title: Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A.
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-PK(CS)(-/-) 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.
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand.
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Gu
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Atwell
authors.forename: Sophia B
authors.initials: SB
authors.lastname: Chernikova
authors.forename: J Martin
authors.initials: JM
authors.lastname: Brown
authors.forename: Larry H
authors.initials: LH
authors.lastname: Thompson
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002699:Chlorambucil / Q000494:pharmacology; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009588:Nitrogen Mustard Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004268:DNA-Binding Proteins; D009588:Nitrogen Mustard Compounds; C524170:PR-104A; C095888:xeroderma pigmentosum group F protein; D002699:Chlorambucil; D009251:NADPH-Ferrihemoprotein Reductase; D051135:Rad51 Recombinase; D004720:Endonucleases