Article

Comparison between growth characteristics of an Epstein-Barr virus (eBV)-Genome-Negative lymphoma line and its EBV-Converted subline in vitro

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Abstract

The GC-BJAB cell line, which carries the Epstein--Barr virus (EBV), was derived from an EBV-genome-negative lymphoma line (BJAB) by EBV infection in vitro [G. B. Clements, G. Klein, and S. Povey (1975) Int. J. Cancer, in press]. Both lines grow at a similar rate at 37 degrees but they differ at other temperatures. BJAB grows well at 34 degrees, 37 degrees, and 39 degrees. GC-BJAB grows at 37 degrees and 39 degrees, but grows poorly at 34 degrees. At 37 degrees, GC-BJAB cultures can be maintained at the viable state for a long time after having reached saturation density (approximately 10(6) cells per ml). In contrast, BJAB cultures die very soon after having attained similar maximum density. Since the identity of the two cell lines has been critically established [Clements et al.; E. Svedmyr and M. Jondal (1975) Proc. Nat. Acad. Sci USA 72, 1622--1626; G. Klein, to be published; J. Zeuthen, personal communication] the remarkable differences in their growth properties must be attributed to the EBV genome.

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... It was therefore of interest to determine whether C-type particles could also be induced in the BJAB-1 EBV-DNA-negative lymphoblastoid cell line derived from a patient with typical African Burkitt's lymphoma (Menezes et al., 1975). Infection of BJAB-I cells with EBV from marmoset lymphoblasts (Miller et al., 1972) converted them into EBV-DNA-positive cells, and enhanced their ability to proliferate in vitro (Steinitz & Klein, 1975). As controls we used lymphoblastoid cell lines derived from a patient with infectious mononucleosis, and lines which developed spontaneously from blood samples of non-leukaemic persons with ataxia telangiectasia (AT) and Down's syndrome. ...
... EBV-DNA-negative BJAB-1 cell line (Menezes et at., 1975), derived from lymphoblasts of an African patient with Burkitt's lymphoma, and the GC/BJAB-1 and AW/BJAB-1 cell lines, were kindly supplied by Professor George Klein, Karolinska Institute, Stockholm. The GC/BJAB-1 and AW/BJAB-1 sublines, that are EBV-DNA positive and produce the virus (Steinitz & Klein, 1975;Klein et al., 1975;Clements et al., 1975) resulted from infection of BJAB-1 cells with EBV from the B95-8 marmoset cell line (Miller et al., 1972). These cells were grown in RPMI 1640 medium (Grand Island Biological Co.) containing 10% heat-inactivated foetal calf serum (GIBCO) as suspension cultures in 250 ml glass bottles at an initial concentration of 4-5 x 105 cells/ml. ...
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Retrovirus-like particles can be recovered by arginine deprivation from the BJAB-1 Epstein-Barr virus (EBV) negative cell line derived from an African patient with typical Burkitt's lymphoma. These particles resemble retroviruses in their morphology and in their physicochemical properties. Particles with a similar morphology were obtained from derivative cell lines established by infecting BJAB-1 cells with EBV. On the other hand, retrovirus-like particles could not be induced in EBV-DNA-positive lymphoblastoid cell lines derived from non-leukaemic patients with ataxia telangiectasia and Down's syndrome and from a patient with infectious mononucleosis. Images Fig. 2 Fig. 3 Fig. 4
... BJAB est une lignée cellulaire humaine de lymphome type B, dérivée d'un cas africain exceptionnel de lymphome de Burkitt établie par Menezes et al. en 1975(Steinitz et Klein, 1975. Cette ligne cellulaire a été fournie par l'Institut Pasteur de Guyane (V. ...
... Les rongeurs sont infestés soit avec du sang parasité, soit avec des sporozoïtes. (Steinitz et Klein, 1975;Diller et al., 2005;Bertani, 2006 (Bertani, 2006). ...
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... Conceivably, cells that use the type III program may have an selective advantage in competition with the original EBV-negative BL cells during the prolonged conversion procedure. Our previous study 27 showed that type III BL lines are more resistant to low serum concentration and high saturation density than EBV-negative BL lines. ...
