CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.
The Fas ligand (FasL)/Fas receptor (CD95) pathway is an important mediator of apoptosis in the immune system and can also mediate cancer cell death. Soluble FasL (sFasL), shed from the membrane-bound form of the molecule by a putative metalloproteinase (MP), may function to locally regulate the activity of membrane-bound FasL. Using a replication-defective recombinant adenovirus-expressing FasL (RAdFasL), we identified a variable ability of different carcinoma cells to respond to FasL-induced cytotoxicity and to shed sFasL. Blockade of FasL cleavage with an MP inhibitor significantly enhanced RAdFasL-induced apoptosis suggesting that sFasL may antagonize the effect of membrane-bound FasL. In support of this concept, a recombinant adenovirus expressing a noncleavable form of FasL (RAdD4) was found to be a potent inducer of apoptosis even at very low virus doses. Our results highlight the therapeutic potential of noncleavable FasL as an antitumor agent and emphasize the important role of MP via the production of sFasL in regulating the response of the Fas pathway. Moreover, these findings have general implications for the therapeutic exploitation of TNF family ligands and for the possible impact of MP-based therapies on the normal physiology of Fas/TNF pathways.
Cancer cells may evade immune surveillance as a result of defective antigen processing and presentation. In this study, we demonstrate that CD40 ligation overcomes this defect through the coordinated action of the transcription factors NF-B and interferon regulatory factor 1 (IRF-1). We show that unlike interferon signaling, which triggers the STAT1-mediated transcriptional activation of IRF-1, the ligation of CD40 in carcinomas induces the rapid upregulation of IRF-1 in a STAT1-independent but NF-B-dependent manner. The transcriptional activation of IRF-1 is controlled largely by the recruitment of p65 (RelA) NF-B to the IRF-1 promoter following the engagement of a TAK1/IB kinase /IB␣ signaling pathway downstream of CD40. NF-B and de novo-synthesized IRF-1 converge to regulate the expression of genes involved in antigen processing and transport, as evident from the sequential recruitment of NF-B and IRF-1 to the promoters of the genes encoding transporter for antigen processing 1 (TAP1), TAP2, tapasin, and low-molecular-mass polypeptides LMP2 and LMP10. Moreover, the RNA interference-mediated knockdown of IRF-1 reduced, whereas the inhibition of NF-B abolished, the effects of CD40 on TAP1 and LMP2 upregulation in carcinoma cells. Collectively, these data reveal a novel "feed-forward" mechanism induced by NF-B which ensures that acutely synthesized IRF-1 operates in concert with NF-B to amplify the immunoproteasome and antigenprocessing functions of CD40.Among the nine known members of the interferon regulatory factor (IRF) family of transcription factors, IRF-1 has attracted significant attention as a master regulator of genes involved in the development of innate and adaptive immunity (reviewed in reference 59). Indeed, studies of mice with a null mutation in the irf-1 alleles (irf-1 Ϫ/Ϫ ) have revealed that IRF-1 plays a crucial role in interferon (IFN)-induced antiviral and antibacterial responses. When challenged with pathogens, irf-1 Ϫ/Ϫ mice display compromised Th1 cell differentiation associated with defects in interleukin-12 p35 subunit (IL-12p35) and inducible nitric oxide synthase (iNOS) gene expression in macrophages and concomitant hyporesponsiveness of CD4 ϩ T lymphocytes and natural killer cells to 37). In line with this observation, the promoters of the IL-12p35 and iNOS genes possess functional IRF-1 binding motifs (42). IRF-1 also plays an important role in the transcriptional control of the transporter for antigen processing TAP1 and the immunoproteasome component LMP2 in response to IFN-␥ (41, 68), thereby influencing the presentation of viral and tumor antigens to CD8 ϩ T cells. Moreover, IRF-1 participates in an autoregulatory loop in the context of type I IFN signaling in which IRF-1 is both a target and a transcriptional activator of 20,72).In addition to its role in regulating the immune response to pathogens, IRF-1 has been proposed to function as a tumor suppressor (reviewed in reference 59) and to influence p53 activity (10). Thus, the loss of IRF-1 dramatically exacerbates susceptib...
The interaction of Epstein-Barr virus (EBV) with epithelial cells and the consequent role of the virus in the aetiology of undifferentiated nasopharyngeal carcinoma (NPC) is poorly understood. One important obstacle to work in this area has been the lack of an epithelial cell culture system in which EBV is stably maintained. Using a previously described approach in which CR2-transfected epithelial cells (SVK-CR2) are rendered susceptible to transient EBV infection (Li et al., Nature 356, 347, 1992), we now demonstrate that the pattern of EBV latent gene transcription in these acutely infected epithelial cells differs from that observed in virus-infected primary B cells. In addition, some of these epithelial cells spontaneously entered the EBV lytic cycle. By cloning Akata virus-infected SVK-CR2 cells we generated two stable lines which remained EBV positive for more than 1.5 years at which time further subclones were isolated. These cloned lines carry the EBV genome as an episome and exclusively use the FQp promoter for driving EBNA1 transcription, display no Cp/Wp promoter activity, and express low levels of the LMP mRNAs. Unlike acutely infected SVK-CR2 cells, the cloned lines responded poorly to suspension-induced terminal differentiation and were impaired in their ability to enter the virus lytic cycle. These results, showing similarities between the cloned EBV-positive SVK-CR2 lines and NPC tumour cells, suggest that stable maintenance of EBV in epithelial cells may require an undifferentiated cellular environment.
Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in druginduced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.
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