Published OnlineFirst May 12, 2009; DOI: 10.1158/1541-7786.MCR-08-0483
Aurora-A Overexpression in Mouse Liver Causes
p53-Dependent Premitotic Arrest during
Liver Regeneration
Chao-Chin Li,1,3 Hui-Yi Chu,1 Chu-Wen Yang,2 Chen-Kung Chou,4 and Ting-Fen Tsai1,3
1
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University;
Department of Microbiology, Soochow University, Taipei, Taiwan; 3Division of Molecular and
Genomic Medicine, National Health Research Institute, Zhunan, Taiwan; and
4
Department of Life Science, Chang Gung University, Tao-Yuan, Taiwan
2
Abstract
Aurora-A, a serine-threonine kinase, is frequently
overexpressed in human cancers, including hepatocellular
carcinoma. To study the phenotypic effects of Aurora-A
overexpression on liver regeneration and tumorigenesis, we
generated transgenic mice overexpressing human Aurora-A
in the liver. The overexpression of Aurora-A after
hepatectomy caused an earlier entry into S phase, a
sustaining of DNA synthesis, and premitotic arrest in the
regenerating liver. These regenerating transgenic livers
show a relative increase in binuclear hepatocytes compared
with regenerating wild-type livers; in addition, multipolar
segregation and trinucleation could be observed only in the
transgenic hepatocytes after hepatectomy. These results
together suggest that defects accumulated after first
round of the hepatocyte cell cycle and that there was
a failure to some degree of cytokinesis. Interestingly,
the p53-dependent checkpoint was activated by these
abnormalities, indicating that p53 plays a crucial role during
liver regeneration. Indeed, the premitotic arrest and
abnormal cell death, mainly necrosis, caused by Aurora-A
overexpression were genetically rescued by p53 knockout.
However, trinucleation of hepatocytes remained in the
regenerating livers of the transgenic mice with a p53
knockout background, indicating that the abnormal mitotic
segregation and cytokinesis failure were p53 independent.
Moreover, overexpression of Aurora-A in transgenic liver led
to a low incidence (3.8%) of hepatic tumor formation after a
long latency period. This transgenic mouse model provides
Received 10/14/08; revised 12/18/08; accepted 12/30/08; published OnlineFirst
5/12/2009.
Grant support: National Science Council grants NRPGM 95HC007 and
NSC96-2752-B-010-004-PAE (T-F. Tsai), NSC96-2320-B-031-001-MY2 (C-W.
Yang), and CMRPD160472 (C-K. Chou) and Ministry of Education, Aim for
the Top University Plan.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Requests for reprints: Ting-Fen Tsai, Department of Life Sciences and Institute
of Genome Sciences, National Yang-Ming University, 155 Li-Nong Street,
Section 2, Peitou, Taipei 112, Taiwan. Phone: 886-2-2826-7293; Fax: 886-22828-0872. E-mail: [email protected]
Copyright © 2009 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-08-0483
678
a useful system that allows the study of the physiologic
effects of Aurora-A on liver regeneration and the genetic
pathways of Aurora-A–mediated tumorigenesis in liver.
(Mol Cancer Res 2009;7(5):678–88)
Introduction
Aurora-A is a serine-threonine kinase that plays a series of
important roles in the regulation of mitotic progression in various organisms. Expression of Aurora-A is cell cycle dependent; it is initiated at late S phase and is at a maximum at
G2-M phase (1, 2). The various functions of Aurora-A correlate
with various different cell cycle events, including centrosome
maturation and separation, bipolar spindle assembly, chromosome alignment, and the transition from prophase to metaphase
as well as cytokinesis (1, 2). Aurora-A is activated by upstream
activators and is further autophosphorylated during mitotic entry (3-5). After mitosis is finished, Aurora-A is rapidly degraded by the anaphase-promoting complex-ubiquitin-proteasome
pathway (6, 7) as well as an ubiquitin-independent mechanism
(8) and this is necessary for the cell to complete cytokinesis at
telophase. Accordingly, the proper timing and the correct level
of Aurora-A expression and activation are required for progression through mitosis. Previous studies have indicated that cytokinesis failure is one of the most significant defects induced by
Aurora-A overexpression in a cell culture system and leads to
centrosome amplification and polyploidy after the first round of
the cell cycle (9, 10). In addition, Aurora-A overexpression can
override the checkpoint induced by defective spindle assembly
and DNA damage, which leads to aneuploidy (11). These
events may all contribute to the carcinogenic transforming activity of Aurora-A.
