Burkitt Lymphoma Tissues Research Articles

Integration of Epstein-Barr Virus into Chromosome 6q15 of Burkitt Lymphoma Cell Line (Raji) Induces Loss of BACH2 Expression

Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is a well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50-60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly guanine/cytosine-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent host sequences. EBV was present at intron 1 of the BACH2 gene located on chromosome 6q15. BACH2 was not expressed in the Raji cell line. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis.

Epstein–Barr Virus Infection

The Epstein–Barr virus (EBV) was discovered 36 years ago by electron microscopy of cells cultured from Burkitt's lymphoma tissue by Epstein, Achong, and Barr.1 Four years later, in 1968, EBV was shown to be the etiologic agent of heterophile-positive infectious mononucleosis.2 EBV DNA was detected in tissues from patients with nasopharyngeal carcinoma in 1970.3 In the 1980s, EBV was found to be associated with non-Hodgkin's lymphoma and oral hairy leukoplakia in patients with the acquired immunodeficiency syndrome (AIDS).4,5 Since then, EBV DNA has been found in tissues from other cancers, including T-cell lymphomas and Hodgkin's disease.6,7 EBV is . . .

Association between Epstein-Barr virus and Burkitt's lymphoma in Taiwan.

The association between Epstein-Barr virus (EBV) and Burkitt's lymphoma (BL) in Taiwan is not clear. In this study, the authors attempted to determine the frequency of the occurrence of EBV infection in patients with BL in Taiwan. A retrospective study was performed using a nonisotopic in situ hybridization technique to detect the presence of EBV-encoded small RNAs (EBERs) in paraffin embedded BL tissues. Tissues of other types of B-cell non-Hodgkin's lymphoma (NHL) were used as controls. Immunohistochemical staining was performed to examine the presence of an EBV-encoded protein, latent membrane protein (LMP), and p53 in specimens. EBERs were detectable in 10 of 18 BL specimens. It was present in the cervical lymph nodes (LNs) in six of the seven cases of cervical tumors, in the maxillary region in one case, in one of two cases of axillary LNs, and in the abdominal tumors in two of the seven cases of intraabdominal disease. EBER positive cells were diffusely present in all tumors except in one abdominal BL, in which only a few EBER positive cells were scattered in a small part of the tumor. EBER positive cells were not detected in the case with BL in an inguinal LN and in the seven cases with intraabdominal tumors. Immunohistochemical studies showed that LMP and p53 were expressed in 3 and 4 of the 18 cases, respectively. In another 20 NHLs in peripheral LNs, EBERs were detectable in only 1 case of diffuse large cell histology with numerous reactive T cells in which only large tumor cells expressed EBERs and LMP. EBERs were not detected in any of the ten cases of extranodal NHL. In Taiwan, EBV is frequently associated with BL occurring outside the abdomen but rarely with intraabdominal BL. The overall association between EBV and BL in Taiwan is intermediate compared with other regions of the world. These results support the theory that the frequency of EBV associated with BL is influenced by the endemicity of EBV and/or the socioeconomic status of a country.

Virus-Cell Interactions in a Natural Killer-Like Cell Line From a Patient With Lymphoblastic Lymphoma

Lymphoproliferative disorders involving Epstein-Barr virus (EBV) infected natural killer (NK) cells are reported with increasing frequency, but the nature and role of EBV infection in these cells remains undefined. In this study, we have investigated virus-cell interactions in the EBV-positive YTN10 cell line, an NK-like cell line established from a patient with lymphoblastic lymphoma. Low level expression of the EBV receptor CD21 molecule was detected by FACS and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Immunoblotting and RT-PCR analysis identified a latency II pattern of EBV gene expression, consisting of EBNA-1 transcription from the Qp promoter, in the absence of other EBNA gene expression, and accompanied by LMP-1 and LMP-2A expression. The EBV genome was present in episomal form and there was evidence for lytic viral replication. This latency pattern is typical of EBV gene expression in nasopharyngeal carcinoma and Hodgkin's disease, and differs from the full spectrum of EBV latent gene expression in most posttransplant lymphoproliferative disorders and from the restricted EBNA-1 expression in Burkitt's lymphoma tissues. The interaction between EBV and NK cells described here has important implications for the pathogenesis and treatment of EBV-infected NK malignancies.

Suppression of Burkitt's lymphoma tumorigenicity in nude mice by co-inoculation of EBV-immortalized lymphoblastoid cells.

