Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Feb 10;117(6):1977-85.
doi: 10.1182/blood-2010-05-285791. Epub 2010 Nov 18.

Lymphomas differ in their dependence on Epstein-Barr virus

David T Vereide  1 , Bill Sugden
Affiliations

Lymphomas differ in their dependence on Epstein-Barr virus

David T Vereide et al. Blood. .

Abstract

Epstein-Barr virus (EBV) encodes oncogenic information and, oftentimes concomitant with host immunosuppression, gives rise to malignancies in all major categories of lymphoma defined by the World Health Organization. Here, we conditionally evicted the viral extrachromosomal genome from tumor cells in vitro to examine the role of EBV in different lymphomas, including Burkitt lymphoma (BL) and posttransplant lymphoproliferative disorder. Cells derived from 2 canonical BLs were found to have the least dependence on the virus; some required EBV to prevent the inefficient induction of apoptosis. In contrast, cells derived from a subset of BL, Wp-restricted BL, required EBV to block a robust apoptotic program that involves the up-regulation of the proapoptotic protein Bim. Wp-restricted BL cells also relied on the virus to promote efficient proliferation, a distinction that highlights the multiple contributions EBV makes to affect proliferation of its host cells. Like Wp-BL cells, posttransplant lymphoproliferative disorder cells depended on the virus to inhibit apoptosis. They furthermore required the virus to drive them out of G(1)/G(0). Together, these results reveal a graded dependence on EBV among tumor cells that directly correlates with the number of viral genes expressed in the tumor cell.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Phenotypes that emerge as EBV is evicted from tumor cells. The growth and survival of tumor cell populations were tracked over time as EBV was evicted. Dashed lines represent cells in which dnEBNA1 remains induced; and solid lines, cells in which dnEBNA1 is uninduced. (A) Growth curves of PTLD1 and Oku-BL clones. The average number of doublings from at least 3 independent experiments ± SD is shown. (B) PTLD1 and Oku-BL clones were assayed for the induction of apoptosis by staining cells with a fluorescently labeled peptide that is specifically bound by active caspases (APO LOGIX, Cell Technologies). For each time point, at least 200 cells were analyzed. The average percentage of apoptotic cells from at least 3 independent experiments plus or minus SD is shown. (C) Growth curves of Sav-BL and Dante-BL clones as measured in panel A. One of 3 S14-2 growth curves was halted at 20 days. (D) Apoptotic assays of Sav-BL and Dante-BL clones as measured in panel B. For clone S14-2, day 20 is the average of 2 independent experiments ± SD.
Figure 2
Figure 2
EBV's antiapoptotic function in both PTLD1 and Oku-BL cells is efficiently complemented by Bcl-XL and involves the repression of the proapoptotic protein Bim. (A) Cells were assayed for global cell death by staining cells with trypan blue. Dashed lines represent cells in which dnEBNA1 is induced; solid lines, cells in which dnEBNA1 remains uninduced. (Left graph) PTLD1 cells (clone P5-1 only shown) transduced with a control vector (gray lines) or a Bcl-XL expression vector (orange lines). (Right graph) Oku-BL cells (clone O2-4 only shown). For each time point, at least 200 cells were analyzed. The average of at least 3 independent experiments ± SD is reported. (B) The levels of endogenous Bim isoforms were analyzed by Western blotting in Oku-BL cells 14 days after induction of dnEBNA1 or in PTLD1 cells 12 days after induction. One of 2 independent experiments is shown. The levels of Bim were normalized to α-tubulin levels and then compared with control cells (dnEBNA1 off) whose normalized level was arbitrarily set to one. Bim isoforms L and S are not resolved on these blots.
Figure 3
Figure 3
