Porcine Kidney Cells Research Articles

Identification and characterization of an alternatively spliced variant of the MHC class I-related porcine neonatal Fc receptor for IgG

The neonatal Fc receptor for immunoglobulin G (IgG) (FcRn) functions to transport maternal IgG to the fetal/neonatal animals and protects IgG from catabolism. The present study identified two pFcRn cDNAs (1.071 and 0.795kb) from intestinal epithelial cells. The corresponding mRNA transcripts were detected in porcine kidney cell line LLC-PK1, peripheral blood mononuclear cells, and porcine tissues by reverse transcription-PCR (RT-PCR) and Northern blot. Sequence analysis showed that the 1.071kb cDNA encodes the full-length pFcRn (pFcRn-L), whereas the 0.795kb cDNA codes for a truncated pFcRn (pFcRn-S) with deletion of 92 amino acids matching the alpha2 domain of pFcRn-L. pFcRn-L was constitutively expressed by epithelial cells; however, pFcRn-S was not detectable in porcine tissues and cell lines although its transcript was abundant. Despite the lack of native pFcRn-S, pFcRn-S was readily detected in transfected cells. Recombinant pFcRn-L was confirmed to bind IgG at pH 6.0, but not at pH 7.5; however, pFcRn-S failed to bind IgG at both pH 5.0-6.0 and 7.5. pFcRn-L was expressed on the cell surface and mainly localized in early endosomes. In contrast, pFcRn-S was absent from cell surface and primarily localized in the lysosome and pFcRn-S trafficking to lysosomes was independent of beta(2)-microglobulin (beta(2)m). The accumulation of pFcRn-S in the lysosome may explain the absence of native pFcRn-S protein expression. In addition, the trafficking of pFcRn-S to the lysosomal compartment suggests that in addition to sorting signals in its cytoplasmic tail, the FcRn structural integrity may be important for proper intracellular trafficking and function.

Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

Treatment of porcine kidney (PK-15) cells with either interferon-gamma (IFN-gamma) or endosomal- lysosomal system acidification inhibitors increases replication of porcine circovirus type 2 (PCV2). In the present study, the effect of a combination of these treatments on the number of infected cells and virus yield was tested. The number of PCV2 (Stoon-1010)-infected PK-15 cells increased in cells treated with ammonium chloride (445 +/- 39% increase), IFN-gamma (446 +/- 8%), ammonium chloride + IFN-gamma (1721 +/- 283%), chloroquine diphosphate (1037 +/- 121%), chloroquine diphosphate + IFN-gamma (2199 +/- 255%), monensin (950 +/- 178%) and monensin + IFN-gamma (1948 +/- 60%). Combined IFN-gamma and endosomal-lysosomal system acidification inhibitors increased PCV2 yield by up to 50 times compared to untreated PK-15. This augmented virus replication in PK-15 cells may be helpful in the production of PCV2 vaccines.

Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH(4)Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH(4)Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% +/- 110%, 1,212% +/- 34%, and 1,100% +/- 179%, respectively, in porcine kidney (PK-15) cells; by 128% +/- 7%, 158% +/- 3%, and 142% +/- 11% in swine kidney cells; by 160% +/- 28%, 446% +/- 50%, and 162% +/- 56% in swine testicle (ST) cells; and by 313% +/- 25%, 611% +/- 86%, and 352% +/- 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.

A Novel Role of Classical Swine Fever Virus Erns Glycoprotein in Counteracting the Newcastle Disease Virus (NDV)-mediated IFN-β Induction

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.

Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line

Post-weaning multi-systemic wasting syndrome (PMWS) has emerged as a major disease that poses a significant threat to the economics of global swine industry. Porcine circovirus type 2 (PCV2) is the causal agent of PMWS in pigs. Currently, the prevention of PCV2 infection based on vaccines is limited, and the available vaccines are either killed viral vaccines or recombinant protein based vaccines and not cost effective. The PK-15 cells, which is widely used for PCV2 propagation, is not efficient and heterogeneous in terms of permissivity to viral infection. In order to acquire a homogeneous porcine kidney cell line that can reliably produce PCV2 in high titers, cell clones that show high- (PK15-C1) or low-permissive (PK15-A2) phenotype to PCV2 infection were derived from heterogeneous PK15 parent cells by limiting dilution and cell cloning. Maximum virus titers in PK15-C1, PK15-A2 and PK15 parent cells were 10 8, 10 2 and 10 5 tissue culture infective dose 50 (TCID 50)/ml, respectively. The viral proteins of PCV2 were produced and accumulated faster in PK15-C1 cells than those in PK15 parent cells. These results indicate that PK15-C1 cell clone is more permissive to PCV2 infection than PK15 parent cells and thus will be useful for PCV2 replication in vitro, as well as, vaccines, diagnostic and research applications on PCV2.

