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 M) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 M) 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 M 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 PCVmediated 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.

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

“…Accordingly, EBV TK is considered a potential target for the treatment of EBVassociated disease and as a tool for anticancer therapies [9,24,26,45]. Understanding the structure-function relationship of TK enzymes is helpful in designing new antiviral drugs and gene therapy strategies.…”

“…EBV TK, similar to HSV-1 TK, has been proved sensitive to many nucleoside analogues, implying that it can be applied in gene therapy [9,43]. Although HSV-1 TK has broader substrate specificity, EBV TK has a specific advantage in using anti-HIV drugs, such as AZT and D4T, which may have an extra benefit in treatment of AIDS-associated cancer [43].…”

“…The viral DNA polymerase incorporates the metabolites preferentially into the elongating viral DNA, resulting in DNA-chain termination [4,5]. Recently, the herpesviral TK, such as those of HSV (herpes simplex virus), VZV (Varicella Zoster virus), equine herpes virus 4 and EBV (Epstein-Barr virus) [6][7][8][9] also have been found to be useful suicide genes in gene therapy. The genes are introduced into tumour cells, which, subsequently, can be killed selectively by the appropriate nucleoside analogues.…”

Virally targeted therapies for EBV-associated malignancies

“…Tumor cells containing the lytic (but not latent) type of EBV infection express virally encoded kinases (BGLF4 and the viral thymidine kinase) that induce phosphorylation of the prodrug, GCV, converting it to its active cytotoxic form (Moore et al, 2001). Phosphorylated GCV inhibits not only the virally encoded DNA polymerase, but also inhibits the host cell DNA polymerase and is thus cytotoxic (Tiberghien, 1994;Conners, 1995).…”

Short, discontinuous exposure to butyrate effectively sensitizes latently EBV-infected lymphoma cells to nucleoside analogue antiviral agents

“…For example, treatment of EBV positive lymphoblastoid cells or primary central nervous system lymphoma with γ-irradiation has been shown to promote ganciclovir-susceptibility of these cells [21,22]. Other studies successfully used 5-azacytidine, gemcitabine, doxorubicin, and a combination of anti-CD20 monoclonal antibody and dexamethasone to induce lytic-phase gene expression and sensitize EBV-containing tumor cells to ganciclovir or other nucleoside analogues [23][24][25].…”