Carcinogenesis vol.30 no.4 pp.711–719, 2009
doi:10.1093/carcin/bgp049
Advance Access publication February 23, 2009
Modulation of key regulators of mitosis linked to chromosomal instability is an early
event in ochratoxin A carcinogenicity
Melanie Adler, Katja Müller, Eva Rached, Wolfgang
Dekant and Angela Mally
Department of Toxicology, University of Würzburg, Versbacher Strasse 9,
97078 Würzburg, Germany
To whom correspondence should be addressed. Tel: þ49 931 20148894;
Fax: þ49 931 20148865;
Email: [email protected]
Ochratoxin A (OTA) is a potent renal carcinogen, but little is
known regarding the mechanism of OTA carcinogenicity. Early
histopathological alterations induced by OTA in rat kidney include single cell death, stimulation of cell proliferation and prominent karyomegaly indicative of blocked nuclear division during
mitosis. Based on these observations, it has been suggested that
disruption of mitosis by OTA may be the principal cause of cell
death and subsequent trigger for cell proliferation to compensate
for cell loss. To gain further insight into the molecular mechanism
of OTA toxicity, we used targeted quantitative real-time polymerase chain reaction arrays to investigate the expression of genes
involved in cell cycle control and mitosis in kidneys of male F344
rats treated with 0, 21, 70 and 210 mg/kg body wt OTA for up to 90
days. Treatment with OTA resulted in overexpression of key regulators of mitosis, including the mitotic protein kinases Polo-like
kinase 1, Aurora B and cyclin-dependent kinase 1 (Cdk1Cdc2),
several cyclins and cyclin-dependent kinase inhibitors, topoisomerase II and survivin. Immunohistochemical analysis confirmed
upregulation of Cdk1, p21WAF1/CIP1, topoisomerase II and survivin in S3 proximal tubule cells, from which OTA-induced tumors
in rats arise, and demonstrated increased phosphorylation of histone H3, a target of Aurora B. Importantly, many of the genes
found to be deregulated in response to OTA have been linked to
chromosomal instability and malignant transformation, supporting
the hypothesis that aberrant mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition, may play a critical role in OTA carcinogenicity.
Introduction
The mycotoxin and food contaminant ochratoxin A (OTA) is one of the
most potent renal carcinogens studied to date. In a 2 year carcinogenicity bioassay in F344 rats, relatively low doses of OTA (70 and 210
lg/kg body wt) were shown to induce high incidences of adenomas and
carcinomas (1,2). Moreover, tumors induced by OTA were characterized by an unusual, aggressive phenotype and high potential to metastasize (1,2). The molecular events leading to renal tumor formation by
OTA and the reason for the aggressive nature of OTA-induced tumors
are still poorly understood. While direct genotoxicity via formation of
covalent DNA adducts does not appear to play a role (3–6), there is
evidence to suggest that OTA may promote genomic instability and
tumorigenesis through interference with cell division. In several mammalian cell lines and primary cells, OTA was shown to inhibit cell cycle
progression by arresting cells at G2/M phase (7–9) and to increase
micronucleus formation, whereby both aneugenic and clastogenic effects were observed (10–13). Recent cytogenetic analyses revealed
a significant increase in endoreduplicated cells, indicating two successive rounds of DNA replication without intervening mitosis (14). In
Abbreviations: BrdU, 5-bromo-2-deoxyuridine; Cdk1, cyclin-dependent kinase
1; CIN, chromosomal instability; mRNA, messenger RNA; OTA, ochratoxin A;
PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Plk1, Pololike kinase 1; qRT, quantitative real-time.
immortalized human kidney epithelial cells, OTA blocked metaphase–
anaphase transition and led to the formation of aberrant metaphases
with misaligned chromosomes and giant cells with abnormally enlarged and/or multiple nuclei, which sometimes remained connected
by chromatin bridges (15). Although immunostaining of the mitotic
apparatus revealed defects in spindle formation in the form of asymmetric and sometimes multipolar mitotic spindles, OTA did not inhibit
microtubule assembly in a cell-free polymerization assay at relevant
concentrations (15), suggesting that OTA may disrupt mitosis through
mechanisms other than direct binding to tubulin.
In vivo, early effects of OTA at doses known to cause renal tumor
formation after long-term treatment (70 and 210 lg/kg body wt) consist
of cell loss accompanied by prominent nuclear enlargement and a doseand time-related increase in 5-bromo-2-deoxyuridine (BrdU) labeling
within the straight segment of the proximal tubule epithelium (P3) in
the outer stripe of the outer medulla, from which OTA-induced tumors
arise (16,17). While it is well established that regenerative cell proliferation in response to cytotoxicity is an important feature of many
non-genotoxic carcinogens in the kidney (18), OTA-induced kidney
pathology appears to be significantly different from that induced by
other renal carcinogens operating through a non-genotoxic/cytotoxic
mode of action, in that simple tubule hyperplasia, which frequently
occurs as a result of compensatory cell proliferation, does not occur
in response to OTA treatment, whereas the frequency and early onset of
karyomegaly appear to be unique to OTA. Disruption of cell division,
leading to the production of polyploid nuclei instead of simple tubule
hyperplasia, may provide a potential explanation for the apparent disparity between kidney pathology induced by OTA compared with other
renal carcinogens, and it appears likely that blocked mitosis may even
represent the principal cause of cell degeneration and subsequent trigger for cell proliferation to compensate for the presumed deficit in
daughter cells. These findings are consistent with the mitotic aberrations observed in immortalized human kidney epithelial cells treated
with OTA in vitro and support the hypothesis that it is the failure of cells
to complete mitosis in the presence of OTA that causes both cell death
and nuclear abnormalities (15).
