c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Abstract

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin K locus composed of a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype ofEBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern ofviral and cellular genes rather than its germinal center origin.

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

“…14 We applied the same construct in the present study for transient transfection of EREB2-5 cells. To this end, EREB2-5 cells were transfected with different amounts of KH348-5 DNA together with 15 g of a plasmid allowing high expression of a truncated nerve growth factor receptor (NGFR).…”

“…14 In brief, cells (1 ϫ 10 6 /tube) were harvested, washed twice with PBS containing 2 % FCS (FACS-medium), and incubated with 50 l of the first antibody for 30 min on ice. In general, antibodies were used at a concentration of 2 g/ml.…”

“…As a third step, estrogen has been withdrawn and cells have been selected that continue to proliferate even in the absence of estrogen. 14 These cells (designated A1) express high MYC levels and proliferate autonomously in the absence of functional EBNA2 and EBNA2-regulated genes such as latent membrane proteins 1 and 2 (LMP1 and LMP2). Similarly to BL cells, these c-myc driven autonomously proliferating cells grow more rapidly than EBV immortalized lymphoblastoid cells (LCLs).…”

“…Importantly, they grow in single cell suspension rather than in large clumps and have adopted the phenotype of Burkitt lymphoma cells, i.e., they have downregulated activation markers and adhesion molecules expressed in LCLs and have upregulated CD10 and CD38, which are not detectable on the surface of LCLs. 14 In keeping with the loss of costimulatory and adhesion molecules, the cells have lost their ability to act as allostimulatory cells for T cells in a mixed lymphocyte reaction similarly to BL cells. 15 The system described has allowed us to reconstruct a number of important features of BL cells in vitro.…”

Oncogenic DNA viruses

“…The regulation of viral gene expression by cellular factors has been less intensively studied in comparison to human papillomaviruses. Yet, it is an interesting observation that overexpression of the cellular myc gene results in a Burkitt-like phenotype of the respective cells, suppressing the expression most EBNA genes, but permitting the expression of EBNA 1 (Polack et al, 1996).…”