Expression of SH2D1A in five classical Hodgkin's disease-derived cell lines

The Src homology 2 domain protein 1A (SH2D1A) is a small, 128-amino acid protein consisting of a single SH2 domain; it is probably involved in signal regulation. It is expressed in activated T and natural killer (NK) cells, but not in B lymphocytes. It was discovered in studies on the rare hereditary condition X-linked lymphoproliferative disease (XLP). Individuals with this condition either lack or carry an altered protein. The serious symptoms (fatal mononucleosis) present almost exclusively at the first encounter with Epstein-Barr virus (EBV). The absence of SH2D1A in B cells, which are the targets of EBV, has to be reconciled with this clinical situation. In an earlier search for B lymphocytes expressing SH2D1A, we detected it in EBV-carrying type I Burkitt's lymphoma (BL) lines. We now show SH2D1A in 5 EBV-negative classical Hodgkin's disease (HD)-derived cell lines. Two lines belong to the T lineage and 3 to the B lineage. One B-HD line, which originated from nodular lymphocyte-predominant Hodgkin's lymphoma and differed in phenotype, was SH2D1A-negative. This finding is in accordance with the previously reported abundant SH2D1A mRNA in Hodgkin and Reed-Sternberg (HRS) cells. We thus found SH2D1A expression in lines of malignant origin assigned to the B lineage. Its presence in HRS cells may lead us closer to an understanding of the pathophysiology of the serious syndrome connected with EBV infection in XLP patients, because HRS-like cells have been detected in the lymphoid tissue of patients with infectious mononucleosis. It is likely therefore that in addition to the demonstrated functional defect of T and NK cells imposed by the SH2D1A mutation, the behavior of certain EBV-infected B lymphocytes is also modified.     

SH2D1A and SLAM protein expression in human lymphocytes and derived cell lines

The gene defect responsible for the X-linked lymphoproliferative disease (XLP) is associated with an impaired control of Epstein-Barr virus (EBV) infection. The gene has been recently identified and the encoded protein (designated SH2D1A, DSHP or SAP) was characterized. It is a 128 amino acid (aa) protein, containing a single Src homology 2 (SH2) domain. It interacts with signaling lymphocytic activation molecule (SLAM) expressed on the surface of activated T and B cells. We show that activated T, but not activated B, cells express the SH2D1A protein. NK cells express the protein as well. Tumor lines originating from B, T or NK cells exhibited similar SH2D1A protein expression as the corresponding normal cells, with some notable exceptions. EBV-carrying, tumor phenotype representative (type I), but not EBV-carrying lymphoblastoid cell line (LCL)-like (type III) or EBV-negative Burkitt lymphoma (BL) lines expressed SH2D1A. The phenotypic switch from type I to type III in the EBV-carrying BL line Mutu was associated with a down-regulation of SH2D1A and up-regulation of SLAM. In contrast to normal ex vivo and long-term activated NK cells, 2 of 3 NK leukemia lines expressed SLAM. All 3 lines expressed SH2D1A, like their normal counterparts.     

Two Epstein-Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma

Epstein-Barr virus (EBV) contributes to the development of several human cancers including the endemic form of Burkitt's lymphoma (BL). In culture, EBV induces the continuous proliferation of primary B cells as lymphoblastoid cell lines (LCLs) and if EBV-negative BL-derived cells are infected with EBV, latency-associated viral factors confer resistance to various inducers of apoptosis. Nuclear proteins EBNA3A and EBNA3C (but not EBNA3B) are necessary to establish LCLs and their expression may be involved in the resistance of BL cells to cytotoxic agents. We have therefore created recombinant EBVs from which each of the EBNA3 genes has been independently deleted, and revertant viruses in which the genes have been re-introduced into the viral genome. Infection of EBV-negative BL cells with this panel of EBVs and challenge with various cytotoxic drugs showed that EBNA3A and EBNA3C cooperate as the main determinants of both drug resistance and the downregulation of the proapoptotic Bcl-2-family member Bcl-2-interacting mediator of cell death (Bim). The regulation of Bim is predominantly at the level of RNA, with little evidence of post-translational Bim stabilization by EBV. In the absence of Bim, EBNA3A and EBNA3C appear to provide no survival advantage. The level of Bim is a critical regulator of B cell survival and reduced expression is a major determinant of lymphoproliferative disease in mice and humans; moreover, Bim is uniquely important in the pathogenesis of BL. By targeting this tumour-suppressor for repression, EBV significantly increases the likelihood of B lymphomagenesis in general, and BL in particular. Our results may also explain the selection pressure that gives rise to a subset of BL that retain expression of the EBNA3 proteins.     

