NQO1 Localizes to the Mitotic Spindle Figure 4. NQO1 inhibitors do not prevent binding of NQO1 to mitotic spindles. Immunostaining for NQO1 (red) in BXPC3 cells pretreated with DMSO (panel A), ES936 (500 nM, panel B) or dicoumarol (50mM, panel C). Cells were pretreated with inhibitors 2 hr before immunostaining. Arrows indicate mitotic cells. doi:10.1371/journal.pone.0044861.g004 The ability of NQO1 to associate with the mitotic spindles in human cells while still maintaining catalytic activity has implica- tions for both quinone detoxification and activation reactions in close proximity to critical mitotic apparatus. The reduction of quinones by NQO1 could result in protection of vulnerable microtubules from attack by electrophilic quinones. The benzene metabolite p-benzoquinone has been shown in cell free systems to react readily with sulfhydryl groups on microtubule proteins and inhibit assembly into microtubules [22]. Alternatively, NQO1 could be the source of high levels of reactive oxygen species due to redox cycling of unstable hydroquinones formed in close proximity to the mitotic apparatus. An alternative catalytic function of NQO1 may be to aid in DNA repair during mitosis through the generation of NAD+ an essential cofactor for proteins including the family of poly (ADP ribose) polymerase (PARP) enzymes and the sirtuins. PARP enzymes have been shown to play an important role in the repair of single strand DNA breaks [23] while SIRT6 has been proposed to aid in DNA repair under conditions of oxidative stress by binding directly to PARP1 and stimulating PARP1 poly-ADP- ribosylase activity [24]. From our studies it is unclear whether NQO1 binds directly to microtubules in mitotic spindles or binds to a protein complex that associates with the mitotic spindles. NQO1 has many character- istics of a microtubule binding protein including a net positive charge and multiple domains containing clusters of positively charged amino acids [25,26,27]. Alternatively, NQO1 could be binding to another protein or protein complex that is tethered to the mitotic spindle. In addition to its major role in quinone metabolism NQO1 has emerged as a regulator of 20 S proteasomal degradation [28]. A growing number of proteins have been shown to be protected from ubiquitin-independent 20 S proteasome degradation by NQO1 including p53, p63, p73 and ornithine decarboxylase [14,29,30,31]. Interestingly, immunoflu- orescence studies have previously demonstrated co-localization of p53 with centrosomes and mitotic spindles in human cell lines [32] and co-localization of the 20 S proteasome to centrosomes and mitotic spindles in rat Schwann cells [33]. Whether NQO1 interacts with these proteins on the spindle to facilitate in the stabilization and redistribution of selected proteins such as p53 during mitosis is currently under investigation. Recently, it has been shown that persistent expression of NQO1 via p62-mediated Nrf2 activation facilitated p53-dependent mitotic catastrophe supporting a role for NQO1 in the stabilization of p53 during mitosis [34]. In summary, these data report the association of NQO1 with mitotic spindles in human cells using immunofluorescent staining and suggest that the association of NQO1 with spindles may involve protection against electrophic quinones, aid in the generation of oxidized pyridine nucleotides or shield selected proteins from 20 S degradation. Materials and Methods Reagents Anti-NQO1 mouse monoclonal antibody (IgG1, clone A180) was developed in our laboratory against full-length recombinant human NQO1 protein and hybridoma supernatant (RPMI1640 containing 10% fetal bovine serum) from clone A180 was used for these studies (dilution 1:5–1:20). Affinity purified anti- NQO1 monoclonal antibody from clone A180 was obtained commercially from Cell Signaling Technologies (Danvers, MA) and used at a dilution of 1:200. Anti-alpha tubulin rabbit polyclonal antibody was purchased from Abcam, (Cambridge MA, Cat#15246, dilution 1:10,000). DyLight 488 and DyLight 544 conjugated goat anti-mouse IgG and DyLight 488 conjugated goat anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove PA). 49,6-diamidino-2-phenylindole (DAPI,) dicoumarol and doxy- cycline were obtained from Sigma (St. Louis MO). The NQO1 mechanism-based inhibitor ES936 (5-methoxy-1,2-dimethyl-3- [(4-nitrophenoxy)methyl]indole-4,7-dione) was synthesized in the laboratory of Dr. Christopher J. Moody, School of Chemistry, University of Nottingham, U.K [35]. Cell Lines and Tissues BXPC3 and Panc-1 human pancreatic cancer cell lines were obtained from ATCC (Manassas, VA) and grown in RPMI 1640 containing 5% (v/v) fetal bovine serum and 1% (v/v) penicillin/ streptomycin. Panc-1 cells have previously been genotyped as homozygous for the NQO1*2 polymorphism and are NQO1null [17]. Panc-1/C5 cells stably express wildtype NQO1 and were created from parental Panc-1 cells as described [17]. Primary cultures of human umbilical cord vein endothelial cells (HUVEC) PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44861