A growing body of evidence indicates that aberrant cell division,
aneuploidy and cancer may occur as a result of misregulation of
mitosis-related genes, which govern faithful replication and segregation of chromosomes. For instance, untimely inactivation of cyclindependent kinase 1 (Cdk1cdc2) during mitosis has been shown to cause
premature exit from mitosis without nuclear division and cytokinesis
(mitotic slippage), leading to polyploidy and nuclear aberrations (19)
similar to those observed in OTA-treated immortalized human kidney
epithelial cells. Likewise, Polo-like kinase 1 (Plk1), which is known
to be involved in spindle assembly, centrosome maturation and separation, chromosome segregation and cytokinesis, has been shown to
exihibit oncogenic potential, and overexpression of Plk1 has been
associated with multinucleation, chromosomal instability (CIN), aneuploidy and poor prognosis in tumor patients (20). Depletion of Plk1
by small interfering RNA has been shown to reduce cell survival in
human cancer cells and inhibit colony formation in soft agar and tumor
growth in mice, demonstrating that Plk1 acts as an oncogene (21–23).
Aurora B, a serine/threonine kinase and part of the chromosomal passenger complex with distinct functions in chromosome condensation,
segregation and cytokinesis through regulation of microtubule kinetochore associations, is frequently overexpressed in various tumors, including renal cell carcinomas, and often correlates with poor prognosis
and high metastatic potential (20,24–27). Experimental evidence for
a role of Aurora B in malignant transformation has also come from
studies demonstrating Aurora B upregulation in early foci of estrogeninduced Syrian hamster kidney tumors, suggesting that elevated expression of Aurora kinases leading to centrosome amplification,
Ó The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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M.Adler et al.
chromosomal instability and aneuploidy may drive estrogen-mediated
oncogenesis (28), as well as from studies showing chromosome number
instability in Chinese hamster embryo cells in response to forced overexpression of Aurora B (29).
Considering the close link between mitotic defects and genetic instability, the nuclear aberrations observed in response to OTA treatment and the malignant phenotype of OTA-induced tumors in rats, we
speculated that the mechanism of OTA carcinogenicity may involve
altered function of key regulators of mitosis, resulting in blocked or
asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition and subsequent tumor formation. To gain further
insight into the early molecular changes involved in OTA carcinogenicity, we used quantitative real-time (qRT) polymerase chain reaction
(PCR) arrays, which combine the quantitative performance of qRTPCR with the multiple gene profiling capabilities of microarrays, supported by immunohistochemistry and immunoblotting, to analyze the
time- and dose-related expression of a focused panel of genes critically
involved in cell cycle control and progression through mitosis in kidneys of male F344 rats treated with OTA at doses of 0, 21, 70 and 210
lg/kg body wt for up to 90 days. The arrays were specifically designed
to cover genes involved in all stages of the cell cycle (G1/S, G2 and M
phase) and included cyclin-dependent kinases, cyclins, cyclin-dependent kinase inhibitors, a range of oncogenes and tumor suppressor
genes that control cell proliferation and cell survival, genes related to
the DNA damage and spindle assembly checkpoints, as well as genes
encoding for growth factors and growth factor receptors.
Materials and methods
Reagents
OTA was purchased from Axxora (Grünberg, Germany). Primary antibodies
used were rabbit anti-GAPDH (Sigma–Aldrich, Deisenhofen, Germany),
mouse anti-Cdk1cdc2 (17; Santa Cruz Biotechnology, Heidelberg, Germany),
mouse anti-p21 (clone SMX30; BD Biosciences PharMingen, Heidelberg,
Germany), rabbit anti-p-histone H3 (Ser10; Santa Cruz Biotechnology), goat
anti-cyclin A (D-19; Santa Cruz Biotechnology), rabbit anti-cyclin B1 (Acris
Antibodies GmbH, Hiddenhausen, Germany), rabbit anti-cyclin E1 (AbD Serotec, Düsseldorf, Germany), goat and mouse Plk (N-19 and F-8; Santa Cruz
Biotechnology), mouse anti-Plk1 (clone 35-206; Abcam, Cambridge, UK),
rabbit anti-Plk1 pT210 (Rockland Immunochemicals, Gilbertsville, PA), rabbit
anti-Aurora B (Abcam and Cell Signaling Technology, Beverly, MA), rabbit
anti-survivin (Novus Biologicals, Littleton, CO), rabbit anti-topoisomerase II
alpha (Epitomics, Burlingame, CA), rabbit anti-separase (Abcam) and mouse
anti-BubR1 (clone 9; BD Transduction Laboratories, Lexington, KY). Secondary antibodies were obtained from Santa Cruz Biotechnology and streptavidinhorseradish peroxidase and 3,3#-diaminobenzidine substrate kit were
purchased from BD Biosciences PharMingen.
Tissue samples
Tissue samples were obtained from a 90 day oral gavage study in which male
F344 rats were treated with OTA dissolved in corn oil at doses of 0, 21, 70 and
210 lg/kg body wt for 14 and 90 days, 5 days/week as described previously
(16). The study was conducted in accordance with guidelines for the humane
treatment of animals after approval by the appropriate authorities. At necropsy,
kidneys and livers were removed, aliquoted and flash frozen in liquid nitrogen
and stored at 80°C until analysis. For immunohistochemical studies, aliquots
of kidney and liver were fixed in 10% neutral buffered formalin and embedded
in paraffin.
RNA isolation
RNA was extracted from kidney and liver tissue using Total RNA Isolation
Reagent (Abgene, Hamburg, Germany), followed by DNA digestion and further purification using RNeasy Mini Kit (Qiagen, Hildesheim, Germany) according to the manufacturer’s instructions. RNA was quantified by measuring
the absorbance at 260 nm. RNA integrity was determined by 1.2% agarose gel
electrophoresis. Purified RNA samples were stored at 80°C until use.