Oku-BL and PTLD1 tumor cells differ in their dependence on EBV for proliferation. (A) The growth of tumor cell populations was measured over time as EBV was evicted. Dashed lines represent cells in which dnEBNA1 remain induced; solid lines, cells in which dnEBNA1 is uninduced. (Left graph) PTLD1 cells (clone P5-1 only shown) transduced with a control vector (gray lines) or a Bcl-XL expression vector (orange lines). (Right graph) Oku-BL cells (clone O2-4 only shown). The percentage of EBV-negative Oku-BL cells was determined by fluorescence in situ hybridization analysis. The average number of doublings from at least 3 independent experiments plus or minus SD is shown. (B) The percentage of cells in each stage of the cell cycle was determined by propidium iodide staining and fluorescence-activated cell sorting analysis. Cells transduced with a Bcl-XL expression vector were analyzed 20 days (Oku-BL) or 12 days (PTLD1) after induction of dnEBNA1 (the cell populations marked in orange). The average of 3 independent experiments ± SD is shown.
Figure 4
Figure 4
MycER, in the presence of Bcl-XL, is not sufficient to drive the proliferation of PTLD1 cells. (A) The level of MycER expression was analyzed by Western blot in Bcl-XL–protected PTLD1 cells and found to be comparable with the level of Myc in several BL cell lines (Oku-BL and Dante-BL) in which Myc is misregulated. (B) The growth of cell populations was measured over time in cells losing EBV but gaining MycER function. Dashed lines represent cells in which dnEBNA1 is induced; solid lines, cells in which dnEBNA1 is uninduced. Gray lines trace cells in which MycER is inactive, red lines cells in which MycER is activated (beginning 4 days after dnEBNA1 was induced). All cells were previously transduced with a Bcl-XL expression vector (Bcl-XL-protected). The average of 4 independent experiments ± SD is shown. The difference in the total number of doublings by day 16 between cells in which MycER is inactive or active is statistically significant (P = .04, one-sided Wilcoxon rank-sum test). (C) The percentage of cells in each stage of the cell cycle was determined by propidium iodide staining and fluorescence-activated cell sorting analysis. PTLD1 cells were analyzed 16 days after dnEBNA1 induction or mock induction and 12 days after activation or mock activation of MycER. The average of 3 independent experiments ± SD is shown. The reduction in the percentage of cells in G1/G0 in the presence of active MycER (from 87% to 82%) is statistically significant (P = .02, one-sided Wilcoxon rank-sum test). (D) RT-PCR of Myc target genes in cells in which EBV is evicted and MycER is active or inactive (day 16 from panel A). Target gene levels were normalized to β-actin levels and then compared with the control (inactive Myc) levels, which were arbitrarily set to one. The average of 3 independent experiments ± SD is shown.
Figure 5
Figure 5
A hypothesis for EBV-induced lymphomagenesis. EBV transforms B lymphocytes, providing cells with much potentially oncogenic information. However, the viral genes these EBV-positive “proto” tumor cells express are immunogenic, placing the cells under strong negative selection by the immune system. In response, tumor cells evolve to express fewer viral genes by gaining cellular mutations that replace the functions of viral oncogenes. Different tumor cells express distinct sets of latent viral genes reflecting their in vivo evolution away from dependence on the virus and toward dependence on cellular mutations. The lengths of the lines for each tumor cell line reflect the hypothesized extent of this evolution.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jaffe ES, Harris NL, Stein H, Isaacson PG. Classification of lymphoid neoplasms: the microscope as a tool for disease discovery. Blood. 2008;112(12):4384–4399. - PMC - PubMed
    1. Kelly GL, Rickinson AB. Burkitt lymphoma: revisiting the pathogenesis of a virus-associated malignancy. Hematology Am Soc Hematol Educ Program. 2007:277–284. - PubMed
    1. Andreone P, Gramenzi A, Lorenzini S, et al. Posttransplantation lymphoproliferative disorders. Arch Intern Med. 2003;163(17):1997–2004. - PubMed
    1. Niedobitek G, Agathanggelou A, Rowe M, et al. Heterogeneous expression of Epstein-Barr virus latent proteins in endemic Burkitt's lymphoma. Blood. 1995;86(2):659–665. - PubMed
    1. Kilger E, Kieser A, Baumann M, Hammerschmidt W. Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J. 1998;17(6):1700–1709. - PMC - PubMed

Publication types

MeSH terms