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. 2000 Nov;74(21):10223-8.
doi: 10.1128/jvi.74.21.10223-10228.2000.

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis

I K Ruf  1 , P W Rhyne, C Yang, J L Cleveland, J T Sample
Affiliations

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis

I K Ruf et al. J Virol. 2000 Nov.

Abstract

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.

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Figures

FIG. 1
FIG. 1
Restoration of EBER expression in EBV-negative Akata BL cells. EBER expression in EBV-positive Akata (A.15) cells and in EBV−/EBER+ cell lines derived from two clonal EBV-negative Akata lines (A.2 and 3F2) was assessed by Northern blot analysis with a probe spanning the EBER-1 and EBER-2 genes. Each lane contained 10 μg of total cellular RNA. The level of EBER expression is reported as a percentage of EBV-positive Akata cells (A.15) and was determined by phosphorimage analysis and normalization of values to GAPDH mRNA expression, which served as a loading control. EBER-1 and EBER-2 are not distinguishable on the blot due to their similar lengths; however, when the blot was sequentially stripped and rehybridized to EBER-specific probes, the ratios of EBER-1 to EBER-2 expression were identical in EBV-positive and EBV−/EBER+ Akata cells (data not shown).
FIG. 2
FIG. 2
EBERs fail to support BL cell survival following serum deprivation. (A) EBV−/Vector (A.2) and EBV−/EBER+ (A.2 E6 and A.2 E12) Akata BL cells were seeded in complete growth medium (containing 10% serum) at 2.5 × 105 cells per ml. Cells from the logarithmic (day 2 postseeding) and stationary (day 5 postseeding) phases of growth were washed three times and reseeded (5 × 105 per ml) in growth medium containing 0.1% serum. Cells (106) were then harvested immediately (day 0) and daily for 3 consecutive days for immunoblot analysis of the cleavage of PARP. Shown are the 113-kDa uncleaved and 89-kDa cleavage fragments of PARP. Detection of actin served as a loading control. In A.2 E6 and A.2 E12, the levels of EBER expression were 127 and 90%, respectively, of the level of expression observed in EBV-positive Akata cells (Fig. 1). (B) EBV-positive, EBV−/Vector and EBV−/EBER+ Akata cells were seeded at 2.5 × 105 per ml in growth medium containing 10% serum; when cells had reached the stationary phase of the growth cycle (day 5 postseeding), they were washed and reseeded in triplicate as described above in growth medium containing 0.1% serum. Percent viability was then determined daily by trypan blue dye exclusion. Numbers in parentheses indicate the clonal designation of individual vector control and EBER-expressing cell lines derived from the EBV-negative Akata cell line A.2. Note that the level of EBER expression in these EBV−/EBER+ cell lines (A.2 E6, E8, and E12) ranged from 90% to 127% of expression in EBV-positive Akata cells (Fig. 1).
FIG. 3
FIG. 3
EBER expression is maintained within tumors induced by EBV−/EBER+ BL cells. EBER expression within each EBV-negative 3F2- and A.2-derived cell line (cl) that expressed EBERs at 41 to 127% of EBV-positive (A.15) Akata cells (Fig. 1) was compared to EBER expression within a respective tumor (t) by Northern blot analysis. Blots were also probed for GAPDH mRNA as a control for RNA loading and integrity. The level of EBER expression, normalized to GAPDH, is presented below each lane. When blots were sequentially stripped and rehybridized to EBER-specific probes, the ratio of EBER-1 to EBER-2 expression had not changed in the tumors relative to that in the parental cell lines (data not shown).
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