NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms

Chemico-Biological Interactions 129 (2000) 77 – 97

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NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms David Ross a,*, Jadwiga K. Kepa a, Shannon L. Winski a, Howard D. Beall b, Adil Anwar a, David Siegel a a Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, Box C-238, Uni6ersity of Colorado Health Sciences Center, 4200 East 9th A6enue, Den6er, CO 80262, USA b Department of Pharmaceutical Sciences, School of Pharmacy, Uni6ersity of Montana, Missoula, MT 59812, USA

Abstract NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NQO1 (NAD(P)H:quinone oxidoreductase 1); Quinone; Vitamin E; Ubiquinone; Antitumor quinones; Gene regulation; Antioxidant response element (ARE); Polymorphisms

* Corresponding author. Tel.: + 1-303-3156077; fax: +1-303-3150274. E-mail address: [email protected] (D. Ross). 0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 1 9 9 - X

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1. Introduction The catalytic properties of NAD(P)H:quinone oxidoreductase 1 (NQO1) were first reported by Ernster and Navazio in 1958 [1]. NQO1 is an obligate two-electron reductase that is characterized by its capacity for utilizing either NADH or NADPH as a reducing cofactor and by its inhibition by dicoumarol [2]. There is considerable data indicating that NQO1 can protect against natural and exogenous quinones. One of the first examples of its protective nature was the discovery that menadione reductase (later discovered to be NQO1) levels were induced in response to low doses of certain carcinogens and this afforded some protection against subsequent treatments [3]. NQO1 reduces quinones to hydroquinones in a single two-electron step. In addition to yielding substrates for Phase II conjugation reactions and promoting excretion, this two-electron process bypasses the potentially toxic semiquinone radical intermediates. Not all hydroquinones are redox-stable, however, and in some cases metabolism by NQO1 yields a more active product. Redox-labile hydroquinones can react with molecular oxygen to form semiquinones and generate reactive oxygen species, or semiquinones can be generated via comproportionation reactions [4]. In addition to potentially causing oxidative stress through this mechanism, the reduction of the quinone moiety can produce a compound that is capable of alkylating nucleophilic sites including DNA (Fig. 1). This process has been termed ‘bioreductive alkylation’ and has formed the basis for research into the design of NQO1-directed antitumor agents.

2. NQO1 catalytic cycle and substrate specificity NQO1 is an obligate two-electron reductase that is characterized by its capacity for utilizing either NADH or NADPH as reducing cofactors and its potent inhibition by dicoumarol [2]. It is primarily a cytosolic enzyme (\ 90%) and exists as a homodimer with one molecule of FAD per monomer. Study of the X-ray

Fig. 1. Activation and deactivation resulting from NQO1-mediated reduction of quinones.

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crystal structure confirmed that NQO1 functions via a ping pong mechanism where the reduced pyridine nucleotide binds, reduces the flavin co-factor and the oxidized pyridine nucleotide is released prior to binding of the substrate [5]. The pyridine nucleotide and quinone binding sites were found to have significant overlap suggesting a molecular explanation for this mechanism [5]. Reduction to the hydroquinone occurs in a single step bypassing semiquinone radical intermediates. This mechanism has been supported by ESR experiments which failed to detect semiquinone radicals during the metabolism of benzoquinone and naphthoquinone substrates [6] and confirmed by stop-flow and steady-state kinetic methods [7]. The mechanism of catalysis has been proposed to involve a hydride transfer between the NADH and FAD cofactors and from FADH2 to the quinone substrate [5]. NQO1 is capable of reducing a very broad range of substrates including quinones, quinone-imines, glutathionyl-substituted naphthoquinones, dichlorophenolindolphenol, methylene blue, azo and nitro compounds [2,8,9]. Both ortho and para quinones are substrates for NQO1 [10]. Metabolism is not limited to quinones and the enzyme functions efficiently as a nitro-reductase utilizing substrates such as dinitropyrenes, nitrophenylaziridines and nitrobenzamides [11–13]. In addition to two-electron reduction, NQO1 is also capable of performing four-electron reduction of azo dyes and nitro compounds [14,15]. Using heterodimers of NQO1, it was determined that the NQO1 subunits function independently in metabolizing twoelectron substrates and in a dependent fashion with four-electron substrates [16].

