Zhang et al.
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Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic
abnormalities and hyperplasia in mammary glands of mouse models
Dongwei Zhang1, Toru Hirota1, Tomotoshi Marumoto1, Michio Shimizu2, Naoko
Kunitoku1, Takashi Sasayama1, Yoshimi Arima1, Liping Feng1, Misao Suzuki3,
Motohiro Takeya4 and Hideyuki Saya1
1
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences,
Kumamoto University, Kumamoto, Japan
2
3
Department of Pathology, Saitama Medical School, Ohmiya, Japan
Division of Transgenic Technology, Center for Animal Resources and Development,
Institute of Resource Development and Analysis, Kumamoto University, Kumamoto,
Japan
4
Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto
University, Kumamoto, Japan
Correspondence: H. S., Department of Tumor Genetics and Biology, Graduate School of
Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.
E-mail: hsaya@gpo.kumamoto-u.ac.jp
Running title: Mouse models for Aurora-A overexpression in mammary glands
Keywords: Aurora-A; mitosis; mammary gland; p53; Cre-loxP
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Abstract
Aurora-A, a serine-threonine mitotic kinase, was reported to be overexpressed in
various human cancers, and its overexpression induces aneuploidy, centrosome
amplification and tumorigenic transformation in cultured human and rodent cells.
However, the underlying mechanisms and pathological settings by which Aurora-A
promotes tumorigenesis are largely unknown. Here we created a transgenic mouse
model to investigate the involvement of Aurora-A overexpression in development of
mammary glands and tumorigenesis using a Cre-loxP system. The conditional
expression of Aurora-A resulted in significantly increased binucleated cell formation
and apoptosis in the mammary epithelium. The surviving mammary epithelial cells
composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression
in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant
mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein
were remarkably increased in those Aurora-A-overexpressing cells, and the apoptosis
was significantly suppressed by deletion of p53. Given that no malignant tumor
formation was found in the Aurora-A-overexpressing mouse model after a long latency,
additional factors, such as p53 inactivation, are required for the tumorigenesis of
Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse
model is a useful system to study the physiological roles of Aurora-A and the genetic
pathways of Aurora-A-induced carcinogenesis.
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Introduction
Chromosomal instability and aneuploidy are remarkable hallmarks of human cancers
(Cahill et al., 1999; Lengauer et al., 1998). In most cancers, high rates of chromosome
gains/losses leading to aneuploidy have been observed. The causes of aneuploidy
involve failure in various critical mitotic events, including centrosome separation,
chromosome alignment, chromosome segregation and completion of cytokinesis. The
error-free mitosis that is important to genomic integrity is regulated by phosphorylation
reactions driven by several evolutionarily-conserved serine/threonine kinases, known as
mitotic kinases. Mitotic kinases include cyclin-dependent kinase 1 (Cdk1), Polo-related,
NimA-related, Aurora-related and Warts-related kinases (Nigg, 2001).
Mutations in genes encoding Aurora-related kinases induce abnormal mitotic
phenotypes from yeast to higher eukaryotes (Bischoff & Plowman, 1999; Nigg, 2001).
The founding members of this family are Ipl1p from S. cerevisiae and Aurora from
Drosophila melanogaster. Ipl1 mutants display missegregation of chromosomes and
aneuploidy in S. cerevisiae (Chan & Botstein, 1993; Francisco & Chan, 1994). In
Drosophila, mutations of Aurora alleles cause a mitotic arrest with characteristics of
circular monopolar spindles around large centrosome and result in pupal lethality
(Glover et al., 1995). In mammals, three members of this kinase family, Aurora-A, -B
and -C were identified. Recently observations have revealed that Aurora-A kinase
activity is required for various events during mitosis, such as G2-M transition,
centrosome separation, chromosome alignment and cytokinesis (Hirota et al., 2003).
Given that not only elevated expression of Aurora-A but also depletion of Aurora-A
leads to mitotic failure and multinucleation, it is speculated that the proper timing and
amplitude of Aurora-A expression is important for accurate chromosome segregation
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and fidelity of chromosome transmission (Anand et al., 2003; Marumoto et al., 2003;
Meraldi et al., 2002).
The human Aurora-A gene is mapped to chromosome 20q13, a region
frequently amplified in many human cancers (Kallioniemi et al., 1994; Schlegel et al.,
1995). The amplification of Aurora-A gene has been found in approximately 12% of
primary breast tumors and in many human tumor cell lines (Sen et al., 1997; Zhou et al.,
1998), and Aurora-A mRNA and protein are frequently overexpressed in various human
cancers, including breast, colorectal, pancreatic, ovarian and gastric cancers (Bischoff et
al., 1998; Gritsko et al., 2003; Li et al., 2003; Miyoshi et al., 2001; Sakakura et al.,
2001; Tanaka et al., 1999). Furthermore, overexpression of Aurora-A was reported to
transform Rat1 fibroblasts, and these cells formed micronuclei that represented
chromosome instability and grew as tumors in nude mice (Bischoff et al., 1998). Given
that its overexpression leads to centrosome amplification, aneuploidy and tumorigenic
transformation in mammalian cells, Aurora-A is considered to be a potential oncogene.
