Biological characterization of an Epstein-Barr nuclear antigen-positive American Burkitt's tumor-derived cell line.

Two distinct cultures derived from a lymphoid cell line designated NAB were characterized immunologically, morphologically, and cytogenetically. Both cultures were positive for Epstein-Barr nuclear antigen. NAB I cultures were negative for virus capsid antigen and early antigen and were not affected by treatment with 5-iododeoxyurdine. NAB II cultures were positive for virus capsid antigen and early antigen, which increased with 5-iododeoxyuridine treatment. Both cultures were superinfected with virus prepared from P3HR-1 cells. Cell-free virus concentrates prepared from both cultures were inactive for transformation and infectivity. NAB I and NAB II cells were lymphoid as determined by light and electron microscopy. NAB II cells showed morphological alterations characteristic of herpes infection. 5-iododeoxyuridine-treated cells from both cultures revealed ultrastructural characteristics of cells infected with herpes-viruses but without particles. In addition, the induction of tubuloreticular structures within the endoplasmic reticulum was observed. Cytogenetic analysis of both cultures revealed a rearranged chromosome 14 and several other chromosome aberrations, three of which may be used as a reliable means of identifying NAB cultures.     

Characterization of the Epstein-Barr virus isolated from a cell line derived from a patient with American Burkitt's lymphoma

Epstein-Barr viral (EBV) DNA is nearly always detectable in African Burkitt's lymphoma (BL) but is infrequently found in the histologically indistinguishable American BL. We have derived a tumor cell line from a patient with American BL which produces EBV, and we have compared this virus isolate [JLP(c)] with African BL EBV. The American JLP(c) virus immortalizes human umbilical cord lymphocytes in vitro, and its DNA is indistinguishable from African BL EBV DNA by nucleic acid hybridization and preliminary restriction endonuclease cleavage analysis.     

State of Epstein-Barr virus DNA in an American Burkitt's lymphoma line.

A human lymphoma cell line, positive for the Epstein-Barr virus (EBV)-associated nuclear antigen, was recently established from a North American Burkitt's lymphoma. This cell line, SU-AmB-2, contained EBV DNA both in the form of circular, nonintegrated DNA molecules of viral genome length, present in multiple copies per cell, and as integrated sequences. Having DNA present in both of these forms, it resembled cell lines established from African Burkitt's lymphomas. In studies on EBV strain differences, the episomal viral DNA in Burkitt's lymphoma cells may now be compared with viral DNA in nonmalignant cells with the use of cell lines from Burkitt's lymphoma patients of similar geographic origin.     

Detection of Epstein-Barr virus DNA in American Burkitt's lymphoma.

Twenty American patients with Burkitt's lymphoma (BL) were studied serologically and biochemically to detect an association with Epstein-Barr virus (EBV). A wide range of antibody reactivities was encountered. Five patients had serological profiles similar to those of African patients with BL: one and possibly two of these patients harbored EBV-DNA in their tumors. Fifteen patients had low or absent antibody to EBV: tumors from 10 of these patients lacked evidence of EBV-DNA. An unexplained association between elevated antibody (greater than or equal to 1:80) to EBV virus capsid antigen (VCA) and a favorable prognosis was noted in this series; this prognostic observation requires further substantiation. Thus, it appears that EBV-associated BL is inversely proportional to tumor endemicity. An etiological role for EBV in BL remains speculative.     

Expression of the Epstein-Barr virus genome in a nasopharyngeal carcinoma epithelial tumor cell line

An epithelial tumor cell line was recently established from a biopsy specimen of a nasopharyngeal carcinoma (NPC), and designated HONE-I. Uncloned (parental) HONE-I and HONE-I clone (C)-40 cells were found to contain latent Epstein-Barr virus (EBV). Expression of the latent EBV genome in HONE-I C-40 cells has been examined. It was possible to detect a small percentage of cells spontaneously synthesizing EBV early antigen (EA) and virus capsid antigen (VCA) by immunofluorescence (IF). In addition, the EBV nuclear antigens (EBNA-I and EBNA-2), as well as the EBV latent membrane protein (LMP) were detected in the HONE-I cells. Attempts were made to induce the latent EBV genome in these cells with iododeoxyuridine (IUdR). We observed a significant increase in the number of EA/VCA-positive cells, an increase in EBV DNA, the synthesis of virus particles, and the rescue of infectious virus after treatment of HONE-I C-40 cells with IUdR. The HONE-I C-40 cells should facilitate studies of the expression and regulation of the EBV genome in NPC epithelial tumor cells, which have not previously been available.     

