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.
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 approximately 1 microM 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 microM) 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.
Tomicic
Maja T
MT
Institute of Toxicology, Medical Faculty, University of Mainz, Mainz, Germany.
Friedrichs
Claudia
C
Christmann
Markus
M
Wutzler
Peter
P
Thust
Rudolf
R
Kaina
Bernd
B
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D016678:Genome; D014645:Herpesvirus 3, Human / Q000201:enzymology; D048031:JNK Mitogen-Activated Protein Kinases; D008562:Membrane Glycoproteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009336:Necrosis; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000502:physiology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000502:physiology; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D000998:Antiviral Agents; D002352:Carrier Proteins; C505533:FADD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D018808:Transcription Factor AP-1; C020235:brivudine; D004247:DNA; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D001973:Bromodeoxyuridine