Moreover, in recent studies of cancer predisposition, human Aurora-A and its mouse
homologue were identified as candidate tumor-susceptibility genes (Ewart-Toland et al.,
2003). Those evidences strongly suggest that Aurora-A plays a role in development of
human malignant tumors. However, the underlying mechanisms and pathological
settings by which Aurora-A promotes tumorigenesis are understood poorly.
In this study, we have generated a transgenic mouse model to investigate the
involvement of Aurora-A overexpression in the development of mammary glands and
tumorigenesis. Using a Cre-loxP system, we achieved the conditional expression of
Aurora-A specifically in the mammary epithelium of adult mice during pregnancy and
lactation. Elevated Aurora-A expression resulted in mitotic failure, leading to
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p53-dependent postmitotic G1 arrest and apoptosis. P53 function is potentially a crucial
factor for suppressing Aurora-A-induced tumorigenesis.
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Results
Mammary gland-specific expression of Aurora-A by a Cre-loxP system in
transgenic mice
To study the function of Aurora-A in mouse mammary gland development, we used a
Cre-loxP-mediated gene-switch approach. We initially generated transgenic mice
carrying a construct of pCAG-loxP-CAT-loxP-Aurora-A (designated
CAG-CAT-Aurora-A mice), which was designed to induce human Aurora-A expression
by Cre-mediated recombination. The CAG-CAT-Aurora-A transgenic mice developed
normally without any abnormalities of the mammary gland or other tissues. To induce
Aurora-A expression specifically in the mammary gland, the CAG-CAT-Aurora-A
transgenic mice were bred with transgenic mice that carried Cre genes under control of
the Wap (whey acidic protein) gene promoter. The WAP-Cre transgene is expressed
almost exclusively in mammary epithelial cells during pregnancy and lactation (Wagner
et al., 1997). In the CAG-CAT-Aurora-A;Wap-Cre transgenic mice, Cre-mediated
excision removes the CAT gene from the construct, allowing expression of the Aurora-A
gene (Figure 1a). PCR analyses revealed that Wap-Cre-mediated recombination was
detected in the mammary glands of late pregnant and lactating
CAG-CAT-Aurora-A;Wap-Cre mice, while no recombination was found in other tissues,
including tail, skin, lung, heart, kidney, spleen (Figure 1b). No recombination was
detected in the mammary glands of virgin CAG-CAT-Aurora-A;Wap-Cre mice. However,
a very low level of recombination was detected in brain tissue.
The Cre-mediated Aurora-A expression in mammary glands was confirmed by
RT-PCR and western blot analyses (Figure 2a and b). Aurora-A mRNA and protein
were expressed specifically in the mammary glands of lactating mice, but not in virgin
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mice. The immunohistochemical analysis also revealed that Aurora-A was specifically
expressed in mammary gland epithelial cells of CAG-CAT-Aurora-A;Wap-Cre mice for
a long period of time (Figure 2c). These data indicate that the
CAG-CAT-Aurora-A;Wap-Cre mice are suitable for analyzing role of Aurora-A in
mammary gland development and tumorigenesis.
Aurora-A overexpression results in the formation of multinucleated cells in
mammary gland
To investigate the effect of Aurora-A overexpression in mammary gland development,
we compared histopathological findings between the CAG-CAT-Aurora-A (control) and
the CAG-CAT-Aurora-A;Wap-Cre mice after the first pregnancy. In both control and
CAG-CAT-Aurora-A;Wap-Cre mice, normal morphological changes due to pregnancy
were observed. These included an increased number of acini per lobule, vacuoles within
the epithelial cell cytoplasm, secretory material within distended lumens, and a hobnail
appearance. These changes were not seen in most mice 12 months after the first
pregnancy, when lobular regression was observed. The CAG-CAT-Aurora-A;Wap-Cre
mice at 3 months after the first pregnancy frequently showed focal hyperplastic lesions
having prominent secretions, a slight loss of cellular cohesion, and foci of cellular
aggregates (Figure 3a), although these appeared to be different from the hyperplastic
alveolar nodules found in MMTV-infected mice (Cardiff & Wellings, 1999). Although
hyperplastic foci were observed frequently in mammary glands that overexpressed
Aurora-A, malignant tumor formation has not been found in 14
CAG-CAT-Aurora-A;Wap-Cre mice or 10 control CAG-CAT-Aurora-A mice over long
periods of follow-up (longer than 15 months after the first pregnancy).
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Histopathological analysis of 10-day lactating CAG-CAT-Aurora-A;Wap-Cre
mice frequently identified foci of cell aggregates and binucleated cell formation (Figure
3b).To confirm whether binucleated cells increase in mammary glands overexpressing
Aurora-A, we examined the mammary epithelium using immunofluorescent analysis for
E-cadherin counterstained with ToTo-3 to label DNA (Figure 3c). We observed that a
significant population of the multinucleated mammary epithelial cells was generated in
10-day lactating CAG-CAT-Aurora-A;Wap-Cre mice (11.9±2.0%), which is 13-fold
greater than control (CAG-CAT-Aurora-A) mice (0.9±1.0%). Interestingly, most of the
multinucleate cells contained two nuclei. The cells containing three nuclei or more are a
very small portion of the whole (Figure 3d). These results suggest that upregulation of
Aurora-A may result in cytokinesis failure, leading to the formation of cells with two
nuclei in vivo.