Epstein-Barr virus status and tumour cell phenotype in sporadic Burkitt's lymphoma

Burkitt's lymphoma (BL) biopsy cells and derived cell lines can be grouped according to their patterns of reactivity with 6 selected monoclonal antibodies (MAbs) against B cell-associated surface antigens. Group I cells react only with MAbs J5 and 38.13, recognising the common acute lymphoblastic leukaemia antigen and a BL-associated antigen respectively; group II cells react with J5 and 38.13 and with one or more of a set of MAbs (Ki-24, MHM6, AC2, Ki-1) against "lymphoblastoid" antigens; group III cells react only with these anti-"lymphoblastoid" MAbs. Tumour biopsy cells from 17 cases of sporadic BL, 9 positive for the Epstein-Barr (EB) virus genome and 8 negative, have been analysed during the process of cell line establishment in vitro. In early passage the EB virus-negative BL cells showed either a group I phenotype or gave an additional reactivity with MAb Ki-24 which placed them in group II; these phenotypes remained essentially stable with continued growth of the cell lines for up to 50 passages. By contrast the EB virus-positive BL cells were much more susceptible to phenotypic change in vitro. Although such cells displayed a group I or group II phenotype in early passage, many of the lines soon moved into group III whilst retaining the karyotypic markers indicative of their malignant origin. These observations suggest that a resident EB virus genome can drive the in vitro progression of BL cells towards a more "lymphoblastoid" phenotype. This was confirmed in subsequent experiments where virus-negative BL cell lines were converted to EB virus positivity by in vitro infection. Clearly, therefore, phenotypic analysis of long-established lines can lead to false distinctions being drawn between the EB virus-positive and -negative forms of sporadic BL; both may derive from the same sub-population of target B cells in vivo.     

Variant translocation in a non endemic case of Burkitt's lymphoma: t (8;22) in an Epstein-Barr virus negative tumour and in a derived cell line

After clinical and histopathological diagnosis of Burkitt's lymphoma (BL) in a male Caucasian child with jaw and abdominal tumours, both Epstein-Barr virus (EBV) and cytogenetic studies were performed. The absence of EB viral markers within the tumour cells and the normal EBV serological profile of the patient indicated that this non-endemic BL case was not associated with the virus. Cytogenetic examination of the tumour cells showed a t(8;22) translocation, suggesting that this case represents an example of the variant translocations newly observed in cases of BL. It indicates that chromosome 8 rearrangement with a break-point at 8q23 is a characteristic feature of this particular lymphoma.     

EB病毒对伯基特淋巴瘤细胞系宿主基因表达的影响

背景与目的：Epstein—Barr病毒（Epstein—Barrvirus,EBV）以潜伏状态存在于Burkitt’s淋巴瘤（Burkitt’s lymphoma,BL）细胞中,其基因表达呈高度限制模式,只有少数肿瘤细胞存在病毒裂解性复制。BL细胞株可被诱导进入病毒裂解周期,通过刺激表面免疫球蛋白分子启动病毒复制过程。此过程中,有许多EBV基因表达,这些基因可能影响宿主基因的表达。本实验目的在于鉴定BL细胞中由EBV所调节的宿主基因及由表面IgG连接所调节的宿主基因。方法：利用微阵列检测EBV阳性Akata细胞和EBV阴性AK31细胞中差异性表达的基因。结果：Akata和AK31细胞中共有91个人类基因呈差异性表达,细胞受刺激进入裂解性复制后,检测到198个差异性表达基因。其中myd88基因的差异性表达与TLR9信号通路受损有关。结论：EBV下调了在裂解周期激活早期阶段由表面Ig交联作用所调节的大部分基因。这些基因与细胞存活、信号转导、转录控制以及可能调节B淋巴细胞和其他细胞（如可能影响病毒复制的HDAC4）EBV转化的免疫应答过程有关。     

Epstein-Barr virus (EBV) genome carrying monoclonal B-cell lymphoma in a patient with adult T-cell leukemia-lymphoma.

A Japanese patient with adult T-cell leukemia-lymphoma (ATL) showed a disease progression from the smoldering type to the chronic type and finally to the acute type. The patient was variously treated, including 2'-deoxycoformycin, with some beneficial effects. During the chronic type he developed a composite lymphoma consisting of T-cell lymphoma (ATL) of medium-sized cells and B-cell lymphoma of diffuse large cell type. At that time, he also suffered from miliary tuberculosis and adenovirus type 11-induced hemorrhagic cystitis, indicating that he was in a marked immunodeficient state. Southern-blot analysis revealed that the two malignancies have distinct clonal origin on the basis of the following results: (1) clonally rearranged T-cell receptor beta-chain gene (TcR-beta gene) and germline configuration of immunoglobulin heavy chain gene (IgH gene) in ATL leukemic cells, (2) clonal rearrangement of IgH gene in lymphoma cells, indicating a monoclonal B-cell lymphoma, (3) monoclonal integration of HTLV-I provirus in ATL leukemic cells, (4) definite presence and monoclonal origin of EBV genome in lymphoma cells. This is the first report of secondary EBV genome carrying monoclonal B-cell lymphoma in an ATL patient. It is suggested that the immunodeficient state in the patient with ATL allows the emergence of EBV-related B-cell lymphoma.     

Frequent association of Epstein-Barr virus in Japanese patients with Burkitt's lymphoma.

