p53 Plays an Important Role in Cell Fate Determination after Exposure to Microcystin-LR

Background Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear. Objective We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure. Methods We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-α, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3β. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-α and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR. Conclusions This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.


Research
Microcystin-LR is a cyclic peptide released by several bloom-forming toxic cyanobacteria (Carmichael and Falconer 1993). Understanding the toxicity of microcystin-LR is of paramount importance because of both the potency of its acute cyto toxicity and its tumor-promoting activity in hepato cytes of animals and humans (Dietrich and Hoeger 2005;Gehringer 2004). Microcystin-LR toxicity is primarily caused by inhibition of the serine/threonine protein phosphatases PP1 and PP2A, thereby influencing the regulation of the balance between cellular protein phosphorylation and dephosphorylation (Dietrich and Hoeger 2005;Gehringer 2004). Several PP1 and PP2A inhibitors, such as okadaic acid, nodularin, and microcystin-LR, are classified as tumor promoters (Fujiki and Suganuma 1999). Several epidemiological studies have indicated that consumption of cyanobacteria-contaminated water can favor the growth of human hepato cellular carcinoma (Falconer and Humpage 2005;Ueno et al. 1996;Yuan et al. 2006). Microcystin-LR has poor cell membrane permeability except for the membranes of hepato cytes (Chong et al. 2000;Eriksson et al. 1990;Nong et al. 2007;Runnegar et al. 1995). Recently, we ) and others (Fischer et al. 2005;Monks et al. 2007) reported that the expression of the human hepato cyte uptake transporter OATP1B1 or OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. However, the intra cellular regulatory mechanisms that determine whether a cell will die or survive in response to microcystin-LR have not been clarified.
The molecular basis of the tumor-promoting ability of microcystin-LR is unclear but most likely involves protein phosphatase inhibition leading to hyperphosphorylation of many cellu lar proteins and, consequently, destruction of cell-cycle control (Fujiki and Suganuma 1999;Guy et al. 1992;Messner et al. 2006). Several signaling pathways have been implicated in cellu lar effects of microcystin-LR. Previous studies have demonstrated that microcystin-LR can stabilize Xenopus and zebrafish β-catenin protein levels by suppressing glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase that phosphorylates β-catenin, resulting in its proteasomal degradation (Li et al. 2001;Wang et al. 2005). β-Catenin is a multi functional protein that plays an important role in the transduction of wingless int (Wnt) signals, which contributes to hyperplasia and tumorigenic progression, and in cellular adhesion by linking the cytoplasmic domains of cadherin to each other (Grimes and Jope 2001;Olmeda et al. 2003;Orford et al. 1999;Wang et al. 2005). In general, a low cyto plasmic level of β-catenin is maintained through inter action with a protein complex consisting of adenomatous polyposis coli, Axin, PP2A, and GSK-3β (Ding et al. 2000). Recently, p53 has been reported to induce proteasomal degradation of β-catenin through the transactivation of seven in absentia homolog 1 (siah-1), which encodes an E3 ubiquitin ligase (Jang et al. 2005;Jung et al. 2007;Kim et al. 2004;Park et al. 2006). Accumulation of β-catenin can induce the expression of various genes, such as cyclin D1 and c-myc, which promote cell prolifera tion and transformation of many cell types (Seki et al. 2006). Phosphorylation of GSK-3β at Ser 9 , which results in an inactive form of GSK-3β, is mediated by activated Akt, which is a serine/threonine protein kinase that is a well-established anti apoptotic protein (Frame and Cohen 2001;Grimes and Jope 2001). Akt regulates many cellular processes implicated in tumori genesis (Altomare and Testa 2005;Lu et al. 2007). Recently, constitutive activation of Akt was reported in microcystin-transformed immortalized colorectal crypt cells (Zhu et al. 2005). We also recently reported that microcystin-LR induces apoptosis through activation of mitogenactivated protein kinase (MAPK) . Moreover, there is increasing evidence of a functional relationship between p53 and MAPK. Thus, MAPK that is phosphorylated after exposure to cellular stress then phosphorylates p53 and, consequently, induces apoptosis (Guan et al. 2008;Karunakaran et al. 2008;Liu et al. 2009;Shimada et al. 2003).
p53 functions as a transcription factor that, in response to a variety of cellu lar stresses, including DNA damage and hypoxia, regulates its downstream genes and p53-mediated pathways in an attempt to repair DNA damage through induction of cell-cycle arrest. p53 is involved in G 1 and G 2 cell-cycle arrest through activation of the p21 cyclin-dependent kinase inhibitor (Burns and El-Deiry 1999).