Elevated Aurora-A expression induces apoptosis
Histopathological analysis of the lactating CAG-CAT-Aurora-A;Wap-Cre mice revealed
that chromatin appears condensed beneath the nuclear membrane in a significant
number of mammary gland cells (data not shown), suggesting that Aurora-A
overexpression induces apoptosis in mammary epithelial cells. To verify this hypothesis,
we performed TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assays (Figure
4a, b, c). Given that mammary epithelial cells are known to undergo programmed cell
death at the end of lactation (Lund et al., 1996), we investigated transgenic mice that
were in the early phase of lactation. In 10-day lactating CAG-CAT-Aurora-A;Wap-Cre
mice, a significant number of apoptotic cells (14.9 + 2.3%) was observed among
mammary epithelial cells while a very small number of apoptotic cells (0.4 + 0.1%)
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were detected in control mice (Figure 4d). These findings indicate that overexpression
of Aurora-A leads to not only mitotic failure but also to apoptosis in mammary
epithelium.
Induction of Aurora-A overexpression leads to mitotic abnormalities and apoptosis
in mouse embryonic fibroblasts (MEFs)
The effects of Aurora-A overexpression on mitotic progression and cell death that we
observed in mammary epithelial cells of CAG-CAT-Aurora-A;Wap-Cre mice were
confirmed using immortalized MEFs established from CAG-CAT-Aurora-A mice. The
growth rate of CAG-CAT-Aurora-A MEFs was indistinguishable from that of MEFs
derived from normal C57BL/6 mice (data not shown). To induce Aurora-A
overexpression, CAG-CAT-Aurora-A MEFs were infected with AxCANCre (Kanegae et
al., 1995), an adenovirus that encodes the Cre enzyme with an artificial nuclear
localization signal. The Aurora-A protein was first detected at 12 hours after the virus
infection, and their protein levels peaked at 48 h after infection (Figure 5a). Induction
of Aurora-A overexpression resulted in significantly increased numbers of cells having
two nuclei (Figure 5b). The number of CAG-CAT-Aurora-A MEFs with two nuclei
increased to more than 10% at 72 h after virus infection (Figure 5c). Staining by
propidium iodide showed frequent abnormal mitotic events after Aurora-A
overexpression, including chromosome misalignment and chromosome missegregation
(Figure 5d).
Fluorescence-activated cell sorting (FACS) analysis revealed that the
proportion of cells at G1/S was reduced while G2/M cells were increased up to
approximately 50% for Aurora-A-overexpressing MEFs compared with control cells
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(CAG-CAT-Aurora-A MEFs infected with adenoviral luciferase) at 48 h after virus
infection (Figure 5e). Furthermore, overexpression of Aurora-A induced a marked
reduction in the proportion of MEFs at G2/M that was also concomitant with an
increase in the size of the subG1 population at 72 h after virus infection, suggesting that
a large number of cells at G2/M phase underwent cell death as a result of the prolonged
expression of Aurora-A (Figure 5e). Based on these findings, we hypothesized that the
binucleated cells induced by Aurora-A overexpression tend to undergo apoptosis. To
address this issue, the fate of Aurora-A-overexpressing binucleated MEFs was followed
by the time-lapse microscopy. At 72 hours after AxCANCre infection, most of the
binucleated CAG-CAT-Aurora-A MEFs died (Supplemental movie 1). Taken together,
these data indicate that overexpression of Aurora-A induces formation of binucleated
cells and subsequently leads to apoptosis.
Overexpression of Aurora-A induces p53-dependent apoptosis
It was shown previously that disruption of chromosome segregation during mitosis or
failure of cytokinesis activates a p53-dependent checkpoint that normally acts in
postmitotic G1 to arrest tetraploid cells. Thus, we speculated that mitotic errors induced
by Aurora-A overexpression may activate the postmitotic checkpoint, resulting in
accumulation of cells arrested in tetraploid G1, followed by cell death. To test this
hypothesis, we analyzed p53 expression in CAG-CAT-Aurora-A;Wap-Cre mice. Levels
of p53 protein detected by western blot analysis were remarkably increased in
mammary epithelium of the lactating CAG-CAT-Aurora-A;Wap-Cre mice but not in
other tissues of the CAG-CAT-Aurora-A;Wap-Cre mice and the mammary glands of
lactating CAG-CAT-Aurora-A and Wap-Cre mice (Figure 6a). Immunohistochemical
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analysis also showed that p53 is accumulated in nuclei of the Aurora-A-overexpressing
mammary epithelial cells, especially in the binucleated cells (Figure 6b). Moreover,
when Aurora-A overexpression was induced in the CAG-CAT-Aurora-A MEFs by
AxCANCre infection, the nuclear accumulation of p53 was specifically detected in the
binucleated cells (Figure 6c). Based on these results, we speculated that p53 is
responsible for the increased apoptosis in the mammary glands that overexpress
Aurora-A. To test this hypothesis, we generated CAG-CAT-Aurora-A;Wap-Cre mice
carrying null alleles for p53 (CAG-CAT-Aurora-A;Wap-Cre;p53-/- mice). TUNEL
staining of the mammary glands of the 10-day lactating mice revealed that apoptosis
was significantly suppressed in the Aurora-A-overexpressing transgenic mice that lack
p53 (Figure 6d, e). These data indicate that p53 plays a crucial role in postmitotic G1
arrest and subsequent apoptosis of the Aurora-A-overexpressing normal cells.
