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Carcinogenesis Advance Access originally published online on June 29, 2007
Carcinogenesis 2007 28(11):2398-2403; doi:10.1093/carcin/bgm146
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Nrf2 and p53 cooperatively protect against BBN-induced urinary bladder carcinogenesis

Katsuyuki Iida1,2, Ken Itoh2, Jonathan M. Maher2, Yoshito Kumagai3, Ryoichi Oyasu4, Yukio Mori5, Toru Shimazui1, Hideyuki Akaza1 and Masayuki Yamamoto2,*

1 Department of Urology, Institute of Clinical Medicine
2 Center for Tsukuba Advanced Research Alliance (TARA), Institute of Basic Medical Sciences and Japan Science and Technology Agency (JST)-Exploratory Research for Advanced Technology (ERATO) Environmental Response Project
3 Institute of Community Medicine, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan
4 Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611-3008, USA
5 Institute of Biological Pharmacy, Gifu Pharmaceutical University, Mitahora-higashi 5-6-1, Gifu 502-8585, Japan

* To whom correspondence should be addressed. Tel: +81 298 53 6158; Fax: +81 298 53 7318; Email: masi{at}tara.tsukuba.ac.jp

Correspondence may also be addressed to Ken Itoh. Tel: +81 172 39 5158; Fax: +81 172 39 5157;Email: itohk{at}cc.hirosaki-u.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2), a transcription factor that regulates inducible expression of detoxifying enzymes, is critical in preventing N-nitrosobutyl(4-hydroxybutyl)amine (BBN)-induced urinary bladder carcinogenesis. To explore whether Nrf2 and the tumor suppressor p53 cooperatively act in tumor prevention, we investigated the susceptibility of Nrf2–/–::p53+/– mice to BBN-induced urinary bladder carcinogenesis. The incidence of BBN-induced urinary bladder carcinoma was 63.0% in Nrf2–/– mice (P = 0.115), 75.8% in p53+/– mice (P < 0.01) and 89.6% in Nrf2–/–::p53+/– mice (P < 0.01) compared with 37.9% in wild-type. Higher incidence of carcinoma was observed in Nrf2–/–::p53+/– mice when compared with either Nrf2–/– (P < 0.01) or p53+/– mice (P = 0.382). Similarly, muscular invasive carcinoma incidence was higher in Nrf2–/–::p53+/– mice (62.0%) than either wild-type (6.9%, P < 0.01), p53+/– (38.0%, P = 0.110) or Nrf2–/– mice (3.7%, P < 0.01). Furthermore, urinary concentrations of N-nitrosobutyl(3-carboxypropyl)amine, a proximate carcinogen of BBN, were only increased when Nrf2 but not p53 was disrupted. These results demonstrate that tumor susceptibility is synergistically exacerbated in Nrf2–/–::p53+/– mice due to poor detoxification and accelerated proliferation in comparison with either single mutant alone. BBN administration increased p53-mediated expression of p21, Mdm2 and Bax, and the inducible expression of p21 was significantly enhanced in Nrf2–/– mice. Conversely, modest increases in NAD(P)H dehydrogenase, quinone 1 (NQO1) and uridine diphosphate (UDP) glucuronosyltransferase 1A6 (UGT1A6) expression were observed in p53+/– compared with those of wild-type mice after BBN exposure. These results thus reveal potential interactions between p53 and Nrf2 and their gene batteries, and indicate that both factors cooperatively contribute to tumor prevention.

Abbreviations: BBN, N-nitrosobutyl(4-hydroxybutyl)amine; BCPN, N-nitrosobutyl(3-carboxypropyl)amine; mRNA, messenger RNA


