EBV-negative Burkitt Lymphoma Cell Line Research Articles

EBV and 1q Gains Affect Gene and miRNA Expression in Burkitt Lymphoma.

Abnormalities of the long arm of chromosome 1 (1q) represent the most frequent secondary chromosomal aberrations in Burkitt lymphoma (BL) and are observed almost exclusively in EBV-negative BL cell lines (BL-CLs). To verify chromosomal abnormalities, we cytogenetically investigated EBV-negative BL patient material, and to elucidate the 1q gain impact on gene expression, we performed qPCR with six 1q-resident genes and analyzed miRNA expression in BL-CLs. We observed 1q aberrations in the form of duplications, inverted duplications, isodicentric chromosome idic(1)(q10), and the accumulation of 1q12 breakpoints, and we assigned 1q21.2-q32 as a commonly gained region in EBV-negative BL patients. We detected MCL1, ARNT, MLLT11, PDBXIP1, and FCRL5, and 64 miRNAs, showing EBV- and 1q-gain-dependent dysregulation in BL-CLs. We observed MCL1, MLLT11, PDBXIP1, and 1q-resident miRNAs, hsa-miR-9, hsa-miR-9*, hsa-miR-92b, hsa-miR-181a, and hsa-miR-181b, showing copy-number-dependent upregulation in BL-CLs with 1q gains. MLLT11, hsa-miR-181a, hsa-miR-181b, and hsa-miR-183 showed exclusive 1q-gains-dependent and FCRL5, hsa-miR-21, hsa-miR-155, hsa-miR-155*, hsa-miR-221, and hsa-miR-222 showed exclusive EBV-dependent upregulation. We confirmed previous data, e.g., regarding the EBV dependence of hsa-miR-17-92 cluster members, and obtained detailed information considering 1q gains in EBV-negative and EBV-positive BL-CLs. Altogether, our data provide evidence for a non-random involvement of 1q gains in BL and contribute to enlightening and understanding the EBV-negative and EBV-positive BL pathogenesis.

B-cell receptors of EBV-negative Burkitt lymphoma bind modified isoforms of autoantigens.

Burkitt lymphoma (BL) represents the most aggressive B‐cell‐lymphoma. Beside the hallmark of IG‐MYC‐translocation, surface B‐cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra‐nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom‐made virome array and against self‐antigens, including post‐translationally modified antigens.An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1‐BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1‐BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1‐BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1‐BYSL‐ETA’ immunotoxin, produced by a two‐step intein‐based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1‐BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt‐lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed.These results indicate a pathogenic role of autoreactivity evoked by immunogenic post‐translational modifications in a subgroup of sporadic BL including two EBV‐negative BL cell lines.

Expression of the Chemokine Receptor CCR1 in Burkitt Lymphoma Cell Lines Is Linked to the CD10-Negative Cell Phenotype and Co-Expression of the EBV Latent Genes EBNA2, LMP1, and LMP2.

Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs.

The JAK2 inhibitor TG101209 exhibits anti-tumor and chemotherapeutic sensitizing effects on Burkitt lymphoma cells by inhibiting the JAK2/STAT3/c-MYB signaling axis

Constitutive activation of JAK2/STAT3 is a major oncogenic signaling event involved in the development of Burkitt lymphoma (BL). In the present study, we investigated the antilymphoma activity of TG101209, a specific JAK2 inhibitor, on EBV-positive and EBV-negative Burkitt lymphoma cell lines and primary BL cells. The results showed that TG101209 had a significant antilymphoma effect by inhibiting BL cell growth and inducing apoptosis along with cell differentiation toward mature B cells in vitro. We also found that TG101209 displayed significant synergistic action and a sensitizing effect on the anti-Burkitt lymphoma activity of doxorubicin. In vivo experiments indicated that TG101209 could suppress tumor growth and prolong the overall survival of BL cell-bearing mice. The mechanistic study indicated that TG101209, by suppressing the JAK2/STAT3/c-MYB signaling axis and crosstalk between the downstream signaling pathways, plays an antilymphoma role. These data suggested that TG101209 may be a promising agent or alternative choice for the treatment of BL.