3. Detoxification reactions catalyzed by NQO1

3.1. Detoxification of substrates by two-electron reduction Numerous studies have proposed a role for NQO1 in the detoxification of redox-cycling quinones such as menadione. Two-electron reduction by NQO1 directly competes with cellular one-electron reductases for menadione [17–22]. Reduction of menadione by NQO1 results in the formation of a stable hydroquinone that can be readily conjugated and excreted [23]. Alternatively, reduction of menadione by one-electron reductases results in the formation of a semiquinone which in the presence of molecular oxygen redox-cycles to form reactive oxygen species [17,22,24,25]. This hypothesis is supported by recent studies in mice where disruption of the NQO1 gene resulted in increased menadione toxicity [26]. Additional evidence for the role of NQO1 in menadione detoxification has come from studies using compounds that induce NQO1 such as BHA, BHT and dimethyl fumarate. Induction of NQO1 was shown to protect against menadione-induced hemolytic anemia, but interestingly, in the same study induction of NQO1 was shown to potentiate the toxicity of 2-hydroxy-1,4-naphthoquinone [27]. These data highlight the role of NQO1 as either a detoxification enzyme or an activation enzyme depending upon the stability of the hydroquinone generated following reduction.

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Fig. 2. The role of NQO1 in regeneration of antioxidant forms of ubiquinone and Vitamin E.

Another role for NQO1 in quinone detoxification is the removal of potentially arylating quinones. Quinones can readily undergo addition and substitution reactions and can react directly with protein thiols. In co-transfection studies with cytochrome P450 reductase, NQO1 has been shown to decrease benzo[a]pyrene 3,6-quinone-induced DNA adduct formation [28]. In these experiments, the authors suggested that the active DNA binding species was the semiquinone formed by a one-electron reduction catalyzed by cytochrome P450 reductase. NQO1-mediated two-electron reduction of benzo[a]pyrene 3,6-quinone resulted in formation of a stable hydroquinone. Our studies have shown that NQO1 participates in the detoxification of the benzene-derived metabolite 1,4-benzoquinone. Hydroquinone was selectively bioactivated in, and toxic to, marrow macrophages rather than fibroblasts in bone marrow stroma because of increased levels of peroxidases (leading to increased bioactivation) and lower levels of NQO1 (decreased deactivation) in macrophages relative to fibroblasts [29–31]. Transfection of human promyeloblastic leukemia cells with NQO1 significantly decreased benzenetriolDNA adduct formation [32]. In addition, we have shown that in human promyeloblastic leukemia cells and human CD34+ hematopoietic progenitor cells, NQO1 induction by prior exposure to non-lethal concentrations of hydroquinone protects cells from apoptosis induced by higher concentrations of hydroquinone [33].

3.2. NQO1 as an antioxidant enzyme Recent work with NQO1 has suggested that the enzyme may play an antioxidant role via the reduction of endogenous quinones and these compounds, when reduced, help protect cellular membranes against oxidative damage (Fig. 2). Experiments have demonstrated that rat liver NQO1 can catalyze the reduction of ubiquinone analogs (coenzyme Q) to their ubiquinol forms in liposomes and rat hepatocytes [34,35]. The rate of reduction of coenzyme Q derivatives was dependent upon the length of the carbon side-chain; short-chain homologs were reduced more efficiently than long-chains. In these studies it was shown that the ubiquinol formed