The effects of Aurora-A overexpression have been investigated intensively in cell culture; these studies suggested that
additional genetic defects might be necessary and that they cooperate with Aurora-A to induce tumorigenicity (10, 12, 13). A
previous study has shown that Aurora-A protein directly interacts with p53. The expression of p53 was found to suppress
Aurora-A–induced centrosome amplification and cellular
transformation (14). On the other hand, Katayama et al. (15)
have shown that Aurora-A directly phosphorylates p53 at
Ser315, leading to its ubiquitination by Mdm2 and proteolysis.
In addition, Liu et al. (16) reported that Aurora-A–phosphorylated p53 at Ser215 abrogates p53 DNA binding and transactivation activity. Accordingly, disruption of this mutual
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Aurora-A Overexpression in Mouse Liver
suppression mechanism between Aurora-A and p53 might trigger checkpoint abnormalities and chromosome instability.
When examined in an animal study, conditional expression
of Aurora-A in a transgenic mouse model using the Cre-loxP
system resulted in aberrant mitosis, increased binucleated cell
formation, and apoptosis in the mammary epithelium. Moreover, hyperplasia but not malignant mammary tumor was found
in the transgenic mice after a long latency (17). In another
transgenic mouse model carrying a mouse mammary tumor
virus-Aurora-A transgene, ∼40% of the mice developed mammary tumors at 20 months of age. Specifically, Aurora-A overexpression led to genetic instability and activation of the AKT
pathway at stages before tumor formation (18). These in vivo
results from the mouse mammary tumor virus-Aurora-A transgenic mice established Aurora-A as an oncogene that causes
malignant transformation of the mouse mammary gland.
The mRNA and protein of Aurora-A have been found to be
frequently overexpressed in various human cancers, including
hepatocellular carcinoma (1, 19). In addition, Aurora-A overexpression is significantly associated with higher-grade hepato-
cellular carcinoma and a poor prognosis (20). Given that
aberrant expression/activity of Aurora-A has been found in hepatocellular carcinoma, the in vivo analysis of Aurora-A in a
mouse model should provide insight into the mechanisms by
which Aurora-A promotes liver tumorigenesis in a physiologic
context. In addition, although Aurora-A is a potential oncoprotein, its effects on hepatocyte cell cycle progression are not
known and its involvement in liver regeneration has not been
explored. In this study, we have generated transgenic mice
overexpressing human Aurora-A protein in the liver and for
the first time studied the phenotypic effects of such overexpression on liver regeneration and tumorigenesis.
Results
Aurora-A Is Expressed in Fetal Liver and Regenerating
Liver of Wild-Type Mice
To identify the potential regulatory roles of Aurora-A in
liver, we examined the temporal expression pattern of AuroraA mRNA by Northern blot hybridization. For fetal liver, high
FIGURE 1. Expression patterns of mouse endogenous Aurora-A mRNA in fetal, postnatal, and adult livers as well as regenerating livers. A. Northern blot
analysis of Aurora-A and Pcna mRNA expression for fetal livers at embryonic stages E11.5 to E18.5, postnatal day 1 (P1), and postnatal day (P7) and adult
mice at 10 wk of age. B. Northern blot analysis of Aurora-A mRNA expression during liver regeneration of wild-type mice after PH. The intensity of the 28S
and 18S rRNA was used as internal control for total RNA loading. C. Western blot detection of Aurora-A protein during liver regeneration of wild-type mice
after PH. The same blot was detected for Hsp70 antibody as a protein loading control. D. Representative photomicrographs of IHC staining of Aurora-A
protein for liver sections prepared from 0, 2, 3, and 4 d (D) after PH. Arrow, mitotic hepatocyte undergoing chromosome segregation. Original magnification,
×200.