Hypermethylation of the tumor suppressor gene PRDM1/Blimp-1 supports a pathogenetic role in EBV-positive Burkitt lymphoma.

PRDM1/Blimp-1 is a tumor suppressor gene in the activated B-cell subtype of diffuse large B-cell lymphomas. Its inactivation contributes to pathogenesis in this setting by impairing terminal B-cell differentiation induced by constitutive nuclear factor-κB activation. The role of PRDM1 in Burkitt lymphoma (BL) lymphomagenesis is not known. Here we identified hypermethylation of the promoter region and exon 1 of PRDM1 in all six Epstein–Barr virus (EBV)-positive BL cell lines and 12 of 23 (52%) primary EBV-positive BL or BL-related cases examined, but in none of the EBV-negative BL cell lines or primary tumors that we assessed, implying a tumor suppressor role for PRDM1 specifically in EBV-associated BL. A direct induction of PRDM1 hypermethylation by EBV is unlikely, as PRDM1 hypermethylation was not observed in EBV-immortalized B lymphoblastoid cell lines. Treatment of EBV-positive BL cells with 5′ azacytidine resulted in PRDM1 induction associated with PRDM1 demethylation, consistent with transcriptional silencing of PRDM1 as a result of DNA methylation. Overexpression of PRDM1 in EBV-positive BL cell lines resulted in cell cycle arrest. Our results expand the spectrum of lymphoid malignancies in which PRDM1 may have a tumor suppressor role and identify an epigenetic event that likely contributes to the pathogenesis of BL.

Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines

AbstractAbstract 4452The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Gene Array Identification of Epstein Barr Virus-Regulated Cellular Genes in EBV-Converted Burkitt Lymphoma Cell Lines

Epstein Barr virus (EBV) is associated with various B-cell neoplasms such as post-transplant lymphoproliferative disease or Burkitt lymphoma. B-lymphocyte reprogramming by EBV involves the control of numerous cellular genes. To identify such EBV-deregulated genes, we have compared the gene expression profile of EBV-negative Burkitt lymphoma cell lines (BL) (BL2, BL30, BL70) with their EBV-converted counterpart (BL2-B95, BL30-B95, BL70-B95) by cDNA array. Statistical analysis of the results was made using Ward's cluster analysis method. Results showed that the expression of up to 26% of the 1176 cellular genes analyzed may be modified in EBV-converted BL cells. Within this set of genes, a subset of genes markedly regulated in EBV-converted BL cells was defined as those for which expression in EBV+ cells was increased or decreased more than 2-fold. Expression of various genes was modulated in agreement with their previously reported regulation by EBV or by transcription factors activated by EBV. Numerous genes were newly identified as modulated in EBV-converted BL cells. Some of these results were verified by both semiquantitative RT-PCR and Western blotting, and were consistent with functional studies. Functional classification of EBV-regulated genes gave a comprehensive picture of cellular reprogramming by EBV in BL, by pointing out cellular modules such as cell cycle, apoptosis, and signal transduction pathways, including BCR and TNF receptor family and interferon pathways. Furthermore, and perhaps most importantly, cDNA array results point to three families of transcription factors, Rel/NF-κΒ, STAT1, and Ets-related proteins Spi-B, Elf-1, and Ets-1 as putative cellular targets of EBV.

Epstein-Barr virus latent membrane protein-1 activates CD25 expression in lymphoma cells involving the NFkappaB pathway.

