ToxSci Advance Access originally published online on April 26, 2006
Toxicological Sciences 2006 92(1):78-86; doi:10.1093/toxsci/kfj213
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Ochratoxin A: Apoptosis and Aberrant Exit from Mitosis due to Perturbation of Microtubule Dynamics?
* Department of Toxicology, University of Würzburg, 97078 Würzburg, Germany; and Institute of Applied Biosciences, Section of Food Chemistry and Toxicology, University of Karlsruhe, 76128 Karlsruhe, Germany
1 To whom correspondence should be addressed at Department of Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany. Fax: +49-931-20148865. E-mail: dekant{at}toxi.uni-wuerzburg.de.
Received March 3, 2006; accepted April 13, 2006
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ABSTRACT |
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Ochratoxin A (OTA) is a potent nephrotoxin and causes high incidences of renal tumors in rodents. The molecular events leading to tumor formation by OTA are not well defined. Early pathological changes observed in kidneys of rats treated with OTA in vivo include frequent mitotic and abnormally enlarged cells, detachment of tubule cells, and apoptosis within the S3 segment of the proximal tubule, suggesting that OTA may interfere with molecules involved in the regulation of cell division and apoptosis. In this study, treatment of immortalized human kidney epithelial (IHKE) cells with OTA (0–50µM) resulted in a time- and dose-dependent increase in apoptosis and activation of c-Jun N-terminal kinase. At the same time, OTA blocked metaphase/anaphase transition and led to the formation of aberrant mitotic figures and giant cells with abnormally enlarged and/or multiple nuclei, sometimes still connected by chromatin bridges. Immunostaining of the mitotic apparatus using an
-tubulin antibody revealed defects in spindle formation. In addition, OTA inhibited microtubule assembly in a concentration-dependent manner in a cell-free, in vitro assay. Interestingly, treatment with OTA also resulted in activation of the transcription factor nuclear factor kappa B (NF
B), which has recently been shown to promote cell survival during mitotic cell cycle arrest. Based on these observations, we hypothesize that the mechanism by which OTA promotes tumor formation involves interference with microtubuli dynamics and mitotic spindle formation, resulting in apoptosis or—in the presence of survival signals such as stimulation of the NFB pathway—premature exit from mitosis. Aberrant exit from mitosis resulting in blocked or asymmetric cell division may favor the occurrence of cytogenetic abnormalities and may therefore play a critical role in renal tumor formation by OTA. Key Words: ochratoxin A; carcinogenicity; apoptosis; mitosis; microtubule.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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The mycotoxin and food contaminant ochratoxin A (OTA) (N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]-carbonyl}-3-phenyl-L-alanine) is a potent nephrotoxin and causes high incidences of renal tumors in rodents (Boorman et al., 1992
; NTP, 1989). The molecular events leading to tumor formation by OTA are not well defined, and both genotoxic and nongenotoxic mechanisms may be involved. Chemically induced renal tumors in rats frequently occur as a result of direct interaction of the compound or its metabolite with DNA, generation of reactive oxygen species leading to oxidative DNA damage, or sustained regenerative cell proliferation in response to cytotoxicity (either directly or mediated via 2u-globulin accumulation) (Lock and Hard, 2004). However, the mechanism by which OTA functions to induce tumors does not appear to fit into any one of these categories. Several studies using radiolabeled OTA indicate that OTA does not form covalent DNA adducts (Gautier et al., 2001; Gross-Steinmeyer et al., 2002; Mally et al., 2004; Schlatter et al., 1996), and although OTA has been shown to induce DNA strand breaks presumably via oxidative stress, the contribution of these lesions to renal tumor formation remains unclear (Mally et al., 2005b). Furthermore, accumulation of 2u-globulin, sustained cytotoxicity, and compensatory cell proliferation do not play a role in OTA carcinogenicity (JECFA, 2001; Rasonyi et al., 1999).
