Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation.
Although ganciclovir (GCV) is most often used in suicide anticancer gene therapy, the mechanism of GCV-induced cell killing and apoptosis is not fully understood. We analysed the mechanism of apoptosis triggered by GCV using a model system of CHO cells stably transfected with HSV-1 thymidine kinase (HSVtk). GCV-induced apoptosis is due to incorporation of the drug into DNA resulting in replication-dependent formation of DNA double-strand breaks and, at later stages, S and G2/M arrest. GCV-provoked DNA instability was likely to be responsible for the observed initial decline in Bcl-2 level and caspase-9/-3 activation. Further decline in the Bcl-2 level was due to cleavage of the protein by caspase-9, as demonstrated by use of caspase inhibitors and transfection with trans-dominant negative caspase expression vectors. Bcl-2 cleavage resulted in the appearance of a pro-apoptotic 23 kDa Bcl-2 fragment and in excessive cytochrome c release, dephosphorylation of BAD, cleavage of PARP and finally DNA degradation. Since Fas/CD95 and caspase-8 were only slightly activated we conclude GCV-induced apoptosis to occur in this cell system mainly by activating the mitochondrial damage pathway. This process is independent of p53 for which the cells are mutated. Caspase-9 mediated cleavage of Bcl-2 accelerates the apoptotic process and may explain the high potential of GCV to induce apoptosis. Data are also discussed as to implications for HSVtk gene therapy utilizing GCV.
Tomicic
Maja T
MT
Division of Applied Toxicology, Institute of Toxicology, Medical Faculty, University of Mainz, Obere Zahlbacher Str. 67,D-55131 Mainz, Germany.
Thust
Rudolf
R
Kaina
Bernd
B
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D016466:CHO Cells; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D006224:Cricetinae; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D015774:Ganciclovir / Q000494:pharmacology* / Q000633:toxicity; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism
D003574:Cytochrome c Group; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor; D011065:Poly(ADP-ribose) Polymerases; D013937:Thymidine Kinase; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; D015774:Ganciclovir