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The urinary bladder is exposed to many water-soluble chemicals, thus this tissue is highly susceptible to tumorigenesis that arises from water-soluble carcinogens. N-nitrosodibutylamine was first identified as a rat bladder carcinogen (1) and is also considered carcinogenic to human (2). Although concentrations are generally low, N-nitrosodibutylamine has been detected in tobacco smoke, corrosion inhibitors, foods and rubber products. Metabolism of N-nitrosodibutylamine occurs primarily in liver, and tumor formation in rat bladder is dependent on the generation of the {omega}-oxidized metabolite, N-nitrosobutyl(4-hydroxybutyl)amine (BBN; ref. 3). Oral administration of BBN to rodents induces cancer specifically in urinary bladder (4). BBN itself is either activated by alcohol/aldehyde dehydrogenase-mediated oxidation to yield N-nitrosobutyl(3-carboxypropyl)amine (BCPN) or detoxified by uridine diphosphate-glucuronosyltransferase-catalyzed conjugation to form BBN-glucuronide (5). BCPN and BBN are chemically cleaved to their respective alkylcarbonium ions and bind covalently to DNA (6). Furthermore, BCPN itself has been shown to have carcinogenic effects on urothelial cells (7). Conversely, BBN glucuronidation in the liver suppresses carcinogenesis in the urinary bladder by decreasing urinary BCPN concentrations (8).

Mutations in p53 are one of the most common genetic lesions found in human cancer, with >50% of tumors examined having a single or compound mutation or deletion in the p53 gene (9). In bladder cancer, a single G to A transition mutation in the CpG island of p53 accounts for 25% of all p53 mutations (10). Furthermore, 65% of bladder carcinoma in situ cases contain a p53 mutation, and 27% involve allelic loss of human chromosome 17, which contains the p53 gene. Not only are p53 mutations common but also they are associated with increased malignancy and disease severity. In patients suffering from bladder cancer, nuclear accumulation of mutant p53 is directly associated with a decreased survival rate (11,12), and loss of a p53-bearing chromosome is an early indication of carcinoma in situ progression and malignancy (13). In stark contrast, p53 mutations are detected in only 3% of low-grade, superficial papillary tumors (14).

Alterations in the p53 gene are also associated with carcinogenesis in rodent models. Homozygous p53 knockout mice are viable, but highly susceptible to spontaneous tumorigenesis, particularly lymphomas, at an early age. These carcinomas that arise in p53+/– mice show higher indices of malignancy by histopathological examination. Mice heterozygous for the p53 gene (p53+/–) are quite vulnerable to BBN-induced bladder cancer (15). Heterozygous p53 mice exposed to BBN appear to undergo a higher rate of cell proliferation than normal, with no observed alterations in metabolism or excretion of BBN. Thus, p53+/– mice seem to develop tumors with the frequency, ease and malignancy observed in spontaneous tumors observed in clinical cases, and p53+/– mice seem to be an ideal model for examining urinary bladder susceptibility to BBN.

High expression of several phase II detoxification enzymes, such as UDP-glucuronosyltransferases, aid in protection from carcinogens by increasing water solubility and excretion of genotoxic chemicals. Several of these enzymes, such as UGT1A6, are regulated by cis-acting antioxidant response elements or electrophile responsive elements in the promoter regions of these genes (16–18). The transcription factor Nrf2 can bind to antioxidant response element/electrophile responsive element sequences and mediate the expression of these phase II enzymes via heterodimerization with small Maf proteins (19–21). Nrf2–/– mice have attenuated basal and inducible expression and activity of phase II detoxification enzymes, thus these mice are sensitive to genotoxic chemicals such as BBN (8,22). Poor expression of phase II enzymes in Nrf2–/– mice leads to BCPN accumulation in urine due to decreased conjugation and excretion, and this is thought to lead to tumor formation. Nrf2-mediated induction of detoxifying enzymes occurs in peripheral urothelial cells and in liver, enhancing detoxification and decreasing oxidative stress. Because Nrf2 has a known role in regulating such processes, Nrf2 may serve as an important component in prevention of BBN-induced bladder carcinogenesis.

Thus, p53 and Nrf2 are transcription factors involved in cellular protection from BBN-induced carcinogenesis, albeit through two seemingly different mechanisms. However, it is not known whether these mechanisms overlap or are independent pathways, and whether the protection is synergistic or additive in nature. To address these issues, we generated an Nrf2–/–::p53+/– double mutant mouse line and attempted to determine whether Nrf2 deficiency further accelerates urinary bladder carcinogenesis in p53-deficient mice and whether there are unique interactions between the Nrf2 and p53 pathways.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Reagents
BBN was from Tokyo Kasei Co. (Tokyo, Japan). UDP-glucuronic acid was purchased from Sigma (St Louis, MO). BCPN was prepared as previously described (23).

