title: Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir.
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.
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI 48109-5633, USA.
authors.forename: B
authors.initials: B
authors.lastname: Ladd
authors.forename: J J
authors.initials: JJ
authors.lastname: O'Konek
authors.forename: L J
authors.initials: LJ
authors.lastname: Ostruszka
authors.forename: D S
authors.initials: DS
authors.lastname: Shewach
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D000071877:Checkpoint Kinase 1; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D015774:Ganciclovir / Q000633:toxicity*; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051135:Rad51 Recombinase / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; D013937:Thymidine Kinase; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase; D015774:Ganciclovir