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following reduction by NQO1 was an effective antioxidant protecting membrane phospholipids from oxidative damage. a-Tocopherolquinone, a product of a-tocopherol (vitamin E) oxidation has been shown to have antioxidant properties following reduction to a-tocopherolhydroquinone [36–38]. In experiments with purified human NQO1 it was shown that this enzyme could effectively catalyze the reduction of a-tocopherolquinone to a-tocopherolhydroquinone [39]. We have also demonstrated that Chinese hamster ovary cells transfected with human NQO1 generated higher levels of a-tocopherolhydroquinone and were more resistant to lipid peroxidation than cells lacking NQO1 [39]. An additional role for NQO1 in a-tocopherol metabolism has been postulated where NQO1 maintains physiological levels of a-tocopherol from the reduction of a-tocopherones by NQO1 [40]. Oxidation of a-tocopherol by peroxyl radicals yields 8a-(alkyldioxy) tocopherones which may either hydrolyze to a-tocopherolquinone or may be reduced to regenerate a-tocopherol [41]. Regeneration of a-tocopherol from a-tocopherone by NQO1, however, remains to be demonstrated. A role for NQO1 as an antioxidant enzyme is further supported by recent immunohistochemical studies in humans that have shown NQO1 protein is expressed in many tissues requiring a high level of antioxidant protection [42–44]. These include the epithelial cells of lung, breast and colon, vascular endothelium, adipocytes, corneal and lens epithelium, retinal pigmented epithelium, optic nerve and nerve fibers. The high levels of NQO1 suggest that NQO1 may function primarily in an antioxidant capacity in these cells. Interestingly, biochemical and immunological-based assays have failed to detect significant levels of NQO1 expression in human liver [42,45] suggesting that in humans, unlike most species, NQO1 does not play a major role in hepatic xenobiotic metabolism.

4. Bioactivation by NQO1

4.1. Bioacti6ation reactions The chemical properties of the hydroquinone formed after NQO1 mediated reduction of a quinone determines whether NQO1 catalyzes activation or deactivation. Not all hydroquinones are chemically stable and in some cases metabolism by NQO1 yields a more active product which can autoxidize to produce reactive oxygen species (Fig. 1) or undergo rearrangement to generate alkylating species. An example of this is the reduction and activation of nitro compounds found in cooked foods such as 4-nitroquinoline-1-oxide (4NQO). Sugimura and colleagues reported the activation of 4NQO to 4-hydroxyaminoquinoline-1-oxide by a rat liver enzyme [46]. It was discovered later that the predominant enzyme responsible for this was NQO1 [47]. As with most substrates that are bioactivated by NQO1, naphthoquinones (NQ) and dinitropyrenes (DNP) can either be activated or deactivated by NQO1 depending on substituent groups and their location. Induction of NQO1 with butylated hydroxyanisole, protected rats from the toxic effects of 2-methyl-1,4-NQ but

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potentiated the toxicity of 2-hydroxy-1,4-NQ [27]. For DNPs which are found in diesel exhaust, the positioning of the nitro groups greatly influences the clastogenic potential of the activated compounds. In addition NQO1 increases mutagenicity of 1,6- but not 1,3- or 1,8-DNP in Ames Salmonella typhimurium assay [48].

4.2. Enzyme directed antitumor agents Drugs that can produce alkylating metabolites after reduction have been termed ‘bioreactive alkylating agents’ [49]. The result of bioreduction is either the production of alkylating species or active oxygen species depending on the chemical properties of the compounds undergoing enzymatic reduction (see Ref. [4] for discussion). Selective toxicity of bioreductive alkylating agents to tumors was based on the premise that hypoxic cells near the necrotic cores of tumors would have a greater propensity for reductive metabolism [49]. Enzyme-directed antitumor drug development, however, exploits bioactivating enzymes that are expressed at high levels in tumors relative to uninvolved tissue. NQO1 is expressed at high levels throughout many human solid tumors [9,50] and is one possible candidate for the enzyme-directed approach. A detailed review of enzyme-directed drug discovery focused on quinones has recently been published [51]. Since NQO1 is present is present in uninvolved tissues as well as human tumor tissue [41], it is possible that toxicity too normal tissue may be an issue in therapy with NQO1-directed antitumor quinones. Mechanistically, DNA is thought to be the target of such enzyme-directed alkylating agents, so the higher growth fraction of tumors may still offer opportunity for selective toxicity. The development of quinones which can be efficiently bioactivated by NQO1 as potential antitumor agents has focuded on aziridinylbenzoquinones, indolequinones, motosenses, pyrrolobenzimidazolequinones and cyclopropamitosenses (Fig. 3). Understanding NQO1 bioactivation in complex cellular systems is complicated by the presence of other reductases. To combat this problem, newer model systems are now being employed including gene targeting [52] and enzyme induction [53] approaches together with the use of NQO1-transfected cell lines [54–56] and specific NQO1 inhibitors [57,58]. This work has provided unequivocal evidence that NQO1 is capable of bioactivation of antitumor quinones in cellular systems and currently work is ongoing to determine the role of NQO1 in vivo in both xenograft models and eventually in clinical settings.

