Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells.

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 drug-induced 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.

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.

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.

In vitro production of stable Epstein-Barr virus-positive epithelial cell clones which resemble the virus:cell interaction observed in nasopharyngeal carcinoma.

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.

Epstein-Barr virus infection of CR2-transfected epithelial cells reveals the presence of MHC class II on the virion.

Epithelial cell lines transfected with the Epstein-Barr virus (EBV) receptor CR2 are susceptible to infection by EBV. Following infection with certain EBV strains we found that these cells became positive for MHC class II. The class II was confirmed as being of viral and not target cell origin by immunostaining with HLA-specific monoclonal antibodies. Electron microscopic immunogold staining confirmed the presence of MHC class II on the surface of the virion. While some MHC class I was also found on the EB virion, other cell surface molecules were absent. Dual color immunofluorescence and confocal microscopy analysis demonstrated colocalization of class II with EBV-encoded structural proteins (MA and VCA) in infected epithelial cells. However, preincubation of EBV with antibodies against either MHC class I or MHC class II failed to affect either EBV binding or EBV infection. The presence of MHC on the surface of the EB virion may be a consequence of the intracellular pathways through which productive virus exits from the cell and may influence the target cell tropism of EBV.