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TOXICOLOGY
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland
Received October 6, 2005; accepted December 2, 2005.
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Abstract |
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; Whitcomb and Block, 1994). Acute exposure of large doses of APAP can also cause liver damage (Thomas, 1993), whereas APAP toxicities caused by overdose and long-term addictive usage are not rare (Shayiq et al., 1999; Lee, 2004). Although N-acetyl-p-benzoquinoneimine (NAPQI), a toxic metabolite of APAP produced by various cytochrome P450 isozymes such as CYP2E1, CYP1A2, and CYP3A (Thomas, 1993; Sinclair et al., 2000), is believed to cause the toxicity, the mechanism for APAP-induced toxicity is still under investigation. Despite strong correlations between the levels of NAPQI-protein adducts and the severity and regional location of the tissue damage (Cohen et al., 1997), other factors such as lipid peroxidation, Ca2+ homeostasis, and reactive oxygen and nitrogen species (Knight et al., 2002) may also play a role in APAP-mediated toxicity. Recent data suggest that mechanisms other than NAPQI may also be important in causing toxicity, because NAPQI-protein binding was still observed after hepatic damage was significantly alleviated (Michael et al., 1999) and that 3-hydroxyacetanilide, a noncytotoxic analog of APAP, also exhibited patterns of protein binding similar to that of NAPQI (Myers et al., 1991; Fountoulakis et al., 2000).
The p53 gene encodes a 393-amino acid protein that possesses five conserved regions: an N-terminal transcriptional activation domain, an SH3 domain, a specific DNA binding domain, a C-terminal oligomerization domain, and a basic domain. p53 protein is a transcription factor that plays a critical role in regulating cell growth, DNA repair, and apoptosis in response to stressful conditions (Lakin and Jackson, 1999; Vogelstein et al., 2000). However, p53 is a short-lived protein under complex regulation, including reversible cycles of post-translational modification such as phosphorylation, acetylation, and ubiquitination. Under physiological conditions, p53 can be conjugated with ubiquitin, primarily by the murine double minute (mdm2) protein (Lakin and Jackson, 1999; Ogawara et al., 2002), a major ubiquitin ligase for p53, leading to its degradation in a ubiquitin-dependent manner. After DNA damage or under apoptotic conditions, p53 is phosphorylated at several sites, including Ser15 and Ser37, near the binding site for mdm2. Phosphorylation of p53 at its N terminus reduces the interaction between p53 and mdm2, resulting in p53 stabilization because of decreased ubiquitin binding. Depending on the severity of DNA damage from various stressful conditions, p53 is activated to induce growth arrest to allow cells to repair damaged DNA. When DNA damage is too severe and thus impossible to be repaired, p53 can promote apoptosis to eliminate damaged cells (Giaccia and Kastan, 1998; Vogelstein et al., 2000). Growth arrest induced by p53 was shown to be mediated by the downstream targets of p53 such as p21 and Gadd45, both of which regulate cell cycling through modulation of cyclin-dependent kinases and regulatory proteins (Zhan et al., 1998). Alternatively, p53 could promote cell death by up-regulating proapoptotic Bax protein upon withdrawal of a neurotrophic factor (Aloyz et al., 1998). Furthermore, a p53-mdm2 feedback loop exists to control the levels of p53 and mdm2 (Lev Bar-Or et al., 2000).
We previously reported that APAP can cause apoptosis (Bae et al., 2001) and oxidative DNA damage in C6 glioma cells (Wan et al., 2004). Therefore, we hypothesized that the level of p53 would be up-regulated by APAP, similar to the levels observed after exposure to chemotherapeutic agents and other DNA-damaging agents (Vogelstein et al., 2000), because APAP can induce apoptosis (Bae et al., 2001) and wild-type p53 is present in C6 glioma cells (Asai et al., 1994). Contrary to this hypothesis, APAP treatment markedly decreased the level of p53. Therefore, in this study, we investigated the mechanism of p53 reduction and determined the level of p21, a downstream target of p53, upon exposure to APAP. We also studied whether phosphorylation of p53 at its N terminus affects the APAP-induced reduction of p53 protein. Finally, we also studied the differential effects of APAP and its nontoxic analog 3-hydroxyacetanilide on the p53 levels in C6 glioma and LLC-PK1 porcine kidney cells.
