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Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation

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Abstract

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.

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Acknowledgements

We are grateful to Dr Vincenz (Univesity of Michigan, USA) for the generous gift of the ICE–LAP6–Flag, ICE–LAP6–mt-Flag and AU1–Yama-mt constructs. We thank Dr Markus Christmann for helpful discussion and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (grants KA 724/7-1 and 7-3 to B Kaina and TH 670/1-2 to R Thust) and the Stiftung Rheinland-Pfalz.

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Authors and Affiliations

  1. Division of Applied Toxicology, Institute of Toxicology, Medical Faculty, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany

    Maja T Tomicic & Bernd Kaina

  2. Institute for Antiviral Chemotherapy, Medical Faculty, University of Jena, Winzerlaer Str. 10, D-07745 Jena, Germany

    Rudolf Thust

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  1. Maja T Tomicic
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  2. Rudolf Thust
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Correspondence to Bernd Kaina.

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Tomicic, M., Thust, R. & Kaina, B. Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation. Oncogene 21, 2141–2153 (2002). https://doi.org/10.1038/sj.onc.1205280

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  • DOI: https://doi.org/10.1038/sj.onc.1205280

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