Zhang et al.
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Discussion
Mitotic kinases are key regulators of mitotic progression, and the timing of expression
and activity of those kinases are regulated precisely during the cell cycle to maintain
genomic integrity in mammalian cells. Aurora-A kinase is localized mainly at the
centrosome and mitotic apparatus in normally proliferating cells, and the expression and
activity of Aurora-A are controlled to peak during late G2 to M phase, similarly to other
mitotic kinases (Bischoff et al., 1998; Hirota et al., 2003; Marumoto et al., 2002;
Marumoto et al., 2003). However, immunohistochemical analyses of clinical samples
have revealed that various epithelial cancer cells overexpress Aurora-A, which was
stained diffusely in the cytoplasm of both interphase and mitotic cells (Gritsko et al.,
2003; Li et al., 2003; Tanaka et al., 1999). In addition to these pathological observations,
evidence that overexpression of Aurora-A overrides the cell cycle checkpoint (Anand et
al., 2003; Marumoto et al., 2002) and induces transformation in immortalized rodent
fibroblasts (Bischoff et al., 1998; Zhou et al., 1998) suggests that dysregulation of
Aurora-A expression and activity is a direct cause of sporadic malignant tumors. To
address this issue, we developed a transgenic mouse that conditionally overexpresses
Aurora-A in adult mammary glands using the Cre-loxP recombination system under the
control of the Wap promoter.
Mammary glands that overexpress Aurora-A showed the following findings:
(1) in 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mice, the numbers of binucleated
cells and cell aggregates were increased significantly compared to control
(CAG-CAT-Aurora-A) mice; (2) in 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mice,
apoptosis of mammary epithelial cells was increased; and (3) although hyperplastic
lesions frequently were found in mammary glands of the CAG-CAT-Aurora-A;Wap-Cre
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mice 3 months after pregnancy, no pathological findings indicating malignant
transformation have been found. The conditional expression system employed in the
present study provides insight into the importance of Aurora-A function in mitosis,
apoptosis and tumorigenesis.
Downregulation of Aurora-A is required for completion of mitosis
It has been shown previously that Aurora-A activation is required for multiple events in
mitosis, such as G2-M transition, centrosome maturation, centrosome separation,
metaphase chromosome alignment and cytokinesis (Hirota et al., 2003; Marumoto et al.,
2003; Zhou et al., 1998). We have reported that microinjection of anti-Aurora-A
antibodies into metaphase cells that had completed centrosome separation and
metaphase chromosome alignment subsequently fail to complete cytokinesis, suggesting
that Aurora-A needs to be active for regulating cytokinesis even after the
metaphase-anaphase transition (Marumoto et al., 2003). However, the present study
showed that overexpression of Aurora-A also induces cytokinesis failure, resulting in
binucleated cell formation in both mammary epithelial cells and cultured MEFs. This
finding is consistent with the recent observation, using time-lapse analysis, that MEFs
transfected with Aurora-A frequently fail to undergo cytokinesis (Anand et al., 2003).
Therefore, the activation and the subsequent inactivation of Aurora-A may be required
for completion of cytokinesis. The levels of Aurora-A protein are regulated by
proteasome-mediated degradation. Aurora-A is reportedly degraded in vitro in late
mitosis/early G1 by the Cdh1/Fizzy-related form of the anaphase-promoting
complex/cyclosome (APC/C) (Castro et al., 2002; Honda et al., 2000). Thus, the rapid
degradation of Aurora-A at late anaphase-telophase may be an important event for
Zhang et al.
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completion of cytokinesis. The molecular mechanism by which Aurora-A regulates
cytokinesis is currently under investigation. Our laboratory is attempting to identify
certain specific substrates that are phosphorylated by Aurora-A at metaphase-anaphase
transition and dephosphorylated toward the end of M phase.
Aurora-A overexpression induces p53-dependent postmitotic G1 checkpoint and
subsequent apoptosis in mammary epithelial cells and MEFs
We have shown here that induction of Aurora-A overexpression elicits binucleated cell
formation and that those cells have p53 accumulation. It was previously reported that
the replication of DNA in tetraploid cells that have entered the subsequent G1 phase
without cell division is usually blocked by p53- and pRB-dependent cell cycle arrest,
which is referred to as the postmitotic G1 checkpoint (Borel et al., 2002; Margolis et al.,
2003). Therefore, cytokinesis failure induced by Aurora-A overexpression may trigger
the p53-dependent postmitotic G1 checkpoint to avoid further cell cycle progression.
