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

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

The transcriptional program of a human B cell line in response to Myc.

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Burkitt lymphoma in the mouse.

Chromosomal translocations juxtaposing the MYC protooncogene with regulatory sequences of immunoglobulin (Ig) H chain or kappa (Ig kappa) or lambda (Ig lambda) L chain genes and effecting deregulated expression of MYC are the hallmarks of human Burkitt lymphoma (BL). Here we report that lymphomas with striking similarities to BL develop in mice bearing a mutated human MYC gene controlled by a reconstructed Ig lambda locus encompassing all the elements required for establishment of locus control in vitro. Diffusely infiltrating lymphomas with a typical starry sky appearance occurred in multiple founders and an established line, indicating independence from positional effects. Monoclonal IgM(+)CD5(-)CD23(-) tumors developed from an initially polyclonal population of B cells. These results demonstrate that the phenotype of B lineage lymphomas induced by MYC dysregulation is highly dependent on cooperativity among the regulatory elements that govern expression of the protooncogene and provide a new system for studying the pathogenesis of BL.

Cell cycle activation by c-myc in a burkitt lymphoma model cell line.

The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors.

Epstein-Barr virus (EBV)-based expression vectors were tested for cytokine gene transfer-mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B-cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitt's lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFalpha transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non-transfected cells. By comparison, mIL 4 transfected BL cells also were non-tumorigenic. However, co-inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non-transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti-lymphoma immune response of the host.

Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes.

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(32/36A) cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)

Activation of c-myc promoter P1 by immunoglobulin kappa gene enhancers in Burkitt lymphoma: functional characterization of the intron enhancer motifs kappaB, E box 1 and E box 2, and of the 3' enhancer motif PU.

Deregulated expression of the proto-oncogene c- myc in Burkitt lymphoma (BL) cells carrying a t(2;8) translocation is mediated by a synergistic interaction of the translocated immunoglobulin (Ig) kappa gene intron (kappaEi) and 3' (kappaE3') enhancers and characterized by a strong activation of the promoter P1. We have investigated the functional role of distinct kappa enhancer sequence motifs in P1 activation on both mini-chromosomes and reporter gene constructs. Stable and transient transfections of BL cells revealed critical roles of the kappaEi and kappaE3' elements kappaB and PU, respectively. Joint mutation of kappaB and PU completely abolished P1 activity, implying that an interaction of kappaB- and PU-binding factors is essential for the enhancer synergism. Mutation of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient but not in stable transfection experiments. Co-expression of the NF-kappaB subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in Drosophila melanogaster SL2 cells synergistically enhanced promoter activity. Our results support a model which proposes cross-talk between promoter and enhancer binding factors as the basic mechanism for kappa enhancer-mediated c- myc activation in BL cells.

Control of cell growth by c-Myc in the absence of cell division.

The c-Myc protein (Myc) is a transcription factor, and deregulated expression of the c-myc gene (myc) is frequently found in tumours. In Burkitt's lymphoma (BL), myc is transcriptionally activated by chromosomal translocation. We have used a B-cell line called P493-6 that carries a conditional myc allele to elucidate the role of Myc in the proliferation of BL cells. Regulation of proliferation involves the coordination of cell growth (accumulation of cell mass) and cell division [1] [2] [3]. Here, we show that division of P493-6 cells was strictly dependent on the expression of the conditional myc allele and the presence of foetal calf serum (FCS). More importantly, cell growth was regulated by Myc without FCS: Myc alone induced an increase in cell size and positively regulated protein synthesis. An increase in protein synthesis is thought to be one of the causes of cell mass increase. Furthermore, Myc stimulated metabolic activities, as indicated by the acidification of culture medium and the activation of mitochondrial enzymes. Our results confirm the model that Myc is involved in the regulation of cell growth [4] and provide, for the first time, direct evidence that Myc induces cell growth, that is, an increase in cell size, uncoupled from cell division.

Tumor promoters in commercial indoor-plant cultivars of the Euphorbiaceae.

Certain decorative indoor-plant cultivars are derived from toxic wild plant species. Native members of the Euphorbiaceae (spurge) contain highly irritating and tumor-promoting diterpene esters. Plant breeders and gardeners are constantly searching for less toxic cultivars of the popular Euphorbiaceae indoor plants. In this investigation, 22 commercial cultivars of Euphorbiaceae indoor plants were examined for tumor promoter contents by high-performance liquid chromatography (HPLC). Cultivars of E. milii (E. lomii hybrids), and in particular E. leuconeura, contained ingenol derivatives, whereas cultivars of E. pulcherrima and Codiaeum variegatum were devoid of these compounds. Tumor-promoting activity was assessed by induction of a luciferase reporter gene, which was placed under the control of an Epstein-Barr virus early antigen promoter. The response was closely correlated with ingenol ester content; the latex of the two E. leuconeura cultivars tested gave the strongest response. The HPLC and bioassay methods used in this study provide a basis for the development of nontoxic indoor-plant cultivars and perhaps for consumer-oriented labeling.

