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Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir

Cancer Gene Therapy volume 18, pages 751–759 (2011)Cite this article

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Abstract

The herpes simplex virus thymidine kinase (HSV-TK) is the most widely used suicide gene in cancer gene therapy due to its superior anticancer activity with ganciclovir (GCV) compared with other HSV-TK substrates, such as 1-β-D-arabinofuranosyl thymine (araT). We have evaluated the role of DNA damage as a mechanism for the superiority of GCV. Using γ-H2AX foci as an indicator of DNA damage, GCV induced ⩾sevenfold more foci than araT at similar cytotoxic concentrations. The number of foci decreased after removal of either drug, followed by an increase in Rad51 foci indicating that homologous recombination repair (HRR) was used to repair this damage. Notably, only GCV produced a late and persistent increase in γ-H2AX foci demonstrating the induction of unrepairable DNA damage. Both drugs induced the ATR damage response pathway, as evidenced by Chk1 activation. However, GCV resulted in greater activation of ATM, which coincided with the late induction of γ-H2AX foci, demonstrating the presence of DNA double-strand breaks (DSBs). The increase in DSBs after Rad51 induction suggested that they occurred as a result of a failed attempt at HRR. These data demonstrate that the late and unrepairable DSBs observed uniquely with GCV account for its superior cytotoxicity and further suggest that inhibition of HRR will enhance cytotoxicity with HSV-TK/GCV.

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Abbreviations

araT:

1-β-D-arabinofuranosyl thymine

BrdUrd:

5-bromo-2′-deoxyuridine

DSB:

double-strand break

GCV:

ganciclovir

GCVMP:

ganciclovir monophosphate

HRR:

homologous recombination repair

HSV-TK:

herpes simplex virus thymidine kinase

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Acknowledgements

This work was supported in part by grants CA076581 and CA083081 from the NIH. The project described was supported by grant number GM007767 from NIGMS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS. The immunohistochemistry work was performed in the Microscopy and Image-analysis Laboratory (MIL) at the University of Michigan, Department of Cell & Developmental Biology with the assistance of Shelley Almburg.

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  1. B Ladd and J J O'Konek: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA

    B Ladd, J J O'Konek & D S Shewach

  2. Accuri Cytometers, Ann Arbor, MI, USA

    L J Ostruszka

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  1. B Ladd
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  2. J J O'Konek
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  4. D S Shewach
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Correspondence to D S Shewach.

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Ladd, B., O'Konek, J., Ostruszka, L. et al. Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir. Cancer Gene Ther 18, 751–759 (2011). https://doi.org/10.1038/cgt.2011.51

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