DT-diaphorase (NADPH:quinone oxidoreductase,NQO1) was discovered in the late Professor Ernster’slaboratory, and the first reports of this discovery werepublished in 1958 [1,2]. The enzyme was detected acci-dentally during studies of NAD and NADP dehydroge-nases, and the historical aspects of the discovery havebeen described elsewhere [3]. NQO1 has attracted inter-est over the years as an enzyme involved in the detoxi-fication of xenobiotics such as quinones and quinone-
imines [4–6] and an enzyme associated with protectionagainst mutagenesis and carcinogenesis [7–10]. Re-cently, NQO1 has been characterized as being capable ofgenerating antioxidant forms of ubiquinone and vitaminE after free radical attack [11,12], providing conclusiveevidence that this enzyme forms part of the body’s an-tioxidant defense system.
The role of NQO1 in chemoprotection has also beensupported by the increased incidence of disease andxenobiotic-induced toxicity in individuals carrying apolymorphism in NQO1. The polymorphism is a ho-mozygous C to T change at position 609 of the cDNA,which codes for a proline to serine change in the aminoacid structure of the enzyme. The homozygous C609Tchange results in either nondetectable or, at best, traceamounts of mutant NQO1 protein and a lack of NQO1activity [13,14]. Because the homozygous change at po-sition 609 results in an essentially null phenotype, it
This work is dedicated to the memory of Professor Lars Ernster, whoprovided us with enthusiastic support, scientific insight, and constantencouragement in our many interactions.
Address correspondence to: Dr. David Ross, Department of Phar-maceutical Sciences, School of Pharmacy C238, University of Colo-rado Health Sciences Center, 4200 East Ninth Ave., Denver, CO80262, USA; Tel: (303) 315-6077; Fax: (303) 315-0274; E-Mail:[email protected].
provides a convenient molecular tool with which to as-sess the role of NQO1 in vivo. The NQO1 609 polymor-phism has been associated with an increased risk ofurothelial tumors [15], therapy-related acute myeloidleukemia [16], urolithiasis [17], cutaneous basal cellcarcinomas [18], and pediatric leukemias [19], as well aswith increased benzene-induced hematopoietic toxicity[20]. Recent work suggests that a lack of NQO1 due tothis polymorphism is not a risk factor for prostate cancer[21]. The role of NQO1 in susceptibility to many otherforms of cancer is currently under investigation, and ourown work suggests it is a risk factor for pharyngealcancer in a Japanese population (M. Yano, D. Siegel, andD. Ross, unpublished results).
Many enzymes which have been generalized as de-toxification systems may also bioactivate certain sub-strates, and NQO1 also falls into this category. NQO1has been demonstrated to bioactivate nitropyrenes con-tained in tobacco smoke [22], and this has been sug-gested as a possible explanation of an association of thewild-type NQO1 allele with an increased incidence oflung cancer in certain ethnic groups [23,24]. NQO1 hasbeen found to be expressed at high levels in many humantumors [25,26–29], which has led to the possibility ofdeveloping compounds bioactivated by NQO1 as che-motherapeutic agents [30–33]. Chemotherapeutic agentsthat are bioactivated by NQO1 include mitomycin C,streptonigrin [33,34], and newly developed agents thatare highly efficient substrates for NQO1, such as theaziridinylbenzoquinone RH1 [35]. RH1 is currently un-der consideration for clinical trial, and efforts are under-way to correlate clinical response of established agents,such as mitomycin C, to patient NQO1 status.
Despite the extensive interest in NQO1, there is littleinformation regarding its distribution in either normalhuman tissues or in human tumors. We have recentlylocalized NQO1 in human lung tumors and uninvolvedtissues by using immunohistochemistry [36]. In thismanuscript, we have extended these data to many othertissues and tumors as a step toward defining the distri-bution of NQO1 in humans.
MATERIALS AND METHODS
Human tissues
Archival samples of formalin-fixed, paraffin-embed-ded tissues (thyroid, adrenal, breast, ovary, lung) weresupplied by the University of Colorado Cancer Center.Archived paraffin blocks of normal eye and cornealtumors were supplied by Dr. Matthew W. Wilson, De-partments of Ophthalmology and Pathology, Universityof Colorado Health Sciences Center. Fresh frozen aortaand myocardial tissues were supplied by Dr. Kathleen
Stringer, Department of Pharmacy Practice, School ofPharmacy, University of Colorado Health Sciences Cen-ter. Human liver cytosol samples were purchased fromthe Human Cell Culture Center (Laurel, MD, USA).
