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 1α promoter produces a bicistronic message containing the genes for humanNQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation inNQO1. 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 theNQO1-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.
Footnotes
- Received April 8, 2000.
- Accepted July 6, 2000.
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Send reprint requests to: Prof. Paul Workman, CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Block E, 15 Cotswold Rd., Sutton, Surrey, SM2 5NG, UK. E-mail:paulw{at}icra
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This research was supported by UK Cancer Research Campaign (CRC). P.W. is a CRC Life Fellow.
- The American Society for Pharmacology and Experimental Therapeutics
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