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... Lysis buffer was composed of 100mM Tris-HCl (pH8.0), 300mM NaCl, 0.04% sodium azide, 0.2% sodium dodecyl sulphate (Sigma-Aldrich), 2% nonidet P-40 (BDH), 1% sodium deoxycholate (BDH), 2mM EDTA, 100mg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich), and 1mg/ml leupeptin (Sigma-Aldrich) negative (Steinitz et al., 1975). BJAB cells were a kind gift from Dr. Rolf Renne (University of Florida Shands Cancer Center, Florida, USA). ...
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The original dissertation included a number or articles which were excluded from the digital file for copyright reasons. This is a list of the articles:
... Lysis buffer was composed of 100mM Tris-HCl (pH8.0), 300mM NaCl, 0.04% sodium azide, 0.2% sodium dodecyl sulphate (Sigma-Aldrich), 2% nonidet P-40 (BDH), 1% sodium deoxycholate (BDH), 2mM EDTA, 100mg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich), and 1mg/ml leupeptin (Sigma-Aldrich) negative (Steinitz et al., 1975). BJAB cells were a kind gift from Dr. Rolf Renne (University of Florida Shands Cancer Center, Florida, USA). ...
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In Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) the role of EBV in the translocation and deregulation of the MYC oncogene remains unknown. By utilizing an EBV-negative BL (BJAB) and several EBV-positive sublines derived from it by in vitro infection, it was possible to show that the presence of the virus was associated with altered expression and copy number of MYC. In the EBV-negative BJAB line, the level of MYC transcripts declined progressively as cells approached the stationary phase of growth. In contrast, in EBV-infected BJAB cells MYC expression remained elevated as cells entered stationary phase. This effect on MYC expression was reversibly linked to the presence of the virus. Furthermore, following EBV infection of BJAB cells by two different strains of EBV, amplification of MYC in association with the appearance of a homogeneously staining region on chromosome 8 at the mapped location of MYC had occurred. These studies suggest that both the deregulation of MYC transcription and the chromosomal rearrangement in the region of the MYC locus in this B-cell line may have occurred as a result of EBV infection.
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Fifteen of 16 lymphoma-derived cell lines and the Faji and P3HR1 cell lines were characterized with regard to certain surface markers, particularly immunoglobulins, complement receptors, Epstein-Barr virus (EBV) receptors, and Fc receptors. Ten lines positive for EBV nuclear antigen (EB-pos) were stained weakly or not at all by antihuman immunoglobulin fluorescein isothiocyanate conjugates, whereas EBV nuclear antigen negative (EB-neg) cell lines stained brightly. EB-pos lines frequently manifested Fc receptors, particularly for 7S antibody, whereas EB-neg lines did not. Receptors for the C3b component of complement and for EBV, which correlated significantly with each other, were expressed to a much lesser extent by EB-neg lines than by EB-pos lines. These findings are pertinent to an understanding of the infrequent association of this virus with American undifferentiated lymphomas of the Burkitt's and non-Burkitt's types.
Article
EBV-conversion of the originally EBV-negative Ramos and BJAB lymphoma lines has led to an increased frequency of C3 receptor positive and a decreased incidence of Fc receptor positive cells in the population. The concentration of C3 receptors per cell has increased as well. This finding supports the view that EBV-conversion leads to a modification of surface architecture which, in turn, may explain the changes in nutritional and other biological properties.
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Lactoperoxidase iodination and two-dimensional electrophoresis of the labeled proteins have demonstrated well-characterized cytoskeletal proteins (actin and tubulins) on the surface of human lymphocytes undergoing blastogenic transformation and of certain malignant human cells. Such proteins could not be detected on the surface of normal resting human lymphocytes. The most prominent cytoskeletal protein identified on the surface membrane of mitogen-transformed T and B lymphocytes was actin. In Epstein-Barr virus genome-positive Burkitt's lymphoma and lymphoblastoid cell lines and in two leukemia cells, the major iodinated membrane protein components were actin and alpha 1-, alpha 2-, and beta-tubulins. These proteins were firmly connected to the cytoplasmic skeleton and could not be removed by Triton X-100. Concurrent immunofluorescence studies with specific antibodies and F(ab')2 fragments confirmed the appearance of cytoskeletal components on the biochemical data, and indicated that such cytoskeletal proteins formed distinctive patterns on the cell surface, ranging from small patches to large projections. Five-hour labeling with [35S]methionine indicates that all such cells released large quantities of labeled actin and tubulins into the culture medium. These materials were not readsorbed to the membrane surfaces of the cells.