Transient expression of the Epstein-Barr virus LMP1 gene in human primary B cells induces cellular activation and DNA synthesis.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) and Epstein-Barr virus nuclear antigen 2 (EBNA2) are expressed in EBV-immortalized human B cells. It has previously been shown that transfection of the LMP1 and EBNA2 genes into Burkitt's lymphoma cell lines results in the up-regulation of CD23, CD21, ICAM-1 and LFA-1 cell-surface proteins. In the present study, the effects of transient expression of the LMP1 and EBNA2 genes were studied in normal primary human B cells pretreated with UV-inactivated EBV particles. To identify and purify cells which express the transfected DNA we used a gene encoding a surface molecule, CD2, as a co-transfection marker. We show that transient expression of the LMP1 gene, from heterologous promoters, is sufficient to induce cellular enlargement and up-regulation of surface expression of the activation markers CD23, CD21, ICAM-1 and LFA-1 in primary B cells. Most importantly, we show that transient expression of the LMP1 gene is sufficient to induce DNA synthesis in human primary B cells. Transient EBNA2 expression enhanced the effect of transient LMP1 expression on CD21 and CD23 cell-surface expression but, under our experimental conditions, inhibited the induction of DNA synthesis by LMP1. We conclude that activation of primary B cells with inactivated EBV particles, followed by transient expression of only two viral genes, EBNA2 and LMP1, is sufficient to reconstitute some of the early events of B-cell immortalization by EBV.     

Stable transfection of a human lymphoma line by sub-genomic fragments of Epstein-Barr virus DNA to measure humoral and cellular immunity to the corresponding proteins

An Epstein-Barr virus (EBV)-negative human lymphoid B-cell line, DG75, was stably transfected with recombinant selection vectors that carry a subfragment of the BamHI WYH region (nucleotides 44664 to 50628), the BamHI K fragment, or a subfragment of the EcoRI D region (nucleotides 166614 to 170149) of B95-8 EBV DNA. These fragments contain the coding exons for the EBV-determined nuclear antigens EBNA2 and EBNA1, and the membrane antigen LMP, respectively. Antigen expression of the cells was detected by immunofluorescence. EBNA2 was expressed in 80–100% of the transfected cells, in contrast to EBNA1 which was expressed in only 25%, and LMP in only about 5% of the cells. Humoral antibody responses were measured by immunofluorescence and compared to cellular immunity as determined by the leukocyte migration inhibition (LM1) technique. Extracts from transfected cell lines expressing EBNA1, EBNA2 or LMP elicited an LMI response with cells from healthy EBV-seropositive individuals whereas the extract from the parental DG75 cell line did not, The results demonstrate the value of stably transfected cell lines expressing a defined EBV antigen for the monospecific analysis of host responses to the EBV-encoded antigen complex in growth-transformed cells.     