Complementary DNA synthesis and expression profiling
Complementary DNA was synthesized from 1 lg total RNA by using RT2 PCR
array first strand kit (SA Bioscience, Frederick, MD) according to the manufacturer’s protocol. Samples were quality controlled using RT2 RNA QC PCR
arrays (SA Bioscience) and subsequently used as template for qRT-PCR using
custom made RT2 Profiler PCR arrays (SA Bioscience). A PCR array of 96
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primers was designed to analyze the expression of 84 selected genes implicated
in cell cycle control and mitosis along with five housekeeping genes and several
controls including a ‘no template’ and a ‘no reverse transcription’ control. qRTPCR was carried out on a ABI PrismÒ 7000 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany) in 25 ll PCR mixture, consisting of
12.5 ll RT2 Real-TimeTM SYBR Green/ROX PCR master mix (SA Bioscience),
11.5 ll double-distilled H2O and 1 ll of template complementary DNA. PCR
amplification was performed by polymerase activation (10 min at 95°C), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Specificity of PCR
products was examined by melting curve analysis. Amplification efficiency
between samples was estimated using standard curves. The Ct values were
normalized to five housekeeping genes (Rplp1, ribosomal protein, large, P1;
Hprt, hypoxanthine guanine phosphoribosyl transferase; Rpl13a, ribosomal protein L13A; Ldha, lactate dehydrogenase A and Actb, b-Actin) and changes in
gene expression relative to controls were calculated using the DDCt method.
Data are expressed as mean fold change relative to control animals (n 5 3).
Statistical analysis was performed by analysis of variance and Dunnett’s post hoc
test. Statistically significant changes are indicated as P , 0.05, P , 0.01
and P , 0.001. A 2-fold change in expression level or a P-value ,0.05 were
considered as biologically significant. A heat map visualizing relative expression
of all genes on the array was generated using the Cluster and TreeView software
(30), and principal component analysis was performed using SIMCA-P version
11.5 (Umetrics, Umea, Sweden).
Immunohistochemistry
Immunohistochemical studies were performed on 5 lm sections of paraffinembedded kidneys. Sections were deparaffinized, hydrated and washed in phosphate-buffered saline (PBS) or PBS containing 0.1% Tween-20. Antigen
retrieval was performed by heating for 4 min in citrate buffer (pH 6.0 and pH
4.0) using a pressure cooker. Endogenous peroxidase was blocked by 3% H2O2
in PBS for 15 min. Subsequently, sections were washed in PBS and blocked with
normal serum (10% in PBS) for 1.5 h, followed by incubation with avidin and
biotin solution (0.001% in PBS) for 15 min, with several wash steps in between.
Then, slides were incubated with primary antibody in 1% bovine serum albumin
in PBS overnight at 4°C in a humidified chamber. Dilutions of antibodies were as
follows: anti-Cdk1cdc2 and anti-p21, 1:50; anti-topoisomerase II alpha, 1:200;
anti-p-histone H3 (Ser10), 1:100; anti-survivin, 1:200 and anti-Plk1, anti-Aurora
B and anti-separase from 1:50 to 1:500. After washing with PBS or PBS containing 0.1% Tween-20, the sections were incubated for 1.5 h at room temperature with biotinylated secondary antibody and were subsequently washed three
times. Following incubation with streptavidin-horseradish peroxidase for 30 min,
immunoreactivity was visualized using 3,3#-diaminobenzidine for 1–3 min
(BD Biosciences PharMingen). The sections were counterstained with hematoxylin, dehydrated and finally mounted in Eukitt mounting medium (Sigma–
Aldrich). Images were acquired at 200 magnification using an Olympus CH-2
microscope fitted with an Olympus E330 digital camera.
Western blot
Kidneys were homogenized in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM
NaCl, 2 mM ethylenediaminetetraacetic acid, 1% Nonidet P40 and 0.1%
sodium dodecyl sulfate), supplemented with 1 mM NaF, 1 mM Na3VO4 and
protease inhibitor cocktail (Sigma) and centrifuged at 8000g for 15 min at 4°C.
The protein concentration of the lysate was determined using the DC Assay
method (Bio-Rad, Munich, Germany). Aliquots of 20–50 lg protein in
Laemmli buffer (Sigma) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes at
100 V for 75 min at 4°C using transfer buffer (25 mM Tris, 192 mM
glycine and 20% methanol). Membranes were blocked in 5% non-fat dry
milk in TBST buffer (50 mM Tris–HCl, 150 mM NaCl and 0.1% Tween-20, pH
7.4) for 2 h at room temperature following incubation with primary antibodies
diluted in blocking buffer overnight at 4°C [anti-cyclin B1, 1:500; anti-cyclin A,
1:1000; anti-Plk1 (F-8, 1:200; N-19, 1:1000; pT210, 1:1000); anti-Aurora B,
1:200; anti-cyclin E1, 1:2000; anti-GAPDH, 1:20 000 and anti-BubR1, 1:500].
After several washing with TBST, horseradish peroxidase-conjugated antibodies
were incubated at a dilution of 1:5000 in blocking buffer for 1 h at room temperature and detection of proteins was performed using ECL detection system
(GE Healthcare, Munich, Germany). Densitometric analysis was performed using the Bio-Rad Gel Doc 2000 system (Bio-Rad, Munich, Germany).