Animals
A colony of Nrf2-null mice on an ICR/129SV background (22) was backcrossed for nine generations with C57BL/6 mice from CLEA Japan (Tokyo, Japan). p53+/– mice on a C57BL/6 genetic background were from the Jackson Laboratory (Bar Harbor). Nrf2–/–::p53+/– double mutant mice were generated by crossing Nrf2–/– mice with p53+/– mice. Mice were housed in stainless steel cages in an animal room kept at 24°C, which is maintained with a 12 hour light/dark cycle. Mice were fed a purified AIN-76A diet (Oriental MF; Oriental Yeast Co., Tokyo, Japan) and water ad libitum.

BBN-induced bladder carcinogenesis
A 0.05% concentration of BBN was dissolved in H2O, and was supplied ad libitum for 8 weeks in dark-colored bottles. After this experimental period, the mice were analyzed. Urinary bladders were inflated and then fixed in 10% buffered formalin. Each bladder was sectioned sagittally and each cup-shaped area was cut into four pieces. These eight strips of bladder tissue were serially embedded in a single paraffin block, cut into thin sections and stained with hematoxylin and eosin. Bladder lesions were histologically diagnosed according to the criteria of Oyasu et al. (24).

RNA blot analysis
Total RNA from whole urinary bladders was extracted with Isogen (Nippon Gene, Toyama). RNA (10 µg) was separated by 1.5% agarose gel electrophoresis containing 2.2 M formaldehyde and transferred to a nylon membrane. Mouse cDNA probes for UGT1A6 and NQO1 were as previously described (8,25). p53-target genes were amplified by reverse transcriptase–polymerase chain reaction, using mouse liver RNA as a template. The primer used for each gene was as follows: p21, forward primer is 5'-CGG TGG AAC TTT GAC TTC GT-3' and reverse primer is 5'-CAC AGA GTG AGG GCT AAG GC-3'; Bax, forward primer is 5'-TGC AGA GGA TGA TTG CTG AC-3' and reverse primer is 5'-GAG GAC TCC AGC CAC AAA GA-3'; Mdm2, forward primer is 5'-CAG CTT CGG AAC AAG AGA CTC-3' and reverse primer is 5'-CTG CTC TCA CTC AGC GATGT-3'.

Immunoblot analysis
Total cell extracts from mouse urinary bladder were solubilized with sodium dodecyl sulfate sample buffer without loading dye and 2-mercaptoethanol. Protein concentrations were estimated by bicinchoninic acid (BCA) protein assay (Pierce Rockford, IL). Proteins were separated by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto an Immobilon membrane (Millipore Tokyo, Japan). Anti-p21 (SC-397) and anti-NQO1 (SC-16464) antibodies were used (both from Santa Cruz Biotechnology Santa Cruz, CA; ref. 26). Immunoreactive proteins were detected using horseradish peroxidase-conjugated anti-Immunoglobulin G (IgG) antibody and enhanced chemiluminescence (ECL) (Amersham Biosciences Arlington Heights, IL).

Determination of BCPN concentration
The urinary concentration of BCPN was measured as described previously (8,15). The urine sample (0.1 ml) was diluted to 0.5 ml with distilled water before starting the assay. A 3.3 µl aliquot of 12 M HCl was added, and the sample was extracted with 0.5 ml of ethyl acetate three times. The organic layer was collected after a 5 min centrifugation at 10,000g, and dried using a speed vacuum concentrator with a cooling trap set below 30°C. The residue was dissolved in ethyl acetate, spotted onto a silica gel 70 F254 pre-coated plate (Wako, Osaka, Japan) and developed with chloroform/methanol/acetic acid (18:1:1, by volume) in the dark. The bands corresponding to BBN or BCPN (retardation factor (Rf)) = 0.86–0.72) were scraped off and eluted from the silica with 4 ml acetone. Eluate was concentrated by speed vacuum concentrator and diluted with acetonitrile to a final volume of 0.2 ml. Samples were filtered through a MINISART RC4 filter (0.2 mm pore size; Sartorius Aubagne, France) and analyzed by high-performance liquid chromatography. Urinary BCPN levels were determined using a SHIMAZU LC9A apparatus (SHIMAZU, Kyoto, Japan) on a Jasco Finepak SIL C18 column (250 x 4.6 mm internal diameter) at 239 nm. Separation was performed with a mobile phase consisting of a 3:7 mixture (v/v) of acetonitrile and 20 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 ml/min. Under these conditions, the retention time of BCPN was 7.8 min. The recovery rate of BCPN was determined in each experiment as the ratio of BCPN recovered from the urine of BBN-untreated mice in which known amount of the pure BCPN was included. The recovery rate of BCPN from the urine was ~60% in our assay conditions.