However, recent reports have indicated that cytokinesis failure alone is not sufficient to
activate the postmitotic “tetraploidy” checkpoint (Uetake & Sluder, 2004) and that
activation of spindle checkpoint is required for this postmitotic checkpoint activation
(Vogel et al., 2004). It is thus possible that Aurora-A overexpression may induce not
only cytokinesis failure but also spindle checkpoint activation, leading to activation of
p53-dependent postmitotic G1 checkpoint.
We have also observed that Aurora-A overexpression induces apoptosis in both
mammary epithelial cells and cultured MEFs. Several studies have indicated that
expression of activated oncogene products, such as myc, generally induce apoptotic cell
death in MEFs (Baudino et al., 2003; Evan et al., 1992; Hermeking & Eick, 1994;
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Wagner et al., 1994). Myc-overexpressing MEFs proliferated less well and had a
considerably higher apoptotic index (10%-15% TUNEL-positive) (Zindy et al., 1998).
Apoptotic cell death induced by activated myc expression in normal cells has been
demonstrated to be p53-dependent. Similarly to the experience in myc-overexpressing
cells, we have found that p53 levels were increased markedly in
Aurora-A-overexpressing mammary epithelial cells and MEFs and that apoptosis was
suppressed significantly in the mammary tissues that lack p53 expression. These
observations suggest that Aurora-A overexpression induces p53-dependent apoptosis.
Recent studies have shown that Wap-Cre-mediated conditional mutation of BRCA1 in
mammary epithelial cells results in increased apoptosis, with a low frequency of tumor
formation which is accelerated in a p53-null background (Xu et al., 1999). Taken
together, activation of p53 appears to play an important role in prevention of oncogeneand antioncogene-associated tumorigenesis.
Aurora-A, p53 and tumorigenesis
Overexpression of Aurora-A promotes changes in ploidy and generates focal
hyperplastic lesions in mammary epithelium. This implies that overfunction of
Aurora-A induces mitotic abnormalities and consequent chromosome instability,
contributing to tumorigenesis. However, no malignant tumor formation was found in
mammary glands overexpressing Aurora-A over 15 months of observation, indicating
that not only Aurora-A overexpression but also further changes are required for tumor
formation.
Recent studies have revealed direct relationships between Aurora-A and p53. It
was reported that p53 interacts with Aurora-A and suppresses the oncogenic activity of
Zhang et al.
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Aurora-A, such as centrosome amplification and cellular transformation (Chen et al.,
2002). These data may partly explain why Aurora-A overexpression alone does not
induce the tumor formation in a short period of the time of observation. On the other
hand, another recent study has demonstrated that the Aurora-A kinase directly
phosphorylates p53 at Ser 315, facilitating Mdm2-mediated ubiquitination and
destabilization of p53 in cancer cell lines, such as H1299 and MCF7 (Katayama et al.,
2004). This finding suggests that Aurora-A overexpression suppresses the p53 tumor
suppressor function, which seemingly contradicts our data. These apparently conflicting
observations regarding the role of Aurora-A in p53 expression might be due to
differences between normal and cancer cells. Alternatively, they may be attributable to
differences in the mode of p53 regulation between normal and cancer cells.
Based on our observations, we postulate that p53 is an important factor that
inhibits tumor progression in Aurora-A-overexpressing mammary glands. Loss of the
p53 function can facilitate genetic instability by leading cells to override the apoptotic
pathway and continue through subsequent cell cycles and is required for Aurora-A to
induce tumorigenesis. Further work may benefit from a focus on proving the correlation
of p53 and Aurora-A in mammary tumorigenesis by long-term follow-up of
CAG-CAT-Aurora-A;Wap-Cre;p53-/- mice.
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Materials and methods
Plasmid construction and production of CAG-CAT-Aurora-A transgenic mice
The transgene vector pCAG-CAT-Aurora-A, which contains a chicken β-actin gene
(CAG) promotor-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-Aurora-A
region, was constructed from pCAG-loxP-CAT-loxp-lacZ by replacing the lacZ gene with
human Aurora-A cDNA. The pCAG-CAT-lacZ plasmid was a gift from Kimi Araki
(Araki et al., 1995). The construct was purified by electrophoresis and elution from
NACS PREPAC (BRL) and used for microinjection. The CAG-CAT-Aurora-A transgenic
mice were produced as described elsewhere (Hogan et al., 1994).
Generation of CAG-CAT-Aurora-A;Wap-Cre transgenic mice and
CAG-CAT-Aurora-A;Wap-Cre;p53-/- mice
CAG-CAT-Aurora-A mice (strain C57BL/6) were mated with Wap (whey acidic
protein)-Cre mice to generate CAG-CAT-Aurora-A;Wap-Cre double transgenic mice.
