title: Establishment of an isogenic human colon tumor model for NQO1 gene expression: application to investigate the role of DT-diaphorase in bioreductive drug activation in vitro and in vivo.
abstract: Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:quinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase deactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase expression. An expression vector was used in which the human elongation factor 1alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation in NQO1. Two clones, BE2 and BE5, were selected that were shown by immunoblotting and enzyme activity to stably express high levels of DT-diaphorase. Drug response was determined using 96-h exposures compared with the BE vector control. Functional validation of the isogenic model was provided by the much greater sensitivity of the NQO1-transfected cells to the known DT-diaphorase substrates and bioreductive agents streptonigrin (113- to 132-fold) and indoloquinone EO9 (17- to 25-fold) and the inhibition of this potentiation by the DT-diaphorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, EO7 and EO2, that are poorer substrates for DT-diaphorase (5- to 8-fold and 2- to 3-fold potentiation, respectively), and there was no potentiation or protection with menadione and tirapazamine. Exposure time-dependent potentiation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2, 5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiation, there was no difference in the response to mitomycin C when BE2 and BE vector control were treated as tumor xenografts in vivo. This isogenic model should be valuable for mechanistic studies and bioreductive drug development.
authors.affiliation: CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom.
authors.forename: S Y
authors.initials: SY
authors.lastname: Sharp
authors.forename: L R
authors.initials: LR
authors.lastname: Kelland
authors.forename: M R
authors.initials: MR
authors.lastname: Valenti
authors.forename: L A
authors.initials: LA
authors.lastname: Brunton
authors.forename: S
authors.initials: S
authors.lastname: Hobbs
authors.forename: P
authors.initials: P
authors.lastname: Workman
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology* / Q000627:therapeutic use; D003110:Colonic Neoplasms; D004195:Disease Models, Animal; D015870:Gene Expression / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016685:Mitomycin / Q000494:pharmacology / Q000627:therapeutic use; D008954:Models, Biological; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis / Q000235:genetics*; D009368:Neoplasm Transplantation; D019163:Reducing Agents / Q000494:pharmacology; D013308:Streptonigrin / Q000494:pharmacology / Q000627:therapeutic use; D014183:Transplantation, Heterologous; D016896:Treatment Outcome; D014407:Tumor Cells, Cultured
chemical_list: D000903:Antibiotics, Antineoplastic; D019163:Reducing Agents; D013308:Streptonigrin; D016685:Mitomycin; D016660:NAD(P)H Dehydrogenase (Quinone)