Kidney tumors in OTA-exposed rats arise from the straight segment of the proximal tubule and are characterized by a relative rapid onset, aggressive behavior, tendency toward an uncommon anaplastic phenotype, and high potential to metastasize (JECFA, 2001; Lock and Hard, 2004). Early histopathological alterations, which occur even after short-term OTA treatment, consist of disorganization of the tubule arrangement and a high incidence of apoptosis and mitosis (Mally et al., 2005c). Abnormally enlarged and aberrant mitotic figures, karyocytomegaly, and increasing polyploidy were frequently observed in kidneys of OTA-exposed animals, indicating that DNA replication occurred in the absence of cytokinesis (Maaroufi et al., 1999; Mally et al., 2005c). These changes were most prominent in tubule cells located in the outer stripe of the outer medulla and appear to be a specific feature of OTA toxicity. Moreover, the presence and severity of nonneoplastic changes were found to correlate with tumor incidence in the 2-year bioassay, suggesting that these early changes may play a critical role in tumor formation mediated by OTA (Boorman et al., 1992). However, the molecular events leading to these histopathological alterations remain unclear.
In vitro, OTA has been shown to induce apoptosis in a variety of cell lines (Gekle et al., 2000; Gennari et al., 2004; Horvath et al., 2002; Kamp et al., 2005; Schwerdt et al., 1999a; Scibelli et al., 2003). Mitogen-activated protein (MAP) kinases, which are involved in a wide variety of cellular events including cell death by apoptosis, have been implicated in OTA toxicity. Results from several studies demonstrate that apoptosis induced by OTA is associated with activation of the stress-activated protein kinase c-Jun N-terminal kinase (JNK) and p38 (Gekle et al., 2000; Horvath et al., 2002; Sauvant et al., 2005a). In addition, treatment of kidney epithelial cells with OTA resulted in activation of the extracellular signal–regulated kinase (ERK) 1/2, which is generally activated in response to mitotic stimuli and is thought to promote cell survival (Horvath et al., 2002; Sauvant et al., 2005a). It has, therefore, been suggested that OTA toxicity may be mediated at least in part by modulation of MAP kinase signaling, yet little is known with respect to the initial damage which triggers activation of these pathways.
JNK is known to be activated in response to a wide variety of stimuli including proinflammatory cytokines, UV radiation, reactive oxygen species, genotoxicity, and cellular stress. Recent evidence indicates that loss of cell anchorage may also provide a strong stimulus for JNK activation, suggesting a role of JNK in detachment-induced apoptosis (Frisch et al., 1996a). Apoptosis induced by loss of cell adhesion has been shown to occur in renal epithelial cells in response to nephrotoxicants in vitro (van de Water et al., 1999) and has been put forward as an interesting hypothesis to explain apoptotic cell death caused by OTA, particularly since detachment of tubule cells from the basement membrane was frequently observed in kidneys of rats treated with OTA in vivo (Mally et al., 2005c; Rasonyi et al., 1999). In COS cells, treatment with OTA resulted in cell detachment and inhibition of focal adhesion kinase (FAK), a central component of integrin-mediated survival signaling, suggesting that disruption of cell adhesion may contribute to JNK activation and apoptosis in response to OTA (Scibelli et al., 2003). However, other prominent changes induced by OTA treatment in vivo such as karyomegaly and aberrant mitosis may not be easily explained by loss of cell adhesion, and the possibility that disruption of cell-matrix interactions may be the consequence rather than the cause of OTA toxicity needs to be considered. In this study, we have utilized a human kidney epithelial cell line derived from the proximal tubule to characterize early effects of OTA on cell division, cell adhesion, and cell death by apoptosis to gain further insights into the mechanism of OTA toxicity and carcinogenicity.
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MATERIALS AND METHODS |
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Chemicals.
Acetone, ethanol, and methanol were purchased from Merck, Darmstadt, Germany. OTA was purchased from Sigma-Aldrich, Taufkirchen, Germany (lot-no. 38H4120) and from Prof. Peter Mantle, Imperial College of Sciences, London, United Kingdom. Purity of OTA was > 99.9% as assessed by HPLC with UV and fluorescence detection. Primary antibodies used were monoclonal mouse anti–phospho-JNK (Thr 183/Tyr 185) (Cell Signaling, Beverly, MA), mouse anti–phospho-FAK (Tyr 397) (BD Biosciences, Heidelberg, Germany), mouse anti–phospho-ERK (Santa Cruz, Heidelberg, Germany), mouse anti–
-tubulin (Sigma, Taufkirchen, Germany), and polyclonal rabbit anti–nuclear factor kappa B (NFB) (p65) (Santa Cruz, Heidelberg, Germany). Unless otherwise indicated, all other chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany).
