ToxSci Advance Access originally published online on September 25, 2006
Toxicological Sciences 2006 94(2):272-280; doi:10.1093/toxsci/kfl115
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p53-Independent Induction of Rat Hepatic Mdm2 following Administration of Phenobarbital and Pregnenolone 16
-Carbonitrile
Discovery Toxicology, Bristol Myers Squibb Co, Princeton, New Jersey 08543
1 To whom correspondence should be addressed at Bristol Myers Squibb Co, Route 206 and Province Line Road, Princeton, NJ 08543. Fax: (609)-252-7046. E-mail: lois.lehman-mckeeman{at}bms.com.
Received July 5, 2006; accepted August 28, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16
-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.
Key Words: Mdm2; carcinogenesis; phenobarbital; pregnenolone 16-carbonitrile.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Phenobarbital (PB) and pregnenolone 16
-carbonitrile (PCN) have been extensively studied as prototypical agonists of the rodent nuclear hormone receptors, constitutive androstane receptor (CAR; NR1I3) and pregnane X receptor (PXR; NR1I2), respectively, and their effects on xenobiotic metabolism are well characterized (Goodwin et al., 2002
; Handschin and Meyer, 2003; Qatanani and Moore, 2005; Swales and Negishi, 2004). In addition to effects on xenobiotic metabolizing enzymes, PB and PCN increase hepatocyte proliferation and induce hepatocellular hypertrophy and hyperplasia (Japundzic et al., 1974; Whysner et al., 1996), and PB is a rodent hepatocarcinogen that is frequently used as a promoting agent in initiation-promotion models for studying mechanisms of hepatocarcinogenesis (Holsapple et al., 2006; Whysner et al., 1996). Recent evidence also suggests that CAR activation is required for liver tumor promotion by PB and the potent CAR agonist, 1,4-bis[2-3,5-dichloropyridyloxy)]benzene (TCPOBOP), in mice (Huang et al., 2005; Yamamoto et al., 2004). In contrast, no long-term carcinogenicity studies have been reported for PCN, but it does promote thyroid follicular cell tumors in rodents (Vansell et al., 2004).
As model agents for studying transcriptional activation associated with binding and nuclear translocation of CAR and PXR, numerous reports on microarray analyses of hepatic gene expression profiles after PB or PCN treatment are also available. In many cases, these studies have focused on xenobiotic metabolizing enzymes and transporters (de Longueville et al., 2002; Ejiri et al., 2005; Gerhold et al., 2001) or have been used to delineate those genes, particularly related to xenobiotic metabolism, that are transcriptionally regulated through CAR (Ueda et al., 2002) or PXR (Rosenfeld et al., 2003). In other cases, broader evaluation of patterns of gene expression changes following PB treatment has guided the development of profiles that can be used to classify and distinguish the biochemical or toxic effects of compounds (Hamadeh et al., 2002). Finally, there are a few examples in which microarray analyses have specifically been used to evaluate gene expression changes that are likely to be involved in liver hyperplasia or regeneration (Locker et al., 2003).
One recent finding that portends a direct relationship to hepatocarcinogenic outcome with agents such as PB and TCPOBOP is evidence that these agents increase hepatic murine double minute 2 (Mdm2) expression in mouse liver through a CAR-dependent mechanism (Huang et al., 2005). Mdm2 is a p53-dependent gene that regulates p53 levels by catalyzing the ubiquitination and proteolytic degradation of p53 (Li et al., 2003; Momand et al., 1992). Through a direct effect on p53, Mdm2 contributes to a network that is critical to the regulation of DNA repair, cell cycle control, and cell differentiation. Furthermore, there is evidence that Mdm2 can also be induced by p53-independent mechanisms and can function as an oncoprotein in p53-independent pathways (Ganguli and Wasylyk, 2003). In either case, Mdm2 could play an integral and central role in early changes leading to hepatic tumor development in rodents. A significant role for Mdm2 in carcinogenesis was confirmed in transgenic mice engineered to overexpress Mdm2, as these mice exhibit a higher incidence of spontaneous tumor formation than the wild-type strain, regardless of the functional status of p53 (Jones et al., 1998). However, the available data for PB or TCPOBOP do not provide evidence as to whether changes in Mdm2 occurred along with or separate from changes in p53 regulation. This is a particularly important distinction since TCPOBOP appears to increase the expression of several p53-dependent genes in mouse liver (Locker et al., 2003). Furthermore, given the general susceptibility of both mice and rats to develop spontaneous and chemically induced liver tumors (Gold et al., 2005), Mdm2 induction may represent a common pathway by which agents like PB increase tumor outcome in both species. Accordingly, the objectives of the present work were to determine whether PB and PCN induced Mdm2 in rat liver and, if so, to determine how induction of Mdm2 affected the regulation of p53 levels. In addition, the effects of PB and PCN on Mdm2 induction in rats and mice was compared in order to confirm previously published results and to determine whether Mdm2 induction represents a common event in response to these compounds in both species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Chemicals and reagents.