Virucidal efficacy of nine commercial disinfectants against porcine circovirus type 2

A number of commercially available disinfectants are commonly used on pig breeding farms and are authorised by the French Agricultural Ministry. However, the efficacy of these disinfectants is unknown with regard to the emergent porcine circovirus type 2 (PCV2). The virucidal efficacy of nine disinfectants was evaluated by testing a suspension of PCV2 isolated in France. The assays were performed at 20 °C and the efficacy determined after 30 min contact time between virus and disinfectant. After this time, the mixture was passed through a detoxification column and then diluted to remove compounds toxic to the virus and the porcine kidney cell line. The filtrate was serially diluted and inoculated onto cell culture. The infectivity of PCV2 was determined by an immunoperoxidase monolayer assay. No reduction in PCV2 titre was demonstrated with iodine and phenolic products. Significant PCV2 titre reductions (⩾1.61 log 10) were noted for the seven other products. For five disinfectants, namely a product composed of potassium monopersulfate, two products comprising a quaternary ammonium with one or three aldehyde(s), sodium hypochlorite, and sodium hydroxide, the concentration that significantly reduced the PCV2 titre was equal or 1.5–4 times lower than the authorised use concentration. Only two disinfectants, one composed of potassium monopersulfate, the other containing peracetic acid with hydrogen peroxide, reduced the PCV2 titre with a product concentration at best equal or two times higher than the authorised use concentration.

TGF-β signaling and its effect on glutaminase expression in LLC-PK1-FBPase+cells

During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK(1)-FBPase+ cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a two- to threefold increase in GA mRNA when transferred to acidic medium (pH 6.9). Transforming growth factor-beta (TGF-beta), a potent activator of MAPK and Smad signaling cascades, also causes extensive renal hypertrophy. Thus the potential role of TGF-beta in the renal response to metabolic acidosis was investigated. Western blot analyses established that in LLC-PK(1)-FBPase+ cells, TGF-beta activated the ERK1/2, p38 MAPK, and Smad1/5/8 pathways, but not the JNK and Smad2/3 pathways. Addition of TGF-beta to cells cultured in normal medium (pH 7.4) produced a steady increase in GA mRNA, resulting in a twofold induction after 18 h. Western blot analysis indicated that treatment with either TGF-beta or acidic medium resulted in an increased level of fibronectin. However, the effects of the two treatments on both GA mRNA and fibronectin levels occurred with different time courses and were additive. In addition, the rates of ammonia production were decreased slightly by addition of TGF-beta. Finally, a GA-luciferase reporter construct, which is activated 3.5-fold by treatment with acidic medium, is not affected by TGF-beta. Therefore, TGF-beta and metabolic acidosis activate some of the same signaling pathways in LLC-PK(1)-FBPase+ cells, but produce separate effects on GA expression.

Synergistic inhibition of pseudorabies virus replication by porcine alpha/beta interferon and gamma interferon in vitro.

Interferon (IFN) is crucial for initiating the innate immune response and for the generation of the adaptive response. IFN, in most species, comprises IFN-alpha (IFN-alpha), IFN-beta (IFN-beta) and IFN-gamma (IFN-gamma). In this study, we compared the capacity of porcine IFN-alpha, -beta and -gamma, or a combination of them, to protect IBRS-2 cells (porcine kidney cells) from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-beta (PoIFN-beta) was the most efficient of the three IFNs in conferring resistance PRV infection; 100 U/mL PoIFN-beta inhibited PRV plaque formation 5.3-fold. Compared with PoIFN-beta, porcine IFN-gamma (PoIFN-gamma) was less capable of inhibiting PRV plaque formation (3.3-fold inhibition). Porcine IFN-alpha (PoIFN-alpha) had the least capability of the three PoIFNs, and inhibited PRV plaque formation only 1.26-fold. The inhibitory capacity increased to only 2.3-fold with a treatment of 12,800 U/mL PoIFN-alpha. A combination of PoIFN-gamma and PoIFN-alpha or PoIFN-beta inhibited PRV plaque formation 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-alpha/beta and PoIFN-gamma inhibited PRV replication 29- or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early (IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-alpha/beta and PoIFN-gamma (23.8-133.0-fold) compared with vehicle-treated cells. All the findings indicate that PoIFN-gamma acts synergistically with other PoIFNs (PoIFN-alpha and -beta) to potently inhibit PRV replication in vitro.