Measurement of BBN-glucuronide in vitro
The protocol for measuring BBN-glucuronide activity in mouse liver has been previously described (8). Briefly, microsome fractions were prepared from mouse liver as described (27). A typical reaction mixture consisted of 100 mM potassium phosphate buffer (pH 7.4), 1 mM BBN, 5 mM UDP-glucuronic acid, 0.05% Brij58 and microsome preparation (600 µg protein) in a final volume of 1 ml. Reactions were initiated by the addition of BBN, and incubations were performed at 37°C for 30 min. bovine serum albumin (1 mg) and 24% trichloroacetic acid (0.1 ml) were added to the incubation mixture to terminate the reaction. After centrifugation at 10,000g for 5 min, the supernatant (0.1 ml) was injected into the high-performance liquid chromatograph as described above. Separation of BBN and BBN-glucuronide was accomplished using a mobile phase consisting of a 2:8 mixture (v/v) of acetonitrile and 20 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 ml/min.

Statistical analyses
Data were expressed as the mean ± standard error. The Student's t-test was used to determine the statistical difference among groups. The values for urinary bladder incidence were analyzed using the {chi}2 or Fisher's exact probability test. A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Higher susceptibility of Nrf2–/–::p53+/– mice to BBN-induced carcinogenesis
In order to clarify whether Nrf2 and p53 cooperatively function in protection against chemical carcinogenesis, we examined the susceptibility of Nrf2–/–::p53+/– mice to BBN-induced urinary bladder tumor formation. Table 1 summarizes the incidence of urinary bladder cancer after BBN treatment. In this analysis, one wild-type, two p53+/–, one Nrf2–/– and two Nrf2–/–::p53+/– mice died within the experimental period from unknown reasons. Conversely, one Nrf2–/– and one p53+/– mouse seemingly died from BBN-induced tumors, as the autopsy revealed renal and lymphatic masses in these mice. Bladder lesions were diagnosed histologically according to the criteria described previously (24). All non-invasive carcinomas were nodular rather than papillary in shape. The term ‘cancer’ applies to both transitional and squamous cell carcinomas because most of the lesions contained both components. No pathological difference in the cancer type of non-invasive and invasive tumors was recognized between the p53+/–, Nrf2–/– and Nrf2–/–::p53+/– mice (Figure 1 and data not shown).


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  		Table I. BBN-induced carcinogenesis of the urinary bladder in wild-type, Nrf2–/–, p53+/– and Nrf2–/–::p53+/– mice

 


Figure 1
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  		Fig. 1. Histopathological analysis of BBN-induced carcinoma in wild-type, Nrf2–/–, p53+/– and Nrf2–/–::p53+/– mouse urinary bladders. Tissue sections of urinary bladder were analyzed with hematoxylin and eosin staining. Non-invasive carcinomas (left panels) and muscle-invasive carcinomas (right panels) are shown for each genotype. Bar indicates 250 µm.