In the present study, we used wild-type p53-expressing HEK293 human embryonic kidney cells stably transfected with OATP1B3, to demon strate that a lethal concentration of microcystin-LR stimulated not only cellular signal transduction, leading to cell death, but also cell survival signals. These data suggest that if cell damage inflicted by microcystin-LR cannot be repaired, then p53 probably promotes cell apoptosis. However, if p53 is inactivated, chronic exposure to a low dose of microcystin-LR might lead to cell proliferation through activation of Akt signaling.

Reagents and antibodies.
Reagents and antibodies used are described in the Supplemental Material (doi:10.1289/ehp.1001899).
Preparation of nuclear fractions. Cells were resuspended in buffer A (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 1.5 mM MgCl 2 , 60 µM phenylmethylsulfonyl fluoride, and a proteinase inhibitor cocktail) at 4°C, incubated for 20 min on ice, and homogenized for 30 sec. The homogenate was centrifuged at 3,000 × g for 5 min, and the post nuclear supernatant was clarified by centrifugation for 30 min at 15,000 × g. The first pellet was washed twice with buffer A and resuspended in buffer A containing 1% Nonidet P-40, and this suspension was homogenized for 30 sec. Homogenates were collected by centrifugation for 30 min at 15,000 × g, and supernatants were collected as the nuclear fraction. Prepared nuclear fractions were analyzed by immunoblotting as previously described (Komatsu et al. 2000. For details of immuno blotting, see Supplemental Material (doi:10.1289/ehp.1001899).
Immunoprecipitation. After incubation with microcystin-LR, cells were lysed with Lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM KCl, 1 mM EDTA, 1% Nonidet P-40, 1% protein inhibitor cocktail,and 1 mM NaF). Following homogenization, the lysates were centrifuged at 10,000 × g for 30 min at 4°C. One milliliter of cell lysate was incubated overnight at 4°C with 5 µL of agarose-conjugated anti-p53 antibody. The pellet was washed four times with Lysis buffer and then suspended in SDS-polyacrylamide gel Laemmli sample buffer. After SDS/PAGE and immunoblotting with the respective phospho-p53 antibodies, phosphorylation of p53 at Ser 6 , Ser 9 , Ser 15 , Ser 20 , Ser 37 , Ser 46 , and Ser 392 was analyzed in the same samples. After stripping with stripping buffer (0.5 M Tris-Cl, pH 6.8 containing 1% 2-ME) for 30 min at 50˚C, the blots were reprobed with an anti-p53 antibody.
Detection of ubiquitination. HEK293-OATP1B3 cells were treated with 50 nM microcystin-LR for 12 hr under serum-free conditions. The cells were treated with 10 µM lactacystin for 2 hr before cell harvest to inhibit proteasomal degradation of β-catenin. Whole-cell lysates from harvested cells were then analyzed by immuno blot analy sis.
Knockdown of p53 mRNA. HEK293-OATP1B3 cells were transiently transfected with the small hairpin RNA (shRNA) plasmid pSuppressorNeo p53 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol to generate p53 small inter fering RNA (siRNA). Cells were then incubated for 72 hr. To determine p53 expression by immuno blotting, 4 × 10 6 cells in 10 mL MEM/10% FCS were inoculated into 100-mm dishes. After 24 hr, the cells were harvested and the cell lysates were analyzed. For MTT analysis, exponentially growing transfected HEK293-OATP1B3 cells were trypsinized and harvested, and equal numbers of cells (1.6 × 10 4 ) in 180 µL MEM/10% FCS were then inoculated into each well of a 96-well micro plate and assayed using the MTT assay.
