Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A

Yongchuan Gu, Adam V. Patterson, Graham J. Atwell, Sophia B. Chernikova, J. Martin Brown, Larry H. Thompson, William R. Wilson

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparingsensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D -/- cells but not of XPD-/- or DNA-PKCS -/- cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restartingcollapsed forks. However, even under anoxia, the hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D-/- cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D-/- cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linkingby its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity.

Original languageEnglish (US)
Pages (from-to)1714-1723
Number of pages10
JournalMolecular Cancer Therapeutics
Volume8
Issue number6
DOIs
StatePublished - Jun 2009
Externally publishedYes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Fingerprint Dive into the research topics of 'Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A'. Together they form a unique fingerprint.

  • Cite this

    Gu, Y., Patterson, A. V., Atwell, G. J., Chernikova, S. B., Brown, J. M., Thompson, L. H., & Wilson, W. R. (2009). Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A. Molecular Cancer Therapeutics, 8(6), 1714-1723. https://doi.org/10.1158/1535-7163.MCT-08-1209