Interaction of Human NAD(P)H:Quinone Oxidoreductase 1 (NQO1) with the Tumor Suppressor Protein p53 in Cells and Cell-free Systems

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1) has been proposed to stabilize p53 via a redox mechanism involving oxidation of NAD(P)H as a consequence of the catalytic activity of NQO1. We report that treatment of HCT-116 human colon carcinoma cells with the NQO1 inhibitor ES936 had no effect on the levels of p53 protein. ES936 is a mechanism-based inhibitor of NQO1 that irreversibly blocks the catalytic function of the enzyme. This suggests that a redox mechanism involving NQO1-mediated NAD(P)H oxidation is not responsible for the stabilization of p53. We also examined the ability of the NQO1 protein to associate with p53 using co-immunoprecipitation experiments. Results from these experiments demonstrated co-immunoprecipitation of NQO1 with p53 and vice versa. The association between p53 and NQO1 was not affected by treatment of HCT-116 cells with ES936, demonstrating that the association was not dependent on the catalytic activity of NQO1. A comparison of isogenic HCT-116 p53؉/؉ and HCT-116 p53؊/؊ cells demonstrated an interaction of NQO1 and p53 only in the p53؉/؉ cells. Experiments performed in an in vitro transcription/translation system utilizing rabbit reticulocyte lysates confirmed the interaction of NQO1 and p53. In these experiments a full-length p53 coding region was used to express p53 in the presence of recombinant NQO1 protein. An association of p53 and NQO1 was also observed in primary human keratinocytes and mammary epithelial cells. In studies where mdm-2 co-immunoprecipitated with p53, no association of mdm-2 with NQO1 was observed. These data demonstrate an association between p53 and NQO1 that may represent an alternate mechanism of p53 stabilization by NQO1 in a wide variety of human cell types.

Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines

“…Dicoumarol has been reported to have various functions through inhibition of NQO1, including accumulation Dicoumarol-mediated enhancement of CDDP cytotxicity J Watanabe et al of intracellular reactive oxygen species (ROS) (Lewis et al, 2004) or abrogation of p53 protein (Asher et al, 2001;Anwar et al, 2003). Ubiquitous expression of NQO1 was confirmed in the examined cell lines, and these cells also presented the almost similar sensitivity to dicoumarol.…”

“…3-3 0 -Methylene-bis [4-hydroxycoumarin] (dicoumarol) was characterized as an enzymatic inhibitor of NQO1 and was shown to inhibit proliferation of pancreatic cancer cells through an accumulation of oxidative stress (Madari et al, 2003;Lewis et al, 2004). In addition, dicoumarol induced p53 degradation through NQO1 inhibition and modulated p53-mediated apoptosis in g-irradiated normal thymocyte (Asher et al, 2001;Anwar et al, 2003). In the present study, to assess the possibility of NQO1 as a modulator for anticancer agents, we investigated the cytotoxic effects of dicoumarol in combination with CDDP, using cell lines derived from bladder and prostate cancers.…”

The NQO1 rs1800566 Polymorphism and Risk of Bladder Cancer: Evidence from 6,169 Subjects

“…This twoelectron reduction prevents the formation of semiquinone free radicals and reactive oxygen species (ROS), thus protecting cells against oxidative stress, cytotoxicity, and mutagenicity (Tsvetkov et al, 2010). Furthermore, both in vivo and in vitro studies have confirmed that NQO1 regulates the stability of the tumor suppressor p53 (Asher et al, 2002;Anwar et al, 2003). NQO1-deficient mice show reduced p53 induction and apoptosis, increased sensitivity to chemically induced tumors (Long et al, 2000;Iskander et al, 2005).…”

p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm

“…Microarray identification of candidate p63 target genes containing REs conforming to the p63 DBS Primary human mammary epithelial cells (HMECs) normally express the p63 isoform DNp63a, have a functional p53 pathway (Anwar et al, 2003) and therefore are a suitable model system to distinguish shared and distinct targets genes of the p53 family. To this end, we transduced HMECs with adenoviruses expressing p53, TAp63g or GFP, and performed comparative global gene expression analysis using Affymetrix microarray technology.…”