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. 1998 Nov;72(11):9150-6.
doi: 10.1128/JVI.72.11.9150-9156.1998.

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

J Komano  1 , M Sugiura, K Takada
Affiliations

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

J Komano et al. J Virol. 1998 Nov.

Abstract

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.

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Figures

FIG. 1
FIG. 1
EBV gene expression in EBV-reinfected Akata cells. (A) Reinfected Akata cell clones are positive for EBNA-1 but negative for EBNA-2, EBNA-3s, and LMP1 by Western blot analysis (arrows at left). The LCL, which represents type III latency, and parental Akata cells (EBV positive), which represent type I latency, are used as positive controls, and EBV-negative cells are used as a negative control. Molecular masses of protein markers are shown at the right. (B) Three EBV-reinfected clones are also positive for BARF0 and EBER1 and weakly positive for LMP2A as demonstrated by RT-PCR. They utilize the Q promoter for transcription of EBNA-1 mRNA but not the C or W promoter. Consequently, reinfected clones were proved to have type I latency. LCL cells are positive for all latent gene expression tested by RT-PCR, and they utilize C and W promoters to transcribe EBNAs.
FIG. 2
FIG. 2
Growth in soft agar of neoR-transfected, EBV-reinfected, and EBNA-1-transfected Akata cell clones. Three sets of clones originating from four independent EBV-negative Akata clones were examined in this study (A, B, C, and D). EBV-reinfected cells could form colonies, but neoR-transfected and EBNA-1-transfected clones could scarcely do so. It is clear that EBNA-1 is not solely responsible for the growth of Akata cells in soft agar. Each dot represents the number of visible colonies of a single clone formed in soft agar. A total of 104 cells were seeded per well. The number of clones tested is noted underneath the graph. Values between the indicated groups (Mann-Whitney U test): ∗, P < 0.005; ∗∗, P < 0.05; ∗∗∗, P < 0.01.
FIG. 3
FIG. 3
Apoptosis in Akata cells. (A) Typical morphology of apoptotic cells induced by cycloheximide in EBV-negative Akata cells. Cells were stained with acridine orange (2 μg/ml) and photographed by laser scanning fluoromicroscopy (Molecular Dynamics). (B) DNA laddering occurs in response to the treatment with UV radiation (UV), cycloheximide (CHX), and glucocorticoid (Gluc). DNA from 4 × 105 cells was subjected to 2% agarose gel electrophoresis and stained with ethidium bromide. Incubation times are given above the gels. The intensity of the DNA ladder of EBV-negative cells [Akata(−)] is stronger than that from EBV-positive cells [Akata(+)], suggesting that EBV-negative cells are more liable to die of apoptosis than are EBV-positive cells.
FIG. 4
FIG. 4
EBV-infected cells are resistant to apoptosis. EBV-negative [EBV(−)] and EBV-positive [EBV(+)] clones derived from the parental Akata cells (A) and neoR-transfected, EBV-reinfected, and EBNA-1-transfected clones derived from an EBV-negative Akata clone (B) were exposed to apoptotic stimuli. The average relative survival rates (percent) and error bars (standard errors of the means) are shown. (A) The average relative survival rate of the EBV-positive clones is significantly higher than that of the EBV-negative clones (∗, < 0.001), suggesting that EBV-positive cells are resistant to apoptosis because of the presence of EBV in cells. The data presented here are typical results from three independent experiments. (B) The average relative survival rates of reinfected clones are significantly higher than those of NeoR- and EBNA-1-transfected clones (∗∗, < 0.005; ∗∗∗, < 0.05), suggesting that EBV-infected cells are resistant to apoptosis because of the presence of EBV in cells. The data presented here are typical results from three independent experiments. The numbers of clones tested are noted beneath the graph. Significance values between the indicated groups were determined by unpaired Student’s t test.
FIG. 5
FIG. 5
Western blot analysis of LMP1, BHRF1, and bcl-2 protein. (A and B) LMP1 (A) and BHRF1 (B) (arrows) are detected only after cells are treated with anti-IgG, which induces the virus lytic cycle, indicating that these proteins are lytic gene products rather than latent gene products. Minor bands appear to be degraded proteins. (C) bcl-2 protein expression (arrow) of EBV-positive and -negative cell clones derived from parental Akata cells (four clones each). (D) bcl-2 protein expression (arrow) of EBV-reinfected and neoR-transfected clones derived from an EBV-negative Akata clone (three clones each). bcl-2 protein is expressed at a higher level in EBV-infected clones than in uninfected clones. Molecular masses of protein markers are shown at the left of each panel.
FIG. 6
FIG. 6
Detection of EBNA-1 protein in an EBV-reinfected Akata clone and three EBNA-1-transfected Akata clones by Western blot analysis. EBNA-1 (arrow) is expressed in the EBNA-1-transfected clones in an amount nearly equal to that of the EBV-reinfected clone. Molecular masses of protein markers are shown at the left.
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