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

  1. Yongchuan Gu1,
  2. Adam V. Patterson1,
  3. Graham J. Atwell1,
  4. Sophia B. Chernikova2,
  5. J. Martin Brown2,
  6. Larry H. Thompson3 and
  7. William R. Wilson1
  1. 1Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; 2Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University, Stanford, California; and 3Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, California
  1. Requests for reprints: William R. Wilson, Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Phone: 64-9-3737599, ext. 86886; Fax: 64-9-3737571. E-mail: wr.wilson{at}auckland.ac.nz

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 comparing sensitivity 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 restarting collapsed 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-linking by 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. [Mol Cancer Ther 2009;8(6):1714–23]

Footnotes

  • Grant support: Health Research Council of New Zealand (01/276); Technology in Industry Fellowship from the Foundation for Research Science and Technology, New Zealand (Y. Gu); and NIH grant P01 CA67166 (S.B. Chernikova and J.M. Brown).

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵4Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

    • Received January 14, 2009.
    • Revision received March 16, 2009.
    • Accepted March 18, 2009.
« Previous | Next Article »Table of Contents

This Article

  1. Published OnlineFirst June 9, 2009, doi: 10.1158/1535-7163.MCT-08-1209 Mol Cancer Ther June 1, 2009 8, 1714
  1. » Abstract
  2. Full Text
  3. Full Text (PDF)
  4. Supplementary Materials, Gu et al

Classifications

:: AACR Publications Home ::

Current Issue

  1. October 2009, 8 (10)
  1. Current Issue
  1. Alert me to new issues of Molecular Cancer Therapeutics Archive

Most