The F1 offspring that carried the two transgenes were genotyped by PCR using
following primers. The primers for detecting Aurora-A were P1
(5’-AAAGAGCAAGCAGCCCCTGC-3’) and P2
(5’-GAATTCAACCCGTGATATTCTT-3’), and yielded a 750 bp product. The primers
for detecting Cre were Cre1 (5’-AGGTTCGTTCACTCATGGA-3’) and Cre2
(5’-TCGACCAGTTTAGTTACCC-3’), and yielded a 235 bp product. The PCR cycling
profile consisted of 35 cycles of 30 s at 95°C, 30 s at 56°C, and 45 s at 72°C.
CAG-CAT-Aurora-A;Wap-Cre female mice were mated at eight weeks of age and kept
with males for continuous mating. Cre-mediated recombination was detected by PCR
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analysis. Another P3 primer (5’-CTGCTAACCATGTTCATGCC-3’) was used to detect
Cre-mediated recombination. The recombined P3/P2 PCR product was 1.2 kb and the
non-recombined 2.8kb.
CAG-CAT-Aurora-A;Wap-Cre transgenic mice carrying null alleles for p53
were typically generated from crosses of CAG-CAT-Aurora-A;p53+/- mice and
Wap-Cre;p53+/- mice. Mice carrying a null allele of p53 were obtained (Tsukada et al.,
1993) and back-crossed seven generations into a C57BL/6 background prior to crosses
with the CAG-CAT-Aurora-A and Wap-Cre transgenic lines.
Histology and immunofluorescence
For conventional histological analysis, mammary glands were fixed in 10%
phosphate-buffered formaldehyde for 24 h and embedded in paraffin. 3-μm thick
sections were stained with haematoxylin and eosin using standard techniques. For
cryosections, tissues were fixed with 4% paraformaldehyde for 6 h, then transferred to a
30% sucrose solution for 24 h before 8-μm sections were prepared.
Immunohistochemical analysis was performed following the manufacturer’s manual
(Nichirei, HISTOFINE SAB-PO(R) kit). Immunofluoresence was performed as follows:
sections were washed once with PBS, permeabilized with 0.5% Triton X-100 in PBS for
1 h, blocked for 1 h with 5% BSA in PBS, and incubated with primary antibodies in
0.3% BSA in PBS. A rat monoclonal antibody to E-cadherin (Takara) was used at a
dilution of 1:1000. Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies
(Biosource) were used as secondary antibodies. Slides were counterstained with ToTo-3
(Molecular Probes) prior to being mounted under glass coverslips and analyzed by
confocal microscopy (FV300, Olympus).
Zhang et al.
Additional materials and methodology including references can be found in
“Supplemental materials and methods”.
19
Zhang et al.
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Abbreviations
chicken beta-actin (CAG); chloramphenicol acetyltransferase (CAT); TdT-mediated
dUTP-biotin nick end-labeling (TUNEL); mouse embryonic fibroblasts (MEFs);
fluorescein isothiocyanate (FITC); fluorescence-activated cell sorting (FACS).
Acknowledgments
We thank Dr. Kimi Araki (Kumamoto University) for providing pCAG-CAT-lacZ
plasmid; Mr. Takenobu Nakagawa (Kumamoto University) for technical assistance; Drs.
Izumu Saito and Yumi Kanegae (University of Tokyo) for providing adenoviral
luciferase and AxCANCre virus; members of the Saya lab for valuable suggestions; and
members of the Gene Technology Center at Kumamoto University for their
contributions to the technical assistance. This work was supported by the Research for
the Future program of the Japan Society for the promotion of Science and by a grant for
Cancer Research from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan (to H.S.).
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Figure legends
Figure 1 Structure of the pCAG-loxP-CAT-loxP-Aurora-A transgene and
Wap-Cre-mediated recombination in CAG-CAT-Aurora-A;Wap-Cre mice. (a) Plasmid
construction and production of transgenic mice. The conditional transgenic Aurora-A
transgene consists of a CAG promotor, a loxP-flanked CAT gene, followed by human
Aurora-A cDNA. Wap-Cre-mediated recombination excises the floxed CAT gene,
resulting in the expression of Aurora-A. Arrows represent primers used in PCR
recombination analysis. (b) PCR analysis of Wap-Cre-mediated recombination in
CAG-CAT-Aurora-A;Wap-Cre mice. Top: The panels are the 750 bp P1/P2 Aurora-A
PCR product in different tissues from 10-day lactating and 7-week-old virgin
CAG-CAT-Aurora-A;Wap-Cre mice. Bottom: PCR recombination analysis. The
non-recombined P3/P2 PCR product is 2.8 kb (arrowhead) and the recombined is 1.2 kb
(arrow). T, tail; Sk, skin; L, lung; H, heart; K, kidney; Sp, spleen; M, mammary gland;
B, brain.