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FIGURE 2. Generation of the Aurora-A transgenic mice. A. The PEPCK-Aurora-A transgenic construct. The human Aurora-A cDNA was driven by the
human PEPCK promoter. The AscI and XhoI were used to excise the 4.7-kb insert for pronuclear microinjection. The 1.8-kb EcoRI fragment containing the
intron and human Aurora-A cDNA was used as probe for Southern and Northern analyses. B. Southern blot analysis of transgene (Tg) copy number for
transgenic lines A79 and A80. Genomic DNA was digested with EcoRI for Southern blot hybridization. Wt, wild-type. C. Northern blot analysis of the human
Aurora-A mRNA expression in transgenic lines A79 and A80. The same blots were stripped and rehybridized with mouse Pepck cDNA probe to compare the
mRNA expression of endogenous Pepck gene and the transgene that was driven by the human PEPCK promoter. The expression pattern of the PEPCKAurora-A transgene is slightly different from that of the mouse endogenous Pepck gene; this may be attributable to species difference in terms of the promoter
activity. The intensity of the 28S and 18S rRNA was used as internal control for total RNA loading. D. Expression of Aurora-A protein detected by Western blot
and IHC staining in transgenic mice. Liver extracts for Western blotting were prepared from transgenic mice (lines A79 and A80), wild-type mice, and wild-type
mice at 2 d after hepatectomy. Liver sections for IHC staining were prepared from wild-type and transgenic mice (line A79) without any treatment. Original
magnification, ×200. All of the liver samples were obtained from 2-mo-old male mice.
expression level of Aurora-A mRNA was sustained until embryonic day 15.5 (E15.5). The signals declined gradually thereafter
in the embryonic livers and postnatal newborns until expression
was barely detectable in the adult liver (Fig. 1A). Interestingly,
the temporal expression pattern of Aurora-A coincided with the
expression of the proliferation marker proliferating cell nuclear
antigen (Pcna; Fig. 1A), implying that Aurora-A expression was
correlated with ongoing hepatocyte proliferation in mouse liver.
Although most hepatocytes are arrested at quiescent stage in
adult liver, quiescent hepatocytes are able to reenter the cell cycle during liver regeneration, which is inducible by partial hepatectomy (PH; ref. 21). Our data indeed showed that expression
of the Aurora-A mRNA was transiently induced at 2 days after
hepatectomy, which coincides with G2-M phase during hepatocyte cell cycle progression, and then rapidly decreased to basal
level 3 days after hepatectomy (Fig. 1B). A peak induction of
the Aurora-A protein was also detected at 2 days after hepatectomy and gradually decreased thereafter when visualized by
Western blot analysis (Fig. 1C). Immunohistochemical (IHC)
staining revealed that Aurora-A protein was mainly localized
to the nuclei of the hepatocytes at 2 days after hepatectomy,
but Aurora-A did show additional staining in the cytoplasm at
3 to 4 days after hepatectomy (Fig. 1D).
Generation of Aurora-A Transgenic Mice
We successfully generated transgenic mice overexpressing
human Aurora-A in the liver. The plasmid used to generate
the transgenic mice is shown in Fig. 2A. Two founders of the
PEPCK-Aurora-A transgenic mice were generated. The transgenic copy numbers of lines A79 and A80 are about 2 and 7,
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Aurora-A Overexpression in Mouse Liver
respectively, by Southern analysis (Fig. 2B). Strong expression
of human Aurora-A mRNA was detected in the transgenic livers of lines A79 and A80, with a lower expression level detected in the other tissues (Fig. 2C). Elevated levels of the
Aurora-A protein were shown by Western blot analysis and
IHC staining (Fig. 2D). There was no observable change in
liver development of the Aurora-A transgenic mice up to adulthood at around 8 weeks old. There was no significant difference in nuclear polyploidy (Supplementary Fig. S1A) and the
baseline proliferation of hepatocytes revealed by IHC staining of the Ki67 marker (Supplementary Fig. S1B) in the
9-week-old Aurora-A transgenic and wild-type mice before the
PH surgery.