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Materials and Methods |
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Cell Culture and Immunoblot Analyses of Various Proteins. C6 glioma or LLC-PK1 cells (1 x 107 cells/150-mm diameter culture dish) were grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum and antibiotics before treatment with different concentrations (up to 5 mM) of APAP or 3-hydroxyacetanilide as described previously (Bae et al., 2001). To prepare proteins for immunoblot analyses, harvested C6 and LLC-PK1 cells were homogenized in the lysis buffer as described previously (Bae et al., 2001). Cell debris and particulate fractions were removed by centrifugation at 5000g for 10 min at 4°C. Equal amounts of protein in the 5000g supernatant fractions or whole homogenates were separated by 10 or 12% SDS-PAGE, transferred onto PVDF-Immobilon membranes (Millipore Corporation, Bedford, MA), and subjected to immunoblot analysis using the respective antibody against p53, phospho-p53, Akt, phospho-Akt, mdm2, p21, actin, or ubiquitin. Immunoreactive proteins were subsequently detected with appropriate secondary antibodies conjugated with horseradish peroxidase and enhanced chemiluminescence kits.
RT-PCR Analysis for p53 mRNA Expression. Total RNA was isolated by using the TRIzol reagent kit. Purity and concentration of RNA were determined by measuring UV absorbance at 260 and 280 nm. RT-PCR was performed using SuperScript one-step RT-PCR kit (Invitrogen) following the manufacturer's instruction. Total RNA (400 ng/assay) was used for each RT-PCR using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA), one cycle of reverse transcription at 37°C for 30 min, 94°C for 2 min, followed by 26 cycles of PCR at 94°C (20 s), 55°C (45 s), and 68°C (60 s). DNA sequences of the oligonucleotide primer set for rat p53 mRNA (Soussi et al., 1988) were: sense, 5'-TCTGTCATCTTCCGTCCCTTCTC-3'; and anti-sense, 5'-AACACGAACCTCAAAGCTGTCCCG-3'. The primers used for amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 194 bp) transcript were the same as described previously (Soh et al., 1996), and 23 cycles of PCR were used to amplify rat GAPDH transcript (as a loading control). Amplified DNA (10 µl of PCR mixture) was resolved on 1% agarose gel for electrophoresis and visualized under UV illumination.
Immunoblot Analyses of Immunoprecipitated p53. To immunoprecipitate p53 protein, specific antibody to p53 was incubated for 2 h with the soluble proteins (500 µg/sample) from C6 cells treated with APAP for different times as indicated. To facilitate immunoprecipitation of p53, protein G-bound agarose (0.1 ml/sample) was added to each sample and incubated for another 4 h before centrifugation at 10,000g for 10 min. The immunoprecipitated p53 was washed twice with 1x phosphate-buffered saline (PBS) and subjected to 10% SDS-PAGE followed by immunoblot analysis using the specific antibody against p53, ubiquitin, or mdm2. In addition, the same membrane used for the first immunoblot for p53 was extensively washed with a buffer containing 62.5 mM Tris-HCl (pH 6.8), 100 mM 2-mercaptoethanol, and 2.0% SDS. The second immunoblot analysis was then performed to determine the level of p53-bound ubiquitin.
Data Processing and Statistical Analysis. The density of immunoreactive proteins or mRNA transcript was quantified using NIH image 1.61 software (Bethesda, MD). The relative densities of p53, Akt, phospho-Akt, phospho-p53, ubiquitin, mdm2, and p21 to actin were calculated and compared for all samples with different treatments. Statistical analyses were performed using the Student's t test, and p < 0.05 was considered statistically significant. All the data represent the results from at least three separate experiments unless stated otherwise. Other materials and methods not described here were preformed as described previously (Bae et al., 2001; Bae and Song, 2003).
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Results |
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), we expected up-regulation of p53 content. Contrary to this expectation, treatment with 5 mM APAP for 24 h markedly decreased the level of p53 in C6 glioma cells (Fig. 1A, top) compared with the DMSO-treated control in an APAP concentration-dependent manner. Immunoblot results revealed that 1.25 mM APAP treated for 24 h markedly decreased the level of p53. In general, greater reduction in p53 content was observed after exposure to higher concentrations of APAP than lower concentrations (Fig. 1A, top panel). The levels of p21 (Fig. 1A, middle panel) and Gadd45 (data not shown), downstream targets of p53, markedly decreased 24 h after treatment with APAP. In contrast, the level of actin (Fig. 1A, bottom panel), used as a loading control, did not change after APAP exposure. Our results indicate that the reduced p21 level probably results from the decreased level of p53 protein following exposure to APAP.
