex. Death-inducing signaling complex-associated FLICE signals proteolytic activation of downstream caspases, ultimately leading to apoptosis (reviewed in Ref. 1). FADD mutant containing only the death domain or FLICE containing two DEDs can act as a dominant negative inhibitor of apoptosis (2-4). Because ligand activation of a death receptor does not lead to apoptosis in all cell types, it has been suggested that natural cell death inhibitory molecules may exist in certain cells. Indeed, FLICE-inhibitory proteins (FLIP, CASH, I-FLICE, and FLAME-1) containing two sequence motifs with significant homology to DEDs have been identified (5-9). FLIPs contain two DEDs in the amino terminus and are repres...

SCC-S2/GG2-1/NDED is a recently discovered antiapoptotic molecule induced by the activation of the transcription factor NF-kappaB. Here we have examined a role of SCC-S2 in cell growth regulation in vitro and in vivo. Western blotting using an antipeptide antibody revealed endogenous SCC-S2 as a approximately 21 kDa cytosolic protein in human breast cancer cells (MDA-MB 231) and renal carcinoma cells (RCC-RS). The immunofluorescence detection method showed the cytosolic localization of FLAG-tagged human SCC-S2 in COS-1 transfectants. MDA-MB 435 human cancer cells stably transfected with the FLAG-tagged SCC-S2 cDNA exhibited increased growth rate as compared to control vector transfectants, as measured by the cell viability (>twofold; n=3; P<0.005) and thymidine-labeling procedures ( approximately sixfold; n=3; P<0.0001). SCC-S2 transfectants also displayed an increase in cell migration in collagen I as compared to control transfectants ( approximately twofold; n=3; P<0.005). In athymic mice, SCC-S2 transfectants showed significantly enhanced tumor growth as compared to control transfectants (mean tumor volumes, day 16: control, 56.86+/-19.82 mm(3); SCC-S2, 127.54+/-18.78 mm(3); n=5; P<0.03). The examination of a limited number of clinical specimens revealed higher expression levels of SCC-S2 protein in certain human tumor tissues as compared to the matched normal adjacent tissues. Taken together, the present studies demonstrate SCC-S2 as a novel oncogenic factor in cancer cells.

The critical pathways through which ionizing radiation induces malignant transformation and cell death are not well defined. Raf-1, a cytoplasmic serine-threonine protein kinase, mediates the transmission of mitogenic signals initiated at the cell membrane to the nucleus, resulting in the activation of transcription factors that regulate cell growth and proliferation. Moreover, Raf-1 overexpression and activation increases the survival response of mammalian cells to the toxic effects of ionizing radiation by an as-yet unknown mechanism (refs 3, 4 and V. Soldatenkov et al.; manuscript submitted). Somewhat analogous to mitogen-induced signalling, radiation stimulates protein-tyrosine kinase(s) and transcription factors. No direct biochemical link has been established, however, between radiation-stimulated protein tyrosine phosphorylation and downstream signals. Here we report a series of radiation-responsive events in which protein-tyrosine phosphorylation is followed by membrane recruitment, then tyrosine phosphorylation and activation of Raf-1 in vivo. Our results show that radiation-stimulated protein-tyrosine kinase(s) modify Raf-1, and implicate Raf-1 in the ionizing-radiation signal-transduction pathway.

SCC-S2/GG2-1/NDED (approved gene symbol TNFAIP8) is a transcription factor NF-kappaB-inducible, antiapoptotic, and oncogenic molecule. In this study, we examined the role of SCC-S2 in invasion and experimental metastasis. We demonstrate that expression of SCC-S2 cDNA in MDA-MB 435 human breast cancer cells is associated with enhanced invasion in vitro and increased frequency of pulmonary colonization of tumor cells in athymic mice. Systemic treatment of athymic mice with a cationic liposomal formulation of SCC-S2 antisense oligo led to decreased incidence of pulmonary metastasis and inhibition of SCC-S2 expression in vivo. Antisense inhibition of endogenous SCC-S2 expression correlated with decreased expression of VEGF receptor-2 in tumor cells and human lung microvascular endothelial cells and loss of endothelial cell viability. In addition, downregulation of SCC-S2 expression in tumor cells was associated with decreased expression of known metastasis-related molecules MMP-1 and MMP-9. These results demonstrate a novel role for SCC-S2 in tumor progression, involving multiple effectors, and provide a basis for SCC-S2-targeted cancer gene therapy.

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