Overexpression of Aurora-A Caused an Earlier Entry into
S Phase, a Sustaining of DNA Synthesis, and Premitotic
Arrest in the Regenerating Livers
To investigate the effect of Aurora-A overexpression on
liver regeneration, we analyzed hepatocyte cell cycle progression during liver regeneration induced by PH. To avoid possible artifacts attributed to the different integration sites of the
transgene, both of the transgenic lines (A79 and A80) were
analyzed. DNA synthesis was assessed by bromodeoxyuridine
(BrdUrd) incorporation and detected by IHC staining. In the
wild-type mice, the majority of the BrdUrd-positive hepatocytes were detected at 1.75 to 2 days; furthermore, there
was a peak at 2 days and no or very few BrdUrd-positive
cells could be detected at 3 days and thereafter after hepatectomy (Fig. 3A). In the Aurora-A transgenic mice, interestingly, there was a significantly increased percentage of the
BrdUrd-positive hepatocytes detected at 1.75 days, which indicates an earlier entry into S phase. In addition, the duration
of the DNA synthesis was extended to 4 days after hepatectomy (Fig. 3A). In the wild-type mice, the number of mitotic
figures peaked at 2 days and decreased thereafter until 4 days
after hepatectomy (Fig. 3B). Notably, a significant decrease in
the mitotic figures was observed over the mitotic period from
2 to 4 days after hepatectomy in both transgenic lines (A79
and A80; Fig. 3B), which suggest that hepatocyte cell cycle
progression was likely to be partly blocked before the mitotic
phase in the regenerating livers of the Aurora-A transgenic
mice.
To further study the abnormal hepatocyte cell cycle progression, we measured the mRNA expression of various cell
cycle–associated markers, including Cyclin D1 (G1 phase),
cyclin-dependent kinase (Cdk) 2 (G1-S phase), Pcna (S-G2M phase), Cyclin B1 (G2-M phase), Cdk1 (G2-M phase),
and mouse endogenous Aurora-A (G2-M phase). Abnormalities in the induction and temporal expression for both Sphase–associated and G2-M-phase–associated markers were
observed in the regenerating livers of the Aurora-A transgenic
mice. For the S-phase marker, a transient peak of induction
was detected at 2 days and the mRNA had decreased to a
basal level at 3 days after hepatectomy in the wild-type mice
(Fig. 3C). In the Aurora-A transgenic mice, there was an earlier induction of the S-phase markers at 1 day and these
peaked at 1.75 days after hepatectomy. In addition, there
was a second wave of the S-phase marker induction at 3 to
4 days after hepatectomy in the Aurora-A transgenic mice
FIGURE 3. Earlier entry of S phase, prolonged DNA synthetic period,
and premitotic arrest in the regenerating livers of the Aurora-A transgenic
mice. A. DNA replication as monitored by BrdUrd incorporation. About
1,000 hepatocytes in random fields of BrdUrd staining slides were examined for the presence of BrdUrd-positive staining for each mouse. Three to
five mice per group were used for BrdUrd examination. B. Mitotic indices
of the regenerating livers. About 1,000 hepatocytes in random fields of liver
sections were examined for the presence of mitotic figures for each
mouse. Three to five mice per group were used for examination of mitotic
figures. The mean for each time point is expressed as a percentage of total
hepatocytes counted. Columns, mean; bars, SD. C. Expression of the cell
cycle–associated genes Cyclin D1 (G1), Cdk2 (G1-S), Pcna (S-G2-M), Cyclin B1 (G2-M), Cdk1 (G2-M), and mouse endogenous Aurora-A (G2-M) in
the wild-type and Aurora-A transgenic (line A79) mice after PH. Total RNA
(5 μg) from each sample was used for slot blot hybridization using mouse
cDNA as the probes and 28S rRNA was used as a control for all RNA
hybridization. Quantification of the hybridization signal was analyzed using
ImageQuant software from Molecular Dynamics. The results are presented
as the fold of induction relative to PH 0 day. Points, mean; bars, SD. *, P <
0.05; **, P < 0.01.
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(Fig. 3C). These S-phase marker expression profiles are consistent with the DNA synthesis results monitored by BrdUrd
incorporation (Fig. 3A). For the G2-M markers, a transient
peak of induction was also detected at about 2 days and the
mRNA levels decreased to a basal level from 3 days after
hepatectomy in the wild-type mice (Fig. 3C). Notably, in
the Aurora-A transgenic mice, there was an earlier induction
of the G2-M markers at 1.75 days and a significantly higher level of the G2-M gene expression was sustained until 3 to 4 days
after hepatectomy (Fig. 3C). Because a significantly lower percentage of the mitotic hepatocytes was observed over the complete mitotic period from 2 to 4 days after hepatectomy in the
transgenic mice (Fig. 3B), these results indicated that Aurora-A
overexpression accelerated hepatocytes entering into G2 phase,
but these cells showed an impaired progression across the G2-M
transition and this resulted in premitotic arrest.