This talk will be limited to a consideration of lymphoma and leukemia development (or certain types) in mice and men where there is extensive evidence for the role of the specific genetic changes recognizable at the chromosomal level. To start with the conclusion, it is clear that lymphoma development can be initiated by a variety of agents. In all probability, the initiation process creates long-lived preneoplastic cells, which are frozen in their state of differentiation and capable of continued division. These cells constitute the raw material for the subsequent cytogenetic evolution that converges towards a common, distinctive pattern. The nature of this pattern as it appears in the overt lymphomas depends on the subclass of the target lymphocyte rather than on the initiating (“etiologic”) agent.
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In this brief review we will discuss some recent trends in work on regulatory mechanisms involved in the selective expression of differentiated markers in human B-cell lines as well as the types of controls involved in the expression and function of Epstein-Barr virus (EBV) associated markers in human cells. A problem of special interest in this connection is the relation of the expression of EBV functions to the expression of “transformation”.
Article
Human B cell lymphoma and murine T cell leukemia can be initiated by several agents. The present paper formulates some thoughts on the role of cytogenetic changes in the subsequent neoplastic process. Initiation creates long-lived preneoplastic cells. In some respects, they are comparable to in vitro-transformed ("immortalized") cell lines that maintain a diploid karyotype and are not tumorigenic in vivo. The development of a tumorigenic ("autonomous") clone is dependent on additional changes at the genetic level. In human B and murine T cell lymphoma, there are characteristic nonrandom chromosomal changes. The 14q+ marker appears to play a key role in human B cell lymphomas. The reciprocal 8;14 translocation in Burkitt lymphoma is a specialized subclass within this category. In murine T cell leukemia, trisomy 15 is the predominant change. The clustering of these nonrandom changes to tumors derived from a certain cell type rather than to tumors induced by a given etiological agent has important implications for the understanding of the genetic control of cellular responsiveness to growth-regulating forces in vivo.
Article
In vitro converted, Epstein-Barr (EBV) genome-carrying cell lines showed a reduced ability to shed surface-bound antibodies, compared to their EBV-free parental B lymphoma lines. On the other hand, antibody degradation was higher with EBV-positive cells. Analysis of antibody-coated single cells revealed that cells which failed to redistribute or cap their ligand-bound surface components at 37 degrees C, were also capable of shedding bound antibody. This suggests that capping and shedding are essentially independent phenomena, although both may be influenced by the same virally induced change in the lateral mobility of membrane constituents.
Article
We have compared the EBV-receptor concentration of two originally EBV-negative human B-cell lymphoma lines, after in vitro conversion with the transforming B95-8 or the cytopathic P3HR-1 EB-viral substrain, respectively, into permanent EBV-carrying sublines. Receptors were measured by the quantitative EBV-absorption bioassay of Sairenji and Hinuma (1973). EBV receptor concentration of all P3HR-1 virus-converted sublines was significantly reduced, in comparison with the B95-8 virus-converted sublines. This suggests that cells with a low receptor concentration are more likely to survive the initial infection with the P3HR-1 viral harvest. The results further confirm the biological differences between the two EBV substrains.
Article
Exposure of the two EBV-negative Burkitt lymphoma lines, BJAB and Ramos, to the abortively cytopathic P3HR-1 substrain of EBV led to an increased expression of C3 receptors within the first twelve weeks. At the twelfth week, 100% of cells carried a high concentration of C3 receptors in both lines. Compared with the receptor pattern of BJAB and Ramos after chronic virus exposure it was seen that after the twelfth week some of the C3 receptors vanished while a certain number of Fc receptors reappeared. These changes in the surface markers are regarded as part of the multiple changes found during exposure to P3HR-1 virus. Apparently, the superinfection initiates a progressive maturation of the cells and the increase of C3 receptors is regarded as an expression of a ‘switch to the right’ in the B-cell differentiation pathway.
Chapter
Epstein-Barr Virus (EBV) transforms human B lymphocytes so that they grow as continuous cell lines. B lymphocyte lines transformed in vitro by EBV (LCL) and B lymphocyte lines derived from EBV positive and EBV negative Burkitt’s lymphomas (BL) release a 15–25 K growth enhancing supernatant (GE-S) factor(s) which functions similarly to macrophage derived Interleukin-1 (IL-1). GES and purified IL-1 were compared and both enhanced growth of LCL and BL similarly. Preliminary data show growth of Ramos, an EBV negative BL, is not changed by GE-S or 1L-1, whereas growth of EBV-converted Ramos sublines is enhanced by the addition of either. These initial findings suggest that EBV infection induces the development of growth factor sensitivity in lymphoma cells.
