A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C

Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a vari ety of cancers including head and neck cancer. Among the various enzymes, d icoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking o f the DNA and cytotoxicity. However, the role of NQO1 in metabolic activati on of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic ac tivation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanentl y express higher levels of cDNA-derived NQO1. These cells showed significan tly increased protection against menadione toxicity. However, they failed t o demonstrate higher cytotoxicity due to exposure to MMC under oxygen (norm al air) or hypoxia, as compared to the wild-type control CHO cells. Disrupt ion of the NQO1 gene by homologous recombination generated NQO1-/- mice tha t do not express the NQO1 gene resulting in the loss of NQO1 protein and ac tivity. The cytosolic fractions from liver and colon tissues of NQO1-/- mic e showed similar amounts of DNA cross-linking upon exposure to MMC, as obse rved in NQO1+/+ mice. The unique cytosolic activity that activated MMC in c ytosolic fractions of liver and colon tissues of NQO1-/- mice was designate d as cytosolic MMC reductase. This activity, like NQO1, was inhibited by di coumarol and immunologically related to NQO1. (C) 2000 Cancer Research Camp aign.