Epstein-Barr virus (EBV) is associated with several human malignancies including Burkitt's lymphoma (BL), Hodgkin's disease (HD) and nasopharyngeal carcinoma. A variety of cytokines and receptors have been described to be activated by EBV. Here we show that the IL-2 receptor (IL-2R) alpha-chain, which is weakly expressed on normal resting lymphoid cells, is activated by EBV. Comparison of EBV-negative BL cell lines and their EBV convertants showed an enhanced CD25 expression in EBV-positive BL cells. Transient expression of the oncogenic virus protein latent membrane protein-1 (LMP1) in L428 Hodgkin's lymphoma cells and in Burkitt's lymphoma cells (BL2, BL41, BL30) cells leads to enhanced CD25 expression. Both C-terminal activating regions (CTARs) of LMP1 are involved in CD25 activation. Inhibition of LMP1-mediated NFkappaB enhancement by a constitutive repressive form of IkappaB-alpha resulted in decreased CD25 surface expression, indicating that NFkappaB is involved in CD25 gene regulation. Furthermore, LMP1-mediated CD25 activation was associated with enhanced levels of the soluble form of CD25 (sCD25) in L428 Hodgkin's lymphoma cells but not in BL cells. LMP1 associated enhanced expression of membrane CD25 and soluble CD25 may have immunomodulatory functions and could be involved in biology of EBV-associated diseases.

EBNA promoter usage in EBV-negative Burkitt lymphoma cell lines converted with a neomycin-resistant EBV strain.

Latent Epstein-Barr virus (EBV) uses two alternative strategies to express the Epstein-Barr nuclear antigens (EBNAs). Resting normal B cells harboring latent virus and Burkitt's lymphoma (BL) cells use monocistronic messages generated from the Q promoter (restricted strategy). EBV-transformed immunoblasts express all EBNAs by using giant messages generated from the W/C promoter (full program). Whether the virus establishes the restricted program on primary infection of a BL cell (or its progenitor) or, alternatively, whether such cells are generated by phenotypic down-regulation from the immunoblast is unclear. We found previously that conversion of EBV-negative BL lines to EBV-positive sublines required repeated exposure to large virus doses. The converted sublines used the full program. However, the possibility that cells with a full program had a selective advantage during the long period of in vitro passage could not be excluded. We therefore infected EBV-negative BL lines with recombinant EBV carrying a neomycin resistance marker. Most convertants of the 12 lines tested were positive for YUK splicing, indicative of the full program, but some were also positive for the restricted QUK splice program. One convertant DG75 line showing both YUK and QUK was cloned and gave rise to stable QUK users. We conclude that EBV infection of established BL lines can give rise to subclones with either the full or the restricted program. The fact that all EBVs carrying BL lines use the restricted program in vitro may be a consequence of immunoselection.

Absence of IL-6 receptor expression in fresh childhood Burkitt's lymphoma cells and induction of IL-6 receptors by Epstein-Barr virus in vitro.

Interleukin-6 (IL-6) is a cytokine which is necessary for the differentiation of activated B cells and growth of early haemopoietic progenitors. It is used for ex-vivo expansion of myeloid progenitors in the setting of high-dose chemotherapy with autologous bone marrow transplantation (BMT). Expression of the IL-6 receptor (IL-6R) was examined in six fresh Burkitt's lymphoma (BL) cell preparations and 12 BL cell lines by reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Inducibility of IL-6R mRNA expression by Epstein-Barr virus (EBV) was studied by comparing two uninfected cell lines with the same cell lines infected by EBV The phenotype of the BL cells lines was analysed by FCM and by proliferation assays in the presence of anti-IgM antibodies. None of the fresh BL cells expressed the IL-6 receptor. The BL cell lines expressed varying degrees of IL-6R mRNA and protein. In vitro infection of EBV-negative BL cell lines resulted in up-regulation of IL-6R mRNA. There was no proliferative response of the BL cell lines to IL-6, including the cells that expressed the receptor. Compared to uninfected BL cell lines, EBV-infected cell lines and lymphoblastoid cell lines (LCLs) showed a weaker or no response to anti-IgM antibodies, indicating a more mature phenotype of these cells. We conclude that the IL-6 receptor is not expressed in fresh childhood BLs, but only in BL cell lines. EBV infection in vitro leads to an up-regulation of IL-6R mRNA but not to increased proliferation. This makes growth stimulation of contaminating BL cells in the setting of autologous BMT unlikely.