Antibodies and reagents
Anti-NQO1 monoclonal antibody (IgG1) –secretinghybridomas (clones A180 and B771) were derived froma BALB/c mouse immunized with purified recombinanthuman NQO1 protein. Control (nonspecific IgG1 secret-ing) hybridoma (clone C100) was derived from aBALB/c mouse. All hybridoma cell lines were grown inspinner flasks in RPMI 1640 medium containing 50units/ml penicillin, 50mg/ml streptomycin, 1% L-glu-tamine, and 10% fetal bovine serum in 5% CO2 at 37°Cto a concentration of 106 cells/ml. Hybridoma tissueculture supernatants were prepared by centrifugation at1800 rpm for 10 min and then stored at280°C. Prior touse, supernatants were centrifuged at 14,000 rpm for 5min. Detection of NQO1 by IHC was performed usingavidin:biotinylated enzyme complex (ABC) methodolo-gies with DAB staining (Vectastain Elite ABC Kit, Vec-tor DAB Substrate Kit, Vector Laboratories, Burlingame,CA, USA). A horseradish peroxidase–conjugated goatanti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) and peroxidase-based en-hanced chemiluminescence (ECL, Amersham Pharma-cia, Buckinghamshire, UK) were used for detection ofNQO1 by immunoblot analysis.
Immunohistochemistry
Immunohistochemistry was performed on tissue sec-tions (4mm) cut from archival paraffin blocks as previ-ously described [36]. Briefly, sections were deparaf-finized in xylene and rehydrated through graded alcoholsto distilled water and then microwaved for two 3 mincycles. Endogenous peroxidase activity was eliminatedby placing sections in 3% hydrogen peroxide for 20 min.Immunodetection of NQO1 was performed using tissueculture supernatants from mouse hybridoma clones A180and B771. Negative controls were performed utilizingsupernatant from control hybridoma clone C100. Serialsections of each tissue sample were incubated with eitheranti-NQO1 or control antibodies for 1 h at27°C. Immu-nodetection was performed using a horseradish peroxi-dase–based Vectastain Elite ABC Kit. 3,3-Diaminoben-zidine (DAB) and hydrogen peroxide were used as thehorseradish peroxidase substrates (brown staining). Sec-tions were then counterstained with hematoxylin. Noimmunostaining was observed in sections that receivedcontrol antibodies. Sections were photographed on aNikon FX microscope with Kodak Ektachrome 64 film.
247Immunodetection of NQO1 in human tissues
Fig. 1. Immunoperoxidase staining (DAB, brown) of NQO1 in normal human tissues. (A) Respiratory epithelium; (B) breast ductalepithelium; (C) thyroid follicle epithelium; (D) colon epithelium; (E) corneal epithelium; (F) lens epithelium; (G) vascular endothe-lium; (H) parasympathetic ganglion (colon); and (I) optic nerve fibers.
248 D. SIEGEL and D. ROSS
Immunoblot analysis
Immunoblot analysis of NQO1 was performed aspreviously described [14]. Aorta and myocardial tissuesamples were homogenized in 5 volumes of 25 mMTris-HCL, pH 7.4, containing 250 mM sucrose and 1mMflavin adenine dinucleotide. Cell homogenates were thensonicated on ice for 15 s, after which they were centri-fuged at 10,0003 g for 30 min. The supernatant wasrecovered and protein concentrations were determinedby the method of Lowry and colleagues [37]. Proteinconcentrations of liver cytosolic samples were also de-termined by the method of Lowry and colleagues [37].Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (minigel) and trans-ferred to 0.4mm polyvinylidene difluoride membrane in25 mM Tris, 192 mM glycine containing 20% (v/v)methanol at 100 V for 1 h. Immunoblot analysis ofNQO1 was performed with mouse anti-NQO1 monoclo-nal antibodies (clones A180/B771). Membranes wereincubated in hybridoma tissue culture supernatant for1 h, followed by the addition of horseradish peroxidase–conjugated goat anti-mouse IgG (1:5000) for 30 min.Protein visualization was performed by using ECL asdescribed by the manufacturer.
