Induction of Epstein-Barr virus kinases to sensitize tumor cells to nucleoside analogues

Abstract

The presence of Epstein-Barr virus (EBV) in the tumor cells of some EBV-associated malignancies may facilitate selective killing of these tumor cells. We show that treatment of an EBV(+) Burkitt's lymphoma cell line with 5-azacytidine led to a dose-dependent induction of EBV lytic antigen expression, including expression of the viral thymidine kinase (TK) and phosphotransferase (PT). Azacytidine treatment for 24 h modestly sensitized the cell line to all nucleosides tested. To better characterize EBV TK with regard to various nucleoside analogues, we expressed EBV TK in stable cell clones. Two EBV TK-expressing clones were moderately sensitive to high doses of acyclovir and penciclovir (PCV) (62.5 to 500 microM) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 microM) compared to a control clone and were shown to phosphorylate GCV. Similar experiments in a transient overexpression system showed more killing of cells transfected with the EBV TK expression vector than of cells transfected with the control mutant vector (50 microM GCV for 4 days). A putative PT was also studied in the transient transfection system and appeared similar to the TK in phosphorylating GCV and conferring sensitivity to GCV, but not in BVdU- or PCV-mediated cell killing. Induction of EBV kinases in combination with agents such as GCV merits further evaluation as an alternative strategy to gene therapy for selective killing of EBV-infected cells.

FIG. 1

Induction of lytic antigens by 5-azacytidine. (A) The EBV⁺ Burkitt's cell line Rael was incubated with the indicated concentrations of 5-azacytidine for 6 h, washed three times, and resuspended in complete medium. Cells were fixed in acetone-methanol 48 h after incubation with 5-azacytidine. Zta and VCA were detected by immunohistochemistry. The percentage of antigen-positive cells was determined by averaging the number of cells expressing antigen in two high-power fields relative to the total number of cells in those fields. (B) Detection of EBV TK protein in 5-azacytidine-induced Rael cells by immunoblot. Rael cells were treated with the indicated concentrations of 5-azacytidine for 6 h and washed three times, and total cellular protein was isolated 48 h later. Fifty micrograms of protein per lane was separated by SDS–7.5% PAGE.

FIG. 2

(A) Immunoblot detection of EBV TK expression in EBV TK-expressing cell clones (TK143b.1 and TK143b.2). One hundred micrograms of total cell protein per lane was separated by SDS–7.5% PAGE. (B) Incorporation of [³H]thymidine in TK-expressing (TK143b.1 and TK143b.2) and control (V143b.1) cell clones. Incorporation into V143b.1 cells is similar to background (medium alone without cells).

FIG. 3

Treatment of EBV TK-expressing cells and control cells with nucleoside analogues in vitro. Cells were seeded into 96-well microtiter plates at 10³ cells/well. After allowing cells to adhere overnight, cells were incubated with GCV (A), PCV (B), ACV (C), BVdU (D), S-BVdU (E), or AZT (F) for 7 days. A colorimetric assay measuring the conversion of MTT to formazan was used to determine the fraction of cells surviving relative to untreated controls (100% viable). Each experiment was repeated three times, with each MTT absorbance value representing replicates of six wells. Each bar represents the average of three experiments, with error bars representing the standard errors of the means.

FIG. 4

Sensitivity of EBV TK-expressing, cellular TK⁺ 293T cells to GCV. 293T cells were transfected with pEBVTK (293-EBV TK), pHSV1TK (293-HSV1 TK), pEBVmutTK (293-EBVmutTK), or control vector DNA (293-p0 cells). Following transfection, cells were seeded into a 96-well plate and treated with GCV for 4 days, and viability was determined by the MTT assay. For each experiment, concentrations were analyzed in replicates of six wells. Bars represent the averages of three experiments. Error bars show the standard errors of the means.

FIG. 5

(A) Phosphorylation of GCV in TK143b and V143b cells as determined by HPLC. Cells were incubated with 2 to 16 μM GCV for 60 h. [³H]GCV was added as a tracer, and phosphorylated products were separated by HPLC. (B) Phosphorylation of GCV in 293T cells transfected with pEBVTK. Following transfection, cells were treated with 8 μM GCV using ³[H]GCV as a tracer, incubated for 36 h at 37°C, and analyzed for GCV phosphorylation by HPLC. Bars represent the averages of three experiments. Error bars show the standard errors of the means.

FIG. 6

EBV PT induction and activity. (A) The EBV PT transcript was detected in Rael cells by Northern blotting after exposure to 1 μM 5-azacytidine overnight. (B) Expression of EBV PT in 293T cells sensitizes cells to GCV cell killing (25 μM for 4 days), as shown by MTT analysis. (C) Phosphorylation of GCV as shown by HPLC analysis. 293T cells expressing EBV PT were treated with 8 μM GCV using [³H]GCV as a tracer as described in Materials and Methods. Bars represent the averages of three experiments. Error bars represent the standard errors of the means.

FIG. 7

Sensitivity of 5-azacytidine-treated cells to nucleoside analogues. In separate experiments, Rael (EBV⁺) and CA46 (EBV⁻) cells were treated with 0.5 μM 5-azacytidine for 24 h. (A) Detection of EBV TK protein in 5-azacytidine-induced Rael cells by immunoblot analysis. Cells were treated and washed three times, and total cellular protein was isolated. Five micrograms of protein per lane was separated by SDS–7.5% PAGE. (B) Cells were seeded into 96-well microtiter plates at 10⁴ cells/well. Cells were then treated with 0.5 μM 5-azacytidine for 24 h. After 24 h, the plates were centrifuged to pellet the cells, the medium was removed, and cells were resuspended in medium containing GCV (a), PCV (b), BVdU (c), S-BVdU (d), or AZT (e). Plates were incubated for an additional 7 days. Viability was determined by the MTT assay. The values of cells treated with drug are presented as the fractions of cells surviving relative to untreated controls (100% viable). Experiments for panels a to d were performed at the same time, while the experiment for panel e was performed at a later date. Each experiment was repeated three times, with each MTT absorbance value representing replicates of six wells. Each bar represents the average of three experiments, with error bars representing the standard errors of the means.

FIG. 7

Sensitivity of 5-azacytidine-treated cells to nucleoside analogues. In separate experiments, Rael (EBV⁺) and CA46 (EBV⁻) cells were treated with 0.5 μM 5-azacytidine for 24 h. (A) Detection of EBV TK protein in 5-azacytidine-induced Rael cells by immunoblot analysis. Cells were treated and washed three times, and total cellular protein was isolated. Five micrograms of protein per lane was separated by SDS–7.5% PAGE. (B) Cells were seeded into 96-well microtiter plates at 10⁴ cells/well. Cells were then treated with 0.5 μM 5-azacytidine for 24 h. After 24 h, the plates were centrifuged to pellet the cells, the medium was removed, and cells were resuspended in medium containing GCV (a), PCV (b), BVdU (c), S-BVdU (d), or AZT (e). Plates were incubated for an additional 7 days. Viability was determined by the MTT assay. The values of cells treated with drug are presented as the fractions of cells surviving relative to untreated controls (100% viable). Experiments for panels a to d were performed at the same time, while the experiment for panel e was performed at a later date. Each experiment was repeated three times, with each MTT absorbance value representing replicates of six wells. Each bar represents the average of three experiments, with error bars representing the standard errors of the means.