Cell culture.
Immortalized human kidney epithelial (IHKE) cells (originally obtained from S. Mollerup, National Institute of Occupational Health, Norway) were kindly provided by Dr Evelyn O'Brien, University of Konstanz, Germany. This cell line, established from normal human embryonic kidney epithelial cells by chemical transformation with nickel(II), has been demonstrated to display morphological and physiological characteristics of the proximal tubule (e.g., presence of apical brush border and various transport systems, and gap junction intercellular communication) (Hirsch et al., 1998, 1999; Jessen, 1994; Jessen et al., 1994, 1996; Rivedal et al., 1996) and has previously been used in a number of studies to assess OTA toxicity in vitro (Benesic et al., 2000; Eder et al., 2000; Schwerdt et al., 1999a,b). Cells were cultured under standard cell culture conditions (37°C, 5% CO2) in Dulbeccos's modified Eagle medium (DMEM)/Ham's-F12 (1:1) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Colbe, Germany), 15mM HEPES, 2mM glutamine, 0.1µM hydrocortisone, 5 mg/l human apotransferrin, 5 mg/l bovine insulin, 10 µg/l human epidermal growth factor, 5 µg/l sodium selenite, 2000 U/l penicillin, and 2 mg/l streptomycin. OTA was freshly dissolved in ethanol and diluted in DMEM supplemented with 5% FCS and 2mM glutamine.
Cytotoxicity.
Cells were seeded in 96-well plates at a density of 1 x 104 cells per well, allowed to grow for 24 h in OTA-free medium (
75% confluency), and subsequently incubated with OTA at concentrations between 0 and 100µM for up to 24 h. For the determination of numbers of cells per well, cells were washed in PBS, 50 µl of 0.2% crystal violet in 0.1M citric acid was added to each well, and stained nuclei were counted using a hemocytometer. Release of lactate dehydrogenase (LDH) into the medium was determined using the Cytotox 96 nonradioactive cytotoxicity assay (Promega, Mannheim, Germany) according to the manufacturer's instructions. The MTT-based in vitro toxicology assay kit (Sigma) was used to measure the activity of living cells via mitochondrial dehydrogenases. The general principle of this cytotoxicity assay is the conversion of the tetrazolium salt (MTT) to the colored product formazan, which can be measured photometrically at 570 nm, by intact mitochondria. Each assay was performed in at least two independent experiments carried out in triplicate.
Mitosis and apoptosis.
The frequency of mitosis and apoptosis was determined by morphological evaluation of cells grown on coverslips and stained with 4',6-diamidino-2-phenylindole (DAPI) as described below (immunofluorescence). Cell preparations were analyzed at x63 magnification using a Leitz Diavert inverted fluorescence microscope fitted with a Nikon DXM 1200 digital camera. Apoptotic cells were identified based on chromatin condensation and fragmentation, morphological features characteristic for apoptosis. Mitotic cells were further categorized into prometaphase/metaphase and anaphase/telophase. Cells with irregular and abnormally enlarged nuclei were classified as aberrant cells. Apoptosis was also determined by flow cytometry after Annexin V/propidium iodide (PI) staining as previously described (Mally et al., 2006).
Immunofluorescence.
Cells were seeded at a density of 2.5 x 104 cells per well onto coverslips in 12-well plates and allowed to grow for 2 days until they reached 75% confluency. Following OTA treatment, cells were washed in PBS, fixed in 10% neutral buffered formalin, and permeabilized by treatment with 0.2% Triton X-100 for 5 min. For immunostaining of -tubulin, cells were fixed in methanol at – 20°C. After fixing, cells were washed in PBS, blocked in 10% goat serum in PBS, and subsequently incubated with primary antibody (1:100 to 1:500 in 1% bovine serum albumin in PBS overnight at 4°C) and FITC-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, Alabama) or goat anti-rabbit IgG (Sigma, Taufkirchen, Germany) (1:50 dilution, 1 h at room temperature [RT]). Coverslips were mounted with Vectashield mounting medium containing DAPI (Vector, Burlingame, CA). Duplicate slides were prepared for each treatment.