Results
Time- and dose-dependent effects on gene expression profiles
Oral administration of OTA at doses known to cause renal tumor
formation after chronic treatment resulted in significant time- and
dose-related changes in the expression of genes of interest in kidneys
Early events in OTA carcinogenicity
Fig. 1. Principal component analysis showing time- and dose-dependent
alterations in gene expression in response to OTA treatment as evidenced by
a clear separation of mid- (90 days) and high-dose (14 and 90 days) animals
from the main cluster.
of male F344 rats (Figures 1 and 2). Of the 84 genes on the array, 20
genes were significantly upregulated after treatment with 70 and 210 lg
OTA/kg body wt for 90 days as compared to controls, whereas only
two genes were downregulated in kidneys of high-dose animals (Figure 2). In contrast, no alterations in gene expression were evident in
kidneys of rats treated with a low dose of OTA (21 lg/kg body wt) or
in the liver (210 lg/kg body wt), which is not a target for OTA
carcinogenicity (Figures 1 and 2). To identify early molecular changes
involved in the mechanism of OTA carcinogenicity, gene expression
profiles were also investigated in kidneys of rats after a 14 day treatment with OTA at doses of 0, 21, 70 and 210 lg/kg body wt, which
caused minor histopathological changes in high-dose animals, consisting of lining cell degeneration in the form of condensed cells with
dense eosinophilic cytoplasm and contracted nuclei and nuclear variability implying a slight increase in nuclear size scattered through
affected tubules (16). The majority of genes (16/22) affected by a 90
day OTA treatment were also found to be deregulated after 2 weeks
administration of 210 lg OTA/kg body wt, albeit to a lesser extent
(Figure 2). In contrast, treatment with 70 lg/kg body wt for 2 weeks
did not induce significant changes in messenger RNA (mRNA) expression levels, consistent with the absence of histopathological
changes at this dose/time point. Genes significantly altered in response to OTA treatment are summarized in Table I. Quantitative data
obtained from all genes on the array are provided as supplement
(supplementary Table 1 is available at Carcinogenesis Online).
Genes significantly deregulated by treatment with OTA for 14 days
Early effects of OTA in rat kidney included changes in the expression
of positive regulators of cell cycle kinases, such as the G1/S-specific
cyclin E1, the S phase/mitotic cyclins A2, B1 and B2 and c-myc
(Table I). In contrast, expression of D-type cyclins (D1 and D2),
which is highly growth factor dependent and drives cell cycle progression through G1 phase in response to extracellular signals,
remained unchanged (supplementary Table 1 is available at Carcinogenesis Online). mRNA levels of cyclin H, the regulatory subunit of
Cdk7, a cyclin-dependent kinase-activating kinase and cyclin G1,
a target of p53, were also not affected by OTA treatment.
In contrast to cyclin-dependent kinases active during G1/S phase
(i.e. Cdk 2, 4 and 6), Cdk1cdc2, which associates with A- and B-type
cyclins and plays a key role during mitosis, was found to be upregulated 3-fold in response to OTA treatment. Likewise, Plk1, which
mediates phosphorylation of cyclin B1 and Cdc25C leading to activation of Cdk1cdc2–cyclin B1 in addition to its role in chromosome
condensation and separation, was induced in kidneys of OTA-treated
Fig. 2. A heat map visualizing time- and dose-dependent changes in gene
expression pattern in kidneys and livers of male F344 rats following exposure
to OTA. Data for each individual animal are presented as fold change relative
to time-matched controls, with red indicating upregulation and green
indicating genes that were downregulated. A similar expression pattern was
observed in kidneys of mid- (70 lg/kg body wt) and high-dose (210 lg/kg
body wt) rats after 90 days treatment, and in high-dose animals after 14 days,
although effects were less pronounced at this early time-point. In contrast, no
significant alterations in gene expression profiles were evident in kidneys of
rats treated with a low dose (21 lg/kg body wt) for up to 90 days or in the
liver after high-dose treatment.
rats as compared to controls. At the same time, OTA treatment resulted in a significant increase in the expression of negative regulators
of cell cycle progression, i.e. p21WAF1/CIP1 and 14-3-3sigma, but not
p53 and Gadd45 (Table I and supplementary Table 1 is available at
Carcinogenesis Online).
Aurora kinase B and survivin, both members of the chromosomal
passenger complex required for mitotic checkpoint function, as well
as the mitotic checkpoint genes Bub1b and Cdc20 were significantly
upregulated in kidneys of OTA-treated animals. In addition, OTA induced the expression of Espl1 (separase), which cleaves sister chromatid cohesion at the onset of anaphase, along with its inhibitor Pttg1
(securin) and topoisomerase II alpha, involved in mitotic chromosome
segregation after DNA replication.
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M.Adler et al.
Table I. Statistically significant changes in gene expression in kidneys of rats treated with OTA (21, 70 and 210 lg/kg body wt) for 14 and 90 days and in livers of
high-dose animals after 90 days relative to controls
Accession no.
