NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells David Siegel*, Jadwiga K. Kepa, David Ross Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America Abstract NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells. Citation: Siegel D, Kepa JK, Ross D (2012) NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells. PLoS ONE 7(9): e44861. doi:10.1371/journal.pone.0044861 Editor: Stefan Strack, University of Iowa, United States of America Received April 12, 2012; Accepted August 8, 2012; Published September 11, 2012 Copyright: 2012 Siegel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant R01ES108943. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: DS, DR and the University of Colorado are the licensor for the anti-NQO1 monoclonal antibody (clone A180). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: david.siegel@ucdenver.edu Introduction NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC 1.6.99.2) is a homodimeric flavoprotein that utilizes either NADH or NADPH and catalyzes the 2-electron reduction of a broad range of substrates most notably quinones [1]. The two- electron reduction of quinones to hydroquinones by NQO1 is believed to be primarily a detoxification reaction since it bypasses the formation of the highly reactive semiquinone [1]. In many cases, however, the reduction of quinones by NQO1 results in the formation cytotoxic hydroquinones and the bioactivation of quinone prodrugs by NQO1 has been utilized as a strategy to target NQO1-rich cancer cells [2]. In normal tissues, NQO1 is expressed at relatively high levels in epithelial tissues, vascular endothelium and adipocytes while in cancer, NQO1 is expressed at high levels in many solid tumors including lung (NSCLC), breast and pancreatic [3,4]. In humans, the NQO1*2 polymorphism plays a major role in governing basal protein levels of NQO1 [5]. The NQO1*2 polymorphism results in a proline to serine amino acid substitution at position 187 in NQO1 and this mutant protein undergoes rapid polyubiquitina- tion by the E3 ubiquitin ligase STUB1/CHIP with subsequent proteasomal degradation [6,7]. Individuals homozygous for the NQO1*2 polymorphism are NQO1 null, while intermediate levels of NQO1 protein are found in individuals with the heterozygous genotype [5]. NQO1 is under transcriptional regulation by the Keap1/NRF2 pathway and upregulation of NQO1 mRNA or protein has been used extensively as a biomarker for NRF2 activation [8,9]. Upregulation of NQO1 may protect the cell from oxidative damage due to the ability of NQO1 to reduce superoxide to hydrogen peroxide and generate antioxidant forms of vitamin E and co-enzyme Q [10,11,12]. In addition to its role as an antioxidant enzyme, NQO1 has been shown to protect a wide range of proteins including p53 from ubiquitin-independent 20 S proteasomal degradation [13,14]. The protection of target proteins by NQO1 from 20 S proteasomal degradation is dependent upon the redox state of NQO1 since treatment with the NQO1 inhibitor dicoumarol has been shown to enhance the 20 S proteasomal degradation of several target proteins [13,14]. NQO1 is predominately located in the cytoplasm but low levels of NQO1 have been found in the nucleus under normal conditions [15]. Under conditions of stress NQO1 has been shown to migrate to the nucleus where it is hypothesized that NQO1 may protect PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44861