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Research ArticleArticle

Establishment of an Isogenic Human Colon Tumor Model forNQO1 Gene Expression: Application to Investigate the Role of DT-Diaphorase in Bioreductive Drug Activation In Vitro and In Vivo

Swee Y. Sharp, Lloyd R. Kelland, Melanie R. Valenti, Lisa A. Brunton, Steve Hobbs and Paul Workman
Molecular Pharmacology November 2000, 58 (5) 1146-1155; DOI: https://doi.org/10.1124/mol.58.5.1146
Swee Y. Sharp
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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Lloyd R. Kelland
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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Melanie R. Valenti
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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Lisa A. Brunton
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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Steve Hobbs
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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Paul Workman
CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom
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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.
  • 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

  • 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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Molecular Pharmacology: 58 (5)
Molecular Pharmacology
Vol. 58, Issue 5
1 Nov 2000
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Research ArticleArticle

Establishment of an Isogenic Human Colon Tumor Model forNQO1 Gene Expression: Application to Investigate the Role of DT-Diaphorase in Bioreductive Drug Activation In Vitro and In Vivo

Swee Y. Sharp, Lloyd R. Kelland, Melanie R. Valenti, Lisa A. Brunton, Steve Hobbs and Paul Workman
Molecular Pharmacology November 1, 2000, 58 (5) 1146-1155; DOI: https://doi.org/10.1124/mol.58.5.1146

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Research ArticleArticle

Establishment of an Isogenic Human Colon Tumor Model forNQO1 Gene Expression: Application to Investigate the Role of DT-Diaphorase in Bioreductive Drug Activation In Vitro and In Vivo

Swee Y. Sharp, Lloyd R. Kelland, Melanie R. Valenti, Lisa A. Brunton, Steve Hobbs and Paul Workman
Molecular Pharmacology November 1, 2000, 58 (5) 1146-1155; DOI: https://doi.org/10.1124/mol.58.5.1146
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