 
The incidence of carcinoma increased from 37.9% in wild-type mice to 63.0% in Nrf2–/– mice (P = 0.115), 75.8% in p53+/– mice (P < 0.01) and 89.6% in Nrf2–/–::p53+/– mice (P < 0.01; Table 1). Moreover, the incidence of carcinoma was increased in Nrf2–/–::p53+/– mice compared with that of either Nrf2–/– (P < 0.01) or p53+/– mice (P = 0.382). Whereas p53+/– mice showed increased incidence of both non-muscle-invasive (P < 0.01) and invasive carcinomas (P < 0.01), Nrf2–/– mice only had increased incidence of non-muscle-invasive carcinomas (P = 0.115) compared with wild-type mice (Figure 1 and Table 1). Furthermore, the incidence of muscle-invasive carcinoma in Nrf2–/–::p53+/– mice markedly increased from 3.7% in Nrf2–/– mice to 62.0% in Nrf2–/–::p53+/– mice (P < 0.01). Also Nrf2–/–::p53+/– mice had increased the incidence from 38.0% in p53+/– mice but was not statistically significant (P = 0.110). However, the ratio of invasive versus non-invasive carcinoma was indeed increased from 1.00 in p53+/– to 2.25 in Nrf2–/–::p53+/– mice (Table 1). These results thus indicate that deficiency of both p53 and Nrf2 causes higher susceptibility and severity to BBN-induced carcinomas.

Urinary BCPN concentrations and BBN glucuronidation activity in Nrf2–/–::p53+/– mice
BCPN is a proximate metabolite of BBN and BCPN and BBN are metabolized through {alpha}-hydroxylation or spontaneous cleavage to the highly reactive alkylcarbonium ion (6). We measured the urinary BCPN concentrations by high-performance liquid chromatography 5 days after 0.05% BBN administration (Figure 2A). Nrf2–/– mice had higher urinary BCPN concentrations than wild-type mice, but BCPN concentrations in p53+/– mice were not altered substantially from wild-type mice (Figure 2A). Basal BBN glucuronidation activity was significantly lower in the hepatic microsomes from Nrf2–/– mice, but not p53+/– mice, which correlates well with previous observations of decreased glucuronidation in the Nrf2–/– mouse liver (8).


Figure 2
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  		Fig. 2. Effects of Nrf2 and p53 deficiency on urinary concentrations of BCPN after BBN treatment, and BBN glucuronidation activity in hepatic microsomes. (A) The mice were supplied drinking water containing 0.05% BBN for 5 days. Urine samples were analyzed by high-performance liquid chromatography for urinary BCPN concentrations. The mean values in wild-type mice are arbitrarily set as 100 and the ratios are represented with ±SE (n = 9). (B) Relative formation of BBN-glucuronide by liver microsomes in male mice of the indicated genotype. Mean values in wild-type mice are arbitrarily set as 100 and the ratios are represented with ±SE (n = 6). *P < 0.05 compared with non-treated wild-type mice.

 
Expression of p53- and Nrf2-target genes in the urinary bladder after BBN treatment
To elucidate the role that p53 plays in attenuating BBN carcinogenesis, we examined the expression of p53-target genes, p21, Mdm2 and Bax, in the urinary bladder after BBN treatment. Wild-type mice were treated with 0.0025, 0.005, 0.0075 or 0.01% BBN in the drinking water for 2 weeks, and messenger RNA (mRNA) levels of the p53-target genes were examined by RNA blotting. Expression of p21, Mdm2 and Bax was increased in a BBN dose-dependent manner (Figure 3A). The increase in p21 was especially marked with expression increasing ~10-fold after 0.01% BBN treatment, and Mdm2 and Bax expression increasing ~4-fold. Expression of these p53-target genes also increased in a time-dependent manner after 0.0075% BBN treatment, up to 14 days (Figure 3B). These results suggest that the BBN treatment activates the p53 pathway through formation of the BBN-induced DNA damage. However, mRNA expression of NQO1, a typical Nrf2-target gene, was not affected by the BBN treatment (data not shown), indicating that Nrf2 was not activated by the BBN administration.


Figure 3
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  		Fig. 3. Effects of BBN on p53-target gene expression in urinary bladder. (A) Dose-dependent effects of BBN on p53-target gene expression in the urinary bladders of wild-type male mice. Mice were treated with 0.0025, 0.005, 0.0075 and 0.01% BBN in drinking water for 2 weeks. The band intensities from RNA blots were quantified by densitometry analysis and normalized with 18S ribosomal RNA. The mean values of vehicle-treated mice were arbitrarily set as 100 and ratios represented with mean ± SE (n = 4). *P ≤ 0.05 compared with non-treated wild-type mice. (B) Time-dependent effects of BBN on p53-target gene expression. BBN was given at a concentration of 0.0075% in drinking water for 4, 7 and 14 days. Data were analyzed and presented as in (A).

