Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

<div><p>Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14^ARF and p16^INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14^ARF and p16^INK4a. By contrast, p16^INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14^ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21^WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21^WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.</p></div

EBNA-3A supports maintenance of Rb hyperphosphorylation and stabilization in Wp-R latency.

<p>Immunoblots of transfected Sal cells harvested at (A) 4 days or (B) 8 days post-transfection were probed with a pan Rb antibody (Rb), a phospho-specific (pRb) antibody that recognizes the phosphorylated residues Ser807/811, and antibodies to detect p53 and Lamin B (loading control). Light and dark refer to film exposure time.</p

Wp-R BL cells have increased tumorigenic potential.

<p>SCID mice were injected with EBV-negative Akata BL cells (Akata⁻) on one flank and either Kem I (Latency I) or Wp-R Sal or Oku BL cells on the opposite flank. The endpoint for tumor formation was set at 2 cm in diameter. No tumors developed on flanks injected with Akata⁻ cells. The time to tumor development for Wp-R BLs (Sal or Oku) was statistically different (p<0.001 using either student's T test or Mann-Whitney U test) from Latency I (Kem I).</p

Knockdown of EBNA-3A results in G0/G1 cell-cycle arrest.

<p>(A) Cell cycle profiles of GFP-positive Sal cells 3 days post-transfection with oriP-GFP empty vector (top) or oriP-GFP-encoding shRNA3A-1490 (bottom). (B) Data from three independent experiments, each performed in duplicate, were analyzed using the two sample student T-test, and demonstrated statistically significant differences between the percentage of cells in G0/G1 (p<0.001) and S (p<0.001) phases in EBNA-3A positive (oriP-GFP) compared to knockdown cells (shRNA3A-1490).</p

EBNA-3A represses p21^WAF1/CIP1 expression in Wp-R BL.

<p>Cell lysates from Sal BL cells transfected with the empty shRNA expression vector (oriP-GFP) or vector encoding EBNA-3A-specific (1490 and 601), or control (C1 or C2) shRNAs were analyzed by immunoblotting to detect (A) p27 at the onset of growth arrest (4 days post-transfection); and (B) p21^WAF1/CIP1 prior to and coincident with the onset of growth arrest at 2 and 4 days post-transfection, respectively. MCF7 and Saos2 lysates were used as positive controls; GAPDH served as a loading control. unTF, untransfected cells.</p

EBNA-3A does not repress p14^ARF or p16^INK4a expression in in Wp-R BL.

<p>The levels of p14^ARF and p16^INK4a in Sal BL were analyzed at 4 days post-transfection and not detected. Sal cells were transfected in duplicate with expression vectors for either EBNA-3A-specific shRNA 1490 or 601, control shRNA (C2) or empty vector (oriP). Saos2 and MCF7 serve as positive controls for detection of p14^ARF and p16^INK4a; GAPDH, loading control. Knockdown of EBNA-3A was similar to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004415#ppat-1004415-g002" target="_blank">Figure 2A</a>.</p

Reduced EBNA-3A expression is associated with increased expression of Bim and cell death.

<p>(A) The percentage of dead cells was measured following knockdown of EBNA-3A in two independent experiments performed in triplicate, and was determined to differ significantly between groups using Tukey's test on pairwise comparison at each time point. The controls were combined as one group for analysis. Cells expressing shRNA3A-1490 at days 3–7 were statistically different from control group at day 3 (p = 0.024) and days 4–7 (p<0.001). Those expressing shRNA3A-601 were statistically different from the control groups at day 4–8: day 4, p<0.001; day 5, p = 0.005; days 6–8, p<0.001. Cells expressing shRNA3A-1490 at days 5–7 were also statistically different from those expressing shRNA3A-601 at day 4 (p = 0.010), and days 5–7 (p<0.001). Immunoblot detection of (B) PARP and Lamin B (loading control) and (C) Bim, and GAPDH (loading control).</p

TMZ-Se is more apoptogenic than TMZ in tumor cells.

<p>(<b>A</b>) LN229 and T98G cells were treated with 100 µM or 200 µM of TMZ or TMZ-Se for 48 h, and apoptosis was examined by flow cytometric analysis of Annexin V and 7-AAD staining. (<b>B</b>) LN229 and T98G cells were treated with TMZ or TMZ-Se for 48h, and the levels of caspse-9, caspase-3, PARP and survivin were measured by Western blot analysis. Tubulin was used as a loading control. (<b>C</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of Z-VAD, and cell viability was measured by MTT assay. (<b>D</b>) 1205LU and UACC cells were treated with TMZ or TMZ-Se for 48h, and the levels of PARP and survivin were measured by Western blot. Tubulin was used as a loading control. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

TMZ-Se triggers a greater autophagic response than TMZ, and inhibition of autophagy decreases the efficacy of TMZ-Se against glioma cells.

<p>(<b>A</b>) <i>Left panels</i>: LN229 and T98G cells were treated with TMZ or TMZ-Se for 48 h, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. <i>Right panels</i>: LN229 and T98G cells were treated with TMZ-Se for 48 h in the presence or absence of bafilomycinA1, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a GFP-LC3 plasmid, followed by treatment with TMZ-Se for 48h. At the end of treatment, the cells were observed under fluorescence microscope. (<b>C</b>) T98G cells treated with TMZ-Se or vehicle were harvested by trypsinization, fixed and embedded in spur resin. Ninety nm thin sections were cut and examined at 80 Kv with a JEOL 1200EX transmission electron microscope. (<b>D</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of 3-MA or bafilomycinA1, and cell viability was measured by MTT assay. (<b>E</b>) LN229 and T98G cells were transfected with an Atg5-targeted siRNA, and then treated with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

TMZ-Se induces calpain-mediated degradation of Beclin 1.

<p>(<b>A</b>) LN229 and T98G cells were transfected with Beclin1siRNA followed by TMZ-Se treatment, and the levels of LC3, Beclin 1 and PARP were examined by Western blot. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a Beclin 1-targeted siRNA, followed by treatment with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p