RESULTS
Using IHC, we examined cell-specific expression ofNQO1 in normal human tissues. In previous work [36],
we have reported a high level of NQO1 expression inrespiratory epithelial cells (Fig. 1A). In this study, NQO1was also detected by IHC in the epithelium of othertissues. Positive staining for NQO1 was observed inbreast duct epithelium (Fig. 1B); thyroid follicle epithe-lium (Fig.1C); epithelial lining of the colon (Fig. 1D);and in the eye in corneal and lens epithelia (Figs. 1E and1F).
In addition to epithelial expression, NQO1 was alsodetected in vascular endothelium (Fig.1G), including incapillary endothelium and the endothelial lining of theaorta. Immunoblot analysis and activity measurements ofNQO1 in cardiac tissues demonstrated NQO1 activity,and NQO1 protein could readily be detected in the aorta,but in the myocardium, NQO1 activity was only slightlyabove the level of detection, and only a trace amount ofNQO1 protein could be detected by immunoblot analysis(Fig. 2). Further studies using monkey aorta demon-strated positive staining for NQO1 in the endotheliallining (not shown).
Other tissues that stained positive for NQO1 includefat and some nervous tissues. NQO1 was detected inadipocytes from lung, breast, ovary, and colon. NQO1protein was also detected by IHC in parasympatheticganglion located throughout the gastrointestinal tract(Fig. 1H). In the eye, NQO1 was detected by IHC in theoptic nerve and optic nerve fibers (Fig. 1I); this wasconfirmed by immunoblot analysis (not shown).
Analysis of human liver confirmed that NQO1 is nothighly expressed in hepatic tissues. Immunoblot analysisof NQO1 in five human liver samples demonstrated onlytrace levels of protein (Fig. 3); IHC revealed only min-imal NQO1 staining in isolated bile ductal epithelium(not shown).
In addition to normal tissues, we also examinedNQO1 expression by IHC in a variety of solid tumors.NQO1 was detected in solid tumors from thyroid, adre-nal, breast, ovary, colon, cornea, and, as reported else-where [36], from lung (Figs. 4A–4G). In most tumors,staining for NQO1 was uniform throughout the sample.In most tumors, there was no difference in intensity ofstaining for NQO1 in tissue proximal or distal to tumorvasculature or areas of necrosis (Fig 4H). No staining forNQO1 was observed in tumor connective and stromaltissues, however, staining for NQO1 was detectedthroughout tumor vascular endothelium (Fig. 4I).
DISCUSSION
We have previously demonstrated the presence ofNQO1 in human respiratory epithelium and endothelium[36], and our present data extend these observations to anumber of other human tissues. NQO1 was detected inthe epithelium of thyroid, breast, colon, and eye tissues,
Fig. 2. Immunoblot analysis of NQO1 in human aorta and myocardium.Immunoblot analysis with chemiluminescence detection was used toexamine NQO1 protein in aorta and myocardium cytosolic samples (20mg) from two separate donors (1 and 2). rhNQO1, recombinant humanNQO1 standard (5 ng). Film exposure time, 2 min.
Fig. 3. Immunoblot analysis of NQO1 in human liver. Immunoblotanalysis with chemiluminescence detection was used to examineNQO1 protein levels in liver cytosol from five separate donors. rh-NQO1, recombinant human NQO1 standard (5 ng); lanes 1–5, livercytosolic samples (20mg). Film exposure time, 10 min.
249Immunodetection of NQO1 in human tissues
Fig. 4. Immunoperoxidase staining (DAB, brown) of NQO1 in human tumors. (A) thyroid; (B) adrenal; (C) breast; (D) ovarian; (E)colon; (F) cornea; (G) lung; (H) breast, showing necrotic areas; and (I) breast, tumor vascular endothelium.
250 D. SIEGEL and D. ROSS
and immunostaining in these tissues was consistent witha high level of NQO1 expression. We have also observedthat NQO1 is expressed at high levels in vascular endo-thelial cells [36], which is consistent with our findings inthis study. Interestingly, although NQO1 activity andprotein could readily be detected in human aorta, onlytrace levels of NQO1 protein could be detected in themyocardium. Because NQO1 can function as an antiox-idant enzyme via the production of antioxidant forms ofubiquinone and vitamin E, the relative lack of NQO1 inmyocardial tissue may contribute to the sensitivity of thistissue to oxidative stress generated from such agents asdoxorubicin [38].
