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

Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:q uinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase d eactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase e xpression. An expression vector was used in which the human elongation fact or 1 alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human c olon BE tumor line, which has a disabling point mutation in NQO1. Two clone s, 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. Functi onal validation of the isogenic model was provided by the much greater sens itivity 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-dia phorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, E O7 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 potenti ation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2,5-d iaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiati on, 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 mode l should be valuable for mechanistic studies and bioreductive drug developm ent.