Chapter
In this review, we will discuss some aspects of current work related to the control mechanisms involved in the selective expression of B-cell differentiated markers, as well as the control mechanisms involved in the expression and function of Epstein-Barr virus (EBV) associated markers in human cells.
Chapter
Besides Burkitt’s lymphoma (BL) nasopharyngeal carcinoma (NPC) represents a unique model for studying the relationships between Epstein-Barr virus (EBV) infection, immune response, and neoplastic transformation. The association of EBV infection and NPC is even closer than in BL: all nonkeratinizing NPC exhibit significantly elevated anti-EBV antibody titers while extra-African BL only sporadically are seropositive.
Chapter
A wide variety of DNA viruses and a more restricted family of RNA viruses can transform normal cells in vitro. Transformation means either immortalization and/or the appearance of certain phenotypic changes. Although it has been often inferred that in vifro transformation can be essentially equated with malignant transformation, increasing evidence indicates that the latter, reflected by tumorigenicity in vivo, requires additional cytogenetic changes. The evidence will be reviewed for EB virus-associated human malignancy (Burkitt's lymphoma) and the role of the 14q + translocation marker in human B-cell neoplasia. These findings point to an initiating role of viral transformation, reflected by in vitro immortalization, followed by a cytogenetic evolution where chromosome 14- associated changes are essential for the liberation of B lymphocytes from superimposed controls. The contrast of tissue-associated, specific chromosomal changes that bring about malignant transformation after the initiating impact of different agents will be illustrated experimentally for murine T-cell lymphoma. Here, X-ray, DMBA and different virus (RadLV, Gross virus)-induced T lymphomas show the same chromosomal change: trisomy 15. It may be questioned whether viral transformation can ever lead to neoplasia in the absence of subsequent cytogenetic changes.
Chapter
In 1964, M.A. Epstein and Barr (1964) and Pulvertaft (1964a) reported the first successful attempts to establish Continuous lymphoblastoid-cell lines (LCLs) from expiants of Burkitt lymphoma (BL). Subsequent electron-microscopic examination of thin sections of the EB1 Cell line revealed virus particles that were morphologically similar to the herpesvirus group (M.A. Epstein et al., 1964a). In lines derived shortly thereafter from other BL tumors (M.A. Epstein et al., 1964b; Stewart et al., 1965; Rabson et al., 1966; Minowada et al., 1967), viral particles resembling those found in EB1 cells were seen. Such particles were not confined to lines originating from BL tissue, but were also seen in a fraction of cells in lymphoid lines established from patients with various malignancies (Armstrong, 1966; Moore et al., 1966; Zeve et al., 1966), from patients with infectious mononucleosis (IM) (Pope, 1967; Diehl et al., 1968), and from apparently normal individuals (Moore et al., 1967).
Chapter
Transformation of human cells by Epstein-Barr virus (EBV) in vitro is of considerable interest for several major reasons. It provides a basic virologic tool in the form of a practical assay of infectivity of the virus. It allows detailed study of virus-cell relationships, which can be expected to have at least some parallels with the activity of the virus in vivo. It is a useful model for the study of control of cell proliferation and, in addition, cell lines may be readily established from specific donors to provide material for the study of genetic or other diseases.
Chapter
Lymphoid cell lines have been invaluable tools not only in studies of the biologic properties of Epstein-Barr virus (EBV), but also in the fields of oncology, hematology, immunology, genetics, and cell biology. In 1964, Pulvertaft (1964 a) and Epstein and Barr (1964) independently described the first cell lines from tumor biopsies of patients with Burkitt’s lymphoma (BL). Before this, attempts to cultivate normal and malignant hematopoietic tissue in vitro had usually led to a gradual deterioration and subsequent death of the various cell types within weeks or months (Osgood, 1958; Reisner, 1959). Only rarely had permanent cell lines been established from leukemic blood (Osgood and Broke, 1955) and bone marrow (Benyesh-Melnick et al., 1963). In the latter report, the sudden outgrowth of permanent cell lines with a lymphoblastoid morphology was described at a low frequency in fibroblastoid monolayers several weeks after initiation of bone-marrow cultures derived from children with leukemia, infectious mononucleosis (IM) and hemolytic anemia. This remarkable event was termed “lymphoblastoid transformation of fibroblastic bone-marrow cultures.” The meaning of the term “lymphoblastoid transformation” here was thus different from the “lymphoblastoid transformation” used to describe the morphologic changes of normal lymphocytes after exposure to phytohemagglutinin (PHA) (Nowell, 1960).