Construction and characterization of porcine circovirus type 2 carrying a genetic marker strain

Porcine circovirus type 2 (PCV2) is the primary causative agent of post-weaning multisystemic wasting syndrome in pigs. To generate a genetic marker strain of PCV2, the full-length genome of the virus was amplified using PCR, and two copies of the genome were ligated in tandem to construct an infectious molecular clone. A Sal I restriction enzyme site was inserted into the clone as a genetic marker, and the recombinant plasmid was transfected into porcine kidney cells to generate mutant virus. The antigenicity of the recovered virus was confirmed by immunoperoxidase monolayer assay. The viral antigen was visualized in the nucleus and cytoplasm of the virus-infected cells. The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). The mutant virus was stable on multiplication through 60 passages in cell culture; the highest titer reached was 10 6.6 TCID 50/ml. Four 35-day-old unvaccinated piglets were inoculated with the virus by the intranasal and intravenous routes. Two of the virus-infected pigs developed high temperatures, progressive weight loss, and swollen lymph nodes. The viral antigen and nucleic acid were detected in numerous tissues of the pigs. The results indicate that the genetic marker strain should be a useful tool in studies on pathogenesis, vaccination, and molecular diagnosis of PCV2.

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.

Nuclear receptor 4A2 and C/EBPβ regulate the parathyroid hormone-mediated transcriptional regulation of the 25-hydroxyvitamin D3-1α-hydroxylase

1α-Hydroxylase is the enzyme responsible for the production of the active form of vitamin D, 1,25-dihydroxyvitamin D3. 1α-Hydroxylase, found largely in the kidney, is known to be up-regulated by parathyroid hormone (PTH), however the mechanism of action of PTH and any required transcription factors have not been clearly identified. During gene array analysis we observed that NR4A2, a nuclear orphan receptor, is markedly up-regulated in a porcine kidney cell line (AOK-B50) following PTH stimulation. NR4A2 over-expression increases the endogenous induction of 1α-hydroxylase mRNA in the absence of PTH, however optimal stimulation is achieved when both NR4A2 and PTH are present. An unconventional site of action of NR4A2 was localized to a fragment comprising the sequence from −35/+22 of the 1α-hydroxylase promoter at a C/EBP consensus site. Study of the involvement of C/EBPβ in the 1α-hydroxylase regulation revealed that the transcriptional enhancement by NR4A2 on the 1α-hydroxylase promoter is inhibited by C/EBPβ. In addition, C/EBPβ over-expression decreases the endogenous levels of both NR4A2 and 1α-hydroxylase mRNA.

Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors

A potential risk in pig-to-human xenotransplantation is the transmission of PERVs to human recipients. Here we show for the first time the inhibition of PERV expression in primary porcine cells by RNA interference using lentiviral vectors. Cells were transduced with lentiviral vectors coding for short hairpin (sh) RNAs directed against PERV. In all primary porcine cells studied and in the porcine kidney cell line PK-15, expression of PERV-mRNA was significantly reduced as measured by real-time PCR. Most importantly, expression of PERV proteins was almost completely suppressed, as shown by Western blot analysis. Thus, lentiviral shRNA vectors could be used to knockdown PERV expression and create transgenic pigs with a reduced risk of PERV transmission during xenotransplantation.

Effects of 3-methoxy-2(5H)-furanone-containing extracts from Narthecium ossifragum (L.) Huds. on renal tubular cells in vitro

Narthecium ossifragum, a perennial herb of the lily family, causes toxic renal tubular necrosis in several ruminant species. 3-methoxy-2(5H)-furanone (3M2F) has been identified as a nephrotoxin present in N. ossifragum extracts. We studied effects of three different 3M2F-containing fractions isolated from N. ossifragum and synthetic 3M2F on the porcine kidney cell line LLC-PK1. In some of the experiments, we included the glioma cell lines U251 and BT4Cn to compare the effects of the toxin on LLC-PK1 cells to the effect on these cell lines. The synthetic 3M2F was shown to be only mildly toxic, and the most purified fraction from N. ossifragum showed the highest degree of toxicity in our studies. When monolayer cultures were exposed to increasing amounts of 3M2F-containing extract, a dose-dependent increase in cell death was observed. Similarly, reduced neutral red uptake and 3H-thymidine uptake (DNA synthesis) was observed. There was increased apoptotic activity in the LLC-PK1 cells with increasing concentration of 3M2F-containing extract. Multicellular three-dimensional spheroids from LLC-PK1 cells stopped fluid transport, showed degenerative changes and collapsed totally 6 h after extract exposure. Our findings indicate junctional damage, reduced cellular endocytosis and DNA-synthesis as well as induction of apoptosis as possible mechanisms for the acute tubular necrosis observed in ruminant species.