Cytokinesis Failure Seems to Follow G 2 -M Transition
Impairment and the Latter Is Likely to Be Caused by
the p53-Dependent Checkpoint in the Regenerating
Livers
To examine whether Aurora-A overexpression causes cytokinesis failure leading to binucleation in the hepatocytes of the
regenerating liver, we counted the ratio of hepatocytes with
double nuclei. Although binuclear hepatocytes are normally
present in the nonregenerating liver, the percentage of binuclear
hepatocytes was significantly increased at 4 days after hepatectomy in the regenerating livers of the Aurora-A transgenic mice
when compared with the regenerating wild-type control
(Fig. 4A). This indicates that cytokinesis failure may occur in
the hepatocytes with Aurora-A overexpression. This conclusion
is supported by the detection of tripolar segregation and trinucleation of hepatocytes in the Aurora-A transgenic mice after
hepatectomy (Fig. 4B). A frequency of between 0.1% and
0.2% of hepatocytes showed trinucleation in the two AuroraA transgenic lines and no such phenomenon was ever found
in the wild-type mice (data not shown). Our results provide important clues and suggest that Aurora-A overexpression in hepatocytes seems to result in centrosome amplification,
multipolar segregation, and multinucleation after cytokinesis
failure under physiologic conditions.
To evaluate if the p53-dependent checkpoint is induced by
Aurora-A overexpression during liver regeneration, we analyzed the expression levels of p53 protein by Western blot analysis. Our data showed that p53 protein was remarkably
up-regulated at 2 and 4 days after hepatectomy (Fig. 4C).
Quantification revealed that there was a 3.5- and 2-fold increase
in the p53 protein level at 2 and 4 days after hepatectomy, respectively, in the transgenic mice (Fig. 4D). These results implied that p53 acts as the crucial checkpoint in hepatocyte cell
cycle progression during liver regeneration and its expression
would seem to be triggered by the abnormal events mediated
by Aurora-A overexpression in the transgenic mice.
Premitotic Arrest and Necrosis Caused by Aurora-A
Overexpression Were Genetically Rescued by p53
Knockout
To test the hypothesis that the p53 checkpoint plays a pivotal
role in the regulation of hepatocyte cell cycle progression and is
FIGURE 4. Increased numbers of binuclear hepatocytes, tripolar spindle segregation and trinucleation, and up-regulation of p53 protein in the
regenerating livers of the Aurora-A transgenic mice. A. The percentage of
hepatocytes with double nuclei in the regenerating livers of wild-type and
Aurora-A transgenic mice. About 1,000 hepatocytes in random fields of
liver sections were examined for the presence of double nuclei for each
mouse. Three to five mice per group were used for examination of double
nuclei. The mean for each time point is expressed as a percentage of total
hepatocytes counted. Columns, mean; bars, SD. B. Trinucleation and tripolar spindle segregation, as indicated by arrows, observed in the regenerating livers of the Aurora-A transgenic mice. The representative
photomicrographs of BrdUrd staining slides were prepared from transgenic
mice at 4 d after hepatectomy. Original magnification, ×400. C. Western
blot analysis of p53 protein in the regenerating livers of wild-type and Aurora-A transgenic mice. Protein lysates were prepared from three individual mice at the indicated time points after PH. Signals of the Hsp70 protein
were used as internal control for protein loading. D. Quantification of the
p53 protein levels in the regenerating livers. At 2 d after hepatectomy,
there was a 3.5-fold (transgenic mice, 3.53 ± 0.51; wild-type, 1.00 ±
0.33) increase in the p53 protein level. At 4 d after hepatectomy, there
was a 2-fold (transgenic mice, 3.50 ± 0.62; wild-type, 1.80 ± 0.21) increase
in the p53 protein level. The results are presented as the fold of induction
relative to the wild-type mice at 2 d after hepatectomy. Columns, mean;
bars, SD. *, P < 0.05; **, P < 0.005.
