Epstein-Barr virus, the TCL-1 oncogene and Burkitt's lymphoma

Epstein-Barr virus (EBV) is strongly implicated in the pathogenesis of several human malignancies, but notably in all endemic and some sporadic cases of Burkitt's lymphoma (BL). Although the virus's contribution to the development of BL remains unclear, evidence now suggests that a common feature of all BL tumours is the expression of the TCL-1 oncogene. A recent report shows that TCL-1 expression in virus-positive BL tumour cells is dependent on the presence of EBV. This finding suggests that the ability of EBV to induce TCL-1 would circumvent the need for the additional genetic or epigenetic changes that lead to the constitutive expression of this oncogene in EBV-negative BL tumours.     

Primary gastric lymphoma is rarely associated with Epstein-Barr virus

Recently, the association of Epstein-Barr virus (EBV) with undifferentiated lymphoepithelioma-like carcinoma and adenocarcinoma of the stomach has been described. In this study of 55 primary gastric lymphomas, most of them belonging to the group of MALT lymphomas, the question of possible EBV involvement has been addressed using in-situ hybridization (ISH) and blot techniques. EBV DNA and/or DNA sequences were found in only two of 24 centroblastic and B-immunoblastic lymphomas and in one anaplastic large cell lymphoma of null cell phenotype. In a further centroblastic lymphoma, a few positive nontumorous (bystander) cells were identified by ISH.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.     

Malignant transformation of B lymphoma cell line BJAB by Epstein-Barr virus-encoded small RNAs

EBV-encoded small RNAs (EBERs) are non-polyadenylated and abundantly transcribed RNAs, whose functions have not yet been fully elucidated. Here, we report that the EBV-negative B lymphoma cell line, BJAB, was rendered more malignant and resistant to apoptosis by EBERs. EBER-transfected cells exhibited enhanced growth potential in SCID mice as well as in soft agar, and showed resistance to apoptotic stimuli in comparison with the vector control. EBERs inhibited the activity of the double-stranded RNA-dependent protein kinase, PKR, which is reputed to act as a tumor-suppressor. These results suggest that EBERs play an important role in the pathogenesis of EBV-associated malignancies through the inhibition of PKR.     

Replacement of the Epstein-Barr virus plasmid with the EBER plasmid in Burkitt's lymphoma cells

Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.     

Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma

We investigated whether Epstein--Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)-alpha. EBV-negative subclones isolated from EBV-positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN-alpha treatment. On the other hand, EBV-positive counterparts exhibited striking resistance against IFN-alpha-induced apoptosis. Transfection of an individual EBV latent gene into EBV-negative BL cells revealed that EBV-encoded poly(A)(-) RNAs (EBERs) were responsible for IFN resistance. EBERs bound double-stranded (ds) RNA-activated protein kinase (PKR), a key mediator of the antiviral effect of IFN-alpha, and inhibited its phosphorylation. Transfection of dominant-negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV-negative BL cells resistant to IFN-alpha-induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA-binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN-alpha-induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN-induced apoptosis in virus-associated tumors.     

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.     

Analysis of Epstein-Barr virus strains and variants in classical Hodgkin's lymphoma by laser microdissection

Epstein-Barr virus (EBV) seems to have an etiological role in the pathogenesis of classical Hodgkin's lymphoma (cHL). Studies of whole tissue DNA by polymerase-chain reaction (PCR) have shown a considerable number of cHL cases with co-infections by different EBV strains and variants, which apparently contradict the clonality of EBV in cHL previously demonstrated by Southern blot analysis. Due to the paucity of HRS cells in HL tissues, studies on single cell DNA are necessary to identify the specific cellular location (HRS cells and/or bystander B lymphocytes) of the EBV strains and variants present in tissue specimens. In the current study, the presence of EBV was determined by PCR of the 3' end of the LMP-1 gene and EBNA-3C gene in whole tissue and, consecutively, in isolated cells from 26 cases of cHL: 10 HIV-positive and 16 sporadic cHL cases. EBV EBERs were present in all but 2 sporadic cHL cases, which were used as negative controls. At isolated cell level, EBNA-3C gene PCR was more sensitive. Indeed, from the cHL cases in which dual-infection was present, it was observed that, in most of them, HRS cells were infected by type 1 virus, and B lymphocytes were co-infected by both types, which points towards EBV infection occurring early in cHL development. Moreover, the finding of 2 cases with dual-infection in HRS may suggest that, in a small percentage of cHL cases, HRS cells derive from different neoplastic clones, or that HRS cells are superinfected by other viral types after the establishment of the neoplastic clone.     

Cellular and Epstein-Barr virus specific DNA polymerases in virus-producing Burkitt's lymphoma cell lines.

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.     

Epstein-Barr virus serology

Because of the ubiquitous nature of EBV, most people are infected with this virus by the time they are adults. People acquire the virus at an early age, earlier in developing countries and in socioeconomically deprived areas of the United States, where about 80% of 5-year-old children are seropositive. In economically privileged areas, only about 40–50% of children are seropositive by age 5. Infections during childhood are usually asymptomatic. In contrast, 50% of adolescents who become infected with EBV develop the fatigue, fever, pharyngitis, and atypical lymphocytosis characteristic of acute infectious mononucleosis (IM). Heterophil antibodies, which are the basis for screening tests for IM, usually appear in the serum of these patients. However, approximately 10% of patients (more commonly children) with EBV induced IM do not develop heterophil antibodies. For this reason, tests for specific antibody-mediated immune responses to EBV may be necessary for diagnosis.