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Time- and Ubiquitin-Dependent p53 Degradation upon APAP Exposure. It is well established that p53 is rapidly degraded through ubiquitin-mediated proteolysis following interaction with mdm2, a major ubiquitin ligase for p53 (Lakin and Jackson, 1999). Because of the APAP-induced p53 reduction at the protein level, we then investigated whether APAP promotes p53 degradation through ubiquitin-dependent proteolysis. The levels of p53 in the soluble fraction were compared after C6 cells were exposed to APAP in the presence and absence of 1 µM lactacystin, a potent inhibitor of proteasome-dependent proteolysis (Fenteany and Schreiber, 1998). Lactacystin alone slightly affected the level of p53 (Fig. 2A, top panel, lane 4) compared with that of the DMSO-treated control (lane 1). However, lactacystin appeared to prevent APAP-induced p53 degradation (lane 5) compared with the level of p53 in C6 glioma cells treated with APAP alone (lanes 2 and 3), whereas the level of actin, used as a loading control, did not change during each treatment (Fig. 2A, bottom panel). Similar results were also observed with MG132 (up to 1 µM final concentration), another inhibitor of proteasomal proteolysis (Maki et al., 1996) (data not shown).
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Additional immunoblot analysis showed that the levels of p53 and p21 in the whole-cell homogenates did not change at 2 and 4 h after APAP exposure but started to decrease at 8 and 16 h (Supplemental Fig. 1). Thereafter, very low levels of these proteins were observed at 24 h. In contrast, the levels of phospho-Ser15-p53 and mdm2 appeared to increase at 8 h after APAP treatment, and increased amounts of phosphorylated p53 and mdm2 were detected at 24 h (Supplemental Fig. 1).
Effects of Akt Activity and p53 Phosphorylation on APAP-Induced p53 Reduction. The activated form of Akt (phospho-Akt) is known to phosphorylate mdm2 at Ser186, leading to mdm2-mediated ubiquitin conjugation and subsequent degradation of p53 (Ogawara et al., 2002). Because of the increased mdm2 binding to p53 protein, we determined whether the level of Akt or phospho-Akt was elevated after APAP treatment. Immunoblot results showed that APAP did not affect the levels of both Akt (Fig. 3A, middle panel) and phospho-Akt (Fig. 3A, bottom panel), indicating that APAP-induced mdm2 binding to p53 and subsequent reduction of p53 protein were independent from the alterations of Akt and phospho-Akt. These results further suggest that the increased mdm2 binding and ubiquitin conjugation to p53 protein most likely result from direct interaction between p53 and APAP or its reactive metabolite(s).
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; Lakin and Jackson, 1999). Because of the APAP-induced p53 reduction (Figs. 1 and 2), it was hypothesized that APAP might interfere with the phosphorylation of p53. Therefore, we studied the degree of phosphorylation of p53 after C6 cells were exposed to APAP for 24 h compared with that by etoposide or 5-FU used as a positive control. Under our experimental conditions, p53 levels were significantly reduced at 24 h after APAP treatment (Fig. 3B, top panel, lane 2), whereas actin levels did not change (Fig. 3B, bottom panel). Contrary to our expectation, APAP stimulated phosphorylation of p53 at both Ser15 (Fig. 3B, second panel, lane 2) and Ser37 (Fig. 3B, third panel, lane 2) compared with the DMSO-treated control (lane 1). Treatment with etoposide and 5-FU stimulated phosphorylation of p53 at these serine residues (Fig. 3B, second and third panels, lanes 3 and 4, respectively) with increased p53 content (Fig. 3B, top panel, lanes 3 and 4) compared with the DMSO-treated control (lane 1). Immunoblot analysis with a specific antibody that recognizes phospho-Ser20 revealed that APAP, etoposide, and 5-FU, respectively, increased phosphorylation of p53 at Ser20 (data not shown), although the quality of this immunoblot was not as good as other immunoblots (second and third panels).
