TNF-related apoptosis-inducing ligand (TRAIL) is a typical member of the tumor necrosis factor (TNF) ligand family that is expressed as a type II membrane protein (memTRAIL) and signals apoptosis via the death domain-containing receptors TRAIL-R1 and -2. Soluble recombinant derivatives of TRAIL (sTRAIL) are considered as novel tumors therapeutics because of their selective apoptosis inducing activity in a variety of human tumors but not in normal cells. Using antagonistic antigen-binding fragment (Fab) preparations of TRAIL-R1-and TRAIL-R2-speci®c antibodies, we demonstrate in this study that TRAIL-R1 becomes activated by both the soluble and the membrane-bound form of the ligand, whereas TRAIL-R2 becomes only activated by mem-TRAIL or soluble TRAIL secondarily cross-linked by antibodies. Furthermore, we show that the restricted signal capacity of sTRAIL can be readily converted into a fully signal competent memTRAIL-like molecule, i.e. a TRAIL-R2 stimulating ligand, by genetic fusion to an antibody derivative that allows antigen-dependent`immobilization' of the fusion protein to cell surfaces. We conclude that antibody targeting-dependent activation can be used to design selective therapeutics derived of those ligands of the TNF family that are biologically inactive in their soluble form. Oncogene (2001) 20, 4101 ± 4106.

No abstract

Using fluorescent variants of Fas and FasL, we show that membrane FasL and Fas form supramolecular clusters that are of flexible shape, but nevertheless stable and persistent. Membrane FasL-induced Fas clusters were formed in caspase-8– or FADD-deficient cells or when a cytoplasmic deletion mutant of Fas was used suggesting that cluster formation is independent of the assembly of the cytoplasmic Fas signaling complex and downstream activated signaling pathways. In contrast, cross-linked soluble FasL failed to aggregate the cytoplasmic deletion mutant of Fas, but still induced aggregation of signaling competent full-length Fas. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-β-cyclodextrin, whereas Fas aggregation by soluble FasL was blocked. Together, these data suggest that the extracellular domains of Fas and FasL alone are sufficient to drive membrane FasL-induced formation of supramolecular Fas–FasL complexes, whereas soluble FasL-induced Fas aggregation is dependent on lipid rafts and mechanisms associated with the intracellular domain of Fas.

Although nuclear factor-jB (NF-jB) usually exerts anti-apoptotic activity, upon activation by interleukin-1 (IL-1) it enhances ultraviolet-B radiation (UVB)-induced apoptosis. This paradoxical effect is associated with NF-jB-dependent pronounced secretion of tumour necrosis factor-a (TNF) which activates TNF-R1 in an autocrine fashion to enhance UVB-induced apoptosis. We demonstrate that sustained TNF transcription in UVB þ IL-1-treated cells involves complete abrogation of the negative feedback loop of NF-jB preventing IjBa resynthesis, hence allowing uncontrolled NF-jB activity. We show that IjBa is not transcriptionally inhibited but resynthesized protein is immediately marked for degradation due to persistent inhibitor of jB kinaseb (IKKb) activity. Continuous IKKb phosphorylation and activation is caused by UVB-mediated inhibition of the phosphatase PP2A. This study demonstrates that the cellular response to different NF-jB activators may be converted to the opposite reaction when both stimuli act in concert. Our data shed new light on the significance of negative feedback regulation of NF-jB and identifies PP2A as the key regulator of this process. The transcription factor nuclear factor-kB (NF-kB) is involved in many cellular responses. It comprises five proteins: p50/ p105, p52/p100, p65, c-Rel and RelB that exist as homo-and heterodimers, p65/p50 being the most abundant form. In unstimulated cells, NF-kB is sequestered in the cytoplasm through interaction with the inhibitory protein IkBa that masks its nuclear localization signal. 1 NF-kB (p65/p50) is mostly activated by pro-inflammatory mediators, including tumour necrosis factor-a (TNF), interleukin-1 (IL-1) or LPS. Activated receptors mediate activation of a multi subunit inhibitor of kB kinase (IKK) complex consisting of IKKa, -b and -g. Activated IKK acts through phosphorylation of IKKb at Ser177/181, subsequently catalysing phosphorylation of inhibitor of kBa (IkBa) at Ser32/36, leading to its polyubiquitination and proteasomal degradation. Released NF-kB translocates into the nucleus to activate responsive genes, among these the one encoding IkBa. 2 Nuclear export of resynthesized IkBa is more potent than import, allowing cytosolic localization of the inactive complex, thus creating a negative feedback loop. 3 As NF-kB serves many different functions, tight regulation by the negative feedback loop is crucial. Only highly controlled and transient expression of NF-kB-driven genes ensures proper function. Uncontrolled NF-kB activity is linked to transformation, proliferation, suppression of apoptosis and metastasis. 4,5 Thus, strategies interfering with signalling pathways activating NF-kB have become major targets for anticancer interventions. 6 We have previously shown that stimulus-dependent activation of NF-kB can result in completely opposite effects. Stimulation with IL-1 protects keratinocytes and epithelial cells from cytotoxic effects of death ligands in an NF-kB-dependent manner by upregulation of anti-apoptotic cFLIP and cIAPs. 7 In contr...

Successful treatment of melanoma is still challenging, because metastasis remain chemoresistant and radioresistant. Accordingly, combinational treatments involving death ligands are mandatory. In a recent study from our lab, the majority out of 18 melanoma cell lines remained resistant against treatment with the death ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand). Resistance was shown to be mainly due to incomplete processing of caspase-3 into catalytically inactive p21 by binding of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP). Coirradiation with sub-lethal ultraviolet (UV) B caused depletion of XIAP resulting in synergistic sensitization of all but two melanoma cell lines to TRAIL. We show here the XIAP depletion to essentially require initial caspasemediated cleavage, which promotes proteasomal degradation of XIAP. Utilizing specific caspase inhibitors and small interfering RNA-mediated knockdown, we further identified caspase-3 to be responsible for performing the initial cleavage of XIAP after UVB treatment. Additional evidence suggests an accelerated mitochondrial outer membrane permeabilization in response to co-treatment with TRAIL and UVB, which directs the release of XIAP antagonizing factors including Smac. Distraction of XIAP consequently liberates caspase-3 to autocatalytically process into active p17. Activated caspase-3 cleaves XIAP and further enhances its activation in a positive regulatory feedback loop. The molecular mechanism discovered here appears to have broader implications, because cleavage of XIAP was also shown to accompany cisplatin-induced sensitization of melanoma cells to TRAIL.