responsible for the premitotic arrest caused by Aurora-A overexpression, we generated Aurora-A transgenic mice (line A79)
carrying two null alleles for the p53 gene (Aurora-A;p53−/−
mice). Cell proliferation indices, including DNA synthesis
(BrdUrd incorporation) and mitotic index, were analyzed
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Aurora-A Overexpression in Mouse Liver
FIGURE 5. Knockout of p53 genetically rescued the phenotypes of premitotic arrest and abnormal cell death in the regenerating livers of the Aurora-A
transgenic mice. A. DNA replication as monitored by BrdUrd incorporation. The DNA synthesis level of Aurora-A;p53−/− (Y) has returned to the normal
control level (X) at 4 d after hepatectomy. B. Mitotic index of the regenerating livers. Liver samples were prepared from wild-type mice, Aurora-A transgenic
mice (Aurora-A), Aurora-A transgenic mice with p53 homozygous knockout (Aurora-A;p53−/−), and p53 homozygous knockout mice (p53−/−). The methods
for quantification of BrdUrd incorporation and mitotic index are the same as in Fig. 3. The sum of the mitotic events (X) and the sum of the mitotic events (Y)
are approximately equal. C. Liver/body weight for different genotypes of mice at different time points after hepatectomy. *, P < 0.05; **, P < 0.005. D. H&E
staining for the wild-type mice, Aurora-A transgenic mice, Aurora-A;p53−/− mice, and p53−/− mice at 0, 3, and 4 d after hepatectomy. Blue arrows, necrotic
area in the Aurora-A transgenic mice. Please note that the presence of trinuclear hepatocytes (black arrows; insets) in the Aurora-A;p53−/− mice is similar to
the situation in the Aurora-A transgenic mice with p53 wild-type background (Fig. 4B). The transgenic line A79 was used for crossing with the p53 knockout
mice. Original magnification, ×200.
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during liver regeneration and these were compared across the
different mouse genotypes (i.e., wild-type, Aurora-A transgenic, Aurora-A;p53−/−, and p53−/− mice). For DNA synthesis,
notable change was detected at 4 days after hepatectomy
(Fig. 5A). In the Aurora-A;p53−/− mice, the percentage of
BrdUrd-positive hepatocytes was significantly decreased (P <
0.001) compared with the Aurora-A transgenic mice (AuroraA, 8.28 ± 0.99%; Aurora-A;p53−/−, 2.38 ± 0.73%) and the
results were comparable with those found for the wild-type
mice at 4 days after hepatectomy (Fig. 5A). For mitotic index,
the percentage of mitotic cells was remarkably increased (P <
0.001) at 3 days after hepatectomy in the Aurora-A;p53−/−
mice compared with the Aurora-A transgenic mice (Fig. 5B).
Interestingly, the total numbers of the mitotic hepatocytes at
2 and 3 days after hepatectomy in the Aurora-A;p53−/− mice
(1.4 ± 0.65% at 2 days after hepatectomy; 2.59 ± 0.62% at
3 days after hepatectomy) are about equal to that of the wildtype mice (2.47 ± 1.08% at 2 days after hepatectomy; 1.27 ±
0.29% at 3 days after hepatectomy; Fig. 5B). Although the timing of mitosis was delayed slightly in the Aurora-A;p53−/−
mice, these results indeed provide in vivo evidence that the impairment of G2-M transition and premitotic arrest of hepatocyte
cell cycle caused by Aurora-A overexpression is genetically
rescued by a p53−/− background. Furthermore, the observation
that the premitotic arrest was rescued at 3 days after hepatectomy also provided an explanation as to why DNA synthesis returns to a normal level at 4 days after hepatectomy as shown in
Fig. 5A. The overall ability of the liver to regenerate was determined by measuring the liver weight and the ratio of liver to
body weight. There was no significant difference in the rate of
mass reconstitution in the Aurora-A transgenic livers compared
with the wild-type (Supplementary Fig. S2; Fig. 5C). It has
been previously reported that regaining of liver mass is a general phenomenon in mouse models despite the fact that they are
defective in liver regeneration; this mostly likely results from
cell size increase (i.e., hypertrophy of hepatocytes; refs. 2225). Interestingly, the ratios of regenerating liver to body
weight for the Aurora-A;p53−/− mice were significantly higher
than for the other genotypes of mice 5 days after hepatectomy
(Fig. 5C). In addition, the proliferation indices, including DNA
synthesis and mitosis, normally decrease to basal levels in the
wild-type mice 4 days after hepatectomy. However, the proliferation indices were persistently higher and detectable in the
Aurora-A;p53−/− mice 4 days after hepatectomy (Fig. 5A
and B). These results clearly indicated that there is a synergistic
effect between the two genetic events, Aurora-A overexpression and p53 knockout, for promotion of cell cycle progression
in the regenerating liver.