PB (sodium salt), PCN, and diethylnitrosamine (DEN) were purchased from Sigma (St Louis, MO). Microarray analyses were performed on Affymetrix U34A GeneChips (Affymetrix, Santa Clara, CA). All reagents for real-time polymerase chain reaction (RT-PCR), cDNA cloning, immunoprecipitations, or Western blotting were electrophoretic grade or higher. All primers for RT-PCR reactions were obtained from Sigma-Genosys (The Woodlands, TX).
Animals and treatments.
Male and female Sprague-Dawley rats (Crl:CD(SD)IGS BR) and C57BL/6 mice were obtained from Charles River Laboratories, Inc. (Kingston, NY). Animals were housed individually in suspended wire cages and maintained on a diet of Certified Rodent Chow (Purina Mills, St Louis, MO), and all aspects of the experimental protocols were compliant with United States Department of Agriculture Animal Welfare regulations. Animals were approximately 8 weeks old at initiation of dosing in all studies.
For microarray analyses, male and female rats (n = 5 per sex) were dosed orally by gavage with PCN (100 mg/kg), PB (100 mg/kg), or vehicle (0.5% aqueous methylcellulose with 1% Tween 80). PCN was a suspension in this vehicle, whereas PB was in solution. Livers were collected at 6 and 24 h after a single dose or 24 h after 5 consecutive days of dosing (n = 5 animals per sex per time point, immediately flash frozen in liquid N2, and stored at approximately – 70°C pending analysis.
In a separate experiment specifically used for immunohistochemical detection of Mdm2, female Sprague-Dawley rats were administered PB (100 mg/kg), PCN (100 mg/kg), DEN (100 mg/kg), or vehicle (0.5% aqueous methylcellulose with 1% Tween 80) once daily for 4 consecutive days (n = 2 animals per group). PB and PCN were dosed by oral gavage, whereas DEN was dosed by ip injection. Livers were collected 24 h after the last dose and fixed in 10% neutral buffered formalin for 3 days after which they were transferred to 70% ethanol pending analysis.
In studies carried out in mice, PB (100 mg/kg dissolved in water) or PCN (100 mg/kg prepared in 0.5% aqueous methylcellulose with 1% Tween 80) were dosed by oral gavage and control mice received the respective vehicles. Mice (five per sex per treatment) were dosed for 5 consecutive days, after which livers were removed and flash frozen 24 h after the last dose.
RNA isolation and preparation for microarray hybridization.
Total RNA was isolated from frozen rat liver using Trizol Reagent (Invitrogen Corporation, Carlsbad, CA) and further purified using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Total RNA was quantified by UV spectrophotometry, and its integrity and quality were assessed on RNA 6000 Nano LabChips with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Double-stranded cDNA was synthesized, purified, and used to transcribe biotin-labeled cRNA with the BioArray High Yield Transcript Labeling Kit (ENZO Life Sciences, Farmingdale, NY). In all, 20-µg purified cRNA was fragmented, and 15-µg fragmented cRNA was hybridized to Rat Genome U34A GeneChips according to the Expression Analysis Technical Manual. Washing and staining of the hybridized arrays were carried out using the Fluidics Station 400 and arrays were subsequently scanned with the Hewlett Packard GeneArray Scanner.
Microarray data analysis.