4.2.1. Indolequinones and mitosenes Mitomycin C (MMC) has been one of the most successful single agents used in the treatment of NSCLC and is currently used in combination cancer therapies [59]. MMC is bioactivated through reduction and this can be accomplished by NQO1 in vitro [60]. MMC is a relatively poor substrate for NQO1 [61,62] and newer compounds have been designed in the hopes of generating more efficient substrates and therefore better chemotherapeutic agents. In structure–activity relationship studies using indolequinones and mitosenes, a number of features have been identified which impact metabolism and cytotoxicity [56–58,63–69] and these have

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been recently summarized [51]. Depending on the substitution patterns in the indole or mitosene structure, compound design within the structural series can be optimized for potency and selective toxicity to cells containing high NQO1 levels.

4.2.2. Aziridinylbenzoquinones Diaziquone (AZQ) is an aziridinylbenzoquinone that was used for the treatment of glioma [70]. Similar to MMC, AZQ is a relatively poor substrate for NQO1, but the structure-based metabolism studies have yielded several aziridinylbenzoquinones that are better substrates for NQO1 than AZQ [71]. MeDZQ (2,5-diaziridinyl-3,6-methyl-1,4-benzoquinone) was discovered in screening a panel of synthetic aziridinylbenzoquinones. The rate of MeDZQ metabolism by purified NQO1 was reported to be over 100-fold higher than MMC, and cytotoxicity and selectivity to NQO1-containing cells was also increased [61,62,72]. MeDZQ also required enzymatic activation by NQO1 in order to crosslink DNA [72]. Due to potential formulation problems from the low solubility of MeDZQ, RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) was developed as a watersoluble analog. RH1 was a better substrate for purified NQO1 and more cytotoxic to NQO1-expressing cell lines than MeDZQ or MMC. Using NQO1-transfected cell lines [54,56], we have examined the relative cytotoxicity of MeDZQ, MMC and RH1. In NQO1-transfected Chinese hamster ovary (CHO) cell lines, we found that

Fig. 3. Quinones and other compounds considered as NQO1-directed antitumor agents.

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MeDZQ exhibited increased toxicity relative to the parent cell line but this effect was not observed with MMC [54]. A similar human-derived model was obtained by transfecting human NQO1 into human BE colon adenocarcinoma cell lines which lack NQO1 activity due to a genetic mutation [56]. Because of the absence of NQO1 activity in parental cells, BE cells stabley transfected with human NQO1 offer a useful model system in which to examine the selective toxicity of NQO1-directed antitumor agents. In this system, MMC, MeDZQ and RH1 exhibited increased cell killing in the NQO1-transfected BE cells relative to parental controls [56]. The degree of selective toxicity was in the order of RH1 (× 17)\MeDZQ (×7) \MMC (× 3) [56]. RH1 is currently under consideration under consideration for clinical trials by the National Cancer Institute and The Cancer Research Campaign. Its potent cytotoxicity is expected to offset its short halflife in mice [73,74]. Recent pharmacokinetic and metabolic studies in mice suggest that RH1 is likely to exhibit more favourable characteristics than the bioreductive agent EO9 which was disappointing in clinical trials [73].

4.2.3. Other quinones Pyrrolobenzimidazolequinones (PBIs) were discovered in the late 1980s and were designed to alkylate the phosphate backbone of DNA upon reduction which results in its clevage [75 – 77]. PBI substrates for NQO1 can be either activated or deactivated by the enzyme depending on their structure [77,78]. Quinolinequinones [79] and benzoquinone mustards [80] have also been considered as potential agents for NQO1-directed approaches to chemotherapy and these have recently been discussed in more detail [51].