Gene title
Symbol
Fold deregulation after treatment with OTA (lg/kg body wt)
Kidney 14 days
21
NM_019296
NM_053702
NM_171991
NM_001009470
XM_574426
XM_342804
NM_080782
XM_232745
NM_053749
NM_017100
NM_022183
XM_342494
NM_022274
NM_022391
NM_012920
NM_080400
NM_171993
XM_235691
NM_017251
NM_022197
NM_012603
NM_031546
Cell division cycle 2 homolog A
(Schizosaccharomyces pombe)
Cyclin A2
Cyclin B1
Cyclin B2
Cyclin E1
Cyclin E2
Cyclin-dependent kinase inhibitor 1A
14-3-3sigma (stratifin)
Aurora kinase B
Polo-like kinase 1 (Drosophila)
Topoisomerase (DNA) 2 alpha
Budding uninhibited by benzimidazoles
1 homolog, beta (Saccharomyces cerevisiae)
Baculoviral inhibitor of apoptosis
repeat-containing 5 (survivin)
Pituitary tumor-transforming 1 (securin)
Calcium/calmodulin-dependent protein
kinase II alpha subunit
Checkpoint kinase 1 homolog (S.pombe)
Cell division cycle 20 homolog (S.cerevisiae)
Extra spindle poles like 1 (S.cerevisiae),
separin
Gap junction membrane canne protein
beta 1, Cx32
FBJ musine osteosarcoma viral oncogene
homolog
Myelocytomatosis viral oncogene homolog
(avian)
Regucalcin
70
Kidney 90 days
210
21
70
Liver 90 days
210
210
1.6
Cdc2a (Cdk1)
1.6
1.2
Ccna2
Ccnb1
Ccnb2
Ccne1
Ccne2
Cdkn1a (p21)
Sfn_pred
Aurkb
Plk1
Top2a
Bub1b
1.4
1.4
1.3
1.6
1.8
1.0
1.9
1.6
1.0
1.3
1.3
1.2
1.1
1.2
1.2
1.6
1.3
2.1
1.3
1.4
1.3
1.2
3.0
3.5
3.2
3.0
1.8
3.9
2.7
3.8
3.6
2.7
3.3
1.2
1.2
1.3
1.1
1.2
1.5
1.3
1.3
1.2
1.0
1.1
5.9
7.6
6.0
4.8
2.6
6.4
3.6
9.7
7.6
7.2
5.6
5.5
6.0
6.0
5.9
2.1
11.8
5.4
7.5
7.8
6.3
5.3
1.4
1.1
1.3
1.6
1.2
1.6
1.1
1.1
1.1
1.5
1.5
Birc5
1.3
1.1
2.4
1.0
2.8
3.3
1.0
Pttg1
Camk2a
1.3
1.4
1.1
1.2
1.8
1.2
1.1
1.1
2.7
1.7
3.7
3.3
1.7
1.3
Chek1
Cdc20
Espl1_pred
1.3
1.6
1.8
1.2
1.2
1.6
1.7
2.2
2.2
1.1
1.0
1.3
2.4
3.0
2.5
2.3
2.3
3.0
1.3
1.3
1.2
Gjb1
1.4
1.2
1.2
1.0
1.3
1.0
1.2
1.2
2.2
2.7
1.1
Myc
1.5
1.5
1.3
2.0
2.6
1.3
Rgn
1.8
1.2
216.2
1.1
Fos
3.2
1.9
1.2
1.2
1.2
6.0
1.6
21.8
6.2
23.4
1.3
Data are expressed as mean of three individual animals per dose group. Statistically significant changes are indicated by P , 0.05, P , 0.01 and P , 0.001
(ANOVA followed by Dunnett’s post hoc test).
Genes significantly deregulated by treatment with OTA for 90 days
qRT-PCR analysis following 90 days treatment revealed more pronounced increases in the expression of those genes found to be deregulated after 14 days. The most upregulated gene was the Cdk inhibitor
p21WAF1/CIP1, which was increased .11-fold after 90 days, whereas
other Cdk inhibitors, such as p16Ink4a, p57Kip2 or p27Kip1, were not
modulated by OTA (Table I and supplementary Table 1 is available
at Carcinogenesis Online). The mitotic protein kinases Aurora B, Plk1,
Cdk1cdc2, Bub1b and several cyclins (A2, B1, B2, E1) were upregulated
.5-fold in kidneys of high-dose animals relative to controls.
In addition to the gene expression changes observed after 14 days
treatment, continuous administration of OTA (70 and 210 lg/kg body
wt) for 90 days resulted in a significant increase in mRNA levels of
c-fos, cyclin E2 and checkpoint kinase 1 (Chek1), which plays a role
in recognition of DNA damage and replication defects to preserve
genomic integrity.
In contrast to the marked upregulation of most cell cycle and
mitosis-related genes, a highly significant decrease in the expression
of regucalcin (.15-fold) was observed after treatment with 210 lg/kg
body wt OTA for 90 days, in line with a recent microarray study (31).
At the same time, Ca(2+)/calmodulin (CaM)-dependent protein kinase,
which is involved not only in calcium signaling but also in initiation of
centrosome duplication, was significantly increased in response to
OTA. The only other gene found to be downregulated in response
to OTA was the gene encoding for the gap junction membrane channel
protein beta 1 (connexin 32), a tumor suppressor gene involved in gap
junction intercellular communication-mediated growth control.
Interestingly, no changes in the expression of a range of growth factors
(epidermal growth factor, insulin-like growth factor 1, platelet-derived
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growth factor and transforming growth factor beta 1) or their receptors
were detected in kidneys of rats treated with OTA (supplementary Table 1
is available at Carcinogenesis Online), suggesting that mitogenic signaling via overexpression of growth factors/growth factor receptors
may not play a major role in OTA-mediated stimulation of renal cell
proliferation.
Immunohistochemical and western blot analyses
In good agreement with changes in gene expression, immunohistochemical anaylsis revealed time- and dose-dependent alterations in
protein expression in proximal tubule epithelial cells located in the
outer stripe of the outer medulla, from which tumors in OTA-treated
rats arise. Enhanced expression of Cdk1cdc2, the key regulator of
mitosis, was detected in affected tubules in kidneys of OTA-treated
animals. Diffuse cellular staining consistent with its role in G2/M
phase was evident in high-dose animals after 14 days treatment and
in kidneys of animals exposed to 70 and 210 lg/kg body wt OTA for
90 days (Figure 3A), whereas Cdk1cdc2 protein was not detected in
control or low-dose animals. Western blotting did not reveal significant changes in the expression of cyclins A and B, which were not
detectable by immunohistochemistry (Figure 4).
