Marino Schuhmacher

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 Burkitts 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.

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. Int. J. Cancer 87:787–793, 2000.

Dissection of transcriptional programmes in response to serum and c-Myc in a human B-cell line

Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix–loop–helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.

c-MYC activation impairs the NF-κB and the interferon response: Implications for the pathogenesis of Burkitt's lymphoma

Deregulation of the proto‐oncogene c‐myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c‐myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein–Barr viral (EBV) antigens, BL cells are not recognized by antigen‐specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I‐restricted antigens. In contrast, cells of EBV‐driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV‐specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c‐myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV‐immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF‐κB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c‐myc can be regulated at will, we show that c‐MYC negatively regulates STAT1, the central player linking the Type‐I and Type‐II interferon response. Switching off c‐myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type‐I interferons. c‐MYC thus masks an interferon‐inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.

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.

c-Myc and Rel/NF-kappaB are the two master transcriptional systems activated in the latency III program of Epstein-Barr virus-immortalized B cells.

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.

c‐MYC Impairs Immunogenicity of Human B Cells

Deregulation of c-myc expression through chromosomal translocation is essential in the pathogenesis of Burkitts lymphoma (BL). A characteristic feature of BL cells, compared to Epstein-Barr Virus (EBV)-immortalized B cells, is their lack of immunogenicity. To study the contribution of EBV genes and of the c-MYC protein to this phenotype, we have generated a conditional B cell system in which the viral proliferation program and expression of c-myc can be regulated independently of each other. In cells proliferating due to exogenous c-myc overexpression, the cell surface phenotype, the pattern of proliferation in single cell suspension, and the immunological characteristics of BL cells could be completely recapitulated. Yet, it had remained open whether nonimmunogenicity is the default phenotype when EBNA2 and LMP1 are switched off, or whether c-MYC actively contributes to immunosuppression. We provide evidence also for the latter by showing that c-MYC down-regulates genes of the NF-kappaB and interferon pathway in a dose-dependent fashion. c-MYC acts at at least two different levels, the level of interferon induction as well as at the level of action of type I and type II interferons on their respective target promoters. c-MYC does not block the interferon pathway completely, it shifts the balance and increases the threshold of interferon induction and action.

A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFκB 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 NFκB was obtained by induction of overexpression of IκBα, a physiological inhibitor of NFκB. 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 IκBα. 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 IκBα, and pointed out some of the potential meaningful spots characterizing the samples in which NFκB 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 IκBα. The overexpression of IκB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IκBα was not overexpressed and, consequently, in which NFκB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFκB.

Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels.

The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes steadily increased with rising c-Myc levels. Strikingly, steady-state mRNA levels of c-Myc target genes did not saturate even at highest c-Myc concentrations. These characteristics predestine c-Myc levels as a cellular rheostat for the control and fine-tuning of cell proliferation and growth rates.

Epstein-Barr virus antagonizes the antiproliferative activity of transforming growth factor-beta but does not abolish its signaling.

TGF‐β induces apoptosis and inhibits the proliferation of EBV‐negative B‐lymphoma cell lines. In contrast, EBV‐immortalized B cells are resistant to both the proapoptotic and the antiproliferative activities of TGF‐β. We have generated a lymphoblastoid cell line, in which we can switch on and off the EBV‐specific transcriptional program driven by EBNA2. When these cells express the EBNA2‐driven phenotype, they are resistant to TGF‐β‐mediated growth arrest. We used this cell line to readdress the question of how EBV can overcome the antiproliferative TGF‐β activity. We show here that EBV‐driven cells remain TGF‐β‐responsive since TGF‐β target genes are readily induced. Thus, EBV can overcome TGF‐β‐mediated growth arrest without interfering with the core machinery of the TGF‐β signaling pathway, which links ligand binding to the induction of TGF‐β target genes.