To rule out the possibility that the expression of human
Aurora-A cDNA driven by the human PEPCK promoter was
affected in the knockout background of p53 gene because there
was a regulatory role on the PEPCK promoter, we monitored
the mRNA levels of the mouse endogenous Pepck gene. Our
data revealed that there was no significant difference in mouse
Pepck mRNA expression among different groups of mice during liver regeneration (Supplementary Fig. S3). Furthermore,
the Aurora-A protein levels were directly examined by Western
blot. Previously, a transgenic mouse study has revealed that
transgenic Aurora-A protein was protected from degradation
between G2-M but was rapidly degraded and barely detectable
between G1-S despite an elevation of the Aurora-A mRNA
levels (26). Our results were similar and showed a cell cycle–dependent turnover of the Aurora-A protein. Although
the Aurora-A protein level is a little higher in the transgenic
mice than wild-type control in the resting liver, there is a very
significant increase in Aurora-A protein in the regenerating livers and much bigger differences compared with the transgenic
and wild-type mice. Moreover, there was no significant change
in the Aurora-A protein levels during liver regeneration when
transgenic mice with and without the p53 knockout were compared (Supplementary Fig. S4).
To examine whether there is apoptosis occurring in the
regenerating livers of the Aurora-A transgenic mice, we have
done a terminal deoxynucleotidyl transferase–mediated dUTP
nick end labeling (TUNEL) assay. Our data revealed that
there was no detectable TUNEL-positive cells in the liver
sections prepared from transgenic and wild-type control mice
after hepatectomy; this analysis included different time points
of liver samples from day 1 to day 7 after hepatectomy in
both groups of mice (data not shown). However, histopathologic analyses of liver sections revealed that abnormal cell
death, mainly necrosis, was reproducibly detectable in the
Aurora-A transgenic mice at 3 and 4 days after hepatectomy
(Fig. 5D); there is between 5% and 10% of the liver parenchyma with necrotic hepatocytes in their liver sections. In
addition, histologic examination revealed the presence of vacuoles at 4 days after hepatectomy, indicating hepatic fatty
change (microsteatosis; Fig. 5D). Our findings indicate that
overexpression of Aurora-A leads not only to impairment
of the G2-M transition but also to necrosis during liver regeneration. Interestingly, the abnormal histopathology, including necrosis and fatty changes, was absent in the liver
sections of the Aurora-A;p53−/− mice at 3 and 4 days after
hepatectomy (Fig. 5D), showing that elimination of the p53
checkpoint indeed rescues the premitotic arrest and histopathologic abnormalities caused by Aurora-A overexpression during liver regeneration.
Overexpression of Aurora-A in Liver Led to a Low
Incidence of Hepatic Tumor Formation after a Long
Latency Period
To study the potential effect of Aurora-A overexpression on
liver tumorigenesis, we followed different ages of transgenic
mice for tumor formation. Furthermore, to evaluate whether
the genetic background made any difference to liver tumorigenesis, we bred the Aurora-A transgenic mice (line A79) into a
series of different genetic background, including FVB/N inbred, FVB/N mixed with C57BL/6, and BALB/c background.
After 25 months of follow-up, no hepatic tumors were found in
24 Aurora-A transgenic mice with the FVB/N inbred background. Two transgenic mice (AG11 and AH7) with the mixed
genetic background were found to have developed hepatic tumor (Supplementary Table S1; Fig. 6A). Overall, there was an
incidence of 3.8% (2 of 52) for hepatic tumor formation observed in Aurora-A transgenic mice with a variety of different
genetic backgrounds (Supplementary Table S1). Histopathologic examination revealed that the tumors in specimens AG11 and
AH7 were hyperplastic nodules resembling hepatocellular
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Aurora-A Overexpression in Mouse Liver
FIGURE 6. Low incidence of hepatic tumors developed at around 15 to
21 mo of age in the Aurora-A transgenic mice. A. Gross view of the hyperplastic nodules observed in male mice of Aurora-A transgenic line A79.