However, a striking increase in p21WAF1/CIP1 protein was detected
in epithelial cells within the outer stripe of the outer medulla and
medullary rays as early as 14 days after treatment with 210 lg/kg
body wt OTA, and in mid- and high-dose animals after 90 days.
Kidneys of control and low-dose animals showed no immunoreactivity toward p21WAF1/CIP1. p21WAF1/CIP1 immunostaining revealed nuclear localization, predominantly in cells with enlarged nuclei,
consistent with its role in maintaining G1 arrest after aberrant exit
Early events in OTA carcinogenicity
Fig. 3. Immunohistochemical analysis showing altered expression of Cdk1cdc2, p21WAF1/CIP1, topoisomerase II alpha (Top2a), phospho-H3 (Ser10) and survivin in
proximal tubule epithelial cells of male rats after treatment with 210 lg/kg body wt OTA for 14 and 90 days, confirming gene expression data. Similar changes
were evident after treatment with 70 lg/kg body wt for 90 days (data not shown). (A) Representative images showing enhanced expression of Cdk1cdc2 in affected
tubules in response to OTA treatment, evident by diffuse cellular staining (arrow). (B) Time- and dose-dependent induction of p21WAF1/CIP1 protein expression
in affected areas, predominantly in cells with enlarged nuclei (note intense nuclear staining, arrow), but also in degenerate cells (arrowhead). (C) Nuclear
expression of topoisomerase II in an occasional proximal tubule cell in control animals and increased frequency and intensity of topoisomerase II staining in OTAtreated rat kidney, particularly in cells with prominent nuclei (arrow). (D) Increased Ser-10 phosphorylation of histone H3, a target of Aurora B kinase, in aberrant
mitotic cells (arrow) and pyknotic nuclei of degenerate cells detached from the basal membrane (arrowhead). (E) Positive survivin immunolabeling in renal tubular
cells with enlarged nuclei (arrows). Images were acquired at 200 magnification using an Olympus CH-2 microscope fitted with an Olympus E330 digital camera.
from mitosis (Figure 3B). In addition, degenerate cells frequently
exhibited enhanced p21WAF1/CIP1 immunoreactivity (Figure 3B), suggesting that p21WAF1/CIP1 may contribute to mitotic failure via untimely inactivation of Cdk1cdc2.
An occasional cell positively stained for topoisomerase II was noted
in proximal tubule cells of control animals (Figure 3C). However, the
number of cells showing topoisomerase II immunoreactivity was significantly increased in kidney sections of high-dose animals after 14
days and in mid- and high-dose rats exposed for 90 days (Figure 3C).
Topoisomerase II staining localized to the nucleus, whereby cells with
increased nuclear content were frequently positive for topoisomerase II.
In contrast to rat thymus extract (Santa Cruz Biotechnology),
K-ras-transformed normal rat kidney cell lysate (Santa Cruz Biotechnology) and HeLa cells that were used as positive controls, Aurora B,
separase, BubR1 and Plk1 were not detectable in rat kidney by either
immunohistochemistry or immunoblotting even after nuclear enrichment using a range of antibodies (data not shown), presumably due to
the low level of expression as suggested by reports demonstrating the
presence of these proteins in tumors but not in surrounding normal
tissues (32–35). However, increased phosphorylation of histone H3 on
Ser10 indicative of enhanced Aurora B activity was evident in OTAtreated animals. Interestingly, phospho-H3 (Ser10) was evident in
abnormally enlarged mitotic figures but also in pyknotic nuclei of
degenerate cells detached from the basal membrane, which may indicate that cell death in these cells may have occurred during mitosis
(Figure 3D).
Consistent with gene expression data, overexpression of survivin,
a member of the inhibitor of apoptosis family and chromosomal passenger complex, was detected in renal tubule cells of OTA-exposed
rats by immunohistochemical analysis. Enhanced expression of
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M.Adler et al.
Fig. 4. Western blot analysis (A and C) and densitometry (B and D) of cyclin A2 and cyclin B1 expression in kidneys of F344 rats treated with 0, 21, 70 and 210
lg/kg body wt OTA for 90 days. Densitometry data are presented as fold change normalized to GAPDH relative to control animals as determined by at least
three independent experiments. Statistical significance was determined by ANOVA followed by Dunnett’s post hoc test ( P , 0.05, P , 0.01 and
P , 0.001). (A and B) No increase in cyclin A2 protein expression in response to OTA treatment was observed despite modulation at the mRNA level. (C and
D) Lack of significant differences in cyclin B1 protein expression in kidneys of treated versus control rats.
survivin was evident in cells with enlarged nuclei, suggesting that
survivin may contribute to the survival of these genetically unstable,
polyploid cells (Figure 3E).
Discussion
We have shown previously that single-cell degeneration, compensatory
DNA replication and marked nuclear enlargement indicative of blocked
nuclear division during mitosis and subsequent inhibition of cytokinesis
are prominent features of OTA nephrotoxicity under conditions of carcinogenicity (16). Based on these findings, we hypothesized that OTA
may interfere with signaling pathways controlling entry into the cell
cycle and coordinate progression through mitosis, thereby causing mitotic aberrations, proliferation and consequently malignant transformation. In this study, we used low-density real-time PCR arrays, which
allow a more accurate analysis of low-abundance mRNAs than GeneChip microarrays due to their much wider dynamic range, to investigate
changes in the expression of a focused panel of genes selected based on
their known function in cell proliferation and growth control. Our
results show that the early histopathological changes induced by
OTA in rat kidney are associated with significant alterations in the
expression of several key regulators of mitosis, including molecules
involved in centrosome function, G2/M progression, chromosome condensation and segregation, spindle checkpoint and cytokinesis, whereas
genes implicated in G1 progression, G1–S transition or growth factor
signaling were generally not modulated by exposure to OTA (Figure 5).