Role of CYP2E1 in APAP-Induced p53 Reduction. It is well established that APAP-induced toxicity results from P450-mediated metabolism (Thomas, 1993; Dai and Cederbaum, 1995; Holownia et al., 1997) and that pretreatment with P450 inhibitors can markedly reduce the rate of APAP-induced cell damage (Sinclair et al., 2000). Our previous results (Bae et al., 2001) supported this conclusion, which suggests an important role for CYP2E1 in APAP-induced cell damage. Therefore, we studied whether the degradation of p53 is also dependent on CYP2E1-mediated metabolism of APAP in C6 glioma cells. In this study, we determined the effect of a chemical inhibitor of CYP2E1, CMZ (Hu et al., 1994), on the rate of cell death, the activity of CYP2E1, and the levels of p53 and p21. Cell death assay measured by MTT reduction (Bae et al., 2001) showed that less than 7% of DMSO-treated C6 glioma cells died and most cells looked normal under light microscopy (Fig. 4A, panel a). In contrast, approximately 35% of cells died at 24 h upon APAP treatment, with many dead cells observed (Fig. 4A, panel b). However, pretreatment with 20 µM CMZ (Fig. 4A, panel c) significantly reduced the rate of APAP-induced cell death, where approximately 20% of cells died after APAP exposure in the presence of CMZ (p < 0.05, significantly different from the cells exposed to APAP alone). Under these experimental conditions, the baseline activity of CYP2E1 in C6 cells, as determined by N-nitrosodimethylamine demethylase (Bae et al., 2001), was approximately 4.8 pmol of HCHO produced/mg of protein of the whole-cell extract/60 min. CMZ treatment reduced the activity of CYP2E1 by 50% (Fig. 4B). Consistent with the results shown in Figs. 1, 2, 3 and Supplemental Fig. 1, APAP markedly decreased p53 levels (Fig. 4C, top panel, lane 2) compared with that of the DMSO-treated control (lane 1). However, pretreatment with CMZ effectively prevented APAP-induced p53 degradation (lane 3), where CMZ alone did not change the level of p53 or p21 (Supplemental Fig. 2). A similar trend of APAP-mediated p21 reduction (lane 2) and recovery in the presence of CMZ (lane 3) was also observed (Supplemental Fig. 2, middle panel). In contrast, the levels of actin (Supplemental Fig. 2, bottom panel), used as a loading control, were similar in all samples.
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Differential Effects of APAP and 3-Hydroxyacetanilide on Cell Death Rate and p53 Degradation. Consistent with earlier reports (Myers et al., 1991; Bae et al., 2001), APAP caused significantly more cell damage (approximately 35%) than DMSO alone or 3-hydroxyacetanilide, a nontoxic analog of APAP (<10%) (Fig. 5A). Under these conditions, we compared the effect of 3-hydroxyacetanilide or APAP on p53 reduction. Treatment with 5 mM 3-hydroxyacetanilide (Fig. 5B, top panel, lane 2) for 24 h did not reduce the p53 level compared with the DMSO-treated vehicle control (Fig. 5B, top panel, lane 1). In contrast, exposure to 5 mM APAP markedly decreased the level of p53 (Fig. 5B, top panel, lane 3) in C6 glioma cells, where the actin level did not change after each treatment (Fig. 5B, bottom panel).
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; Al-Ghamdi et al., 2004). Immunoblot analysis showed that 2.5 mM (data not shown) or 5.0 mM APAP treated for 24 h caused marked reduction of p53 and p21 contents in LLC-PK1 cells (Fig. 5C, lane 3, top and middle panels), whereas the levels of actin used as a loading control were similar in all lanes (Fig. 5C, bottom panel). In contrast, 5.0 mM 3-hydroxyacetanilide, a nontoxic analog of APAP, did not change the levels of p53 and p21 (Fig. 5C, lane 2, top and middle panels).
Our results observed in both C6 glioma and LLC-PK1 porcine kidney cells strongly suggest that APAP selectively decreased the level of p53 protein, likely through interaction between p53 and NAPQI or another reactive metabolite(s) produced from CYP2E1-mediated metabolism of APAP. In fact, increased levels of APAP moiety bound to p53 protein were recognized with the specific antibody to APAP when immunoprecipitated p53 protein with the p53-specific antibody was used in immunoblot analysis (Supplemental Fig. 3). This protein (54 kDa in lane 2) seemed to be absent in DMSO-treated control sample (lane 1), indicating that APAP and/or its related molecules appeared to bind to p53 protein after APAP exposure.