Human papillomavirus type 16 E7 DNA vaccine: mutation in the open reading frame of E7 enhances specific cytotoxic T-lymphocyte induction and antitumor activity.

A human papillomavirus type 16 E7 DNA vaccine with the open reading frame encoding mutations in two zinc-binding motifs expressed a rapidly degraded E7 protein. This vaccine induced a significantly stronger E7-specific cytotoxic T-lymphocyte response and better tumor protection in mice than did a wild-type E7 DNA vaccine expressing a stable E7 protein.

Expression of cyclin D2 and D3 in lymphoid lesions.

The D-type cyclins, involved in the regulation of G1 progression of the cell cycle, are expressed in a lineage-specific manner. Normal hematopoietic cells express cyclin D2 and/or D3. In order to determine whether their expression pattern changes in lymphoid tumors, we examined cyclin D2 and D3 expression in non-neoplastic and neoplastic lymphoid lesions, using a sensitive immunohistochemical amplification method. Centroblasts in lymphoid follicles of reactive lymph nodes expressed exclusively cyclin D3 and no D2. Interfollicular areas contained scattered cyclin D3 and D2 positive cells. By double staining, cyclin D3 was detected in CD79a positive B cells, CD3 positive T cells and CD68 positive macrophages. Cyclin D2 was present only in CD3 positive T cells. Neoplastic lymphoid lesions included 33 B cell lymphomas, 9 T cell lymphomas and 12 Hodgkin's lymphomas. The B cell lymphomas comprised 9 follicular lymphomas (FL), 1 Burkitt lymphoma (BL), 22 diffuse large cell lymphomas (DL) and 1 chronic lymphocytic leukemia (CLL). All 9 FLs and the single BL expressed exclusively cyclin D3, similarly to germinal center B cells, that represent their cells of origin. Six DLs expressed both cyclin D2 and D3, while 6 expressed only D3. Among the 9 pleomorphic T cell lymphomas, medium and large cell type, 5 expressed cyclin D2. Cyclin D3 was also detected in scattered cells in 4 of 9 cases and was highly expressed in 2 of 9 T cell lymphomas. The majority of Hodgkin's lymphomas expressed both cyclin D2 and D3 in Hodgkin/Reed-Sternberg (HRS) cells. The high frequency of positive cells indicates that both cyclins were expressed in the same cells.

A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFkappaB in lymphoblastoid cell lines.

Strategies are needed for conclusive interpretation of two-dimensional gel electrophoresis (2-D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFkappaB was obtained by induction of overexpression of IkappaBalpha, a physiological inhibitor of NFkappaB. The analytical methodology used relies on the comparison of two sets of 2-D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IkappaBalpha. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IkappaBalpha, and pointed out some of the potential meaningful spots characterizing the samples in which NFkappaB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IkappaBalpha. The overexpression of IkappaB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IkappaBalpha was not overexpressed and, consequently, in which NFkappaB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFkappaB.

Deregulation of the proto-oncogene c-myc through t(8;22) translocation in Burkitt's lymphoma.

In Burkitt's lymphoma (BL) cells the proto-oncogene c-myc is juxtaposed to one of the immunoglobulin (Ig) loci on chromosomes 2, 14, or 22. The c-myc gene becomes transcriptionally activated as a consequence of the chromosomal translocation and shows preferential usage of promoter P1 over P2, a phenomenon referred to as promoter shift. In order to define the responsible regulatory elements within the Ig lambda locus, we studied the effect of the human Ig lambda enhancer (HuE lambda) on c-myc expression after stable transfection into BL cells. A 12 kb genomic fragment encompassing HuE lambda, but not HuE lambda alone, strongly activated c-myc expression and induced the promoter shift. To identify additional elements involved in c-myc deregulation, we mapped DNaseI hypersensitive sites within the 12 kb lambda fragment on the construct. Besides one hypersensitive site corresponding to HuE lambda, three additional sites were detected. Two of these elements displayed enhancer activity after transient transfection. The third element did not activate c-myc transcription, but was required for full c-myc activation and promoter shift. Deletion analyses of the c-myc promoter identified the immediate promoter region as sufficient for activation by the Ig lambda. locus, but also revealed that induction of the promoter shift requires additional upstream elements.

Direct activation of TERT transcription by c-MYC.

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC.

