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.

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.

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-kappaB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-kappaB 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 Burkitt's 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.

C-myc activation impairs the NF-kappaB 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-kappaB 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.

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.

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

TGF-beta 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-beta. 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-beta-mediated growth arrest. We used this cell line to readdress the question of how EBV can overcome the antiproliferative TGF-beta activity. We show here that EBV-driven cells remain TGF-beta-responsive since TGF-beta target genes are readily induced. Thus, EBV can overcome TGF-beta-mediated growth arrest without interfering with the core machinery of the TGF-beta signaling pathway, which links ligand binding to the induction of TGF-beta target genes.