Figure 2 Wap-Cre-mediated Aurora-A expression in the mammary gland. (a) Expression
of Aurora-A mRNA in CAG-CAT-Aurora-A;Wap-Cre mice. Aurora-A mRNA was
detected in tissues taken from 7-week-old virgin and 10-day lactating
CAG-CAT-Aurora-A;Wap-Cre mice by RT-PCR analysis with a pair of primers, P1 and
P2, that yield a 750 bp product. Aurora-A mRNA was found specifically in the
mammary gland (M) but not in the tail (T) of a 10-day lactating mouse. Control
(β-actin) RT-PCR was performed in the same reaction mixtures. (b) Expression of
Aurora-A protein in the mammary glands of CAG-CAT-Aurora-A;Wap-Cre mice by
western blot analysis. The proteins were taken from mammary glands of two
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7-week-old virgin (lane 1 and 2, control) and two 10-day lactating
CAG-CAT-Aurora-A;Wap-Cre mice (lane 3 and 4). α-tubulin was used as a loading
control. Aurora-A overexpression was detected in lactating mice following the Cre
activity (lane 3, 4). (c) Immunohistochemical analyses for Aurora-A protein expression
in the mammary glands of a 10-day lactating CAG-CAT-Aurora-A mouse and
CAG-CAT-Aurora-A;Wap-Cre mice at 10 days (10d), 30 days (30d), 90 days (90d) and
180 days (180d) after the first lactation. We stained the paraffin-embedded mammary
gland sections using anti-human Aurora-A antibodies at a dilution of 1:1500. Scale bars,
100 μm.
Figure 3 Hyperplastic changes and binucleated cell formation in the mammary glands
of CAG-CAT-Aurora-A;Wap-Cre mice. (a) Histological sections of mammary gland
tissues from a 5-month-old lactating CAG-CAT-Aurora-A mouse (control, left) and a
lactating CAG-CAT-Aurora-A;Wap-Cre mouse 3 months after the first pregnancy (right).
The arrow indicates the area of papillary hyperplasia. Scale bars, 50 μm. (b)
Haematoxylin and eosin staining of a paraffin-embedded mammary gland section from
a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mouse. Scale bar, 20 μm. Note that cell
aggregates and binucleated epithelial cells were frequently observed. (c) Confocal scans
of mammary gland sections from a 10-day lactating CAG-CAT-Aurora-A mouse (control,
left) and a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mouse (right). The tissues
were stained with anti-E-cadherin antibodies (green), which highlights cell-cell contact
regions. DNA was stained with ToTo-3 (blue). The multinucleated epithelial cells are
indicated by arrowheads. Scale bars, 20 μm. (d) Graph showing the increased percentage
of multinucleated cells in mammary glands of 10-day lactating
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CAG-CAT-Aurora-A;Wap-Cre mice contrasted to CAG-CAT-Aurora-A mice. Values
shown are the means from three animals per experimental group in which over 1,000
cells were counted.
Figure 4 Aurora-A overexpression induces apoptosis in mammary epithelial cells. (a-c)
TUNEL staining for apoptotic cells of mammary gland tissues from a 10-day lactating
CAG-CAT-Aurora-A mouse (a, control) and a 10-day lactating
CAG-CAT-Aurora-A;Wap-Cre mouse (b). A higher magnification of the section of (b) is
shown in (c). Scale bars, 50 μm. (d) Graph showing the increased percentage of
apoptotic epithelial cells in mammary glands of CAG-CAT-Aurora-A;Wap-Cre mice in
contrast to CAG-CAT-Aurora-A mice. Values shown are the means from three animals
per experimental group in which over 1,000 cells were counted.
Figure 5 Mitotic abnormalities and apoptosis in Aurora-A-overexpressing MEFs. (a)
MEFs from CAG-CAT-Aurora-A mice were infected with adenoviral luciferase (Luc) or
AxCANCre (Cre) virus. Aurora-A overexpression was observed 24 hours after virus
infection by western blot analysis. (-), untreated. α-tubulin was used as a loading control.
(b) Confocal microscopic analysis shows that multinucleated cells were observed at 48
hours after AxCANCre (Cre) virus infection in MEFs. The MEFs were stained with
anti-α-tubulin antibodies (green), counterstained by propidium iodide (red). Scale bars,
20 μm. (c) The number of nuclei of control MEFs (from normal C57BL/6 mice) and
MEFs from CAG-CAT-Aurora-A mice, before and 72 hours after virus infection (Luc or
Cre), were counted by propidium iodide staining. Values shown are the percentage of
binucleated cells from three independent experiments in which over 300 cells were
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counted. (d) Chromosome misalignment (upper panels) and chromosome
missegregation (lower panels) were observed in CAG-CAT-Aurora-A MEFs at 48 hours
after AxCANCre virus infection. (e) Aurora-A overexpression induces hyperploidy and
apoptosis in MEFs from CAG-CAT-Aurora-A mice. MEFs were infected with
AxCANCre (Cre) and adenoviral luciferase (Luc). DNA contents were measured by
FACScan analysis at 0, 48 and 72 hours after virus infection. Percentages of cells at
G1/S, G2/M and hyperploid cells were calculated. Apoptosis was scored using
propidium iodide-based FACScan analysis to quantitate cells with subG1 DNA content.
Data are means ± SD of values from three independent experiments.
Figure 6 Aurora-A overexpression induces p53-dependent apoptosis. (a) Western blot
analysis of p53 in tissues from a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mouse,
a 10-day lactating CAG-CAT-Aurora-A mouse and a 10-day lactating Wap-Cre mouse.