Affymetrix scan data (.cel files) were imported into Rosetta Resolver for analysis (Rosetta Biosoftware, Seattle, WA). Following intrachip normalization and background correction, the software calculated an error-weighted treatment group mean, a fold change of the treated versus control group means, and an error-weighted t-test p value for the observed difference of the treated and control group means for each probe set on the microarray. Microrray data quality was assessed by principal component analysis of all chips and by intragroup chip-to-chip correlation analysis. Principle component analysis (n = 90) of global expression revealed a pattern of segregation based on sex and treatment and identified one outlier, which was omitted from subsequent data analysis. Examination of intradose group correlation revealed minimal variability within groups (Pearson correlation coefficient 
0.975 in all cases). Transcripts were defined as active if they changed with a p value 
0.01 and increased or decreased by greater than 1.5-fold.
RT-PCR analysis of hepatic Mdm2 mRNA.
Total RNA was isolated and purified as described above, after which 5 µg was reverse transcribed into cDNA using the SuperScript II Reverse Transcriptase system and random hexamers (Invitrogen Corporation). RT-PCR was performed on 20 ng of each cDNA sample in duplicate with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green Master Mix (Eurogentec, San Diego, CA). Cycle parameters used were as follows: 2 min at 50°C, 10 min at 95°C (denaturation), 40 cycles of 15 s at 95°C/30 s at 60°C (denaturation/amplification), 15 s at 95°C (denaturation), 15 s at 60°C (annealing), gradual increase to 95°C over 20 min (dissociation stage). The following oligonucleotide primers, designed using Primer Express software (Applied Biosystems), were used at a final concentration of 100nM to quantify Mdm2 mRNA: forward primer 5'-CGG CCT AAA AAT GGT TGC AT-3'; reverse primer 5'-TTT GCA CAC GTG AAA CAT GAC A-3'. Both primers span a region of the Mdm2 mRNA sequence that is identical in rat and mouse. Data were normalized to mRNA levels of 18s rRNA as a housekeeping gene.
Determination of Mdm2 and p53 protein levels by immunoblotting.
Rat livers were homogenized in a lysis buffer containing 50mM Tris (pH 8), 120mM NaCl, 0.5% Nonidet P-40, and Complete Mini Protease Inhibitor tablets (Roche Applied Science, Indianapolis, IN) after which particulates were removed by centrifugation (13,000 x g). Protein in the resulting supernatants was quantified with the bicinchoninic acid assay (Pierce, Rockford, IL) with bovine serum albumin as standard. For SDS-PAGE, 50 µg of total soluble protein was separated on 4–12% Tris-Glycine precast gels (Invitrogen Corporation). Western blotting was performed using the ECF Western Blotting Kit (Amersham Biosciences Corporation, Piscataway, NJ). Rabbit polyclonal antibodies against Mdm2 (SMP14) and p53 (FL-393) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against -tubulin was purchased from Cell Signaling Technology (Danvers, MA). Western blot images were captured with the Storm 860 phosphorimager, and protein levels were determined using ImageQuant v 5.0 software (Amersham Biosciences).
Immunohistochemical detection of Mdm2 in liver.
Fixed liver tissue from female rats treated with DEN, PB, or PCN was processed by routine histological procedures after which heat-induced epitope retrieval was performed in a Decloaking Chamber (Biocare Medical, Concord, CA) using Target Retrieval Solution (DakoCytomation, Carpinteria, CA) for 1 min at 125°C and 22–24 psi. Mdm2 was detected with the SMP14 antibody, and control mouse IgG antibody was used as a negative control. Visualization was achieved with a biotin-labeled secondary antibody system followed by development with diaminobenzediene.
Immunoprecipitation and immunoblotting.
Rat liver was homogenized in lysis buffer as described above, and equivalent amounts of protein (500 µg) were subjected to immuoprecipitation with Mdm2 (SMP14). The resulting immune complexes were collected on protein A-Sepharose beads in the presence of a rabbit anti-mouse secondary antibody (Sigma). Immunoprecipitated proteins were eluted from the sepharose beads by boiling in the presence of 1x Tris-Glycine SDS sample buffer (Invitrogen Corporation) and fractionated by SDS-PAGE for Western blotting analysis of Mdm2 as described previously. A mouse monoclonal antibody against SUMO-1 (21C7) was purchased from Zymed Laboratories, Inc. (South San Francisco, CA).
Cloning of rat Mdm2 cDNA and plasmid construction.