EBV-immortalized B-lymphoblastoid cell lines (LCL) inoculated s.c. into T-cell-deficient nude mice regress completely after a short initial growth period. We tested whether the putative host response underlying this phenomenon might also be directed against progressively growing Burkitt's lymphoma (BL) tumors in nude mice. Outgrowth of BL tumors was suppressed when cells of the highly tumorigenic BL cell line BL 60 were mixed with cells of the autologous LCL IARC 277 before s.c. inoculation into nude mice. Even when the cells were inoculated separately and simultaneously into contralateral flanks of the mice, regression of initially growing BL tumors could be observed, albeit with reduced frequency and dependent on the dose of LCL cells. Tumor growth of BL 60 cells could also be suppressed by co-inoculation with the non-autologous LCL IARC 174 and IARC 277 cells could suppress growth of the non-autologous BL cell line Eli. Pronounced infiltration with murine (m)CD-11b-positive mouse macrophages and mCD-8a-positive mouse lymphoid cells, most probably natural killer cells, was seen in histological tissue sections of regressing BL 60 tumors when LCL cells were inoculated contralaterally. In regressing BL tumors, these mouse cells were present not only in necrotic areas but also in vital BL tissue, indicating that infiltration of mouse cells had taken place before the development of necrosis. Since tumor-infiltrating mouse cells can be activated at least by some human cytokines, we measured cytokine production of BL 60 and IARC 277. High amounts of IL 6 and IL 10 were produced by the LCL cells, whereas IL-6 and IL-10 production by the BL 60 cells was beyond or close to the detection threshold. In addition, IL 8 was secreted up to 5-fold more by the LCL than by the BL cells. The results presented here thus suggest a host response of the nude mouse, which is triggered by cytokines released from the LCL but, once induced, is directed also against BL cells.

Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt's lymphoma.

Deregulation of the proto-oncogene MYC by specific chromosomal translocations has been shown to be essential but not sufficient for the development of Burkitt's lymphoma (BL). To identify other genes which either mark important steps in tumorigenesis or which reflect the cellular differentiation state of BL cells we have compared tumor cells to immortalized lymphoblastoid B cells by subtractive hybridization. We have identified a complementary DNA clone which encodes a novel member of the superfamily of GTP-binding (G) protein-coupled receptors, designated BLR1. The corresponding mRNA is expressed in BL and lymphatic tissues but not in other cell lines either of the B cell lineage or of other hematopoietic or non-hematopoietic origin. This exclusive expression of BLR1 and the oncogenic potential of this receptor class supports the hypothesis that BLR1 exerts a regulatory function in BL lymphomagenesis and/or B cell differentiation. Moreover, the protein sequence is highly related to that of receptors for the cytokine interleukin (IL)-8 and other neutrophil chemoattractants. We conclude that BLR1 may represent a potential candidate involved in the process of physiologic trafficking, cell-cell interactions, and activation of mature B lymphocytes in lymphatic tissues.

Restricted Epstein-Barr virus protein expression in Burkitt lymphoma is due to a different Epstein-Barr nuclear antigen 1 transcriptional initiation site.

Epstein-Barr virus (EBV) expresses six nuclear antigens (EBNAs) and three integral latent membrane proteins (LMPs) in latently infected growth-transformed B lymphoblastoid cell lines (LCLs). In contrast, EBV protein expression in Burkitt lymphoma tissue or in newly established Burkitt lymphoma cell lines is frequently restricted to the EBV genome maintenance protein, EBNA-1. EBNA-1 expression in the absence of other EBNAs and LMP-1 has been an enigma since, in LCLs, all EBNA mRNAs are processed from a single transcript. We now show that the basis for restricted EBV expression in Burkitt lymphoma cells is selective EBNA-1 mRNA transcription from a hitherto unrecognized promoter that is 50 kb closer to the EBNA-1-encoding exon than previously described EBNA-1 promoters. Infected cells with EBNA-1-restricted expression could preferentially persist in vivo in the face of EBV-immune T-cell responses, which are frequently directed against other EBNAs and are also dependent on LMP-1 expression.

Isolation of a herpesvirus-specific DNA polymerase from tissues of an American patient with Burkitt lymphoma.

A DNA polymerase (DNA nucleotidyltransferase) has been partially purified from a neck mass of an American patient with Burkitt lymphoma and separated from the cellular DNA polymerases. The molecular weight of the enzyme was approximately 90,000. The enzyme differs from the cellular DNA polymerases, but resembles herpes-virus-induced DNA polymerase in its primer template preference, high monovalent cation requirement for activity, and sensitivity to phosphonoacetate. Enzyme activity was inhibited specifically by an antibody directed against herpes-simplex-virus-induced DNA polymerase but not by antibodies directed against DNA polymerase alpha of HeLa cells and DNA polymerase gamma of a normal human lymphoblast cell line, NC37. Although serum of the patient with Burkitt lymphoma contained high Epstein-Barr virus titer, addition of the serum to the assay mixture did not have any effect on the activity of Burkitt lymphoma DNA polymerase. Tissues from spleen and liver of the patient with Burkitt lymphoma did not contain the herpes-virus-induced DNA polymerase. Detection of the herpes virus polymerase in the Burkitt lymphoma tissue provides additional evidence for the association of Epstein-Barr virus with this malignancy.