No changes were seen in the liver as a non-target organ. Immunohistochemical analysis confirmed upregulation of mitosis-control proteins
in S3 proximal tubule cells, which represent the target cells of OTA
carcinogenicity, presumably due to preferential uptake and intracellular
accumulation of OTA at this site (36,37).
In recent years, it has become increasingly more evident that genetic and epigenetic alterations in mitotic genes that coordinate equal
distribution of the genetic material between two daughter cells may be
associated with tumor development and act as a driving force during
malignant progression. Evidence for this has come from numerous
reports demonstrating overexpression of mitosis-related genes in
a wide variety of human cancers, as well as from experiments using
genetically engineered mice and cultured cells (38). Moreover, loss of
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mitotic regulation leading to chromosomal instability and alterations
in chromosome number (aneuploidy) frequently correlates with tumor
malignancy and poor prognosis. Carter et al. (39) recently identified
a signature of 70 genes associated with chromosomal instability, overexpression of which predicted clinical outcome in patients. Among
these genes, 29 play key roles in mitosis (38). Importantly, this gene
signature contains several genes that we found to be significantly
upregulated in kidneys of OTA-treated animals as early as after 14
days treatment, including Aurora kinase B, Cdk1cdc2, Cdc20, cyclin
B1, cyclin B2, Espl1 (separase) and Pttg1 (securin), providing strong
evidence for mitotic deregulation and chromosome instability as an
early and perhaps initiating event in tumor formation by OTA. Furthermore, the close link between tumor malignancy and aberrant expression of genes associated with chromosomal instability may help
explain why OTA-induced kidney tumors develop with such a rapid
onset and are characterized by aggressive growth, tendency toward an
uncommon anaplastic phenotype and high potential to metastasize
(1,18).
In our study, we also observed differential expression of a number
of genes that are not part of Carter’s chromosomal instability signature but are also intimately linked to mitosis and growth control and,
indeed, tumor development. These include A- and E-type cyclins,
myc, the mitotic spindle checkpoint gene Bub1b (encoding BubR1),
the mitotic protein kinase Plk1, survivin, p21WAF1/CIP1 and the gene
encoding the gap junction membrane channel protein beta 1 (Cx32).
Although the precise mechanism of how gap junction proteins regulate growth control still remains to be elucidated, there is a wealth of
evidence which shows that connexins function as tumor suppressors
and that sustained inhibition of gap junction intercellular communication provides a tumor-promoting stimulus (40). In renal cell carcinomas from hemodialysis patients, Cx32 is transcriptionally
inactivated (41), and transfection of Cx32 into metastatic renal cell
carcinoma cells lacking endogenous Cx32 suppresses growth, invasion and metastasis of renal tumor cells (42,43). The downregulation
of Cx32 mRNA observed in this study is in line with previous reports
which demonstrate that disruption of gap junction intercellular communication may play a role in the mechanism of tumor formation by
OTA (44,45). However, the fact that significant alterations were evident in high-dose animals only after 90 days treatment indicates that
Early events in OTA carcinogenicity
Fig. 5. Graphical representation of the multiple roles of mitotic regulators found to be modulated by OTA and the cellular consequences of perturbed mitosis in
response to OTA.
loss of Cx-mediated growth control may well be a contributing, but
not primary, event in OTA carcinogenicity. Similarly, expression of
regucalcin (also known as senescence marker protein 30), which decreases with aging and has been shown to play a cytoprotective role
against intracellular calcium elevation and oxidative stress (46), was
only affected following 90 days treatment with the high dose of OTA.
In contrast, several cancer-related genes that function as oncogenes
were significantly upregulated as early as after 14 days OTA treatment. Overexpression of E- and A-type cyclins, which may be mediated by myc, is not only associated with proliferation but is also
considered to be linked to malignant phenotype and chromosomal
instability (47). In late G1 and during S phase, cyclins E and A sequentially activate Cdk2, which has been proposed to act as a key
regulator in the initiation of centrosome duplication (48). Centrosome
duplication, separation and function are essential for formation of
bipolar mitotic spindles and accurate chromosome segregation. Consequently, numeral and functional abnormalities of centrosomes may
result in mitotic aberrations and are thought to be a major cause of
genomic instability. Besides E- and A-type cyclins, a large number of
cancer-associated genes are known to be involved in the regulation of
centrosome number, including myc, CaMKII, Chek1 and Plk1, which
were also found to be deregulated in kidneys of OTA-treated rats. In
addition to regulating centrosome duplication and function, Plk1
plays multiple roles during mitosis, including chromosome condensation and separation (20,49). Plk1 cooperates with Aurora B and
separase (both associated with CIN and also overexpressed in response to OTA) in mediating removal of cohesin, which maintains
sister chromatid cohesion, leading to separation of sister chromatids
(50,51). Interestingly, Mosesso et al. (14) recently showed that OTA
induces aberrant condensation and separation of chromatids, which
may be mediated in part by Plk1, Aurora B and separase. In addition,
overexpression and Plk1-dependent phosphorylation of topoisomerase II alpha (upregulated 2.6- and 6.2-fold after 14 and 90 days OTA
treatment, respectively), which plays crucial roles in mitotic chromosome assembly and organization, may contribute to premature chromosome condensation (52), whereas increased expression of BubR1
(Bub1b), a spindle checkpoint protein that senses lack of attachment
and tension at kinetochores, may block mitotic progression through
inhibition of the anaphase promoting complex APC/CCdc20 (53). Both
BubR1 (Bub1b) and topoisomerase II alpha are frequently elevated in
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M.Adler et al.
various cancers and high expression levels of topoisomerase II alpha
have been linked with poor differentiation and aggressive tumor phenotype (54–59).