Article
Human lymphoid lines derived from normal or neoplastic B cells were assayed for insulin binding. 125I-Labeled insulin was allowed to bind to cells. Bound radioactivity which was inhibited with unlabeled insulin was regarded as specific binding. Among 46 lines tested, 43 bound more insulin than normal peripheral B lymphocytes. The majority of the lines resembled activated lymphocytes, with regard to their insulin binding. More mature cells represented by EBV-transformed lines of normal origin, bound more insulin than the less differentiated Burkitt lymphoma lines. However, even the latter bound significantly more insulin than peripheral blood lymphocytes.
Article
We have compared the growth in agarose medium of two EBV-negative Burkitt lymphoma lines, BJAB and Ramos, and that of their EBV-converted derivatives. Optimal conditions for growth in agarose medium are described. The original EBV-negative lines can grow in agarose to a high efficiency, provided a critical level of cell concentration is attained. Below this level, there is no growth at all. EBV-converted sublines form colonies at a much lower cell concentration, and their maximal plating efficiency is higher. These differences may be related to a super-transformation state induced in these cells by EBV.
Article
Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for binding and complement mediated lysis by Campath-1. Expression of the cell surface antigen was determined with fluorescein isothiocyanate conjugated Campath-1, by ultraviolet microscopy and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between Campath-1 binding and the stage of lymphoid-cell differentiation. The null and T lines bound Campath-1 weakly and the fluorescence intensity was low. In the B lines there was a gradual increase in labelling correlating with the stage of differentiation. The redistribution pattern of Campath-1 on the membrane of null and T lines was in the form of rings and small caps whereas that on the B lines was that of moderate patches. Complement mediated cytolysis occurred with the majority of the cell lines, but did not correlate with the extent of surface antibody binding nor with the stage of lymphoid differentiation. The recovery and proliferative capacity of the residual cells after treatment with Campath-1 and complement was low for the null and T lines. This was variable for the B lines, but some did not regain proliferative capacity, whereas others recovered after treatment. The present results suggest that Campath-1 may have considerable utility in marrow depletion of residual. leukemic cells, more specifically of null and T-cell origin, prior to autologous transplantation. Determination of both the sensitivity of the patient's leukemic: cells to cytolysis and the recovery of proliferative capacity of Campath-1 resistant cells may contribute essential information concerning the possible efficacy of purging with this antibody in patients with significant marrow involvement prior to transplantation.
Article
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Epstein-Barr nuclear antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B cells. EBNA1 transactivates viral promoters for genes that are necessary for immortalization when it is bound to a cluster of 20 cognate binding sites, termed the family of repeats. A region of EBNA1 from amino acids (aa) 40 to 89, termed linking region 1 (LR1), has been identified previously as being sufficient for transactivation. LR1 contains two domains that are conserved in the EBNA1 orthologs of other gamma herpesviruses. The first of these, termed unique region 1 (UR1), corresponds to aa 65 to 89 of EBNA1. UR1 is necessary for transactivation and contains a conserved recognition site for cyclic AMP-dependent protein kinase (PKA), corresponding to serine 78 of EBNA1. We have pharmacologically modulated PKA activity to determine if PKA controls EBNA1's ability to transactivate. Our results indicate that PKA activators and inhibitors do not affect transactivation by EBNA1. In addition, site-directed mutagenesis demonstrates that transactivation is not influenced by the phosphorylation status of serine 78 in the UR1 domain. The second conserved domain within LR1 is a glycine-arginine repeat, corresponding to aa 40 to 54 of EBNA1. This domain, termed ATH1, functions as an AT-hook, a DNA-binding motif found in architectural transcription factors such as HMGA1a. We demonstrate that deletion of the ATH1 domain decreases EBNA1 transactivation ability, which is consistent with a transcriptional role for ATH1. Furthermore, transactivation is restored when ATH1 is replaced by equivalent AT-hook motifs from HMGA1a. Our data strongly indicate a role for AT-hooks in EBNA1's ability to transactivate, a function necessary for EBV to immortalize naïve B-cells.