Transcription of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells

The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with that of cells infected with wild-type virus. Switching to Cp, as well as EBNA1 gene transcription, was observed upon infection of EBV-negative Burkitt's lymphoma (BL) cell lines with EBNA2- EBNA4mut virus, thus establishing a correlation between early EBNA1 gene transcription and upregulation of transcription initiation from Cp. However, in EBV-negative BL cell lines infected with EBNA2- EBNA4mut virus, transcription of the EBNA1 gene at early time points postinfection initiated from Qp, the EBNA1 gene promoter active in group I BL cells (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995), rather than from Wp. The data support a model in which EBNA1 plays an important role in the cascade of events leading to successful switching from Wp to Cp and subsequent immortalization of the infected B cell.

Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors.

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.

Regulation of C‐FGR proto‐oncogene expression in epstein‐barr virus infected B‐cell lines

We and others have previously shown that in vitro conversion of Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) cell lines with the immortalizing B95-8 strain of EBV results in a marked elevation in levels of c-fgr proto-oncogene mRNA. We now show, using a nuclear run-off assay, that this induction results from an increase in the rate of transcription of the c-fgr gene. We also show that BL cell lines freshly converted with the non-immortalizing HR-I strain of EBV do not accumulate higher levels of c-fgr mRNA, suggesting that EBNA-2 and/or LMP, the genes which are deleted in the HR-I strain, may be involved in the pathway which leads to changes in c-fgr gene expression. In order to assess the generality of a role for the c-fgr gene in the response of B-lymphocytes to EBV-infection, which is controversial, we have analysed c-fgr expression in 6 freshly immortalized cell lines established by EBV (B95-8) infection of B-lymphocytes from the peripheral blood of normal adults and of adults with rheumatoid arthritis, from cord blood, and from foetal liver. All 6 cell lines expressed c-fgr mRNA at elevated levels compared to EBV-negative BL cell lines.

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23.

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells induces some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line, including BL markers (cALLA and BLA), proliferation markers (transferrin receptor and BK19.9), and cell adhesion-related molecules (LFA-1 and LFA-3). Increased CD23 expression in cells expressing EBNA-2 was apparent from monoclonal anti-CD23 antibody binding to the cell surface, from immunoprecipitation of the 45-kDa and 90-kDa CD23 proteins with monoclonal antibody, and from RNA blots probed with labeled CD23 DNA. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.

Diploid human lymphoblastoid and Burkitt lymphoma cell lines: susceptibility to murine NK cells and heterotransplantation to nude mice.

Human lymphoid cell lines which had been classified on the basis of studies on clonality and morphological, on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell line (LCL) of presumed non-neoplastic origin and Burkitt lymphoma (BL) lines of proven malignant origin, were tested for susceptibility to natural killer (NK) cells obtained from the spleens of athymic nude mice. The 20 lines included normal diploid LCL and aneuploid BL lines. All cells carried the Epstein-Barr virus (EBV) genome. In addition, two EBV-negative BL lines were tested. The pronase-induced release of 14C-DNA from 14C-thymidine-labelled target cells was used to assess the sensitivity of the cell lines to NK activity. When attempts were made to correlate the growth of the EBV-positive LCL and the EBV-positive BL cell lines in the subcutaneous space of adult nude mice with their susceptibility to NK cells, no significant correlation was observed. The EBV-negative BL cell line, Ramos, however, could be transplanted subcutaneously in nude mice and was more resistant to NK activity than was the EBV-negative BL cell line, BJAB, which cannot be transplanted subcutaneously. Growth of heterotransplanted EBV-converted cell lines in the subcutaneous space of adult nude mice may be influenced by immune effectors other than NK cells.