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. 2012 Jan;86(1):60-8.
doi: 10.1128/JVI.05623-11. Epub 2011 Oct 19.

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma

Nirojini Sivachandran  1 , Christopher W Dawson, Lawrence S Young, Fei-Fei Liu, Jaap Middeldorp, Lori Frappier
Affiliations

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma

Nirojini Sivachandran et al. J Virol. 2012 Jan.

Abstract

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.

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Figures

Fig 1
Fig 1
PML NBs in GC cells are reduced by EBV and EBNA1. Log-phase cells were fixed and stained with anti-EBNA1 (red) and anti-PML (green) antibodies. PML foci were counted for 50 to 100 cells for each sample in three separate experiments, and the average number with standard deviation is shown in the histograms. Images with the same antibody treatment were captured using the same exposure times. (A) AGS cells were compared to AGS-EBNA1 and AGS-EBV cells. (B) AGS-EBNA1 and AGS-EBV cells are shown after treatment with siRNA against GFP (siGFP) or EBNA1 (siEBNA1). (C) AGS cells were treated with siRNA against GFP or EBNA1 or with AllStars negative-control siRNA.
Fig 2
Fig 2
Transient EBNA1 expression induces loss of PML NBs. AGS cells were transfected with a plasmid expressing EBNA1 (OriPE) or empty plasmid (OriP) and then stained 24 h later with antibodies against EBNA1 and PML and counterstained with DAPI. Both EBNA1-expressing and nonexpressing cells are shown for the OriPE transfection. PML NBs were quantified from 3 separate experiments as for Fig. 1.
Fig 3
Fig 3
EBNA1 expression decreases PML protein levels. (A) Equal amounts of cell lysates from AGS, AGS-EBNA1, and AGS-EBV cells were compared by Western blotting using an antibody that recognizes all PML isoforms. Blots for EBNA1 and actin are also shown. (B) AGS-EBNA1 and AGS-EBV cells were transfected with siRNA against EBNA1 (+) or GFP (−), and then equal amounts of cell lysate were analyzed by Western blotting as for panel A. (C) AGS or AGS-EBV cells were transfected with siRNA against GFP or EBNA1 or AllStars negative-control siRNA, and then Western blotting was performed as for panel A. (D) AGS cells were transfected with a plasmid expressing EBNA1 (OriPE) or with the empty plasmid (OriP), and 24 h later whole-cell lysates were compared by Western blotting for PML, EBNA1, and actin.
Fig 4
Fig 4
EBNA1 decreases p53 activation and apoptosis. (A and D) AGS, AGS-EBNA1, and AGS-EBV cells were treated with etoposide (+) or dimethyl sulfoxide (DMSO) (−) for 5 (A) or 48 (D) hours, and then equal amounts of cell lysates were analyzed by Western blotting using antibodies against p53 acetylated on K382, total p53, p21, actin, and caspase-3, as indicated. In panel D, the positions of molecular weight markers are shown for caspase bands. (B) Cells treated with etoposide as for panel A were stained with antibodies against p21 and acetylated p53. All images were captured at the same exposure time. (C) Cells treated with either DMSO or etoposide as for panel D were stained with DAPI to show apoptotic bodies, indicated by the arrowheads. The apoptotic bodies were quantified by counting over 300 cells in 3 separate experiments, and average numbers with standard deviations are shown in the histogram.
Fig 5
Fig 5
Effect of EBNA1 mutations on PML and p53 activation. AGS cells were transfected with a plasmid expressing EBNA1 (OriPE) or EBNA1 mutant Δ387-394 or Δ395-450 or with the empty plasmid (OriP) and then treated with etoposide or DMSO as for Fig. 4A. Equal amounts of cell lysates were then Western blotted for PML, EBNA1, actin, p53 acetylated on K382, and p21.
Fig 6
Fig 6
Effects of EBNA1 on cell proliferation and viability. (A and B) AGS-EBNA1 (A) and AGS-EBV (B) were treated with siRNA targeted against GFP or EBNA1. Cells were harvested immediately (day 0) or at the indicated days after silencing and counted. (C) AGS, AGS-EBNA1, and AGS-EBV cells were treated with etoposide, and cells were harvested and counted at 0, 1, 2, and 3 days posttreatment. For all experiments, average numbers with standard deviations from three separate experiments are plotted.
Fig 7
Fig 7
PML staining is diminished in EBV-positive GC biopsy specimens. Sections of EBV-positive and EBV-negative human gastric carcinoma biopsy specimens were stained for PML using standard immunohistochemistry (brown stain). Sections of the same biopsy specimens were also subjected to in situ hybridization staining for EBER (EBERish) (red stain). All images were captured at the same exposure time.
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References

    1. Bernardi R, Pandolfi PP. 2007. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat. Rev. Mol. Cell Biol. 8:1006–1016 - PubMed
    1. Catalano V, et al. 2009. Gastric cancer. Crit. Rev. Oncol. Hematol. 71:127–164 - PubMed
    1. Cheng TC, et al. 2010. Expression of Epstein-Barr nuclear antigen 1 in gastric carcinoma cells is associated with enhanced tumorigenicity and reduced cisplatin sensitivity. Int. J. Oncol. 36:151–160 - PubMed
    1. de Stanchina E, et al. 2004. PML is a direct p53 target that modulates p53 effector functions. Mol. Cell 13:523–535 - PubMed
    1. Everett R, et al. 1997. A novel ubiquitin-specific protease is dyamically associted with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J. 16:1519–1530 - PMC - PubMed

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