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Discussion |
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) at the same concentration used in other studies (Dai and Cederbaum, 1995; Holownia et al., 1997). Therefore, we hypothesized that the level of p53 would increase after APAP treatment, because p53 normally promotes growth arrest or apoptotic cell death (Vogelstein et al., 2000). However, the level of p53 actually decreased before and during apoptosis of C6 glioma cells (Figs. 1, 2, 3, 4, 5 and Supplemental Fig. 1) after APAP treatment. Therefore, p53 cannot be directly involved in the APAP-induced apoptosis of C6 glioma cells. Based on this view, our current results support the importance of c-Jun N-terminal protein kinase-related cell death pathway in APAP-induced apoptosis (Bae et al., 2001) in a p53-independent manner, similar to many other cases of apoptosis without involvement of p53 (Lieberman et al., 1995; Conner et al., 1999). Consistent with the in vitro data, our preliminary results with alcohol-pretreated BALB/c mice also show that acute exposure of APAP reduces the amounts of p53 and p21 during APAP-mediated necrosis and apoptosis (data not shown). The current data also provide evidence for an important role of CYP2E1 in the metabolic activation of APAP and subsequent loss of p53 and p21, because CMZ, an inhibitor of CYP2E1, significantly prevented the APAP-induced degradation of p53 and p21 (Fig. 4C). The partial protection by CMZ against APAP-mediated cell death rate may result from the incomplete inhibition of CYP2E1 by the concentration of CMZ used in this experiment (Fig. 4B). Alternatively, this could result from the fact that APAP can be also metabolized by other P450 isozymes, besides CYP2E1, present in C6 glioma cells (Geng and Strobel, 1995).
It was shown that many cellular proteins can be covalently modified with NAPQI, an electrophilic metabolite of APAP (Myers et al., 1991; Fountoulakis et al., 2000). Some of these modified proteins became inactivated. For example, the covalent binding of APAP to N-10-formyl-tetrahydrofolate dehydrogenase led to a decrease in its catalytic activity (Pumford et al., 1997). APAP could also bind and reduce the activities of certain mitochondrial enzymes such as glutamate dehydrogenase (Halmes et al., 1996) and aldehyde dehydrogenase (Landin et al., 1996), respectively, although the content of each protein was not evaluated in those studies. In addition, certain P450 enzymes such as CYP2E1 and CYP1A2, involved in the APAP metabolism, were degraded after administration of toxic doses of APAP (Snawder et al., 1994; Sinclair et al., 2000), possibly through interaction between these P450 proteins and NAPQI or other reactive metabolites produced from P450-mediated metabolism of APAP. Furthermore, we recently observed that APAP causes degradation of nuclear Ogg1, a DNA repair enzyme, without changing its mRNA level (Wan et al., 2004). By similar mechanisms, p53 may interact directly with NAPQI or other reactive metabolites of APAP (Supplemental Fig. 3), prior to its degradation despite phosphorylation at its N terminus.
Ray et al. (2001) previously reported that APAP slightly elevated the level of p53 in mouse liver. The level of p53 reported in that study, however, did not correspond with improvement in APAP-induced liver damage and normalization of other parameters such as hepatic lipid peroxidation, serum alanine aminotransferase activity, DNA fragmentation, and the level of Bcl-XL after treatment with chlorpromazine, 4-aminobenzamide, or nicotinamide. Those results suggest that the apoptotic role of p53 elevated by APAP may not be as important as decreased Bcl-XL content in APAP-induced liver damage. The results observed by Ray et al. (2001) are clearly different from our current results of APAP-induced p53 degradation in C6 and LLC-PK1 cells as well as preliminary results in APAP-treated mouse livers. Although the reason for the apparent differences is unknown, these differences can stem from variations in the strain of mice used, presence of ethanol in our treatment, and dosage of APAP. This difference remains to be clarified.