Western blot.
Cells were seeded into 60-mm cell culture dishes at a density of 3–4 x 105 cells per plate, allowed to grow to subconfluency ( 75%) and exposed to OTA for up to 24 h. To obtain total cell lysates, cultures were washed twice with ice-cold PBS and lysed in 500 µl RIPA buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 1% Nonidet P40, 0.24% Na-deoxycholate, 1mM EDTA, 1mM Na3VO4, 1mM NaF, and 1 µl/ml protease inhibitor cocktail [Sigma, containing 104mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 80µM aprotinin, 2mM leupeptin, 4mM bestatin, 1.5mM pepstatin A, and 1.4mM E-64]). Following incubation on ice for 15 min, cell lysates were centrifuged for 15 min at 4°C and 10,000 rpm. Protein concentrations in the supernatant were determined using the Bio-Rad DC protein assay (Bio-Rad, München, Germany). To determine subcellular localization of NFB, nuclear and cytosolic fractions were prepared by lysing cells in 10mM HEPES, pH 7.5, 150mM NaCl, 0.6% Nonidet P40, 1mM EDTA, 1 µl/ml protease inhibitor cocktail (Sigma, Taufkirchen, Germany), 1mM Na3VO4, and 1mM NaF. After incubation for 20 min on ice, nuclei were pelleted by centrifugation at 4°C and 1500 rpm for 15 min and homogenized in nuclear extraction buffer (20mM HEPES, pH 7.9, 420mM NaCl, 1.5 MgCl2, 0.2mM EDTA, 25% glycerin, 5mM dithiothreitol, 1 µl/ml protease inhibitor cocktail, 1mM Na3VO4, and 1mM NaF). Protein concentrations in the cytosolic fraction (supernatant) were determined using the Bio-Rad DC (detergent compatible) protein assay (Bio-Rad, München, Germany), while protein concentrations in the nuclear fraction were measured by the Bio-Rad protein assay (Bio-Rad, München, Germany) due to the high glycerol content of the nuclear extraction buffer, which is not compatible with the Bio-Rad DC protein assay.
Equal amounts of protein (10 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat, dry milk in TBST buffer (50mM Tris, 150mM NaCl, 0.1% Tween 20, pH 7.4) for 2 h at RT and subsequently incubated with primary antibody (overnight at 4°C) and secondary goat anti-mouse IgG–HRP or goat anti-rabbit IgG–HRP (both Amersham Biosciences, 1:2000, in 5% nonfat, dry milk in TBST, 2 h at 4°C). Dilutions of primary antibodies were 1:200 (anti–phospho-ERK, anti-NFB), 1:1000 (anti–phospho-FAK), and 1:2000 (anti–phospho-JNK) in 5% nonfat, dry milk in TBST. Membranes were developed using the ECL western blotting detection system (Amersham Biosciences, Freiburg, Germany) and exposed to Hyperfilm (Amersham Biosciences, Freiburg, Germany). Densitometry was performed using the Bio-Rad Gel Doc 2000 system running with Molecular Analyst software (Bio-Rad, München, Germany). Changes in protein expression in response to OTA treatment are presented as percentage of control.
Microtubule polymerization assay.
Microtubule proteins were prepared from horse brain by two polymerization-depolymerization cycles according to the method of Shelanski et al. (1973). The protein pellet was resuspended in assembly buffer (100mM morpholinoethanesulfonic acid, 0.5mM MgCl2, 1mM EGTA, pH 6.4), and the protein concentrations were determined using the Bio-Rad protein assay. For the assembly assay, OTA was dissolved in DMSO and added to 0.5 ml of assembly buffer containing 1 mg/ml microtubule protein to give a final concentration of 50–400µM. After a 15-min incubation at 37°C, microtubule assembly was started by the addition of GTP (final concentration 0.5mM) and the increase in turbidity in the samples measured at 340 nm for 30 min. Disassembly at 4°C and a second assembly cycle were carried out to distinguish between normal microtubule formation and aggregation. Control incubations were carried out in which OTA was omitted and were used as reference (100% assembly). Colchicine at a final concentration of 2 and 4µM was used as a positive control.