 
Expression of p53- and Nrf2-target genes in the Nrf2–/–::p53+/– urinary bladder
Considering that p53 and Nrf2 seemingly protect from urinary bladder carcinogenesis, we aimed to determine whether crosstalk exists between these two pathways. To this end, mice were treated with 0.0075% BBN for 2 weeks, and mRNA expression levels of Nrf2- and p53-target genes were examined. BBN-inducible expression of p21, Mdm2 and Bax mRNAs all decreased in p53+/– mice compared with those in wild-type mice, although the decrease in Bax expression was not statistically significant (Figure 4A). In contrast, p21 expression was modestly increased in Nrf2–/– mice by ~2-fold, and although the expression of Mdm2 and Bax showed modest increases, neither was statistically significant. In Nrf2–/– mice, UGT1A6 and NQO1 mRNA expression significantly decreased by 50 and 90%, respectively, compared with wild-type mice (Figure 4B). UGT1A6 and NQO1 expression in p53+/– mice significantly increased by ~1.4- and 1.7-fold, respectively, compared with those in wild-type mice after BBN exposure. However, in Nrf2–/–::p53+/– mice this increase was not observed, suggesting that the increased induction is Nrf2 dependent (Figure 4B).


Figure 4
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  		Fig. 4. Effects of the Nrf2–/–::p53+/– mutation on the expression of (A) p53- or (B) Nrf2-target genes in urinary bladder. Mice were treated with BBN at a concentration of 0.0075% in drinking water for 2 weeks. RNA band intensities were quantified by densitometric analysis and normalized by 18S ribosomal RNA. Mean values of vehicle-treated mice were arbitrarily set as 100, and ratios were expressed as mean ± SE (n = 4). a, P ≤ 0.05 compared with wild-type mice. b, P < 0.05 compared with p53–/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The current study aimed to determine whether there are interactions between the Nrf2 and p53 regulatory pathways that prevent carcinogenesis. The number of total carcinomas and invasive cancers were both increased in Nrf2–/–::p53+/– mice compared with mice bearing either Nrf2–/– or p53+/– mutation alone. As has been previously reported, the primary role of Nrf2 in the protection of BBN-induced carcinogenesis is to regulate BBN detoxification genes such as UGTs. In addition, Nrf2 regulates antioxidant genes such as glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM), thereby aiding in oxidative stress prevention during carcinogenesis. Conversely, p53 acts to regulate cell proliferation, apoptosis and DNA repair upon BBN exposure. The results of this study support our contention that these two pathways work cooperatively to protect the bladder from BBN-induced carcinogenesis.

The incidence of invasive carcinoma increased in Nrf2–/–::p53+/– mice compared with p53+/– mice, demonstrating the presence of an additive pathological effect when expression of both factors was disrupted. Indeed, the ratio of invasive versus non-invasive carcinoma was increased from 1.00 to 2.25 (Table 1). Recent reports also suggest that in p53 knockout mice, increased formation of reactive oxygen species occurs, demonstrating that Nrf2 and p53 both have a role in cellular protection from oxidative stress (28). Similarly, formation of reactive oxygen species seemingly aids in carcinogenic progression (29,30), thus it is probable that in bladder epithelia from Nrf2–/–::p53+/– mice, the increased incidence of invasive carcinoma is due to higher levels of reactive oxygen species. We summarized this notion in Figure 5.


Figure 5
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  		Fig. 5. Cooperative network between the Nrf2 and p53 pathways in protection against urinary bladder carcinogenesis. Nrf2-dependent increases in hepatic BBN glucuronidation reduced BBN-induced carcinogenesis by suppressing urinary BCPN excretion. Conversely, p53 regulates expression of genes to repair DNA damage and to induce apoptosis. In Nrf2–/– mice, elevated urinary BCPN concentrations lead to higher levels of DNA damage and enhance p53 activation. In p53+/– mice, the failure to instigate DNA damage repair may lead to Nrf2 activation via the ataxia telangiectasia mutated pathway. Elevated levels of reactive oxygen species in Nrf2–/– or p53+/– mice may contribute to increased incidence of carcinogenesis and tumors.