Statistical analysis. Differences between groups were analyzed using Wilcoxon-Mann-Whitney test. p-Values < 0.05 were considered significant.  To determine if p53 is involved in microcystin-LR-induced apoptosis, we examined p53 protein stability and its phosphorylation in HEK293-OATP1B3 cells by immunoblot analysis (Figures 1 and 2). The level of p53 protein increased after 3.5 hr of exposure to a lethal concentration (50 nM) of micro cystin-LR (Figure 1). After accumulation of p53, the expression level of protein product of its transcriptional target gene p21 also increased (Figure 1). After 3.5-5 hr, we observed phosphorylation of p53 at Ser 15 , which reduces the ability of p53 to bind to its negative regulator, the oncoprotein MDM2, and at Ser 392 , which is increased in human tumors. In both cases phosphorylation coincided with the accumulation of p53 protein ( Figure 2). After these early phosphorylation events, we observed delayed phosphorylation of p53 at Ser 37 , which impairs the ability of MDM2 to bind p53, thus promoting both the accumulation and activation of p53 in response to DNA damage, and at Ser 46 , which is important in regulating the ability of p53 to induce apoptosis. Phosphorylation was slightly detectable at these sites after 3.5 and 5 hr but was considerably stronger after 8-10 hr of exposure to 50 nM microcystin-LR ( Figure 2). In addition, we observed weak phosphorylation of p53 at Ser 6 and Ser 9 , which are mediated by the casein kinases CK1δ and CK1ε, and at Ser 20 , which is induced by DNA damage and leads to reduced interaction between p53 and MDM2, as reflected by accumulation of p53 protein ( Figure 2).

Activation of the Akt pathway by microcystin-LR.
Substrates of PP2A, a target of microcystin-LR, include components of the well-described Akt cell survival signaling pathway. We therefore analyzed the effect of microcystin-LR on the activation of such components, including 3-phospho inositidedependent protein kinase 1 (PDK1), Akt, and GSK-3β. The intensity of PDK1 phosphorylation at Ser 241 , which correlates with PDK1 activity, was slightly and transiently increased 3-6 hr after exposure to microcystin-LR and recovered to control levels after 10 hr. In contrast, phosphorylation of Akt at the activating phosphorylation sites Ser 473 and Thr 308 was considerably enhanced by exposure of the cells to 50 nM micro cystin-LR. However, the expression level of Akt in HEK293-OATP1B3 cells was not changed by treatment with 50 nM micro cystin-LR for up to 10 hr ( Figure 3).
In HEK293-OATP1B3 cells, phosphorylation of GSK-3β increased in a timedependent manner after incubation with 50 nM microcystin-LR for up to 10 hr. In contrast, the expression level of GSK-3β did not change ( Figure 4A). These data suggest that microcystin-LR indirectly regulates the activity of both Akt and GSK-3β.
Correlation of β-catenin with microcystin-LR cytotoxicity. We next determined if microcystin-LR-induced phosphorylation of GSK-3β led to an enhanced nuclear translocation of its substrate protein β-catenin. Surprisingly, the level of β-catenin in the nuclear fraction of cells after up to 24 hr incubation with 50 nM micro cystin-LR indicated that β-catenin levels in the nucleus were equivalent before and after treatment ( Figure 4B). In addition, we detected ubiquitina tion of β-catenin in the presence of 10 µM proteasome inhibitor lactacystin after treatment with micro cystin-LR ( Figure 4C).
Analysis of cyclin D1 and c-myc mRNA expression. To confirm that β-catenin levels in the nucleus were not increased after microcystin-LR treatment, we analyzed the mRNA    levels of the β-catenin targets cyclin D1 and c-myc after treatment with 50 nM microcystin-LR. The mRNA level of cyclin D1 was not changed after microcystin-LR treatment, but, unexpectedly, that of c-myc was considerably up-regulated ( Figure 5A), suggesting that microcystin-LR activated c-myc through signaling pathways other than β-catenin.

Induction of siah-1 by microcystin-LR.