4.2.4. Nitro prodrugs and other agents Cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) occurs through reductive activation and crosslinking of DNA [81]. It was noted in the late 1960s that Walker rat carcinoma cells were particularly sensitive to CB1954 and this sensitivity was later linked to expression of NQO1 [13,82,83]. NQO1 reduces CB1954 to a hydroxylamine derivative that is converted to a bifunctional crosslinking agent by thioesters [13]. In cells with moderate levels of NQO1, crosslinking may play a lesser role and toxicity may be exerted primarily through redox cycling [84]. Newer analogues of CB1954 that target NQO1 are being developed [85]. The bioactivation of tirapazamine (3-amino-1,2,4-benzotriazene-1,4dioxide, TPZ, SR4233) is complex and NQO1 may play a limited role in its metabolism. TPZ is mainly activated through one-electron reduction, but NQO1 can activate TPZ in hypoxic conditions as well [86]. There is evidence that NQO1 deactivates TPZ in two- and four-electron reductions (for review see Ref. [87]). 17-Allylamino, 17demethoxygeldanamycin (17AAG) is an hsp90 inhibitor which has currently entered clinical trials. A positive correlation between NQO1 expression and growth inhibitory of properties of 17AAG in cell culture and xenografts has been reported [88].

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5. NQO1 gene regulation Isolation of the human [89], rat [90] and mouse [91] cDNAs for NQO1 confirmed that NQO1 is a single copy gene and is located on human chromosome 16q22.1 [92– 94] and mouse chromosome 8 [95]. The human [92], mouse [52,96] and rat [97,98] genes have been cloned and structurally characterized. Restriction mapping and sequencing have revealed that the NQO1 gene consists of six exons and five introns for an approximate length of 20 kb. Exon 1 encodes the 5%UT, the first two amino acids and the first nucleotide of the third amino acid, while exons 2–6 encode the remaining 272 amino acids and the 3%UT. There is extensive homology (85%) between the rat and human NQO1 coding regions [91]. Transcriptional start sites differ among the rat, human and mouse genes suggesting potential species-specific regulation of NQO1 [99]. Biochemical studies have already demonstrated that NQO1 activity is induced by a wide range of chemicals including polycyclic aromatic hydrocarbons, azo dyes and phenolic antioxidants [100 –104]. Two distinct regulatory elements in the 5% flanking region of the NQO1 gene that have been studied extensively are the antioxidant response element (ARE), also called the EpRE (electrophile response element), and the xenobiotic response element (XRE), also called the AhRE. The ARE and the XRE have been shown to mediate NQO1 induction as well as repression, in many cellular systems. The structure–function relationships within the NQO1 promoter are now being addressed using functional assays, mutational analysis and transgenic models.

6. ARE induction ARE-mediated NQO1 gene expression is increased by a variety of antioxidants, tumor promoters, and H2O2 [105–107]. Various transcription factors have been described, which bind to the ARE (TMAnnRTGAYnnnGCRwww) in-vitro, suggesting that this is a composite regulatory DNA element. The abbreviations follow standard IUPAC nomenclature; M= A or C, R = A or G, Y =C or T, W = A or T, S= G or C. The composition of the specific proteins binding to the ARE is variable. Because the ARE sequence, GTGACnnnGC, is similar to the AP-1 binding site, TGASTMAG, many reports have suggested that AP-1 and other basic leucine zipper (bZIP) proteins Nrf1 (NF-E2 related transcription factor), Nrf2 and Maf [108] drive the induction of ARE-dependent genes such as NQO1. Others have recently shown that the ERK (extracellular signal regulated protein kinase) pathway also participates in the ARE-mediated induction of Phase II detoxifying enzymes [109]. The ERK pathway mediates the induction of ARE-dependent genes [110] whereas p38 MAPK (mitogen activated protein kinase) negatively regulates induction by tBHQ and sulforaphane [110]. AP-1 proteins are sensitive to activation by hypoxia [111 – 113]. Waleh et al. [114] demonstrated that the hNQO1 ARE was responsive to low oxygen conditions. A useful model to integrate all these findings is one proposed by Wasserman and Fahl [115] which illustrates ARE-medi-

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Fig. 4. Schematic representation of ARE-mediated regulation of hNQO1 (modified from Wasserman and Fahl [115]). Members of the bZip protein family are proposed to bind to the RTGAYnnn portion of the ARE consensus. Binding protein 1 (BP-1) interacts with the bZip proteins and binds to the flanking sequences within the ARE.