The protein encoded by the c-MYC proto-oncogene is a transcription factor that can both activate and repress the expression of target genes, but few of its transcriptional targets have been identified. Here, c-MYC is shown to repress the expression of the heavy subunit of the protein ferritin (H-ferritin), which sequesters intracellular iron, and to stimulate the expression of the iron regulatory protein-2 (IRP2), which increases the intracellular iron pool. Down-regulation of the expression of H-ferritin gene was required for cell transformation by c-MYC. These results indicate that c-MYC coordinately regulates genes controlling intracellular iron concentrations and that this function is essential for the control of cell proliferation and transformation by c-MYC.

Improving stable transfection efficiency: antioxidants dramatically improve the outgrowth of clones under dominant marker selection.

Many cell lines are sensitive to growth at low cell density and undergo apoptosis induced by oxidative stress if the cell density is decreased below a critical threshold. In stable transfection experiments this cell density-dependent growth may be the limiting factor, since during drug selection the cell density falls below the critical threshold, precluding outgrowth of transfected clones. We describe here a simple protocol for the establishment of stably transfected human B cell lines making use of the protective action of antioxidants. The protocol includes: (i) seeding the cells in medium supplemented with sodium pyruvate, alpha-thioglycerol and bathocuproine disulfonate; (ii) delaying the onset of dominant marker selection to improve recovery of the cells after electroporation. Stably transfected clones have thus been obtained from Burkitt's lymphoma lines, which have been regarded as untransfectable. Using this protocol the stable transfection efficiency with episomal plasmids approaches the transient transfection efficiency, indicating that virtually every transfected cell can be established as a stably transfected clone. This protocol should also prove useful for other cell lines, e.g. neuronal cells, having similar sensitivities to oxidative stress.

Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene.

Alpha-N-acetylgalactosaminidase (2-acetamido-2-deoxy-alpha-d-galactoside acetamidodeoxy-galactohydrolase, NAGA; EC 3.2.1.49) deficiency is a recently recognized autosomal recessive lysosomal disease. As a prerequisite for the generation of an animal model, the mouse NAGA gene was cloned and characterized. The NAGA gene was assigned to mouse chromosome 15 band E3, syntenic to the region encompassing the human gene, and NAGA-deficient mutant human cells transfected with the cosmid clone containing the mouse NAGA gene expressed NAGA activity. Comparison of the mouse NAGA nucleotide sequence with the human NAGA sequence predicted that the mouse NAGA gene contains an open reading frame of 1245bp, comprising nine coding exons and spanning a genomic region of 8258bp, and a 3' untranslated region of 0.5kb. The 5' untranslated region was determined in primer extension studies to be 235bp in length. Nucleotide identity between the human and mouse NAGA exons ranged from 67.4 to 89.5%, with better matches for exons 1-7 than for 8 and 9. The overall amino acid identity between the mouse and human deduced NAGA polypeptides was 82.0%, between those of mouse and chicken 72.9%. Homology was found to only one other mouse gene, i.e. the alpha-galactosidase A (GALA; EC 3.2.1. 22) gene. The amino acid identity ranged from 51.6 to 62.1% in the polypeptide regions corresponding to NAGA exons 2-7 and GALA exons 1-6, but little, if any, in the remainder. These analyses gave emphasis to the strong conservation of the NAGA gene and its origin from an ancestor common with the GALA gene, with NAGA exons 8 and 9 and GALA exon 7 being the most divergent regions in the evolution of the two genes.

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines.

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.

Suitability of Epstein-Barr virus-based episomal vectors for expression of cytokine genes in human lymphoma cells.

Plasmids carrying the Epstein-Barr virus (EBV) latent gene EBNA1 and the EBV latent origin of replication (oriP) stay in transfected human cells as autonomously replicating extrachromosomal genetic units. They thus might represent a suitable tool for cytokine gene introduction into human tumor cells with the prospect of therapeutic antitumor vaccination. The aim of this study was to analyze whether such plasmids permit stable and efficient expression of cytokine genes in human non-Hodgkin lymphoma cells. We tested physical stability and expression levels of plasmids carrying EBNA1 and oriP for episomal maintenance, immunoglobulin light chain enhancer elements for augmentation of expression, and cytokine or marker genes after introduction into human NHL cell lines in vitro and in vivo after inoculation into nude mice. Data obtained with these EBV-based vectors were compared with another plasmid, not carrying EBNA1 and oriP. cDNAs coding for GM-CSF, IL6, TNF alpha, the chloramphenicolacetyltransferase (CAT) and the beta-galactosidase (lacZ) gene were transfected into the EBV-positive Burkitt's lymphoma cell line BL60 and the EBV-negative B cell lymphoma cell line BJA-B. EBV-derived vectors permitted a high, host cell independent transfection efficiency and high and host cell independent levels of expression. After removal of the selection pressure (hygromycin B) cytokine expression could be detected for several weeks in vitro and in vivo but, however, declined continuously. These experiments suggest that episomal BC-derived vectors represent an effective tool for cytokine gene transfer in human lymphoma cells.