The lack of an increase in nuclear levels of β-catenin despite the fact that GSK-3β was phosphorylated after microcystin-LR treatment suggested that β-catenin might be ubiquitinated and degraded through another pathway that is activated by microcystin-LR. β-Catenin can be degraded after p53-dependent up-regulation of the E3 ubiquitin ligase gene siah-1 and its protein product. We therefore determined whether siah-1 mRNA levels were modulated after treatment of cells with 50 nM microcystin-LR. Real-time RT-PCR analysis indicated that microcystin-LR treatment led to the up-regulation of siah-1 mRNA ( Figure 5B), suggesting that siah-1--mediated degradation of β-catenin may be responsible for the lack of nuclear accumulation of β-catenin.

Effects of p53 on the cytotoxicity of microcystin-LR.
To verify whether p53 was critically important for microcystin-LR-induced apoptosis, we analyzed the effect of pretreatment with an inhibitor of the transcription factor activity of p53, pifithrin-α, for 5 hr on the cytotoxicity of micro cystin-LR in HEK293-OATP1B3 cells. Pifithrin-α concentrationdependently attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR compared with vehicle (6%DMSO)-treated (control) cells ( Figure 6). The median inhibitory concentrations (IC 50 ) were approximately 2 and 8 times higher in cells that were pretreated with 10 µM and 30 µM pifithrin-α, respectively, than in controls. We further analyzed the acute cyto toxicity of microcystin-LR in HEK293-OATP1B3 cells after knockdown of p53 following transfection with a p53 shRNA plasmid. This plasmid effectively decreased the expression level of p53 protein ( Figure 7A). The susceptibility of HEK293-OATP1B3 cells to microcystin-LR was attenuated to a greater extent by knockdown of p53 than by transfection of cells with a plasmid encoding a scrambled shRNA sequence, which does not show significant homology to rat, mouse, or human gene sequences ( Figure 7B).

Discussion
Microcystin-LR is of great interest because of its acute cytotoxicity and its tumor-promoting activity in hepato cytes of animals and humans (Gehringer 2004;Li et al. 2009;Wang et al. 2005). In our previous studies aimed at clarifying the onset of liver-specific toxicity of microcystin-LR Nong et al. 2007), we demonstrated that transfection of OATP1B1 (SLCO1B1) or OATP1B3 (SLCO1B3) cDNA into HEK293 cells increased their sensitivity to, and their accumulation of, microcystin-LR and, consequently, induced apoptosis in those cells. The use of these cell lines permits exclusive analysis of the effects of low concentrations of microcystin-LR on the cellular balance between phosphatase and kinase activity, which is a crucial regulatory mechanism for cellular homeostasis. PP2A is one of the key proteins in this regulatory mechanism (Junttila et al. 2008;Lin et al. 2007;Yan et al. 1997) and is known to be a target molecule of micro cystin-LR (Gehringer 2004). However, because of the poor membrane permeability of microcystin-LR, a complete understanding of the effect of low concentrations of microcystin-LR on various cell signaling pathways remains unclear. Here, we used HEK293-OATP1B3 cells to clarify the molecular basis of the balance between cell death and cell survival after microcystin-LR treatment. In this study, we focused on the effects of low concentrations of microcystin-LR on the phosphorylation of p53 and on Akt signaling pathways.
In our previous study using HEK293-OATP1B3 cells, microcystin-LR activated MAPKs, including ERK1/2 (extra cellular signal-regulated kinase 1/2), JNK (c-Jun N-terminal kinase), and p38, through inhibition of PP2A ). In the present study, we demonstrated that once MAPKs are activated by micro cystin-LR, they activate downstream kinases that phosphorylate a number of substrates, including p53, which plays a central role in the cellul ar response to various stresses (Wu 2004). Treatment with 50 nM microcystin-LR for  -1 (B) were quantified by realtime RT-PCR, and the mRNA expression level of each sample was calculated relative to that of GAPDH, which was used as an internal control. The data represent mean ± SD of three independent experiments, each performed in triplicate. *p < 0.05, and **p < 0.01, compared with control before incubation with microcystin-LR. 10 hr induced phosphorylation of p53 at Ser 15 , Ser 37 , Ser 46 , and Ser 392 . In contrast, Ser 6 , Ser 9 , and Ser 20 were weakly phosphorylated ( Figure 2). The site at which p53 is phosphorylated is dependent on the kind of stress applied or the cell type assayed (Kurihara et al. 2007;Wu 2004). Phosphorylation of p53 at Ser 15 promotes p53 stabilization by attenuating the inter action between p53 and its negative regulator MDM2. The resulting accumulation of phosphorylated p53 resulted in increased expression of p21 and siah-1 (Figures 1 and 5B). The p21 gene is a transcriptional target of p53 and induces inactivation of the cyclin-dependent kinase complex CDK2/cyclin E that controls the initiation of DNA synthesis (Hegarat et al. 2005). Therefore, elevated levels of p53 and p21 after treatment with micro ystin-LR are probably responsible for induction of cellcycle arrest. These results are consistent with our previously reported flow cytometric analysis, which revealed that the G 1 phase, and the sub-G 1 proportion of HEK293-OATP1B3 cells, increased after exposure to 50 nM microcystin-LR for 24 hr ). Furthermore, the phosphorylation of p53 at Ser 46 correlates well with the induction of apoptosis (Smith et al. 2003;Yoshida et al. 2006). Therefore, these events ultimately induce apoptosis of HEK293-OATP1B3 cells after exposure to microcystin-LR.