A pCDNA3.1 + RatMDM2 plasmid was constructed using standard molecular biology techniques. Total RNA was isolated from the liver of a female Sprague-Dawley rat administered PCN at 100 mg/kg for 5 days followed by reverse transcription into cDNA. The full-length rat Mdm2 open reading frame (GenBank accession number: XM_235169) was amplified by PCR using the following primers: forward primer: 5'-GCG CGG ATC CAT GTG CAA TAC CAA CAT GTC TGT-3' and reverse primer: 5'-GCG CGA ATT CCT AGT TGA AGT ACG TGA G-3'. The PCR product was digested with BamHI/EcoRI and ligated into pCDNA3.1+ (Invitrogen Corporation, Carlsbad, CA) that was digested with the same enzymes. The sequence of pCDNA3.1 + RatMDM2 was confirmed by restriction digestion and direct sequencing.
In vitro translation of rat Mdm2.
In vitro translation reactions were performed using 1 µg of either pCDNA3.1+ vector or pCDNA3.1 + RatMDM2 construct using the TNT T7/SP6 Coupled Reticulocyte Lysate System (Promega Corporation, Madison, WI). In vitro translation products were diluted 1:10 in lysis buffer and fractionated by SDS-PAGE for Western blotting analysis as described above.
Transfection of rat Mdm2 into rat hepatic Clone 9 cells.
Clone 9 cells, derived from normal rat liver, were purchased from the American Type Culture Collection (Catalog No. CRL-1439; Manassas, VA) and grown in Kaighn's modification of Ham's F12 medium supplemented with 2mM L-glutamine, 1.5 g/l sodium bicarbonate, and 10% fetal bovine serum. Clone 9 cells were transfected when approximately 80% confluent using Lipofectamine 2000 Reagent (Invitrogen Corporation). Twenty-four hours after transfection, cells were collected by trypsinization, washed with PBS, and lysed for Western blotting analysis as described above.
Statistical analysis.
RT-PCR data were compared with an unpaired t-test using StatView v 5.0 statistical software (SAS Institute Inc., Cary, NC). The significance level was p < 0.05. Statistical analyses of microarray results were performed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Microarray analysis of hepatic gene expression indicated changes in numerous CAR- or PXR-regulated genes commonly reported for PB and PCN. A comprehensive list of all PB and PCN-induced gene expression changes from this study is available as a Supplementary data file (Supplementary Data Table 1). An abridged summary of statistically significant gene changes observed after 5 days of dosing, with a particular emphasis on phase I, II, and III metabolism, is presented in Table 1. As anticipated, PB and PCN increased transcript levels of cytochrome P450 genes including members of the CYP2B and 3A families and numerous phase II biotransformation enzymes. Increased levels of major transporter genes were also seen, including Mdr1a, Mrp2, Mrp3, and Oatp1a4. In addition, changes in several key genes from this data set, including Ugt1a1, Mrp2, Oatp1a4, and members of the CYP2B and CYP3A families, were validated by RT-PCR (data not shown).
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In addition to the expected induction of CAR and PXR-dependent genes, Mdm2 was induced as early as 6 h after a single dose of PCN in both male and female rats (Table 2). With PCN treatment, maximal induction of approximately 5- and 12-fold was observed in males and females, respectively, following 5 days of dosing. In PB-treated rats, Mdm2 induction was lower in magnitude, with approximately a twofold induction observed in males across all three time points, and a similar twofold increase observed in female rats at the 24-h and 5-day time points. The increased mRNA levels observed after 5 days of dosing with PB and PCN were also confirmed by RT-PCR analysis of Mdm2.
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To determine whether the hepatic induction of Mdm2 mRNA by PB and PCN involved activation of the p53 pathway, the microarray data were queried for alterations in expression of p53 and related genes. Whereas Mdm2 transcript expression was consistently induced following PB and PCN administration, p53 transcript levels were not significantly altered in PB or PCN-treated male and female rats at any of the time points (Table 3 for 5-day data; Supplementary Data Tables 2 and 3 for 6-h and 24-h data, respectively). Furthermore, no changes in other genes transcriptionally regulated by p53 activation in rat liver (Ellinger-Zigelbauer et al., 2004
) were observed, suggesting that PB and PCN treatment did not increase Mdm2 through a p53-dependent response (Table 3; Supplementary Data Tables 2 and 3).
