title: Zinc finger nuclease knock-out of NADPH:cytochrome P450 oxidoreductase (POR) in human tumor cell lines demonstrates that hypoxia-activated prodrugs differ in POR dependence.
abstract: Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.
authors.affiliation: From the Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Jiechuang
authors.initials: J
authors.lastname: Su
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Gu
authors.forename: Frederik B
authors.initials: FB
authors.lastname: Pruijn
authors.forename: Jeff B
authors.initials: JB
authors.lastname: Smaill
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Guise
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011355:Prodrugs / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D011355:Prodrugs; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Flavoproteins; Gene Knock-out; Hypoxia; Hypoxia-activated Prodrug; Mass Spectrometry (MS); Molecular Cell Biology; NADPH:Cytochrome P450 Oxidoreductase; Proteotypic Peptides; Reductase; Zinc Finger Nuclease