Statistical analysis.
Data are expressed as mean ± SD. Statistical analysis was performed by ANOVA and Dunnett's multiple comparison test. Values significantly different from control are indicated as *p < 0.05 and **p < 0.01.
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RESULTS |
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Cytotoxicity of OTA in IHKE cells was assessed over a wide range of concentrations using cell number, mitochondrial activity, and LDH release as end points for toxicity. Cell number was found to be the most sensitive end point for OTA cytotoxicity (Fig. 1). A clear decrease in cell viability was observed in response to treatment with
1µM OTA for 24 h. Consistent with previous results obtained in LLC-PK1 cells (Mally et al., 2005a), OTA did not lead to a significant release of LDH into the culture medium, indicating that loss of cell viability under these conditions was not primarily due to necrosis (Fig. 1). Consistent with these findings, a time- and concentration-dependent increase in the number of cells showing chromosome condensation and fragmentation—morphological features characteristic for apoptosis—was evident following exposure to OTA (Fig. 2). The presence of apoptotic cells was further confirmed by flow cytometry of cells stained with Annexin V/PI, which showed a dose-dependent increase in the number of cells in early and late apoptosis, whereas Annexin–/PI+ cells (necrotic cells) were not increased in response to OTA treatment (data not shown).
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Loss of cell adhesion has previously been suggested to be involved in OTA toxicity, and we, therefore, investigated the effects of OTA treatment on cell contact sites. Similar to results obtained in COS cells (Scibelli et al., 2003
), treatment of IHKE cells with OTA for 24 h resulted in disruption of cell-cell and cell-matrix contacts. This was associated with decreased phosphorylation of FAK, a central component of integrin-mediated cell adhesion and survival signaling, which was clearly evident at concentrations of 10µM and above, as shown by immunofluorescence and western blot analysis (Fig. 3). Consistent with the inhibition of FAK and increased apoptosis, treatment of IHKE cells with OTA lead to activation of the MAP kinase JNK as indicated by enhanced immunoreactivity toward an antibody which specifically detects phosphorylated (activated) JNK (Fig. 4). At the same time, exposure to OTA resulted in activation of the (antiapoptotic) transcription factor NFB through translocation of the NFB subunit p65 into the nucleus, demonstrated by both western blotting and immunocytochemistry (Fig. 4). Consistent with previous results in kidney epithelial cells (Horvath et al., 2002; Schramek et al., 1997), increased phosphorylation of ERK1/2 was also observed in cells treated with 50µM OTA for 24 h by immunoblotting (Fig. 4).
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In evaluating nuclear morphology, we noted that later stages of mitosis (anaphase/telophase) were rarely present in OTA-treated cells in contrast to controls and completely disappeared after incubation with 50µM OTA for 24 h (Table 1, Figs. 5a–5c). This was associated with an overall decrease in the rate of mitosis (Table 1). In addition, aberrant metaphases with apparently misaligned chromosomes were frequently observed in the presence of OTA (Figs. 5b and 5d). More importantly, treatment with OTA resulted in the formation of giant cells with abnormally enlarged or multiple nuclei, which were sometimes still connected by chromatin bridges (Figs. 5e and 5f), indicating defects in chromosome segregation and/or cytokinesis. While enlarged cells were infrequently observed in untreated IHKE cells, the number of aberrant nuclei was significantly increased in OTA-treated cultures in a time- and dose-dependent manner (Table 1). Aberrant chromosome segregation and cell division may result from defects in assembly and function of the mitotic spindle, which can be visualized by
-tubulin staining. While untreated cells showed perfectly normal, symmetric spindles, immunofluorescence analysis revealed the formation of irregular, asymmetric, and sometimes multipolar mitotic spindles in the presence of OTA (Figs. 5g–5i).
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A cell-free, in vitro tubulin polymerization assay was used to determine if defects in mitotic spindle formation were caused by interference with microtubule polymerization. OTA inhibited microtubule assembly in a concentration-dependent manner; however, relatively high concentrations were required to affect tubulin polymerization compared to known microtubule-interfering agents such as colchicine, which was used as a positive control (Fig. 6).