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Mdm2 shuttles between the nucleus and cytoplasm, and its nuclear location is required to export p53 from the nucleus in order to target p53 for proteosomal degradation (Boyd et al., 2000
; Tao and Levine, 1999). Therefore, it was important to establish whether the Mdm2 induced by PB and PCN in rat liver was cytosolic or nuclear in location. Female rats were used for this work because they showed higher levels of Mdm2 induction. By immunohistochemical evaluation, very low levels of Mdm2 were detected in control rat liver, and no nuclear staining was observed (Fig. 1A). In contrast, DEN, which is known to activate Mdm2 through a p53-dependent mechanism, increased Mdm2 in liver which was predominantly nuclear in location (Fig. 1B). With PB or PCN treatment (Figs. 1C and 1D, respectively), both cytoplasmic and nuclear staining of Mdm2 was observed, and although not as apparent as that observed with DEN, a clear pattern of nuclear staining was noted with PB and PCN.
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Given the nuclear staining pattern for Mdm2 protein, it is possible that this induction may cause hepatic levels of p53 to decrease as a result of the regulatory feedback loop. As shown in Figure 2, PB and PCN decreased p53 protein levels after 5 days of dosing with approximately a 35 and 50% reduction in p53 protein, respectively. These results provide additional evidence that the Mdm2 induced by both PB and PCN was functionally active.
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Table 4 summarizes the effects of PB and PCN on hepatic mRNA levels of Mdm2 in C57BL/6 mice. C57BL/6 mice were used because Mdm2 induction with PB and TCPOBOP was previously demonstrated in this strain (Huang et al., 2005
). Unlike the 4- to 10-fold induction of Mdm2 observed with PCN in male and female rats, respectively, PCN had no effect on Mdm2 in mouse liver. Similarly, no increase in Mdm2 was observed with PB after 5 days of dosing. An increase in Mdm2 mRNA levels (approximately 50% over control values) was observed in male and female mice after 2 weekly dosages of TCPOBOP (3 mg/kg; data not shown).
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Mdm2 isolated from human or mouse cell lines or tissues migrates electrophoretically as two bands with molecular weights of 76 and 90 kDa, respectively (Barak et al., 1993
; Saucedo et al., 1999). Although the nuclear localization of Mdm2 and associated decrease in hepatic p53 levels suggest that the Mdm2 induced by PB and PCN is a fully functional protein, the apparent molecular weight of rat hepatic Mdm2 protein was approximately 120 kDa (Fig. 3A). To assess the molecular weight discrepancy between rats and other species, the full-length Mdm2 open reading frame was cloned from rat hepatic mRNA, ligated into a mammalian expression vector, and translated in a cell-free in vitro expression system to evaluate the corresponding protein products. The in vitro translation assay yielded products that migrated at 76 and 90 kDa, consistent with the Mdm2 molecular weights described previously (Fig. 3B). The Mdm2 construct was then transiently transfected into rat hepatic Clone 9 cells to evaluate the expressed protein products in a relevant hepatocyte system. In Clone 9 cells, a 76-kDa protein was observed, but there was a clear shift in molecular weight, with an additional band at approximately 120 kDa observed. There was no 90-kDa protein band detected after transfection (Fig. 3C).