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

Antiviral Activity of Antimicrobial Lipopeptide from Bacillus subtilis fmbj Against Pseudorabies Virus, Porcine Parvovirus, Newcastle Disease Virus and Infectious Bursal Disease Virus in Vitro

Bacillus subtilis fmbj can produce lipopeptide antimicrobial substance, whose main components were surfactin and fengycin. In the study, the antiviral activity of antimicrobial lipopeptides (AMLs) from B. subtilis fmbj (CGMCC No. 0934) against Pseudorabies Virus (PRV), Porcine Parvovirus (PPV), Newcastle Disease Virus (NDV) and Infectious Bursal Disease Virus (IBDV) was evaluated in vitro. The AMLs represented a direct inactivation effect on cell-free virus stocks of PRV, PPV, NDV and IBDV, and it could effectively inhibit infection and replication of the NDV and IBDV, but failed to affect PRV and PPV. The AMLs were represented higher toxicity for the Porcine Kidney (PK-15) cells (50% cytotoxic concentration (CC50) value was 32.87 μM) and lower for the Chicken Embryo Fibroblasts (CEF) cells (CC50 value was 89.16 μM). The Selectivity index of AMLs on PRV, PPV, NDV and IBDV was 1.44, 2.23, 8.40 and 12.19, respectively.

Isolation and partial characterization of a novel porcine astrovirus

Astroviral infection has been described as one of the causes of porcine diarrhoeal disease. Here we describe the detection of astrovirus-like particles by electron microscopy in a diarrhoeal specimen. Furthermore, a cytopathic virus was isolated and propagated in an established porcine kidney cell line, PK-15. Reverse transcription and PCR performed with astrovirus-specific primers amplified a product with the expected size. Sequencing of the PCR product revealed that the virus observed by electron microscopy and propagated in the porcine cell line is an astrovirus, showing 86% identity at the nucleotide level with the only known porcine astrovirus, PAstV. Phylogenetic analysis clustered the novel isolate, Sb4685, together with PAstV in a broad clade comprising mammalian astroviruses.

Classical swine fever virus replicon particles lacking the Ernsgene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK- stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Infectivity of Porcine Circovirus 1 and Circovirus 2 in Primary Porcine Hepatocyte and Kidney Cell Cultures

Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome.

Ubiquitin-Dependent Degradation of p53 Protein Despite Phosphorylation at Its N Terminus by Acetaminophen

We previously reported that acetaminophen (APAP, 4-hydroxyacetanilide) caused apoptosis of C6 glioma cells. Therefore, we hypothesized that the level of p53, which usually stimulates apoptosis, might be increased after APAP exposure. However, APAP exposure for 24 h markedly decreased the p53 content and its downstream target p21 in a concentration-dependent manner. Reduction of p53 was not accompanied by a decrease in p53 mRNA in C6 glioma cells, suggesting that p53 was mainly affected at the protein level. Unexpectedly, APAP stimulated phosphorylation of p53 at Ser15, Ser20, and Ser37, which usually elevates p53 content. However, phosphorylation of these residues did not prevent APAP-induced decrease in p53. The p53 reduction was independent from the level of phospho-Akt, which is known to promote p53 degradation. Immunoblot analysis of the immunoprecipitated p53 revealed that increased amounts of murine double minute 2 (mdm2) and ubiquitin were bound to p53 during its degradation. Lactacystin and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), inhibitors of proteasomal proteolysis, prevented the decrease, supporting the proteasomal degradation of p53 upon APAP exposure. Pretreatment with chlormethiazole, an inhibitor of ethanol-inducible CYP2E1, significantly lowered the CYP2E1 enzyme activity and the rate of APAP-induced cell death while it prevented the reduction of p53 and p21 in C6 glioma cells. A nontoxic analog of APAP, 3-hydroxyacetanilde, did not reduce p53 and p21 contents in C6 glioma cells and LLC-PK1 porcine kidney cells. Taken together, our results show that APAP or its reactive metabolite(s) can directly reduce the p53 content through mdm2-mediated ubiquitin conjugation, despite phosphorylation of p53 at its N terminus.