 
Mutagenic or allelic loss of p53 correlates well with the incidence of urinary bladder carcinogenesis in rodent models (12). In this study, we demonstrated that p53-target gene expression was induced in urinary bladder by BBN in a dose- and time-dependent manner, and this induction was p53 dependent. Thus, uroepithelial cells protect against BBN-induced DNA damage by increasing p21 and Bax expression and regulating cell proliferation and apoptosis, respectively. Because DNA damage activates p53 through ataxia telangiectasia mutated (31), expression of p53-target genes is typically a reflection of the resultant DNA damage. As Nrf2 plays important roles in BBN detoxification and the cellular antioxidant response, uroepithelial cells of Nrf2–/– mice may have increased incidence of DNA damage, and an increased number of cells that have undergone cancer initiation. Conversely, the invasive/non-invasive ratio in Nrf2–/– mice (0.063) was much lower than that in wild-type mice (0.222) (Table 1). The activated p53 pathway in Nrf2–/– mice may decrease the ratio of invasive cancer by increasing apoptosis or cell cycle arrest (Figure 4A). Therefore, it seems plausible that the decrease of p53 activity in conjunction with Nrf2 knockout leads to the increased incidence of invasive cancer in Nrf2–/–::p53+/– mice.

The expression of NQO1 was increased by BBN treatment in p53+/– mice, but not in wild-type mice (data not shown and Figure 4B). On the other hand, we previously demonstrated that UGT1A6 expression was decreased by BBN exposure (8). The expression of UGT1A6 in p53+/– was increased compared with that in wild-type mice after BBN exposure (Figure 4B). Showing agreement with this observation, it was recently reported that p53 suppresses Nrf2-dependent expression of NQO1 and xCT (a component of the cystine/glutamate exchange transport system) genes in a few culture cell lines (32). The latter observation further supports our contention that the Nrf2 and p53 regulatory pathways interact. Whereas the precise mechanisms of how p53-mediated apoptotic response and the Nrf2-mediated antioxidant response remains to be clarified, both pathways seem to function together in cancer chemoprevention.

One plausible explanation for this crosstalk of p53 and Nrf2 during cancer chemoprevention could be due to phosphorylation and activation of protein kinase C {delta} (PKC{delta}) by the ataxia telangiectasia mutated/c-Abl DNA repair and cell cycle regulatory pathways in response to BBN-induced genotoxic stress (33). Because p53+/– mice suffer from increased DNA damage due to reduced DNA repair capacity, activation of Nrf2-target genes by BBN may be caused by PKC{delta} in response to rising levels of DNA damage. A previous study demonstrated a strong correlation between PKC{delta} activity and Nrf2 activation in osteoblasts derived from ataxia telangiectasia mutated or c-Abl-deficient mice, but a firm relationship between the two factors has not been established (34).

In conclusion, our study demonstrates that the Nrf2–/–::p53+/– mutation in mice increases the incidence of BBN-induced carcinogenesis in comparison with those harboring the Nrf2–/– or p53+/– mutation alone. After BBN treatment, the expression of p53-target genes increase in Nrf2–/– mice, probably due to the higher BCPN concentrations and the consequent increase in DNA damage in the urinary bladder. Conversely, mice with decreased p53 expression had higher Nrf2-target gene expression after BBN treatment. Taken together, these results shed light on the interactive and compensatory pathways of p53 and Nrf2 against BBN-induced urinary bladder carcinogenesis.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Japan Science and Technology Agency (JST)-Exploratory Research for Advanced Technology (ERATO); Japanese Society for the Promotion of Science (JSPS) grant # PD5909; the Ministry of Education, Science, Sports, and Technology; Atherosclerosis Foundation.


   Acknowledgments
 
We thank Drs Atsushi Maruyama and Nobuhiko Harada for their help and discussion.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Druckrey H, et al. Organotropic carcinogenic effects of 65 various N-nitroso-compounds on BD rats. Z. Krebsforsch. (1967) 69:103–201.[CrossRef][Web of Science][Medline]
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Received February 1, 2007; revised May 24, 2007; accepted June 18, 2007.


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X.-J. Wang, Z. Sun, N. F. Villeneuve, S. Zhang, F. Zhao, Y. Li, W. Chen, X. Yi, W. Zheng, G. T. Wondrak, et al.
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