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The transient transfection results suggest that in rat, Mdm2 likely undergoes a posttranslational modification in liver that causes it to migrate primarily at 120 kDa. One such modification described for Mdm2 involves the enzymatic coupling with small ubiquitin-like modifier-1 (SUMO-1) in a process termed sumoylation (Muller et al., 2001
; Xirodimas et al., 2002). To determine whether hepatic Mdm2 was sumoylated in rat, a liver homogenate from a PCN-treated rat was subjected to immunoprecipitation of Mdm2, followed by Western blot analysis of either Mdm2 or SUMO-1 protein (Fig. 4). Immunoprecipitation of Mdm2 followed by Western blotting of SUMO-1 revealed an intense band that migrated at approximately 120 kDa. Similarly, a 120-kDa band was detected through immunoprecipitation of SUMO-1 followed by Western blotting of Mdm2 (data not shown). Thus, rat liver Mdm2 is likely to be sumoylated by posttranslational modification, and the addition of one or more SUMO-1 moieties may explain the discrepancy between the observed molecular weight of Mdm2 in this study and the published data for Mdm2 in humans and mice. Collectively, these results provide evidence that the antibody used in these studies is specific for rat Mdm2 and detects the 76- and 90-kDa proteins from the in vitro translation experiments as well as the higher molecular weight protein resulting from posttranslational modification that is detected by immunohistochemistry, immunoblotting, and immunoprecipitation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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It is well established that p53 is an important tumor suppressor gene that, in response to a variety of cellular stresses, mediates cell cycle arrest or apoptosis and inhibits cell transformation by preventing the replication of cells with damaged DNA (Momand and Zambetti, 1997
). Mdm2, the first negative regulator of p53 discovered (Momand et al., 1992), controls p53 levels by functioning as an E3 ubquitin ligase, conjugating ubiquitin monomers to multiple lysine residues within the COOH terminus of p53 and marking the protein for nuclear export and subsequent degradation (Boyd et al., 2000; Li et al., 2003; Tao and Levine, 1999). In rat liver, a predictable induction of Mdm2 has been observed with numerous genotoxic hepatocarcinogens including DEN (Finnberg et al., 2004), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and aflatoxin B1 (Ellinger-Zigelbauer et al., 2004), and in studies using microarray analyses, Mdm2 induction was directly associated with activation of p53 (Ellinger-Zigelbauer et al., 2004). In contrast, the results of the present work indicate that PB and PCN induce Mdm2 in rats, and given the lack of changes in other p53-dependent genes, this induction appears to occur in a p53-independent manner.
Cellular localization plays an important role in determining the biochemical functions of Mdm2 and p53. Both proteins are capable of shuttling between the nucleus and cytoplasm of the cell, and these movements are tightly regulated events. Importantly, ubiquitination of p53 requires that Mdm2 translocate to the nucleus where it mediates nuclear export of p53 protein (Boyd et al., 2000; Tao and Levine, 1999). Consequently, nuclear localization of Mdm2 following PB and PCN treatment provides evidence that it is functioning to modulate p53 levels. This conclusion is further supported by the observed decrease in p53 protein levels in livers of PB- and PCN-treated rats.
Whereas the nuclear location and decreased levels of p53 provided evidence that the Mdm2 induced by PB and PCN was functionally active, the protein detected in rat liver differed in molecular weight from that which has been described in various human and mouse tissues and cell lines (Barak et al., 1993; Saucedo et al., 1999). Mdm2 is a 491-amino acid protein that, although the predicted molecular weight is approximately 54 kDa, typically migrates as two species with molecular weights of 76 and 90 kDa. While faint expression of the 76- and 90-kDa forms were detected in rat liver, the majority of the hepatic-expressed Mdm2 protein reproducibly migrated at approximately 120 kDa. Although there are limited data published on rat Mdm2, forms of Mdm2 larger than 90 kDa have been reported during various stages of fetal development and adulthood in rat liver, kidney, brain, heart, and lung (Ibrahim et al., 1997). There are additional reports of Mdm2 species that migrate by SDS-PAGE with molecular weights greater than 90 kDa as a result of posttranslational modifications. Conjugation of Mdm2 coupled with the SUMO-1 yields a higher molecular weight species migrating at approximately 120 kDa in human H1299 cells (Xirodimas et al., 2002), and in the present work, the 120 kDa form of Mdm2 in rat liver was identified as a sumoylated protein. Presently, the functional consequences and species differences in posttranslational modification of Mdm2 are not fully understood, but modification with SUMO is known to affect localization, overall stability, and function of proteins (Meek and Knippschild, 2003; Muller et al., 2001). Therefore, additional studies are required to determine the molecular basis of sumoylation of Mdm2 and to ascribe functional significance to the extensive sumoylation observed in rats relative to other species.