Recently, crosstalk between p53 and Akt has been hypothesized to play a critical role in the regulation of cell fate determination (Oren 2003), and the Akt signaling pathway plays an important role in promoting cell proliferation (Lu et al. 2007). Zhu et al. (2005) reported that transformation of immortalized colo rectal crypt cells by microcystin is involved in the constitutive activation of Akt and MAPK. In the present study using HEK293-OATP1B3 cells, phosphorylation of Akt at Ser 473 was enhanced after 3 hr incubation with 50 nM microcystin-LR (Figure 3). Subsequently, phosphorylation of Akt at Thr 308 was gradually enhanced to reach a high level of phosphorylation ( Figure 3). Therefore, microcystin-LR treatment may activate Akt to phosphorylate its downstream targets, such as GSK-3β kinase (Frame and Cohen 2001). Indeed, microcystin-LR induced the phosphorylation of GSK-3β at Ser 9 ( Figure 4A), resulting in a loss of GSK-3β kinase activity. Phosphorylation of Akt is regulated by the balance between the activities of PP2A and PDK1, which are upstream of Akt activation (Ivaska et al. 2002;Li et al. 2003). However, analysis of the phosphorylation level of PDK1 indicated that it was slightly and transiently increased and subsequently recovered to control levels after treatment with microcystin-LR for 10 hr, suggesting that PDK1 may not play an important role in microcystin-LR-activation of Akt (Figure 3). In a previous study using unstimulated HEK293 cells, Casamayor et al. (1999) found that PDK1 was strongly phosphorylated at Ser 241 , which is critical for PDK1 activity, and that the level of PDK1 phosphorylation at this site was unaffected by stimulation with insulin-like growth factor-1. Hence, the phosphorylation of Akt observed in the present study is likely due to PP2A inhibition by microcystin-LR.
Because microcystin-LR can activate Akt to phosphorylate and thereby inhibit GSK-3β, we next analyzed the effect of incubation of HEK293-OATP1B3 cells with 50 nM microcystin-LR on the translocation of the GSK-3β substrate β-catenin from the cytoplasm to the nucleus. Inhibition of GSK-3βmediated phosphorylation of β-catenin plays a role in the translocation of β-catenin to the nucleus (Ding et al. 2000;Grimes and Jope 2001). We therefore predicted that β-catenin might accumulate in the nucleus of HEK293-OATP1B3 cells after incubation with 50 nM microcystin-LR. However, under these conditions, nuclear accumulation of β-catenin was negligible ( Figure 4B). In agreement with this finding, the transcription of cyclin D1-one of the transcriptional targets of β-catenin and a key regulator of cell-cycle progression (Alt et al. 2000;Diehl et al. 1998;Seki et al. 2006)-was not enhanced by microcystin-LR ( Figure 5A). We further analyzed the effect of microcystin-LR on the mRNA level of c-myc, which is also a well-known transcriptional target of β-catenin. In contrast to the lack of effect of micro cystin-LR on cyclin D1 expression levels, microcystin-LR strongly up-regulated c-myc mRNA ( Figure 5A). A significant increase of c-myc may play a role in cellular accumulation of p53 (Matsumura et al. 2003). However, the c-myc promoter is targeted not only by β-catenin but also by other transcription factors under the control of multiple signal transduction cascades, including the MAPK cascade (Vervoorts et al. 2006). Therefore, analysis of the mechanism by which c-myc transcription is enhanced remains to be investigated.
