Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas

Two epithelial tumor cell lines were established from biopsy specimens of 2 nasopharyngeal carcinomas (NPC) and designated HNE-1 and HONE-1. Uncloned HNE-1 cells were found to be Epstein-Barr virus (EBV) DNA-positive when examined by Southern blot analysis up to passage 35, after which the EBV genome could no longer be detected. A similar loss of EBV DNA took place in uncloned HONE-1 cells. However, HONE-1 clone 40 cells are still EBV DNA-positive up to passage 42 thus far and cell cultures contain 85-90% EBV nuclear antigen (EBNA)-positive cells. The HNE-1 cell line has been passaged more than 100 times and the uncloned HONE-1 cells more than 90 times. The tumorigenicity of the HNE-1 and HONE-1 cells was demonstrated by tumor induction in nude mice. Karyotypic analysis of the HNE-1 cells demonstrated an aneuploidy with a modal chromosomal number of 74 at passages 5 and 101 at passage 20; 18 marker chromosomes were identified. We have continued to map the EBV genome latently associated with the HNE-1 and HONE-1 cells using the Bam HI, EcoRI or Hind III restriction enzymes. Using EcoRI fragments A-K as probes, we found that HNE-1 EBV DNA is different from B95-8 and HR-1 EBV DNA in the EcoRI-C region. The Bam HI map for HONE-1 EBV DNA is very similar to the B95-8 map; it contains the Bam HI-Y fragment but without Bam HI B' and WI'. Differences were observed between HONE-1 EBV DNA and B95-8 DNA using the Hind III restriction enzyme. There was no evidence of spontaneous expression of the latent EBV genome in HNE-1 cells, and attempts to induce replication of the latent EBV genome and rescue infectious virus have failed, suggesting a tightly restricted virus genome.     

Laboratory markers of tumor burden in nasopharyngeal carcinoma: a comparison of viral load and serologic tests for Epstein-Barr virus

Epstein-Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real-time or end-point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non-NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end-point and real-time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.     

Constitutive expression of HLA class II antigens on EBV positive malignant cells from nasopharyngeal carcinoma: possible involvement in T cell infiltration

Undifferentiated nasopharyngeal carcinoma (NPC) was originally referred to as "lymphoepithelial carcinoma." Two main cell types are constantly associated within these tumors: the malignant epithelial cells which harbor the Epstein-Barr virus (EBV) genome, and the nonmalignant lymphocytes mostly of the T lineage. Three characteristic features of malignant NPC cells might explain T cell migration within NPC tumor tissue: HLA class II molecule expression, IL-1 production, and presence of EBV antigens. Homogeneous suspensions of malignant NPC cells were derived from a nude-mouse transplantable tumor in order to specify the status of HLA class II molecules in these tumors. These suspensions were stained for HLA class II antigens and analyzed by flow cytometry in comparison with other carcinoma cell lines. Three characteristics of transplantable malignant NPC cells were demonstrated: constitutive and coordinate expression of DR, DP, and DQ molecules, and concomitant expression with the CDw40 antigen ("B lymphocyte carcinoma cross-reacting antigen").     

Elevated expression of ICAM1 (CD54) and minimal expression of LFA3 (CD58) in Epstein-Barr-virus-positive nasopharyngeal carcinoma cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is a remarkable entity among human tumors because of its constant association with the Epstein-Barr virus (EBV). Malignant epithelial cells harbor the EBV genome and often express at least 2 species of latent EBV protein (EBNA1 and LMP1). Despite the massive presence of tumor-infiltrating lymphocytes, NPC cells obviously escape immune surveillance directed to EBV antigens. Previous investigations carried out on EBV-positive Burkitt lymphoma (BL) cells have shown that this fact may be partially accounted for by a lack of expression of ICAM1 (CD54) and LFA3 (CD58). ICAM1 and LFA3 have therefore been investigated in fresh NPC biopsies and transplanted NPCs. With only 1 exception out of 9 cases, NPC cells appear to express high levels of ICAM1 and low levels of LFA3. This is a complete inversion of the pattern observed in normal epithelial cells in vivo. Additional investigations will be required to determine to what extent these characteristics affect T-cell interactions with NPC cells, specially in the process of EBV-antigen recognition.     