Article
Two EBV-negative lymphoma lines of human B-cell origin, BJAB and Ramos, were compared with altogether six of their in vitro EBV-converted, EBNA- and EBV-DNA-carrying sublines (four of Ramos and two of BJAB derivation). All converted lines closely resembled the parental line with regard to karyotype and HL-A and B antigen typing. Induction of EBV antigens (EA and VCA) by P3HR-1 virus superinfection was either similar in the converted and the negative lines, or somewhat increased in certain converted lines. These findings argue against a simple, virally determined repressor model and emphasize the role or cellular controls in restricting the EBV cycle in virus-carrying B-lymphocyte lines of human origin. IUdR inducibility varied in the different converted lines. There was a possible relationship between average number of EBV-genome equivalents per cell and inducibility. Converted sublines did not differ from the original negative lines with regard to surface immunoglobulin and Fc receptors. There was a dramatic increase in complement-consuming ability, however, following EBV conversion. Among the EBV-positive lines, there was a linear relationship between complement-consuming and EBV-receptor activity, the latter measured by a quantitative absorption test.
Article
Cells of two EBNA (Epstein-Barr virus nuclear antigen)-negative human lymphoma cell lines, BJAB and RAMOS, were infected with two strains of Epstein-Barr virus (EBV). In two different experiments, B95-8 virus-infected BJAB cells revealed a gradually increasing number of EBNA-positive cells. Twenty weeks after infection almost 100% of the cell population expressed this antigen. In contrast, it has not so far been possible to convert RAMOS cells into an EBNA-positive cell line. The initial proportion of 35% EBNA-positive cells declined to about 10% 20 weeks after infection. The development of EBNA-positive multinuclear giant cells was a characteristic feature of infection with B95-8 virus. EA (early antigen) and VCA (virus capsid antigen) appeared in less than 0.1% of the cell population after induction with IUdR only. Infection of BJAB and RAMOS cells with P3HR-1 virus finally resulted in both cases in EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive cells remained below 1% during the first 6 to 8 weeks. A sudden increase occurred thereafter, bringing the number of EBNA-expressing cells to almost 100% within the following 4 weeks. During this period, BJAB but not RAMOS cells revealed a small number of EA- as well as VCA-positive cells (less than 0.1%). Thus, reinfection by spontaneously released virus may explain the sudden increase in EBNA-positive BJAB cells. Two distinct patterns of EBNA staining in P3HR-1 virus-infected cells were observed. They may suggest a genetic heterogeneity of this virus preparation.
Article
Independently EBV-converted, viral genome carrying sublines of the originally EBV negative Ramos and BJAB lymphoma lines showed decreased serum dependence, in comparison with the progenitor lines. The virus negative lines could not grow in 10% dialysed FCS, unless reconstituted with the dialysate. The converted lines grew well on dialysed serum. Seeding of the EBV negative lines with larger inocula enabled them to “take off” on dialysed serum as well. The EBV converted lines were able to form colonies in soft agar, whereas the original negative lines failed to do so.
Article
Epstein-Barr virus (EBV) DNA (82 genome equivalents/cell) and EBNA (EBV determined nuclear antigen) were found in tumor tissue from an Israeli Arab child with Burkitt's lymphoma. A lymphoma cell line (LB-132) carrying the EBV genome was established from tumor tissue from this patient. This line resembled other previously established and characterised Burkitt's lymphoma lines. Our results suggest that EBV—carrying lymphoma which occurs endemically in Africa occurs sporadically throughout the world.
Article
Most human lymphoid cell lines contain multiple copies of circular, nonintegrated Epstein-Barr virus (EBV) DNA molecules as well as viral DNA sequences with properties of integrated DNA. The physical state of the EBV DNA in a human lymphoma line that only contains one virus genome equivalent per cell has now been studied by three different methods, neutral CsCl density gradient centrifugation, actinomycin D-CsCl gradient centrifugation, and Hirt fractionation. This cell line, AW-Ramos, has been obtained by EBV infection in vitro of the apparently EBV-negative Ramos lymphoma line. The results indicate that the EBV DNA in AW-Ramos is present exclusively in a linearly integrated form. Similar data were obtained with two other EBV-converted sublines of Ramos cells.