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DISCUSSION |
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OTA is one of the most potent renal carcinogens studied by the NTP to date (Lock and Hard, 2004
). However, the primary events leading to tumor formation by OTA are poorly understood. Repeated administration of OTA to rats results in uncommon histopathological alterations which are not consistent with the nonspecific action of reactive intermediates or oxidative stress in the kidney, suggesting that the mechanism of tumor formation by OTA may be highly different from that of most known renal carcinogens (Mally et al., 2005c). Kidney pathology induced by OTA consists of frequent mitotic and abnormally enlarged cells, detachment of tubule cells from the basement membrane, and apoptosis within the S3 segment of the proximal tubule (Boorman et al., 1992; Maaroufi et al., 1999; Mally et al., 2005c). In this study, we found that OTA induces morphological alterations in IHKE cells in vitro, which closely resemble the effects of OTA in rat kidney, and have utilized this model to gain a better understanding of the primary molecular events involved in OTA toxicity and carcinogenicity. Although it is difficult to predict intracellular concentrations of OTA in the target cell population in vivo, concentrations of OTA which induced apoptosis and aberrant mitosis in IHKE cells in this study were in the same range as OTA plasma concentrations obtained after repeated administration of OTA to rats at doses known to cause tumors in the chronic rodent bioassay (Mally et al., 2005c), suggesting that the effects observed in vitro may be highly relevant to the mechanism of OTA carcinogenicity. In humans, mean OTA plasma concentrations resulting from dietary intake within the European Union are at least three orders of magnitude lower (0.18–1.19 ng/ml corresponding to 0.45–2.95nM) (SCOOP, 2002). However, in some countries such as Tunisia, exceptionally high concentrations of up to 1136 ng/ml (2.8µM) have been reported in some individuals suffering from kidney or urinary disorders (Maaroufi et al., 1995). Although a causal link between dietary OTA exposure and human diseases has not been established, it seems reasonable to assume that the morphological and molecular alterations reported here may also occur in individuals exposed to extremely high levels of OTA.
Based on the observation that detachment of proximal tubule cells from the basement membrane was frequently observed in OTA-treated rats and was accompanied by increased apoptosis (Mally et al., 2005c; Rasonyi et al., 1999), we initially speculated that loss of cell adhesion may contribute to OTA-induced apoptosis. Increasing evidence suggests that cells that lose contact with the extracellular matrix undergo apoptosis because they lack pivotal survival signals normally transmitted by cell-matrix interactions, and activation of FAK by tyrosine phosphorylation has been recognized as an important mechanism by which adhesion-mediated survival signals are transferred into the cell (Frisch et al., 1996b). Similar to recent results in COS cells (Scibelli et al., 2003), we observed decreased phosphorylation of FAK in response to OTA treatment at concentrations which also resulted in activation of JNK and cell death by apoptosis. However, at the same time, OTA stimulated activation of the transcription factor NFB and ERK1/2, which are both known to be downstream effectors of activated FAK and mediate signals involved in cell proliferation and survival (Giancotti, 1999). Since activation of these pathways is not consistent with the known effects of FAK inhibition, we concluded that other events must occur in response to OTA treatment which stimulate NFB and ERK1/2 survival signaling in OTA-treated cells.
Analysis of the mitotic index showed that the number of cells in mitosis was significantly reduced in the presence of OTA. While the percentage of cells in (pro)metaphase was not significantly altered, the proportion of cells in anaphase and telophase decreased in a time- and dose-dependent manner. These observations suggest that OTA did not block entry into mitosis but inhibited metaphase-anaphase transition, presumably because of defects in chromosome alignment and malfunction of the mitotic apparatus. Numerous natural compounds and anticancer drugs including several fungal metabolites exert their antiproliferative effects through perturbation of microtubule dynamics and mitotic spindle formation, resulting in metaphase arrest and subsequently apoptosis of mitotically blocked cells (Koizumi et al., 2004; Pfeiffer et al., 1998), and we, therefore, speculated that OTA may inhibit cell division in a manner similar to microtubule-disrupting agents. In support of this hypothesis, formation of aberrant mitotic spindles and inhibition of microtubule assembly were evident in the presence of OTA. Although the inhibitory effects of OTA on microtubule polymerization were weak compared to other known microtubule inhibitors such as colchicine and required relatively high concentrations, similar effects have been observed with citrinin, another mycotoxin produced by Aspergillus and Penicillium species (Pfeiffer et al., 1998). While the exact nature of the interaction between OTA and microtubules remains to be determined, it is possible that OTA may inhibit microtubule assembly through mechanisms other than direct binding to tubulin, e.g., through interaction with microtubule-associated proteins.
