Abstract
The molecular mode of cell killing by the antiviral drug (E)-5-(2-bromovinyl-2′-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 <1% at a concentration of ∼1 μM 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 μM) 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-xL 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.
Footnotes
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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.).
- Abbreviations:
- BVDU
- (E)-5-(2-bromovinyl)-2′-deoxyuridine
- HSV
- herpes simplex virus
- VZV
- varicella zoster virus
- TK
- thymidine kinase
- BVDU-MP
- (E)-5-(2-bromovinyl)-2′-deoxyuridine monophosphate
- BVDU-TP
- (E)-5-(2-bromovinyl)-2′-deoxyuridine triphosphate
- CHO
- Chinese hamster ovary
- TS
- thymidylate synthase
- GCV
- ganciclovir
- ACV
- aciclovir
- AP-1
- activator protein 1
- fmk
- fluoromethyl ketone
- PARP
- poly(ADP-ribose) polymerase
- AIF
- apoptosis inducing factor
- ERK2
- extracellular signal receptor-regulated kinase
- pAb
- polyclonal antibody
- PI
- propidium iodide
- PBS
- phosphate-buffered saline
- FITC
- fluorescein isothiocyanate
- dThd
- deoxythymidine
- DN-FADD
- dominant-negative-Fas-associated death domain
- SCGE
- single cell gel electrophoresis
- BrdU
- 5-bromo-2′-deoxyuridine
- CREB/ATF
- cAMP response element-binding protein/activating transcription factor
- MMS
- methyl methansulfonate
- zXXXX-fmk
- N-benzyloxycarbonyl-XXXX-fluoromethyl ketone (where X is an amino acid)
- Received May 2, 2002.
- Accepted November 4, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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