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Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.

The phenotype of malignant epithelial cells in nasopharyngeal carcinomas (NPC) results from latent infection by the Epstein-Barr virus (EBV) combined to cell gene alterations, especially those affecting the p16/Ink 4 gene. At the site of the primary tumor, NPC are strongly infiltrated by non-malignant EBV-negative T-lymphocytes. There are experimental clues suggesting that these lymphocytes are involved in tumor development, for example by providing anti-apoptotic signals to malignant epithelial cells. The amazing geographic distribution of NPC is accounted for by the conjunction of several risk factors in endemic regions. These risk factors are related to the diffusion of one or several susceptibility genes, the probable existence of viral strains with high oncogenic potential and non-viral environmental factors, especially dietary factors. The perspectives of immunotherapy in NPC are still unclear since viral proteins detected in tumors are poorly immunogenic (EBNA1, LMP1). Targeted molecules designed to interfere with viral and cellular oncoprotein signals will probably have interesting applications for the treatment of NPC.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) protein expressed in EBV-transformed B lymphocytes and in approximately 50% of nasopharyngeal carcinomas (NPCs). LMP1 signaling involves several cellular signaling intermediates, especially TNF receptor-associated factors (TRAFs). We have shown previously that LMP1 is highly concentrated in a cell fraction called glycosphingolipid-rich membrane complexes (GSL complexes). We report here that parallel accumulation of LMP1 and TRAF3, but not TRAF1 or TRADD, was observed in GSL complexes from lymphoblastoid and LMP1-positive NPC cells. In contrast, TRAF3 was not concentrated in GSL complexes from LMP1-negative cells. Binding of LMP1 and TRAF3 in GSL complexes was demonstrated in lymphoblastoid and NPC cells, by co-immunoprecipitation with both anti-LMP1 and anti-TRAF3 antibodies.

The expression and function of CD95 and CD40 were investigated in malignant cells from EBV-positive undifferentiated nasopharyngeal carcinomas (NPCs). Large amounts of CD95 and CD40 expression were detected in 15 of 16 EBV-positive NPC specimens. In contrast, CD95 was not detected in two biopsies from patients with EBV-negative differentiated NPCs. We tested whether the CD95 apoptotic pathway was functional in NPC cells by treating two EBV-positive NPC tumor lines in vitro with a CD95 agonist. In both cases, NPC cells were extremely susceptible to CD95-mediated apoptosis, despite strong constitutive expression of Bcl-x. Combined CD40 and CD95 stimulation was used to investigate the possible anti-apoptotic activity mediated by CD40. The CD40 receptor was activated by incubating NPC cells with murine L cells producing CD154, the CD40 ligand. This treatment resulted in a strong inhibition of CD95-related cytotoxicity. Such an anti-apoptotic effect of CD40 is well known for B lymphocytes, but has not previously been reported for epithelial cells. These data suggest that NPC tumor-infiltrating lymphocytes, which often produce the CD40 ligand in situ, may increase the survival of malignant cells, thereby enhancing tumor growth in patients.