Article
Activation of the alternative complement pathway by human B cell lymphoma lines is correlated with the presence of Epstein Barr virus (EBV) in the cell genome. EBV-negative B cell lymphoma lines produce little activation of the alternative pathway as measured either by C3 deposition on the cell surface or C3 conversion and consumption of alternative pathway activity in the supernatant serum. By contrast, EBV-positive sublines derived by in vitro EBV conversion of EBV-negative parental lines produce considerable activation of the alternative pathway. This membrane-associated complement-activating mechanism reflects an EBV-induced membrane change in these cells and may provide a mechanism whereby EBV-transformed cells are controlled in vivo.
Article
Increased hexose uptake is a marker for viral transformation, as has been shown in non-human fibroblasts transformed by oncogenic viruses. If this phenomenon is a general expression of viral induced transformation it should also apply on different oncogenic virus-cell systems. Recently two human EBV-negative lymphoma lines were converted to a stable EBV-positive state by infection with EBV. According to their biochemical and biological properties they enable us to study events associated with EBV-transformation. We analysed the uptake of (3H) glucosamine and (3H) 2-deoxy-D-glucose into BJAB and Ramos and their EBV-converted sublines and found a clear increase of the rate of uptake of both sugars in the EBV-positive sublines. Control experiments confirmed that the increased uptake was due to alterations on the level of the hexose membrane carriers and not due to increased metabolism. The observation of increased hexose uptake in the only presently available virus transformed human cell system is a strong argument for the general importance of this transformation-associated membrane change.
Article
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Three exceptional cell lines have been tested for the presence of the Epstein-Barr virus genome by nucleic acid hybridization (complementary RNA.DNA) and Epstein-Barr virus-determined nuclear antigen tests. Two lines were derived from Swedish lymphoma cases and one from an African Burkitt-like lymphoma biopsy that was negative for Epstein-Barr virus DNA and the virus-determined nuclear antigen. All three lines apparently lacked the viral genome. Two of the three lines clearly had characteristics of B-cells (bone-marrow-derived).
Article
Over 200 established human hematopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. (1) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus (EBV), polyclonal derivation, differentiation for immunoglobulin production (secretion) and their diploids. (2) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with EBV, variability in the expression of Ig synthesis (no production, or membrane bound Ig, or secretion) and aneuploidy. (3) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of EBV and aneuploidy. (4) The leukemia cell line (MOLT 4) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typical surface ultrastructure, absence of EBV and absence of immunoglobulin production, Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. We conclude that EBV-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells;
Article
Peripheral lymphoid cells, from 12 cases of acute infectious mononucleosis (IM), were tested in a micro chromium-51 release assay for cytotoxic activity against a variety of cell lines that did or did not carry the Epstein-Barr virus (EBV) genome. Unfractionated lymphocytes from these patients were cytotoxic to both types of cell lines, as were lymphocytes from healthy individuals. If, however, lymphocytes bearing complement receptors were removed, the residual IM lymphocyte fraction was specifically cytotoxic for EBV-genome-carrying cell lines. The residual lymphocyte fraction in normal donors had no such effect. Heterophile-positive IM is caused by EBV, and these results indicate that, during the acute phase of this disease, patients harbor killer cells, probably T cells, which specifically kill EBV-genome-carrying B cells in vitro. No such specificity for EBV-genome-psitive target cells was found in normal lymphocytes stimulated in vitro with autologous EBV-genome-positive lymphoblastoid cells. Such stimulated cells were highly cytotoxic to both genome-positive and negative lines after removal of complement receptor-positive lymphocytes.
Article
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.
Article
Twenty-six of 27 African Burkitt lymphomas with histologically confirmed diagnosis contained relatively large amounts of EBV DNA (10–101 viral genomes per cell), as determined by nucleic acid hybridization. Twenty-five of the 26 EBV DNA-positive lymphomas contained the EBV-determined nuclear antigen, EBNA, in the majority of the nuclei. Technical reasons may have accounted for the apparent EBNA-negativity of one EBV DNA-positive biopsy. Four African lymphoma biopsies, one with a definite diagnosis of Burkitt's lymphoma and three with a questionable diagnosis of the same disease, were all EBV DNA- and EBNA-negative. The same was true for a collection of Swedish cases of Hodgkin's disease, lymphocytic lymphoma, chronic lymphatic leukemia and some other lymphoproliferative malignancies. Thus, there is excellent agreement between the presence of EBV DNA and of EBNA in tumor biopsies. The EBNA antigen test therefore appears a relatively simple way of testing for the presence of the virus genome, provided it is carried out with appropriate controls. Several of the EBV-genome and EBNA-negative cases came from patients with high serum titers of EBV antibodies. It is concluded that the virus does not really travel along with malignant lymphomas as a passenger in the seropositive patients. In comparison with other lymphomas, African Burkitt's lymphoma of the high endemic areas is unique in that the tumors (with rare exceptions) represent the proliferation of an EBV-genome carrying clone. These findings stress the necessity to distinguish between EBV-seropositive status and evidence for EBV-genome-carrying neoplastic cells.
