NAD(P)H:quinone oxidoreductase 1 (NQO1) localizes to the mitotic spindle in human cells - PubMed (original) (raw)
NAD(P)H:quinone oxidoreductase 1 (NQO1) localizes to the mitotic spindle in human cells
David Siegel et al. PLoS One. 2012.
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.
Conflict of interest statement
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.
Figures
Figure 1. Immunofluorescent staining of NQO1 in BxPc-3 human pancreatic cancer cells.
Immunostaining for NQO1 reveals cytosolic localization and intense staining of mitotic spindles in cells undergoing division (*).
Figure 2. Knockdown of NQO1 eliminates immunostaining of NQO1 in BxPc-3 cells.
Knockdown of NQO1 in BxPc-3 cells was performed using a doxycyline- inducible anti-NQO1 shRNA methodologies. BxPc-3 cells were immunostained for NQO1 (green) in the absence (panel A) and presence (panel B) of doxycyline pretreatment. DAPI staining was included in panel B to aid in the location of cells. Right panel: immunoblot demonstrating near complete knockdown of NQO1 protein in the presence of doxycycline. Arrows indicate mitotic cells.
Figure 3. Co-localization of NQO1 and alpha-tubulin in mitotic spindles and midbody region in BxPc-3 human pancreatic cancer cells.
Triple immunofluorescent staining for NQO1 (red), alpha-tubulin (green) and nuclei (blue). Association of NQO1 with spindles can be seen in different stages of mitosis including metaphase (*), telophase (**) and the midbody region (arrow) during cytokinesis.
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 (50µM, panel C). Cells were pretreated with inhibitors 2 hr before immunostaining. Arrows indicate mitotic cells.
Figure 5. Co-localization of NQO1 and alpha-tubulin with mitotic spindles in human cells.
Triple immunofluorescent staining for NQO1 (red, panel A); alpha-tubulin (green, panel B); merged (with DAPI,panel C).
Figure 6. Co-localization of NQO1 with mitotic spindles is not observed in cells homozygous for the NQO1*2 polymorphism.
Panc-1 cells which are homozygous for the NQO1*2 polymorphism (NQO1 null), were prelabeled with cell tracker green then mixed with unlabled Panc-1/C5 cells which stably express wild type hNQO1. Following a 24hr coincubation period cells were fixed and immunostained for NQO1 followed by confocal microscopy. Cells were searched until a mitotic Panc-1 cell (green) and a mitotic Panc-1/C5 cell (non-staining) were observed in the same field. Panels A and C, data was collected using 488nm excitation wavelength for cell tracker green fluorescence (green Panc-1 cells). Panels B and D, data was collected using 561nm excitation wavelength (red/NQO1). Co-localization of NQO1 to the mitotic spindles was not observed in NQO1-null Panc-1 cells (green) while co-localization of NQO1 to the mitotic spindle was seen in NQO1 overexpressing Panc-1/C5 cells.
Figure 7. NQO1 localizes to mitotic spindles in human lung cancer cells.
Immunohistochemical analysis of NQO1 (DAB, brown) in archival formalin-fixed human squamous lung carcinoma tissue samples. Mitotic figures are visible due to their intense chromatin staining (hemotoxylin).
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