EBV is necessary for proliferation of dually infected primary effusion lymphoma cells

Epstein Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are found together in approximately 80% of primary effusion lymphomas (PEL), but their contribution to these cancers is unclear. We found that dominant-negative derivatives of EBNA1 inhibited EBV-positive PEL cells from forming colonies. Those rare PEL cells that proliferated after expression of the dominant-negative derivatives usually expressed these derivatives at low or undetectable levels and continued to maintain their EBV genomes. Those proliferating cells expressing higher levels of the derivatives expressed mutant derivatives that could not bind DNA. These findings indicate that EBV is required to sustain proliferation, as measured by colony formation of dually infected PEL cells. The dominant-negative derivatives of EBNA1 had no effect on the colony-forming ability of five EBV-negative, KSHV-negative hematopoietic cell lines. Surprisingly, they did inhibit the colony-forming ability of EBV-negative, KSHV-positive PEL cells. The small fraction of cells that continued to proliferate expressed only mutants of the EBNA1 derivatives that could no longer bind DNA. These findings indicate that the site-specific DNA-binding activity of EBNA1 or its derivatives when expressed efficiently in EBV-negative, KSHV-positive PEL cells inhibits their colony formation possibly through their binding to the KSHV genome.     

Down-regulation of the EBV-encoded membrane protein (LMP) in Burkitt lymphomas

A radioimmunoassay (RIA) has been developed and used to determine the expression of LMP-a membrane protein encoded by the LT3 region of the Epstein-Barr virus (EBV) genome-in cell lines of various origins. The RIA was highly sensitive, specific and reproducible. All EBV-negative cell lines were LMP-negative and 18 of 21 EBV-carrying cells were LMP-positive. LMP concentrations varied widely, ranging approximately from less than 4 ng up to 650 ng/mg protein. In several instances comparisons were made between lymphoblastoid (LCLs) and Burkitt lymphoma (BL) cell lines (EBV-positive or EBV-converted sublines of originally EBV-negative BL) originating from the same patient. In all such cases LMP and LMP-specific mRNA levels were higher in the LCLs. Most of the LMP was found in the cytosol fraction, yet this fraction was negative in immunoblotting tests. However, antiserum preincubated with the cytosol lost its ability to react in immunoblotting with membrane LMP, indicating that the 2 LMP forms (membrane and cytosol) are completely cross-reactive.     

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.     

Concomitant increase of LMP1 and CD25 (IL-2-receptor alpha) expression induced by IL-10 in the EBV-positive NK lines SNK6 and KAI3

Extranodal, nasal NK/T-cell lymphomas are regularly Epstein-Barr virus (EBV)-positive, with a type II latency pattern, expressing thus EBNA-1 and LMP1. The contribution of EBV to the tumor development is not known. Similarly to normal natural killer (NK) cells, cell lines derived from malignancies with a NK phenotype require IL-2 for in vitro proliferation. In our effort to explore the contribution of EBV, particularly the role of the LMP1 protein, to the pathogenesis of the NK lymphoma we found that its expression, studied in the NK-lines SNK6 and KAI3, depended on the supply of IL-2 or other cytokines. In the absence of IL-2 other cytokines, such as IL-10 and IFN-gamma, could maintain LMP1, but the cells did not proliferate. When grown in IL-2, the SNK6 cells produced IL-10 and IFN-gamma, and these cytokines mediated the expression of LMP1. IL-10 treatment enhanced, while IFN-gamma receptor blocking antibody reduced, the expression of CD25 and CD54 in the EBV-positive, but not in the EBV-negative lines. IL-10 treated cells required lower amount of IL-2 for proliferation compared to the untreated cells. This effect was seen only with the EBV-positive NK lines in which LMP1 and CD25 were concomitantly upregulated. By this mechanism EBV could have an important role in the development of NK lymphoma since the inflammatory component in the tumor tissue can provide these cytokines.     

Antiviral agent cidofovir decreases Epstein-Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.     