Seven Japanese patients with Burkitt's lymphoma (BL), residing in Hokkaido, were studied during the period, 1979-1991. Immunological analyses of their lymphoma cells showed all to express surface and/or cytoplasmic immunoglobulins. Chromosomal analysis was performed in five cases. Four of the five lymphomas had the chromosomal translocation, t(8;14), and one had t(2;8). Three patients had extremely high IgG antibody titers to Epstein-Barr virus (EBV) viral capsid antigen (VCA) and to EBV early antigens (EA). Two patients had positive antibodies to EA, and two others had normal antibody patterns comparable to those of EBV-seropositive age- and sex-matched healthy controls. Four of the seven lymphomas (57.1%) were positive for EBV-determined nuclear antigen (EBNA) by anticomplement immunofluorescence and/or EBV DNA using DNA-DNA reassociation kinetics, and/or Southern blot analysis. The frequency of EBV positivity in BL patients residing in Hokkaido was higher than that of cases previously reported in Japan. Three of the four EBV genome-positive BL patients responded well to chemotherapy, radiotherapy and/or surgical treatment, with no significant relapse being observed during the study period. In contrast, EBV genome-negative patients had poor prognoses despite having similar levels of clinical staging at the time of diagnosis.     

Establishment of an Epstein-Barr virus-negative B-cell lymphoma line from a Japanese Burkitt's lymphoma and its serial passage in hamsters.

An Epstein-Barr virus (EBV)-negative lymphoma line (JBL) was established in vitro from pleural effusion of an EBV-seropositive 29-year-old Japanese female with Burkitt's lymphoma. JBL cells as well as her original lymphoma cells bore monoclonal surface IgM with lambda light chains. The JBL line grew in single cell suspension with a doubling time of 30 hours. Attempts were made to serially transplant JBL cells in antilymphocyte serum-treated newborn hamsters; intraperitoneal implantation of 1-3 X 10(7) cells gave rise to invasive tumors in all recipients with death after 10 to 14 days. The hamster-passage line, now in the 9th passage, has been converted to an ascitic form with progression to leukemia in some animals. A "starry sky" pattern closely resembling the human tumor material was preserved in every tumor through serial animal passage.     

Association of Burkitt's lymphoma with the Epstein-Barr virus in two developing countries

The clinical presentation of Burkitt's lymphoma (BL) and it's association with the Epstein-Barr virus (EBV) varies in different geographic areas, BL in developing countries being "intermediate" between the sporadic and endemic types, both in it's clinical presentation and it's association with EBV, which varies from 25-80%. In this study we have analysed the clinical features, EBV association, subtype and prevalence of the deleted variant of the Latent Membrane Protein-1 (LMP-1) of EBV in forty-two cases from two developing countries- India (n = 25) and Argentina (n = 17). In both countries the abdomen was the site most commonly involved while jaw involvement was rare. EBV was detected by in-situ hybridization using the EBER-1 RNA probe. 47% of cases from Argentina and 80% of cases from India were EBER positive. EBV typing using EBNA-3C primers showed a predominance of Type A in both countries (India-13/16 and Argentina-(7/8)). The 30bp deletion of the LMP-1 gene was detected in all evaluated cases from Argentina while the wild type of the gene was seen in all the evaluable Indian cases. Our study highlights the similarities and differences in the clinical presentation and EBV association of BL in two developing countries and also indicates that the subtype of EBV and prevalence of the LMP-1 deletion may reflect the predominant subtype in a particular population.     

Phenylbutyrate induces cell differentiation and modulates Epstein-Barr virus gene expression in Burkitt's lymphoma cells.

Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease.     

Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.

YT cells, originally reported as a natural-killer-like (NK-like) lymphoid cell line, were investigated for Epstein-Barr virus (EBV) genome, gene rearrangement for T-cell receptor (TCR), phenotype and function. The YT cells of the original batch (YT-0) and two subclones (YT2C2 and YTC3) expressed EBV-associated nuclear antigen, and the BamHI-digested DNA showed the 3.4 kb hybridizing band with the BamHIW probe of EBV DNA in Southern blot analysis. When tested with latent-infection membrane protein probe, an identical hybridizing band was shared, indicating that all three sources of YT cells were of monoclonal derivation in terms of the terminal repeat junctional structure of EBV DNA, and that the original YT cells had been infected with EBV before the isolation of the two subclones. The cell-surface antigen analysis revealed the expression of CD7, CD28, CD30, CD45R0, TLiSA and S6F1 antigen besides the originally recorded CD25, CD56 and HLA-DR antigen. Gene rearrangement analysis showed the germ-line genotype, including TCR gamma and delta as well as beta chain. The Northern blot study using the CD3 epsilon and CD3 delta chain gene probes revealed CD3 epsilon, but not CD3 delta RNA. The YT-0 cells exhibited NK and antibody-dependent cellular cytotoxicity activity, but the YT2C2 and YTC3 cells did not. It was not resolved whether the fresh neoplastic NK-like cells of the YT-cell donor carry EBV genome, but YT cells, the first lymphoid cell line found to have EBV genome and non-B lymphoid properties, are valuable for investigating the relationship between EBV and human non-B lymphoid hematopoietic cells.