Non-random chromosome gains in human lymphoblastoid cell lines

HUMAN lymphoblastoid cell lines derived from the peripheral blood lymphocytes of healthy donors are commonly diploid when examined in the early months after establishment but acquire chromosome abnormalities on prolonged culture. Other lines, notably those derived from Burkitt's lymphoma tissue, may display chromosome aberrations from the outset1–5. A partial translocation 8q−14q+ has been demonstrated in the majority of Burkitt lymphoma-derived lines5,6 and data on the karyotypes of some human tumours suggest that non-random gains and/or losses of chromosomes may be a feature, in particular, of certain leukaemias and lymphomas7–13. As the emergence of an aneuploid clone from a previously diploid lymphoblastoid line may be associated with other changes suggesting the development of a more ‘malignant’ phenotype14, it is relevant to compare the chromosome aberrations detected in such lines with the human tumour data. We have therefore undertaken a study of banded karyotypes of eighty EB virus-carrying human lymphoblastoid lines. The first stage of this analysis is concerned only with gains and losses of whole chromosomes or chromosome arms and the data presented here establish that, considering all the lines together, there have been nonrandom gains of five autosomes (numbers 3, 7, 8, 9 and 12) and of the sex chromosomes.

Detection of an antigen associated with acute leukemia.

Antiserums to a purified cell membrane component from a Burkitt's lymphoma tissue culture cell line were produced in rabbits. These antiserums were cytotoxic to peripheral white blood cells from 8 of 15 patients with acute leukemia and 5 of 41 relatives, but not to peripheral white blood cells from leukemia patients in clinical remission or from normal individuals. These antiserums appear to be detecting an acute leukemia associated antigen or antigens.

Herpes-like Epstein-Barr virus in leprosy.

THE herpes-like virus (HLV), first detected1 in lymphoid cell cultures derived from Burkitt's lymphoma tissue, has been suggested as the causative agent of diseases such as Burkitt's lymphoma itself2, infectious mononucleosis3,4, carcinoma of the posterior nasal space5 and sarcoidosis6. It appears that between 70% and 85% of normal adults have antibodies against HLV7,8 suggesting that the agent is widespread, but it is also clear that the pathogenic nature of the virus is uncertain9.

Morphologic and cytogenetic studies in vitro of surface-adherent lymphoreticular cells derived from Burkitt lymphoma tissue.

AbstractA line of lymphoreticular cells (AL‐1‐G) that can adhere to glass surfaces has been derived from suspension cultures of Burkitt lymphoma cells (AL‐1). By light microscopy, the predominant cell type in the AL‐1‐G cultures has features of a neoplastic histiocyte and ultrastructurally these cells have more elaborate development of cytoplasmic organelles, such as elements of the Golgi complex and mitochondria, than the predominant cell types in the suspension cultures of AL‐1. Cytogenetically, the percentage of polyploid cells in the AL‐1‐G cultures increased progressively from 39% in 1967 to 100% in 1968 and 1969, while the percentage of polyploid cells in the suspension cultures remained between 12 and 24% during the same period. No herpesvirus or any other type of viral particle was found in the AL‐1‐G cultures on repeated electron microscopic examination.

Some factors affecting membrane immunofluorescence reactivity of Burkitt lymphoma tissue culture cell lines.

AbstractMembrane immunofluorescence (MIF) reactivity of Epstein‐Barr virus (EBV) carrying cultured Burkitt's lymphoma (BL) cells with BL sera shows certain fluctuations from time to time, even with standard reference sera and apparently constant tissue culture conditions. In order to determine the culture conditions that are critical for this variation and to establish the optimal situation favoring the production of highly reactive cells, we studied two established BL‐derived cell lines under different conditions of initial cell number, cell density per unit volume or bottom unit area in the culture vessel, depth of the fluid phase, proportion of conditioned medium, etc. Although the two lines behaved somewhat differently, they showed better reactivity when maintained at a high concentration, as long as crowding did not entirely prevent cell multiplication. Under identical conditions of medium supply and depth of the fluid phase, more closely packed cells within a smaller bottom area of the culture vessel exhibited a better MIF reactivity than more dispersed cells. Under different conditions of growth, there was an inverse relationship between the rate of cell multiplication and the percentage of viable MIF‐positive cells. Optimal conditions with steady, high‐level reactivity have been worked out for both lines, but were different with regard to quantitative detail.

Production of Immunoglobulins and Their Subunits by Human Tissue Culture Cell Lines

Summary Tissue culture cell lines established from Burkitt's lymphoma tissue or from peripheral blood of patients with various forms of leukemia were examined for their ability to incorporate C14-labeled amino acids into serum proteins. Autoradiographs of immunoelectrophoretic patterns showed that the great majority of these cell lines synthesized immunoglobulin(s). The use of antisera specific for individual classes and types of immunoglobulins demonstrated a wide variety in the immunoglobulins produced by the various cell lines. The characteristics of the globulin produced by any given cell line were remarkably constant. Some lines synthesized more than one class of immunoglobulin, often IgM and IgG, though usually only one type of L-chain was produced. The immunoglobulins were frequently of restricted electrophoretic mobility. Analysis of culture fluids in the ultracentrifuge showed that in many cases subunits rather than intact immunoglobulin molecules were produced. Burkitt's cell lines produced proteins of µ- or L-chain specificities, or combinations of these two, but never proteins with other H-chains. Cell lines derived from patients with lymphatic or myeloid leukemia, however, formed proteins of α-, µ- or γ-, and of L-chain specificities.