An interesting observation in this work was the pres-ence of marked NQO1 protein levels in nervous tissues,such as parasympathetic ganglia located throughout thesmall and large intestine, as well as in the eye, in theoptic nerve and nerve fibers. NQO1 has previously beenshown to be expressed in rat brain where it was found inboth neuronal and glial cells and localized predominantlyto the substantia nigra, the striatum, and the ventraltegmental area [39]. It has been suggested that the pres-ence of NQO1 in dopaminergic neurones may protectsuch cells from toxic dopamine metabolites such as cy-clized dopa-O-quinone [40]. The presence of NQO1 ac-tivity in human adipose tissue from one individual hasbeen observed elsewhere [41], and our study confirmsthis observation by localizing NQO1 by IHC to adipo-cytes. The ability of NQO1 to generate lipophilic anti-oxidant forms of ubiquinone and vitamin E [11,12] mayassist in the protection of adipocytes from oxidativestress generated by lipid radicals. The high levels ofNQO1 in corneal and lens epithelium suggest that NQO1may also have a protective role against UV-induced lipidperoxidation.
The primary enzyme capable of reducing quinones tohydroquinones in human liver has been suggested to becarbonyl reductase rather than NQO1 [42], although thisconclusion has been questioned [26]. The observation ofrelatively low NQO1 activity in human liver [26,42],however, is in marked contrast to the situation in rat liverwhere levels of NQO1 are high, and as a result, rat liverhas been used in the past as a tissue source for purifica-tion of NQO1 [43]. In our work, we observed only tracelevels of NQO1 protein in five separate human livercytosol samples when using immunoblot analysis,whereas immunostaining of human liver tissues revealedthe presence of NQO1 in only a few isolated bile ductepithelial cells. NQO1 is expressed, however, in humanfetal liver (Wiemels and Greaves, unpublished, in [19])and in human hepatic tumors [26,44], suggesting thatthere may be differential regulation of NQO1 expressionin fetal versus adult livers as well as liver cancers.Regulation of expression of NQO1 is complex [45,46],
and we have observed marked differences in expressionin cell types within the lung and in various types of lungtumors. For example, NQO1 levels are high in NSCLC,but in SCLC, they are very low [28,47]. The mechanismsunderlying this differential expression are currently un-der investigation, and whether the lack of NQO1 expres-sion reflects a lack of positive regulatory proteins or thepresence of repressive factors remains to be determined.
In addition, relatively little NQO1 protein expressionhas been detected in cells of hematopoietic origin [48].Recent studies with freshly isolated human bone marrowaspirates confirmed the absence of NQO1 activity andprotein, although NQO1 protein and activity could beinduced in bone marrow by in vitro exposure to benzenemetabolites. [49]. Human bone marrow stroma grown invitro from human bone marrow aspirates, however, con-tains substantial NQO1 protein and activity [50]. It is notclear at present whether this difference in NQO1 expres-sion between primary and cultured human bone marrowis due to induction by in vitro culture conditions or thepresence of adherent cells in the marrow that containNQO1 and are not recovered by aspiration. The absenceof NQO1 activity in the marrow may have clinical con-sequences for NQO1-directed antitumor agents, and inclinical trials of the antitumor quinone E09, significantmyelotoxicity was not observed [51].
The marked NQO1 activity and protein levels thathave previously been observed in some human tumors[26–29] was extended to human breast, ovarian, pancre-atic, thyroid, adrenal, colon, and corneal tumors by usingIHC. There was uniform staining for NQO1 throughoutmost tumors, suggesting little heterogeneity of expres-sion of NQO1. These results are supportive of the use ofNQO1-directed agents for the therapy of tumors withelevated levels of this enzyme and suggest that the ther-apeutic effects of such compounds may be applicable tomany types of tumor. The expression of NQO1 in manynormal tissues, however, suggests that potential sideeffects of these agents should be monitored carefully inpreclinical studies.
Acknowledgement— The work described was supported by NIH grantsRO1 CA 51210 and RO1 ES 09554.
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