Establishment and characterization of three transplantable EBV-containing nasopharyngeal carcinomas

Three transplantable nasopharyngeal carcinoma (NPC) tumors, designated C15, C17 and C18, have been obtained and characterized. C15, derived from a primary NPC tumor, has been propagated in nude mice for 30 passages. C17 and C18, derived from metastatic NPC tissue, have been passaged 10 times. Desmosomes, present in every case, provided confirmation of the epithelial origin of all 3 tumors. The Epstein-Barr virus (EBV) genome is contained in C15, C18 and C17 tumor cells with 30, 12 and 3 copies, respectively. The Epstein-Barr virus nuclear antigen (EBNA) was stained by the classical anti-complement immunofluorescence (ACIF) technique. Fluorescence intensity was strong in C15, moderate in C18, and hardly detectable in C17 cells. No expression of the EA and VCA antigens was detected. Flow cytometry analysis performed on monocellular suspensions showed the absence of detectable CR2 molecules (the EBV receptor on B lymphocytes) in all 3 tumors, and the constitutive expression of HLA class-II antigens in C15 and C17 cells. IL-1 activity was demonstrated in the supernatant of C15 and C17 cells cultivated in vitro for 3 days. These data confirm that the constitutive synthesis of MHC class-II molecules and the release of IL-1-like activities are frequent features of NPC cells. These characteristics could be of importance in relation with the T-cell infiltrate found in NPC primary tumors.     

Site-specific Localization of Epstein-Barr Virus in Pharyngeal Carcinomas

In this study, the correlations of factors with Epstein-Barr virus (EBV)-association were investigated in 50 patients with nasopharyngeal carcinoma (NPC), 61 with oropharyngeal carcinoma (OPC), and 55 with hypopharyngeal carcinoma (HPC) in Okinawa and Osaka prefectures in Japan. The incidence of pharyngeal carcinoma in Okinawa was previously found to be higher than that in Osaka; the incidence of OPC was approximately 6 times higher and that of HPC was two times higher. The EBV genome was detected in the tumor cells of the present patients; 83% of the Okinawa and 92% of the Osaka NPC patients. The EBV genome was not detected in OPC or HPC. A univariate analysis showed that sex, the location of the tumor, histology, and the degree of lymphocytic infiltration correlated with the EBV-positive rate. A multivariate analysis revealed that only the location of the tumor was independently correlated with the EBV-positive rate. Histology and tumor size were factors affecting the prognosis of the patients with NPC. The NPC of poorly differentiated type frequently showed the EBV genome, and NPC with lymphocytic infiltration showed a more favorable prognosis compared to the other NPC types. These findings suggest that latent genes of EBV expressed in cancer cells might trigger a cytotoxic T cell reaction against the cancer.     

THE INTRACELLULAR FORM OF EPSTEIN-BARR VIRUS GENOME IN NASOPHARYNGEAL CARCINOMA

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Epstein-Barr virus in the pathogenesis of NPC

Epstein-Barr virus (EBV) is consistently detected in nasopharyngeal carcinoma (NPC) from regions of high and low incidence. EBV DNA within the tumor is homogeneous with regard to the number of terminal repeats. The detection of a single form of viral DNA suggests that the tumors are clonal proliferations of a single cell that was initially infected with EBV. Specific EBV genes are consistently expressed within the NPC tumors and in early, dysplastic lesions. The viral proteins, latent membrane protein 1 and 2, have profound effects on cellular gene expression and cellular growth, resulting in the highly invasive, malignant growth of NPC tumors. In addition to potential genetic changes, the establishment of a latent, transforming infection in epithelial cells is likely to be a major contributing factor to the development of this tumor.     

Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma

This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in endemic areas.     

Similarity of Epstein-Barr virus early polypeptides induced by various tumor promoters

Epstein-Barr virus (EBV)-harboring non-producer Raji cells were activated to express Epstein-Barr virus early antigen (EA) by the combined use of n-butyrate and various tumor promoters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA), Euphorbiaceae plant extracts and the toxic microbial metabolite, teleocidin. With regard to the structural differences among the inducing agents (promoters), the patterns of EBV early polypeptides were strikingly similar. A common pathway is probably involved in the activation of the latent genomes of the virus in the host cells.     

Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral Latent Membrane Protein 1 and the immunomodulatory protein galectin 9

Background  

Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Establishment of Epstein-Barr Virus-positive Human Gastric Epithelial Cell Lines

We have established two Epstein-Barr virus (EBV)-positive cell lines, GT38 and GT39, derived from human gastric tissues of two patients bearing gastric carcinoma. Both cell lines were positive for cytokeratin, an epithelial marker, but not for lymphocyte-related markers. Unlike GT39 cell line, GT38 cells lacked the property of contact inhibition. EBV genome was detected in both cell lines. The cell lines were positive for latent membrane protein 1, and EBV-determined nuclear antigen 1 (EBNA1). EBNA2 was also detected in GT38. These cell lines should be useful for studying the interaction of EBV with gastric epithelial cells and its role in gastric carcinogenesis.     