It is also interesting to note that—in contrast to most microtubule-binding agents—OTA did not induce prolonged mitotic arrest in IHKE cells. Rather, it seems that cells blocked in early mitosis by OTA treatment rapidly commit to apoptosis (mitotic catastrophe) (Castedo et al., 2004) or exit mitosis without cell division (mitotic slippage), resulting in aberrant cells with increased nuclear content. Although the reasons for this apparent difference remain unknown, the failure of OTA to efficiently block mitosis, thus allowing a subpopulation of cells to escape apoptosis, presents a violation of the "better dead than wrong" principle and may explain why OTA promotes malignant transformation rather than effectively inhibiting mitosis such as microtubule-interfering agents used in cancer therapy. It is becoming increasingly evident that tetraploid cells that arise from failure of chromosome segregation and cytokinesis are genetically highly unstable and frequently progress to become aneuploid (Andreassen et al., 1996). Although little is known regarding the signaling pathways which determine whether a cell transiently arrested in metaphase will exit mitosis rather than commit to apoptosis, NFB activation has been shown to suppress apoptosis and promote cell survival in response to microtubule-disrupting agents (Mistry et al., 2004). In addition, NFB activation appears to promote cell cycle transition from G1 to S phase (Chen et al., 2001) and may thus facilitate reentry of tetraploid cells into the cell cycle despite failure to divide in the previous cycle, thereby increasing polyploidy and genetic instability. Similarly, stimulation of ERK1/2 survival signaling by OTA may promote mitotic slippage by protecting cells from apoptosis. This is supported by recent findings, which show that simultaneous inhibition of ERK by pharmacological inhibitors potentiates OTA toxicity in opossum kidney tubule cells (Sauvant et al., 2005b).
It is striking that many of the intracellular signaling pathways stimulated by OTA are also known to be activated by microtubule inhibitors (Kook et al., 2000; Mistry et al., 2004; Rosette and Karin, 1995; Wang et al., 1998, 1999, 2000). In addition to stimulation of MAP kinase signaling, disruption of microtubules by nocodazole has been shown to induce degradation of several focal adhesion proteins including FAK (Kook et al., 2000), suggesting that alteration of microtubule function may also provide an explanation for the observed decrease of FAK activity.
In summary, results from this study show that OTA blocks mitosis at metaphase-anaphase transition in human kidney epithelial cells and simultaneously activates pro- and antiapoptotic signaling pathways, resulting in mitotic cell death and aberrant exit from mitosis. Blocked or asymmetric cell division may favor the occurrence of cytogenetic abnormalities and may ultimately lead to tumor formation. Although further work is required to identify the primary cellular targets and fully understand the molecular events and signaling pathways leading to disruption of mitosis in kidney tubule cells exposed to OTA, it should be noted that the proposed mechanism may be able to reconcile most if not all aspects of OTA toxicity, including lack of mutagenicity in bacteria, lack of metabolic activation and DNA adduct formation, induction of micronuclei and chromosomal aberrations, apoptosis and mitotic abnormalities both in vitro and in kidneys of OTA-treated rats, and the high incidence and aggressive nature of OTA-induced renal tumors, and may also provide a reasonable explanation for the apparent nonlinear dose response for OTA carcinogenicity.
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SUPPLEMENTARY DATA |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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Parts of this work were supported by the Fifth RTD Framework Programme of the European Union, project no. QLK1-2001-01614, the Deutsche Forschungsgemeinschaft, and the Gesellschaft für Mykotoxinforschung e.V. The authors would also like to thank Ursula Tatsch and Michaela Bekteshi for technical assistance.
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