Nuclear accumulation of β-catenin is highly related to tumor promotion (Cui et al. 2001(Cui et al. , 2003, and PP2A activity also correlates with β-catenin degradation (Bos et al. 2006;Li et al. 2001). The nuclear dephosphorylated β-catenin level is decreased after treatment with okadaic acid compared with control without okadaic acid (Bordonaro et al. 2007). To resolve the lack of correlation between the inactiva tion of GSK-3β and the cellular β-catenin level after exposure to microcystin-LR, we considered the possibility that β-catenin might be degraded via the p53-siah-1 pathway after micro cystin-LR treatment. Indeed, we detected ubiquitination of β-catenin after treatment with microcystin-LR ( Figure 4C). According to previous studies, stabilization of p53 can Figure 8. Model of the p53-Akt cross-regulatory network that mediates the cellular response to microcystin-LR stress in HEK293-OATP1B3 cells. Microcystin-LR simultaneously induces activation of both Akt and p53 signaling. However, p53 signaling is more active than Akt signaling in HEK293-OATP1B3 cells after treatment with 50 nM microcystin-LR. As a result, p53 induces arrest of the cell cycle and apoptosis by the induction of several genes, including p21 and siah-1. Abbreviations: P, phosphorylation; Ub, ubiquitination. Dashed lines indicate proposed pathways; small arrows, transport; large arrows, activation; blunt-end lines (⊥), inhibition. Up (↑) and down (↓) arrows adjacent to protein names indicate activation and inactivation, respectively. Proteins shaded gray were analyzed in the present study and/or in our previous studies  induce GSK-3β-independent proteasomal degradation of β-catenin through the transactivation of the E3 ubiquitin ligase siah-1, which is a transcriptional target gene of p53 (Jang et al. 2005;Jung et al. 2007;Kim et al. 2004;Park et al. 2006). Real-time RT-PCR analysis indicated that siah-1 mRNA significantly increased in a time-dependent manner after exposure to microcystin-LR and that this increase coincided with the accumulation of p53 ( Figure 5B). These results suggest that the p53-siah-1 pathway may play an important role in preventing microcystin-LR-induced β-catenin accumulation and may therefore block cell-cycle progression in HEK293-OATP1B3 cells. In this scenario, if p53 were to be inactivated by, for example, a mutagen such as aflatoxin B1 (Szymańska et al. 2009) or by oxidative stress produced by microcystin-LR itself , chronic exposure to low-dose micro cystin-LR might lead to cell proliferation through activation of Akt signaling.
To verify that p53 is critically related to microcystin-LR-induced apoptosis, we analyzed the cytotoxicity of microcystin-LR in HEK293-OATP1B3 cells in which p53 was inhibited or down-regulated. Both pharmacological inhibition of transcription factor activity of p53 ( Figure 6) and knockdown of p53 with shRNA ( Figure 7) attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR, suggesting that p53, at least in part, is associated with microcystin-LRinduced apoptosis. p53 promotes apoptosis mediated by the mitochondrial pathway (Green and Kroemer 2009). However, caspase-3 is not involved in the apoptosis that occurs after exposure to microcystin-LR (Ding et al. 2002;Komatsu et al. 2007). Further studies are needed to clarify the contribution of p53 to cell fate determination after exposure of HEK293-OATP1B3 cells to microcystin-LR.

Conclusion
The present study has demon strated that HEK293-OATP1B3 cells are a useful tool for analysis of the cellu lar effects of low concentration of micro cystin-LR. We revealed that the fate of HEK293-OATP1B3 cells is determined through activation of various cell signaling pathways, including MAPK pathways, especially the JNK pathway ), as well as through Akt and p53 signaling pathways. We have also shown the importance of p53 in microcystin-LRinduced apoptosis (Figure 8). Our results suggest that conditions of inactivated p53, coupled with chronic exposure to low-dose microcystin-LR, may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.