Apoptosis Induced by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Varicella Zoster Virus Thymidine Kinase-Expressing Cells Is Driven by Activation of c-Jun/Activator Protein-1 and Fas Ligand/Caspase-8

Abstract

The molecular mode of cell killing by the antiviral drug (E)-5-(2-bromovinyl-2Ј-deoxyuridine (BVDU) was studied in Chinese hamster ovary (CHO) cells stably transfected with the thymidine kinase gene (tk) of varicella zoster virus (CHO-VZVtk). The colony-forming ability of the cells was reduced to Ͻ1% at a concentration of ϳ1 M BVDU, whereas for nontransfected cells or cells transfected with tk gene of herpes simplex virus type 1 (CHO-HSVtk), a 1000-fold higher dose was required to achieve the same response. BVDU inhibited thymidylate synthase in CHO-VZVtk but not in CHO-HSVtk and control cells. On the other hand, the drug was incorporated into DNA of VZVtk-and HSVtk-expressing cells to nearly equal amounts. Because coexposure of CHO-VZVtk cells to exogenous thymidine protected them from BVDU-induced cell killing, the cells obviously die because of thymidine depletion. At highly cytotoxic BVDU doses (50 M) and longer exposure times (24 -48 h), VZVtk cells were blocked to some extent in S and G2/M phase and underwent apoptosis (48 -72 h). Not only apoptosis but also necrosis was induced. The findings also show that the drug causes the induction of c-Jun and the activation of activator protein-1 resulting in increased level of Fas ligand (FasL) and caspase-8/-3 activation. Bid and poly(ADP-ribose) polymerase were cleaved by caspases. Expression of Bax increased, whereas Bcl-2/Bcl-x L remained unchanged. Transfection of dominant-negative Fas-associated death domain and inhibition of caspase-8 by N-benzyloxycarbonyl-IETD-fluoromethyl ketone strongly abrogated BVDU-induced apoptosis, indicating Fas/FasL to be crucially involved. Thus, BVDU-triggered apoptosis differs significantly from that induced by ganciclovir, which induces in the same cellular background the mitochondrial damage pathway.(E)-5-(2-Bromovinyl)-2Ј-deoxyuridine (BVDU) is a pyrimidine nucleoside analog exhibiting potent antiviral activity especially against herpes simplex virus (HSV) type 1 and varicella zoster virus (VZV) infections. It is licensed (in Germany) for oral therapy of these virus diseases. Similar to most other antiherpetic drugs, the antiviral activity of BVDU relies on selective monophosphorylation by herpesvirus-encoded thymidine kinases (TKs), whereas cellular thymidine kinases, owing to their much more stringent substrate specificities, do not phosphorylate this nucleoside analog (Cheng et al., 1981). In herpesvirus-infected cells BVDU monophosphate (BVDU-MP) is phosphorylated to the diphosphate by a thymidylate kinase that is associated with certain herpesviral thymidine kinases (e.g., those of HSV-1 and VZV) (Cazaux et al., 1998). The thymidylate kinase function of the viral TK is essential for the antiviral activity of BVDU because the drug is inactive in cells infected with herpesviruses in which TKs lack the thymidylate kinase function (e.g., HSV type 2) (Fyfe, 1982;Mayo, 1982). Finally, BVDU diphosphate The work was supported by Deutsche Forschungsgemeinschaft Grants KA 724/7-3 and 7-4 (to B.K.) and TH 670/1-3 (to R.T.)...

Adaptive upregulation of DNA repair genes following benzo(a)pyrene diol epoxide protects against cell death at the expense of mutations

“…For monitoring drug-induced apoptosis and necrosis within the same cell population, annexin V-FITC/PI double staining combined with flow cytometry was applied. The protocol was conducted as described (32). The metabolic activity was determined by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as already described (33).…”

“…To follow progression of cells through the cell cycle, cells were pulse-labeled with 10 μM BrdU for 30 min and thereafter the thymidine analog was washed out and cells were non-treated or treated with BPDE. After treatment, cells were fixed, incubated with mouse anti-BrdU antibody (BD Biosciences) and subsequently with Alexa Fluor 488-coupled anti-mouse antibody (Millipore), and stained with PI as described (32). Cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences).…”

Topotecan-Triggered Degradation of Topoisomerase I Is p53-Dependent and Impacts Cell Survival

“…Progression of cells through the cell cycle was analyzed by flow cytometry as previously described (18). In brief, cells were continuously treated with topotecan, and after different time points, fixed with ethanol, incubated with 0.1 mg/mL RNase in PBS for 1 hour, and stained with propidium iodide (25 Ag/mL) prior to flow cytometry (CellQuestPro, BD, Heidelberg, Germany).…”

The Anti-Tumor Effect and Mechanisms of Action of Penta-Acetyl Geniposide

“…Activation of JNK is an obligatory event for release of Smac during stress-induced cell death [72]. However, it was suggested that JNK activation is followed by induction of Fas L/Fas signaling, the receptor dependent death pathway which might either activate and translocate mitochondria protein Bid or might be mitochondria independent [73][74][75][76][77][78][79][80][81]. In our previous study, we showed that (Ac) 5 GP mediates tumor cell death via induction of PKCδ/JNK/Fas L cascades, which subsequently activate caspase 8 and caspase 3, thus promoting the apoptotic signal [82] (Fig.…”