Article
Continuous human Iymphoblastoid cell lines (LL), derived from lymphoid tissue or peripheral blood of 20 adults without histories of recent infectious mononucleosis at a frequency close to 100%, were examined for the presence of Epstein-Barr virus (EBV) using immunofluorescence methods for the detection of EBV-dependent cell membrane and EB viral capsid antigens. All lines except one were found to be EBV carriers in the initial tests, but the antigens gradually disappeared in most during the course of an observation period of 4 months.. Only eight lines maintained the initial percentage of antigen-containing cells. The results of the two immunofluorescence tests, performed simultaneously on each cell pool, were concordant in all instances. Sera were available from 16/20 donors. All cell donors, except one, possessed antibodies to EBV as an indication ofprior EBV infections. The growth-promoting role of EBV in the establishment of LL was supported by studies with fetal lymphoid tissue cultured by the same grid method which yielded the high frequency of LL from adults. In sharp contrast to the results obtained with adult lymphoid tissue, no lines were established from 20 fetuses aged 13-20 weeks. However, when such tissue was exposed to a cell-free filtrate prepared from an EBV-carrying LL lymphoblastoid cell, lines were established in some instances. Filtrate from an EBVnegative line inoculated into parallel cultures failed to promote the establishment of LL. The results indicate that EBV infection in vivo or in vitro may be a prerequisite for the indefinite growth of Iymphoblastoid cells in vitro and that EBV infections, as a rule, are not vertically transmitted.
Article
Epstein-Barr virus (EBV) derived from the B95-8 line has transforming activity for cord blood cells, whereas virus derived from the P3HR-1 line lacks such activity. When the two viral preparations were compared for their ability to infect the same EBV-genome-negative lymphoblastoid cell line, BJA-B, they induced approximately the same number of EBV-determined nuclear antigen (EBNA)-positive cells. EBNA is compatibile with continued cell proliferation. No early antigen (EA)-positive cells appeared in the B95-8 virus-infected cultures, whereas P3HR-1 virus-infected cells went on to express EA. EA signals the entry of the cell into the lytic cycle. No late viral antigen (VCA) appeared and, as a consequence, the P3HR-1 virus infection became abortive. In contrast, the EBNA-positive cells induced by the B95-8 virus continued to divide over several weeks. These findings show that different EBV isolates may differ in their biological activity, probably due to their having different degrees of viral dependence on restrictive host cell controls.
Article
Human hematopoietic tissue and lymphocytes separated from 10-20 ml samples ofperipheral blood have been grown in vitro in a lens-paper and a gelatin foam (Spangostan ) grid organ culture. Lymphoblastoid cell lines were established from the lymph nodes, and in one case from the spleen, of 22/23 consecutive, unselected adult individuals without manifest malignancy or infectious mononucleosis. Biopsies from 5/8 patients with malignancy were successful. The blood lines were derived from 5//0 patients with and 4//0 donors without malignancy. The very high frequency of success from normal tissue confirms the assumption made before that the spontaneous establishment of Iymphoblastoid cell lines is unrelated to manifest malignancy of the donor. The results indicate that lymphoid cells with a potential for infinite proliferation (" Iymphoblastoid transformation") are present in almost all adult individuals. The Spongostan grid culture is a superior instrument to select and/or adapt these cells in vitro. All Iymphoblastoid lines produced immunoglobulins. The majority started with a "polyclonal" pattern of immunoglobulin production but changed towards stable " monoclonality " during the course oflong-term cultivation. It is suggested that Iymphoblastoid lines have a polyclonal origin and that the reason for development of a monoclonal line is a selection ofone cell clone either in the organ culture during establishment or in long-term culture.
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