Detection of antigens associated with Epstein-Barr virus replication in extracts from biopsy specimens of nasopharyngeal carcinomas

By using monoclonal antibodies to different Epstein-Barr virus (EBV) polypeptides in combination with immunoblotting, we detected antigens associated with EBV replication in extracts from nasopharyngeal carcinoma (NPC) biopsy specimens. Major polypeptides associated with both the diffuse and the restricted components of the early antigen (EA) complex were found in extracts from nine of nine NPC biopsy specimens. Cells from an additional NPC biopsy specimen, passaged repeatedly in nude mice, were found to be positive for the major EA (restricted) polypeptide. This approach revealed that extracts from three of 14 biopsy specimens form other benign and malignant diseases also expressed these viral polypeptides. Therefore, for the first time, these results conclusively demonstrate the presence of EA polypeptides in extracts from NPC biopsy specimens. This finding provides at least a partial explanation for the reported prognostic value of antibodies to this antigen in patients with this disease.     

Induction of epstein-barr virus by a new tumor promoter,teleocidin, compared to induction by TPA

The effect of teleocidin, a new, naturally occurring tumor promoter, on induction of Epstein-Barr virus (EBV), was compared with that of a known tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Early antigen (EA) and/or capsid antigen (VCA) of EBV was induced in the EBV genome-carrying cell lines C-6 and P3HR-I cells by teleocidin, its effect being maximal at a concentration of 12.5 ng/ml. The production of infectious EBV from P3HR-I cells was enhanced by teleocidin maximally at a concentration of 0.5 to 2.5 ng/ml. The outgrowth of EBV-transformed cells from peripheral lymphocytes of seropositive healthy donors was also enhanced by teleocidin at a concentration of 0.02 to 0.5 ng/ml. TPA tested simultaneously in all experiments exhibited the same activities as teleocidin, and was effective at similar concentrations. Teleocidin enhanced both EA and VCA synthesis in P3HR-I cells additively with n-butyrate, but not with TPA. This suggests that teleocidin and TPA have a common mechanism of action, although their chemical structures are different.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Expression of RANTES and MCP-1 in epithelial cells is regulated via LMP1 and CD40

Epstein-Barr virus (EBV)-associated undifferentiated nasopharyngeal carcinoma (NPC) is characterized by a prominent nonneoplastic lymphoid stroma. The functional role of these inflammatory cells and the mechanism of their recruitment are not fully understood. In B-cells, the EBV-encoded latent membrane protein 1 (LMP1) can induce the expression of chemokines in an NF-kappaB dependent manner. We now show that LMP1 can induce the expression of RANTES and MCP-1 in an epithelial cell line, and that this effect is partially reversible by an inhibitor of NF-kappaB. Since tumor cells of virtually all NPCs show CD40 expression while many cases are LMP1-negative at the protein level, we also investigated the effect of CD40 signaling and demonstrate that CD40 stimulation can transiently induce RANTES and MCP-1 expression in LMP1-negative epithelial cells. In in situ hybridization only rare tumor cells showed expression of these chemokines unrelated to LMP1 expression, a pattern consistent with transient induction through CD40 signaling. Since RANTES and MCP-1 were also detected in the neoplastic cells of oral squamous cell carcinomas lacking a lymphoid stroma it remains uncertain to what extent these CC chemokines contribute to the attraction of inflammatory cells into the NPC microenvironment.     

Sensitivity to EBV superinfection and IUdR inducibility of hybrid cells formed between a sensitive and a relatively resistant Burkitt lymphoma cell line

The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr virus (EBV) superinfection and the inducibility of the latent EBV genome in these lines were studied by somatic cell hybridization. Two non-EBV producer lines; AG R3, an adherent, 8-azaguanine-resistant variant of the Rajiline; and Namalwa, a non-adherent, 8-azaguanine-sensitive line, were fused with the aid of β-propiolactone-inactivated Sendai virus. The resident EBV genome in Raji is inducible with 5-iododeoxyuridine and the line is also sensitive to EBV superinfection. Namalwa is relatively resistant to both super-infection and induction, and synthetizes surface-associated IgM-lambda. Cytological studies showed that hybrid cells appear to be much larger than either parent and attach to culture plates less firmly than the adherent Raji variant parent. Karyological analyses showed that hybrids contain the expected sum of the parental chromosome markers. Membrane immunofluorescence tests also showed that hybrids synthetize IgM. All the hybrid cells appear to be non-EBV producers, but they are sensitive to both EBV super-infection and induction of latent EBV. These findings suggest the following explanations: (1) there is no evidence of any complementation between the two non-EBV producer lines (Raji and Namalwa) to elicit spontaneous EBV production in hybrid cells; (2) Namalwa is deficient in some factors required for the synthesis of EBV-specified early antigens after EBV superinfection and after induction of latent EBV by IUdR; these factors are supplied by the Raji parent in the hybrids; or (3) Raji, Namalwa and hybrid cells or EBV all produce a substance which inhibits activation of a productive viral cycle, but whose action is antagonized in Raji and hybrid cells to allow the synthesis of EBV-specific early antigens.