Results from this study demonstrate that p53 protein is likely to be degraded through increased mdm2 binding and ubiquitin conjugation after exposure to APAP, despite multiple phosphorylations of p53 at its N terminus. The mechanism by which APAP promotes mdm2 binding to phosphorylated p53 is unknown. It is conceivable that binding of NAPQI to p53 may cause conformational change in p53, with the opening up of another binding site(s) for mdm2 (Fig. 2B) in the NAPQI-p53 complex (Supplemental Fig. 3), regardless of the phosphorylation status at p53 N terminus as demonstrated (Fig. 3B). Increased mdm2 binding to NAPQI-p53 complex subsequently led to more ubiquitin conjugation and proteasomal degradation of p53 despite elevated phosphorylation of p53 protein. In contrast, 3-hydroxyacetanilide may not produce a reactive intermediate like NAPQI, which can bind to p53 protein. As a consequence, little degradation of p53 and its downstream target protein p21 took place after exposure to 3-hydroxyacetanilide in C6 glioma cells (Fig. 5B) and LLC-PK1 porcine kidney cells (Fig. 5C). The detailed molecular mechanism for APAP-mediated p53 degradation observed in two different cell lines is unknown but likely results from binding of NAPQI to p53 followed by ubiquitin-dependent p53 degradation after treatment with a relatively high dose (2.5 or 5 mM) of APAP.
It is well established that p53 level is regulated by complex mechanisms. Depending on the nature of the DNA-damaging agents, severity of damage, or cellular status, p53 level is elevated or reduced. For instance, p53 levels can be elevated through phosphorylation at its N terminus after treatment with DNA-damaging or apoptosis-inducing agents (Tishler et al., 1993; Vogelstein et al., 2000). On the other hand, p53 degradation is mainly facilitated through mdm2, the major p53 ubiquitin ligase, leading to ubiquitin-dependent proteasomal degradation of p53 (Lakin and Jackson, 1999; Ogawara et al., 2002). Our results shown in this study particularly differ from the well established relationship between p53 phosphorylation and mdm2 binding and p53 degradation. Therefore, our results represent a novel finding about p53 degradation by APAP. To our knowledge, our data represent the first report that APAP, possibly through the production of a highly reactive metabolite such as NAPQI, can reduce the level of p53 through ubiquitin-dependent proteolytic degradation. However, the biological significance of the reduced levels of p53 and p21 after APAP exposure remains to be determined, because these proteins are known to be involved in many biological functions such as apoptosis, DNA repair, and cell cycle arrest.
In conclusion, the results of this study demonstrate that a large dose of APAP can promote rapid degradation of p53 through mdm2-mediated ubiquitin conjugation but independent from Akt and phospho-Akt proteins, which are known to promote p53 degradation. In addition, APAP-induced p53 reduction is unrelated to the phosphorylation status of its N terminus. APAP also decreased the level of p21 consistent with the reduced level of p53. Comparative experiments with APAP and 3-hydroxyacetanilide in the presence of a CYP2E1 inhibitor suggest that binding of NAPQI, produced during CYP2E1-mediated metabolism of APAP, to p53 is likely to lead to increased mdm2 binding and subsequently ubiquitin-dependent p53 degradation. These results represent another example of p53 modulation by a chemical compound such as APAP.
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Acknowledgements |
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Footnotes |
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Y.-S.L. and J.W. equally contributed to this work.
ABBREVIATIONS: APAP, acetaminophen; NAPQI, N-acetyl-p-benzoquinoneimine; mdm2, murine double minute 2; CYP2E1, ethanol-inducible cytochrome P450 2E1 isoform; DMSO, dimethyl sulfoxide; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; CMZ, chlormethiazole; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair(s); PBS, phosphate-buffered saline; 5-FU, 5-fluorouracil; P450, cytochrome P450; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
1 Current affiliation: Department of Endocrinology, School of Medicine, University of Pennsylvania, Philadelphia, PA.
2 Current affiliation: Department of Neurology, The Chengdu 416 Hospital (The Formal Suzhou Medical College 2nd Hospital), Chengdu, Sichuan, China.
3 Current affiliation: Laboratory of Molecular Pharmacology and Physiology, Korea Research Institute of Chemical Technologies, Daejon, Korea.
Address correspondence to: Dr. B. J. Song, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, 9000 Rockville Pike, Bethesda, MD 20892-9410. E-mail: bjs{at}mail.nih.gov
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