ated expression of genes (Fig. 4). The ARE core sequence, RTGAYnnn, binds the bZIP family of transcription proteins (Jun, Fos, Fra, Nrf, Maf, Raf, NF-E2), while the flanking sequences are critical in mediating transactivation in a cell or tissuespecific fashion. AP-1 proteins may not be necessary for ARE-mediated gene expression. The hNQO1 ARE was induced by BHQ in mouse F9 cells which are known to contain no significant AP-1 activity [116]. The ARE in the hNQO1 gene bears a close resemblance to the NF-E2 (nuclear factor-erythroid 2) and allows binding for other bZip proteins, Nrf1 and Nrf2 [117]. Venugopal et al. [117] showed that Nrf1 and Nrf2 are critical for the induction of specific protein binding to the hNQO1 ARE in extracts of either human liver or monkey kidney cell lines exposed to B-NF or tBHQ. However, overexpression of cFos or Fra-1 repressed basal ARE driven promoter activity. Furthermore, Nrf1 and Nrf2 heterodimerize with several Jun family members in the presence of unknown cytosolic factors, causing induction of NQO1 in human hepatoma cells [118]. Induction through the ARE has primarily been studied in hepatoma cells, however, Moehlenkamp et al. [119] have shown differing responses to tBHQ and TPA in human neuroblastoma cells. Activation of ARE sequences by tBHQ in neuroblastoma cells was shown to be significantly different from HepG2 cells and a complete 5% palindrome within the ARE was necessary for maximal induction. Studies in lung cancer cells [120] have shown that the ARE was also important for mediating constitutive NQO1 expression in NSCLC, but surprisingly not in SCLC. Gel supershift assays with various specific Fos/Jun antibodies identified Fra1, Fra2 and Jun B binding activity in NSCLC cells, whereas SCLC do not appear to express functional Fra or Jun B. Overexpression of cFos is known to repress the ARE-mediated expression of hNQO1 in transfected human liver cell lines [117]. Data from transgenic studies in mice indicate disruption of cFos leads to increased expression of detoxifying enzymes including NQO1 and suggest a negative role for cFos in their regulation [121]. The combinatorial interactions of all of these transcription factors introduce specificity into the transcriptional response to extracellular signals. By dimerizing with different partners under diverse circumstances, bZip proteins may be mixed and matched to assume a greater role in NQO1 gene regulation.

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7. Antiestrogens in regulation of NQO1 Classically, estrogen receptors (ER) enhance transcription by binding to estrogen response elements (ERE) within target genes. While antiestrogens such as tamoxifen, exert little activity at EREs, they can induce significant transcriptional activity at AP-1 sites [122]. Estrogen receptor beta, (ERb) enhances AP-1 activity via interactions with a p160/p300 coactivator complex [123]. Recently, Montano et al. have shown a direct interaction of anti-estrogen liganded ERb with the hNQO1 ARE in the MCF-7 human breast cell line [124]. These observations and the fact that other reported factors, such as Hela cell binding protein 1 (BP-1) [125], that interact with AREs are now beginning to be characterized, raises the possibility that antiestrogens might play a role in regulating other ARE containing genes offering chemoprotective benefits in ER-expressing tissues.

8. XRE induction Induction through the XRE involves the liganded aromatic hydrocarbon receptor, AHR. This intracellular member of the PAS (Per, Arnt, Sim) family of proteins, translocates to the nucleus upon binding to Arnt [126]. The AHR/Arnt dimer that interacts with the DNA sequences known as XREs. The hNQO1 XRE shares significant homology with the human CYP1A1 XRE [127]. Both NQO1 and CYP1A1 genes can be induced by TCDD and polycyclic aromatic hydrocarbons [128], while DeLong et al. [129] and Rushmore and Pickett [130] have suggested that the induction of NQ01 is largely dependent on the ability of bifunctional inducers such as azo dye, Sudan I and B-NF to first undergo conversion to oxidative labile metabolites through a functional P450-dependent mechanism. Recently, Rajendriane et al. [131] reported that TCDD induction of hNQO1 in mouse hepatoma cells was ARE-mediated and not dependent on XRE.