Overall, the results of the present study indicate that PB and PCN increase transcription of Mdm2 in liver within a few hours after a single dose, and this induction is sustained over a 5-day period. Additional studies carried out over longer treatment durations (up to 90 days) suggest that this early effect is sustained over time (data not shown). The data also indicate that the induction observed in response to PCN is greater in magnitude than that observed with PB. In this regard, the present data suggest a possible role for nuclear receptor activation, particularly PXR, in mediating Mdm2 induction in rat liver. A role for nuclear receptor–mediated Mdm2 induction is not without precedent as there is evidence that activation of the thyroid hormone (T3) receptor regulates Mdm2 induction in a p53-independent manner (Qi et al., 1999).
Recently, a role for CAR-dependent Mdm2 induction in mice, particularly in response to TCPOBOP, was reported (Huang et al., 2005). In the present work, no change in Mdm2 mRNA was noted in mice treated with PB or PCN at the same dosages which induced Mdm2 in rats. It is important however, to note that evidence for CAR-dependent Mdm2 induction in mice has been inconsistent as other investigators failed to observe Mdm2 induction in C3H mice treated with PB (Yamamoto et al., 2004). A significant new finding in the present work is that PCN, likely inducing through activation of PXR, markedly increased Mdm2 induction in rat liver, but failed to increase Mdm2 in mouse liver, suggesting possible species differences in Mdm2 regulation.
In addition to the possible role that nuclear receptors may play in Mdm2 induction, the present work has potential significance relative to mechanisms of hepatocarcinogenic outcome in rodents with nongenotoxic agents like PB. It is well established that Mdm2 plays an essential role in the regulation of the tumor suppressor p53 and consequently cell cycle dynamics. Furthermore, rat hepatic preneoplastic foci have been shown to overexpress Mdm2 resulting in a defective response to DNA damage that may support advancing clonal growth and malignant transformation (Van Gijssel et al., 2000). Accordingly, agents like PB may promote tumor progression in the absence of a genotoxic signal by directly amplifying cellular Mdm2 levels and subsequently perturbing p53-mediated cell cycle regulation. Furthermore, Mdm2 also catalyzes the ubiquitination and degradation of the retinoblastoma protein (Rb) tumor suppressor, concurrently activating several transcription factors responsible for promoting cell cycle progression (Uchida et al., 2005). Thus, induction of Mdm2 may increase the likelihood of liver tumor development by abrogating cell cycle arrest in response to spontaneous DNA damage or other cell stress, thereby increasing the chances that spontaneous mutations will occur.
In addition to the critical role that Mdm2 plays in the regulation of p53 or Rb, there is also evidence suggesting that Mdm2 can regulate neoplastic growth through p53-independent mechanisms. Mdm2 is a proto-oncogene that is amplified and overexpressed in a variety of cancers (Momand and Zambetti, 1997). Furthermore, Mdm2 transgenic mice, showing an average fourfold increase in tissue levels of Mdm2, are more prone to spontaneous tumorigenesis. The increased tumor incidence in Mdm2 transgenic mice is observed in the presence or absence of p53 (Jones et al., 1998), thereby demonstrating a direct association between Mdm2 levels and tumor development. Accordingly, the increase in hepatic Mdm2 observed within 24 h of a single dose of PB or PCN may provide a direct stimulus for cell transformation in a tissue that is already prone to a high incidence of spontaneous tumors. Furthermore, increased levels of Mdm2, whether functioning in a p53-dependent or independent manner, are likely to be detrimental when combined with hepatocellular hyperplasia typically observed with agents like PB and PCN.
In summary, the results of the present study demonstrate that PB and PCN induce Mdm2 in rodents, and this induction is likely to occur independent of direct effects on p53 transcription. Mdm2 is found in the nucleus, suggesting normal function in the nuclear export and proteolytic degradation of p53 with a concurrent reduction in constitutive p53 levels. The Mdm2 protein observed in rats appears to be different from that typically seen in mice in that it is subject to posttranslational modification, particularly with SUMO proteins, and sumoylation is likely to further stabilize the Mdm2 protein. Although the molecular mechanisms controlling Mdm2 induction in rats have not yet been determined, a role for nuclear receptors is likely. However, neither PB nor PCN induced Mdm2 in mouse liver, suggesting that, in a manner similar to species difference in posttranslational modification, the molecular regulation of Mdm2 may differ across species. Finally, induction of Mdm2 by PB and PCN suggests that early effects on cell cycle regulation, response to DNA damage or cell transformation may play a critical role in liver tumor development in rats.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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