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Homologous Recombination Is the Principal Pathway for the Repair of DNA Damage Induced by Tirapazamine in Mammalian Cells

¹ Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University, Stanford, California; ² Department of Radiation Oncology, Boston University Medical Center; ³ Department of Radiation Oncology, Massachusetts General Hospital, Harvard University School of Medicine, Boston, Massachusetts; ⁴ Medical Biophysics Department, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada; ⁵ Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; and ⁶ Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand

Requests for reprints: J. Martin Brown, Division of Radiation and Cancer Biology, Stanford University Medical Center, CCSR South, Room 1255, 269 Campus Drive, Stanford, CA 94305-5152. Phone: 650-723-5881; Fax: 650-723-7382; E-mail: mbrown{at}stanford.edu.

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective cytotoxin that has shown significant activity in advanced clinical trials in combination with radiotherapy and cisplatin. The current study aimed to advance our understanding of tirapazamine-induced lesions and the pathways involved in their repair. We show that homologous recombination plays a critical role in repair of tirapazamine-induced damage because cells defective in homologous recombination proteins XRCC2, XRCC3, Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with the involvement of homologous recombination repair, we observed extensive sister chromatid exchanges after treatment with tirapazamine. We also show that the nonhomologous end-joining pathway, which predominantly deals with frank double-strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In addition, we show that tirapazamine preferentially kills mutants both with defects in XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable DNA-topoisomerase I cleavable complexes. We further show that H2AX, an indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle. These observations lead us to an overall model of tirapazamine damage in which DNA single-strand breaks, base damage, and DNA-protein cross-links (including topoisomerase I and II cleavable complexes) produce stalling and collapse of replication forks, the resolution of which results in DSB intermediates, requiring homologous recombination and XPF/ERCC1 for their repair. [Cancer Res 2008;68(1):257–65]

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