α-tubulin was used as a loading control. H, heart; K, kidney; Sp, spleen; L, liver; M,
mammary gland; C, colon. (b) Immunohistochemical analysis for p53 in the mammary
gland of a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mouse. A frozen tissue section
was immunostained by anti-p53 antibody. P53 accumulation was observed in
binucleated mammary epithelial cells (Arrows). Scale bar, 20 μm. (c) Specific p53
accumulation in Aurora-A-overexpressing binucleated MEF. MEFs from
CAG-CAT-Aurora-A mouse were infected with AxCANCre virus then undergone
immunofluorescence analysis for p53 at 72 hours after infection. DNA was labeled by
4’,6-diamidino-2-phenylindole (DAPI). (d) TUNEL staining for apoptotic cells of
mammary gland tissues from a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre mouse
(left) and a 10-day lactating CAG-CAT-Aurora-A;Wap-Cre;p53-/- mouse (right). Scale
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bars, 50 μm. (e) Suppression of apoptosis in CAG-CAT-Aurora-A;Wap-Cre;p53-/- mice.
The percentage of TUNEL-positive apoptotic cells in mammary glands was
significantly lower in 10-day lactating CAG-CAT-Aurora-A;Wap-Cre;p53-/- mice than
in CAG-CAT-Aurora-A;Wap-Cre mice. Values shown are the means from three animals
per experimental group in which over 1,000 cells were counted.
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Supplemental materials and methods
RNA extraction, RT-PCR
Total RNA was extracted from tails and mammary glands by the RNeasy Mini Kit
(Qiagen) according to the method recommended by the manufacturer. cDNA was
synthesized from total RNA using an oligo-dT primer according to the manufacturer’s
manual (GIBCO-BRL, Superscript kit). With the cDNA as a template, Aurora-A cDNA
was amplified by PCR with a pair of primers, P1 and P2, and yielded a 750 bp product.
TUNEL assays
Paraffin sections were deparaffinized, rehydrated, and permeabilized. Apoptotic
mammary gland cells were detected by the TdT-mediated dUTP-biotin nick-end
labeling (TUNEL) method with the in situ Apoptosis Detection Kit (Takara) according
to the manufacturer’s instructions.
Western blot analysis
Mouse tissues were obtained when needed after animals were sacrificed, snap-frozen in
liquid nitrogen, and stored at -70°C. Tissue specimens were lysed with PBS/TDS buffer,
as described (Okamoto et al., 2002), and centrifuged at 14,000 g to remove insoluble
material. Equal amounts of protein were electrophoresed on SDS-PAGE gels and
transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat
milk in 0.1% Tween-20/TBS and then incubated with a primary antibody overnight at
4°C. The following primary antibodies were used: rabbit polyclonal antibodies to Cre
(1:4000; Covance); rabbit polyclonal antibodies to p53 (1:1000; Cell Signaling
Technology); mouse monoclonal antibodies to α-tubulin (B-5-1-2; 1:5000; Sigma).
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Rabbit polyclonal antibodies to Aurora-A were generated as described previously
(Marumoto et al., 2002). Membranes were washed three times with 0.1%
Tween-20/TBS and then incubated for 1 h with secondary antibodies in blocking buffer.
The membranes were detected using a chemiluminescence system from PerkinElmer
life sciences.
Mouse embryonic fibroblasts (MEFs)
MEFs from day-14.5 embryos of CAG-CAT-Aurora-A transgenic mice were isolated, as
described previously (Patel et al., 1998) and propagated in Dulbecco's modified Eagle's
medium (DMEM) plus 10% fetal bovine serum. Immortalized MEFs were cultured
continuously for at least 200 days. The AxCANCre virus was constructed and produced
essentially as described (Kanegae et al., 1995). The cells were infected with adenoviral
luciferase or AxCANCre virus (MOI≥20) for 3 h, and then the medium was changed to
virus-free growth medium. After 48 h or 72 h from infection, the cells were harvested for
western blot analysis and flow cytometry. To prepare for flow cytometry, cells were
trypsinized, fixed with 70% methanol, and the DNA were stained with propidium iodide.
Cells were subjected to flow cytometry on FACScan (Becton Dickinson).
Time-lapse microscopy
Cells were grown in 35 mm Delta-T dishes (Bioptechs) in Leibovitz's CO2-independent
medium (GIBCO). Time-lapse series were generated by collecting images every 5 min,
and single focal planes were acquired in lieu of z sections. Each exposure time was 100
ms. Images were acquired with a Sensys-CCD camera controlled by Metamorph
imaging software (Universal Imaging). Cells were maintained at 37°C in a humid
Zhang et al.
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chamber, and mineral oil was overlaid on the medium to minimize pH changes and to
avoid evaporation.
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Supplemental movie 1
A representative time-lapse image of Aurora-A-overexpressing MEFs. MEFs prepared
from CAG-CAT-Aurora-A;Wap-Cre mouse were infected with AxCANCre virus. At 72
hours after infection, the cells were monitored at 5 min intervals by time-lapse
differential interference contract (DIC) microscopy. Two binucleated cells underwent
apoptosis.