CpG methylation of viral DNA in EBV-associated tumours

In EBV-immortalized lymphoblastoid cell lines (LCLs) a small number of "latent" proteins are expressed. These are the EBV nuclear antigens, EBNAs 1-6, and a latent membrane protein, LMP. We have investigated the expression of these proteins in a variety of EBV-associated tumours and cell lines. Whereas transplant and B-cell lymphomas from cotton-top tamarins appear to express the full range of antigens found in LCLs, we and others have found that in Burkitt's lymphomas (BL) and a nasopharyngeal carcinoma (NPC) isolate, EBNA expression is restricted to EBNA-I. (In NPC, but not in BL, LMP may also be expressed). In order to ask what restricts the expression of EBNA 2-6 in NPC and BL cells it seemed reasonable to consider the possibility that the DNA sequences normally regulating expression of these antigens could be chemically modified. In this analysis, a tight inverse correlation between methylation of CpG dinucleotides in the 5' flanking region of the EBNA-2 gene and the expression of EBNAs 2-6 has been revealed. In the NPC tumour, CpG methylation within the gene is also observed, as is specific methylation over the EBNA-I region I and II binding sites (in oriP). The significance of these observations is considered.     

Epstein-barr virus-positive gastric carcinoma has a distinct protein expression profile in comparison with epstein-barr virus-negative carcinoma.

PURPOSE: EBV has been detected in 2-16% of gastric carcinomas. However, there is little information available about the gene expression profile of EBV-positive gastric carcinomas. EXPERIMENTAL DESIGN: EBV infection was examined using EBV-encoded small RNAs (EBERs) in situ hybridization, and 63 (5.6%) of 1127 consecutive gastric carcinomas were found to be EBV-positive. The expressions of 27 tumor-associated proteins were evaluated immunohistochemically in 63 EBV-positive gastric carcinomas and 287 EBV-negative carcinomas using the tissue array method. In addition, the genotype of EBV was investigated by PCR amplification of LMP1 (latent membrane protein 1), Epstein-Barr nuclear antigen 2 (EBNA2), and EBNA3B genes. RESULTS: EBV-positive gastric carcinomas are characterized by the presence of lymphoid stroma, proximal location, and predominance in males. In comparison with EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal cancer), and some DNA repair proteins (P < 0.05). There was negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA, C-erbB2, and smad7. Using hierarchical cluster analysis, we divided EBV-positive gastric carcinomas into two clusters. Those patients with cluster 1 (42 cases) carcinomas had a better prognosis than those with cluster 2 (12 cases; P = 0.0002) or those with EBV-negative carcinomas (280 cases; P = 0.0251). Fifty-one (92.7%) of 55 EBV-positive carcinomas demonstrated the 30-bp deletion in LMP1 gene, and 53 (96.4%) of 55 cases were type 1 for EBNA2 and EBNA3B genes. CONCLUSION: EBV-positive gastric carcinomas have a distinct protein expression profile as well as distinct clinicopathological features, as compared with EBV-negative carcinomas. The subclassification of EBV-positive carcinomas, by hierarchical cluster analysis, is significantly associated with patient survival.     

EB病毒与结直肠癌的相关性

背景与目的:研究表明,EB病毒(Epstein-Barrvirus,EBV)除与鼻咽癌密切相关外,可能与其它癌症相关。本研究旨在探讨EBV与中国人结直肠癌的关系。方法:采用PCR方法从90例原发性结直肠癌标本及25例配对的癌旁标本检测EBVLMP1(latentmembraneprotein1)基因的第3外显子及BamHⅠW片段的部分序列;采用免疫组织化学方法检测EBNA1(EBVnuclearantigen1)和LMP1的表达,采用原位杂交检测EBERs(EBV-encodedRNAs)的表达。结果:在90例原发性结直肠癌标本中,EBV基因阳性率为27.7%(LMP1外显子3)和32.2%(W片段),而25例癌旁组织中只有1例(4.0%)EBV基因阳性,癌组织和癌旁组织EBV阳性率差异有显著性(P<0.001)。29例W片段阳性的大肠癌标本中,有23例(79.3%)EBNA1表达阳性,只有1例(3.4%)EBERs阳性,阳性细胞主要为癌细胞,阳性信号集中在核内;而在8例W片段阴性的标本中,未检测到EBNA1的表达,也未检测到EBERs;以上标本中均未检测到LMP1的表达。结论:EBV与部分中国人结直肠癌相关。