Relationship between NAD(P)H : quinone oxidoreductase 1 (NQO1) levels in aseries of stably transfected cell lines and susceptibility to antitumor quinones

To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-di aphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adeno carcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphi sm. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined b y the thiazolyl blue (MTT) assay] to known antitumor quinones and newer cli nical candidates. Inhibition of NQO1 by pretreatment with an irreversible i nhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4, 7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES 921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-benzoquinone]. RH1 was ev aluated further by clonogenic assay for cytotoxic response and was more cyt otoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhi bition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-li nks than did BE cells in response to RH1 treatment. DNA cross-linking in BE -NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirmin g that NQO1 activates RH1 to a potent cross-linking species. Further studie s using streptonigrin, ES921, and RH1 were undertaken to analyze the relati onship between NQO1 activity and quinone toxicity. Toxicity of these compou nds was measured in a panel of BE-NQ cells expressing a range of NQO1 activ ity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induce d toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (> 7 7 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antit umor quinones in this system and suggest that a threshold level of NQO1 act ivity is required to initiate toxic events. (C) 2001 Elsevier Science Inc. All rights reserved.