Although most of the genes differentially expressed in kidneys of
OTA-treated rats are cell cycle related, it needs to be emphasized that
upregulation of mitotic genes linked to chromosomal instability is not
considered to be simply a reflection of cell proliferation (38,39). This
is also supported by the fact that significant increases in gene expression, but not BrdU labeling (16) were evident in kidneys of rats
following 14 days treatment with OTA. At this time point, single
proximal tubule cell death was an important feature of OTA nephrotoxicity, and the finding that degenerate cells were frequently positive
for phospho-H3 (Ser10) indicates that cell death is likely to have
occurred during mitosis. Commitment to apoptosis in response to
mitotic disruption might be seen as a protective means to prevent
generation of genetically unstable polyploid cells and subsequent
oncogenesis. After 90 days, however, cell degeneration was less frequent, but the incidence and degree of karyomegaly was increased as
compared to earlier time points, along with a significant increase in
BrdU-labeled cells (16). This suggests that the deficit in daughter cells
being formed may provide a strong and continuous stimulus for cells
to become resistant against apoptosis, thereby causing a shift toward
mitotic exit, cell proliferation and reentry of tetraploid cells into a second round of DNA synthesis. It is possible that the overexpression of
Birc5 (survivin) observed in this study may contribute to cell survival
following continuous exposure to OTA. Survivin, a chromosomal passenger protein and member of the inhibitor of apoptosis gene family,
is upregulated in various tumors, and it has been speculated that tumor
cells may exploit the antiapoptotic function of survivin to evade apoptosis (60). Survivin function is regulated at the transcriptional and
post-transcriptional level and requires sharp cell cycle-dependent
transcription at mitosis and post-transcriptional modification, including phosphorylation by Cdk1cdc2 and Aurora B. On the other hand,
there is also evidence that survivin gene transcription may be driven
by non-cycle-dependent signaling pathways, including Wingless
(Wnt)/T cell factor (TFC)-b-catenin, AKT and nuclear factor-jB signaling, and, indeed, results from a number of in vitro or in vivo studies
suggest that OTA may modulate these pathways (15,31,45,61,62).
In our study, p21WAF1/CIP1, which is commonly considered to possess tumor suppressor function, was significantly upregulated in kidneys of OTA-treated rats, particularly in cells with giant nuclei. It is
thought that post-mitotic G1 arrest in response to spindle damage and
aberrant exit from mitosis is dependent on p21WAF1/CIP1 and serves to
prevent tetraploid cells from re-entering the cell cycle (63). However,
there is accumulating evidence that p21WAF1/CIP1 may inhibit apoptosis
through interfering with stress-signaling pathways (c-Jun N-terminal
kinase and p38) and binding to pro-caspase-3, thereby preventing its
activation (64). Thus, overexpression of p21WAF1/CIP1 in tetraploid/
polyploid cells may block secondary apoptosis after mitotic slippage
and promote the survival of cells with nuclear aberrations. This suggests that aberrant expression of p21WAF1/CIP1 may indeed contribute to
genomic instability and tumor formation (19,65). In addition, we found
p21WAF1/CIP1 was not only expressed in polyploidy cells but also in
degenerate cells. Chang et al. (19) have shown that inactivation of
the Cdk1cdc2–cyclin B1 complex through transient overexpression of
p21WAF1/CIP1 during mitosis may cause transcriptional inhibition
and rapid loss of mitosis-control proteins, leading to growth arrest,
and abnormal mitosis and endoreduplication in recovering cells.
In tumor therapy, DNA endoreduplication and polyploidization in
response to chemotherapeutic agents have been suggested to be a means
of tumor cells to escape cell death. Importantly, however, it has been
shown that these cells, which can survive for weeks, can resume proliferation and play an important role in tumor relapse (66). Thus, the
potential of polyploid cells to survive for prolonged periods of time and
re-enter the cell cycle, thereby increasing the risk of aneuploidy acquisition, may not only be a key event in the mechanism of OTA carcinogenicity but may also have important implications for the time
of exposure required to induce tumor formation by OTA. In view of
human risk assessment of OTA in food, it is also important to empha-
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size that the dose–response of the transcriptional changes after 90 days
is in perfect agreement with the dose–response for nephrotoxicity,
BrdU labeling and, indeed, tumor formation after chronic treatment.
Thus, the absence of changes at the low dose of 21 lg/kg body wt,
which did not result in increased BrdU labeling (16) or tumor incidence
(1), confirms the non-linearity of the dose–response and provides further support for a thresholded mechanism of OTA carcinogenicity.
In summary, the finding that OTA specifically modulates key regulators of chromosome segregation and progression through mitosis
even after short-term treatment provides crucial evidence in support
of a mechanism of OTA carcinogenicity involving disruption of mitosis and chromosomal instability (Figure 5). Although the precise
mechanism as to how OTA functions to perturb the intricate and
tightly controlled process of mitosis remains to be elucidated, it is
well established that interference with the mitotic machinery and subsequent chromosome segregation errors leading to numerical or structural chromosomal aberrations can introduce multiple genetic
alterations simultaneously and act as a driving force in tumorigenesis
and acquisition of a malignant phenotype.
Supplementary material
Supplementary data and Table 1 can be found at http://carcin.
oxfordjournals.org/
Funding
Deutsche Forschungsgemeinschaft (MA 3323/2 and MA 3323/3);
RCC Ltd, Itingen, Switzerland.
Acknowledgements
The authors would like to thank Dr Gordon C.Hard for helpful discussions,
Andreas Strobel for expert artwork and Theresa Ehrlich, Ursula Tatsch and
Elisabeth Rüb-Spiegel for excellent technical assistance.
Conflict of Interest Statement: None declared.
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Received November 14, 2008; revised February 9, 2009;
accepted February 14, 2009
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