9. Polymorphisms in NQO1 We have characterized a single nucleotide polymorphism in NQO1 which has profound phenotypic consequences [132,133]. The polymorphism (NQO1*2 allele) is a C to T change at position 609 of the cDNA which codes for a proline to serine change in the structure of the human protein. Genotype-phenotype studies of the NQO1*2 allele have been performed using both cell systems and tissues. No detectable or only trace levels of mutant NQO1 protein could be observed in cell lines and in saliva, bone marrow or lung samples from individuals with the NQO1*2/*2 genotype [133,134]. Although the mutant NQO1 protein purified from E. coli expression systems has only between 2 and 4% of the activity of the wild-type protein [133] because of a diminished ability to bind FAD [135], the mechanism underlying the lack of NQO1 activity in NQO1*2/*2 individuals appears primarily to be due to a lack of protein [133]. Deficient NQO1 protein

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levels have also been observed in COS-1 cells transfected with mutant NQO1 cDNA [136]. Recently, we have demonstrated that the lack of protein as a result of the NQO1*2/*2 genotype appears to be due to accelerated degradation of the mutant NQO1 protein mediated by the ubiquitin/proteasomal system [137]. The frequency of the NQO1*2/*2 genotype varies across ethnic group from 4% in Caucasians, 5% in African – Americans, 16% in Mexican Hispanics to 22% in Chinese populations [138]. Gaedigk et al. [139] reported that the NQO1*2 allele frequency was 0.16 in Caucasians, 0.4 in Native Indians, 0.46 in Inuits and 0.49 in Chinese. A second polymorphism in NQO1 (NQO1*3 allele) has also been characterized [140,141]. This is a C465T change coding for an arginine to tryptophan substitution in the protein. The implications of this polymorphism for phenotype are variable depending on the substrate and the frequency of the NQO1*3 polymorphism is low. In a recent study, the NQO1*3 allele frequency varied from 0 to 0.05 in different ethnic groups with only one homozygous variant detected in 575 samples tested [139]. Because the homozygous NQO1*2 allele is essentially a null phenotype [132– 134], it provides a convenient molecular tool with which to assess the potential chemoprotective role of NQO1 in-vivo. Previous work on the implications of the null polymorphism in NQO1 have almost exclusively been examined from the perspective of the susceptibility to cancer of individuals carrying the NQO1*2/*2 genotype. The NQO1*2 allele has been associated with an increased risk of urothelial tumors [142], therapy-related acute myeloid leukemia [143], cutaneous basal cell carcinomas [144] and pediatric leukemias [145]. We have also demonstrated that the homozygous NQO1*2 allele is a significant risk factor for the development of benzene-induced hematotoxicity in exposed workers [146]. The NQO1*2 polymorphism does not appear to be a risk factor for prostate cancer [147] and the alternative hypothesis seems to be true in lung cancers with an over-representation of the wild-type NQO1 allele in lung cancer cases relative to controls [148,149]. The NQO1*2 polymorphism may also have relevance for chemotherapy using antitumor quinones. Mitomycin C is currently the only quinone used extensively in chemotherapeutic regimens. Although NQO1 is clearly not the only reductase that can bioactivate mitomycin C, the use of stable transfection, enzyme induction and gene targeting approaches by ourselves and others [52,55,56,150] clearly demonstrate that it is a major determinant of the biological effects of mitomycin C. This suggests that the effectiveness of mitomycin C in therapy would be diminished in individuals carrying the NQO1*2 polymorphism. Important supporting evidence for this hypothesis is beginning to appear in the literature. We recently reported that the response of primary cultures of gastric tumors to mitomycin C was dependent on NQO1 genotype [151] with increased response observed in *1/*1 genotypes. More importantly, Fleming et al. [152] reported significant differences in survival in patients treated with mitomycin C depending on NQO1 genotype. Optimal responses in patients with disseminated peritoneal cancer receiving mitomycin C were associated with the NQO1*1/*1 genotype rather than NQO1*1/*2 or

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*2/*2 genotypes. This is the first report relating clinical response to mitomycin C with NQO1 genotype and builds on considerable in-vitro data demonstrating the lack of effect of mitomycin C in human cell lines carrying the NQO1*2/*2 genotype [153 – 155].

Acknowledgements The authors acknowledge support from NIH grants CA51210 and ESO9954.

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