Sample AH7 was collected at 15 mo of age with a mixed genetic background, namely, FVB/N and C57BL/6 (50%-50%). Sample AG11 was collected at 21 mo of age with a mixed genetic background, namely, FVB/N
and BALB/c (50%-50%). B. Representative photomicrographs of H&E
staining of the hyperplastic nodule of sample AG11. Pathologic examination indicated that the nodule is a benign tumor. Histopathologic analysis of
sample AH7 is shown in Supplementary Fig. S5.
adenoma (Fig. 6B; Supplementary Fig. S5). In specimen
AH7, severe fatty changes were observed in multiple hyperplastic nodules (Supplementary Fig. S5). We also followed the
tumor incidence among Aurora-A transgenic mice with a p53
heterozygous background (Aurora-A;p53+/−) to study their
synergistic effect on liver tumorigenesis. However, no hepatic
tumors were found in 16 Aurora-A;p53+/− mice with the
FVB/N inbred background after 25 months of follow-up.
Among Aurora-A transgenic mice with the p53 homozygous
knockout (Aurora-A;p53−/−), because the p53−/− mice usually die with sarcoma or lymphoma at around 4 to 6 months of
age before hepatic tumor formation, the issue will be answered in a separate study using mice carrying a liver-specific
p53 knockout allele.
Discussion
The level of Aurora-A protein is tightly cell cycle regulated;
it is degraded rapidly toward the end of mitosis and before cytokinesis in mammalian cells (6-8, 27). Previous studies have
revealed that Aurora-A protein is a short-lived protein that has a
rapid turnover rate with a half-life of ∼2 hours (6). The periodic
degradation of Aurora-A protein is crucial for modulating cell
cycle progression and maintaining genomic stability (1, 2).
Here, we show that there was a peak induction of Aurora-A
protein detected at 2 days after hepatectomy, which coincides
with G2-M phase during hepatocyte cell cycle progression, and
the protein rapidly decreased to a basal level 3 days after hepatectomy in the wild-type mice. However, in the transgenic
mice, the total amount of Aurora-A protein (transgenic plus endogenous Aurora-A) in the regenerating livers is significantly
higher than that of wild-type livers from day 1 to day 7 after
hepatectomy (Supplementary Fig. S4). Consequently, the enhanced levels of Aurora-A protein, which are also expressed
at an inappropriate timing, seem to cause the phenotypic effects
in the regenerating liver; these phenotypic abnormalities include earlier entry into S phase, premitotic arrest, and cytokinesis failure. In this regard, the Aurora-A transgenic mice indeed
mimic the situation observed in human cancer cells, which
frequently exhibit overexpression of Aurora-A protein regardless of the cell cycle stage (28).
Our results show for the first time that Aurora-A overexpression seems to cause cytokinesis failure and tetraploidization in the regenerating livers of transgenic mice. During
liver regeneration induced by two-third PH in mice, the regenerating liver theoretically requires 1.66 proliferative cycles per
residual hepatocyte to restore the original number of hepatocytes and complement the original liver mass. The first peak
of the hepatocyte cell cycle is initiated at around 2 days and
the second one appears at 3 to 4 days after hepatectomy (21).
In this study, several abnormal events, including cytokinesis
failure, as indicated by a significant increase in binuclear hepatocytes, tripolar segregation, and trinucleation of hepatocytes,
were observed at 4 days after hepatectomy. This suggests that
there was an accumulation of defects after the first round of cell
cycle and these may lead to pathologic changes, such as necrosis and cell death, in the regenerating livers of the Aurora-A
transgenic mice. Notably, tripolar segregation and trinucleation
of hepatocytes were unique phenomena that were only detectable in the Aurora-A transgenic mice. Centrosome amplification may result in multipolar segregation, which in turn gives
rise to multinucleation. These abnormal events could be regarded as the precursors of aneuploidy and genomic instability
(29, 30).
The p53-dependent checkpoint is likely to be an important
method of preventing cells from transformation by Aurora-A
overexpression (31-33). Our results revealed that the p53
checkpoint was indeed activated in the regenerating liver
at 4 days after hepatectomy and this activation followed multipolar segregation and cytokinesis failure, which were induced by Aurora-A overexpression. Specifically, distinct
trinucleation was still present in the Aurora-A transgenic mice
with the p53 knockout background as shown in Fig. 5D,
indicating that abnormal mitotic segregation and cytokinesis
failure were not p53 dependent. Previously, it was shown
in the mammary gland conditional Aurora-A transgenic
mouse model that cytokinesis failure and multinucleation activated the p53-dependent checkpoint, which in turn acts in
the postmitotic G1 phase to arrest tetraploid cells followed
by cell death (17). Our observations show that, in a similar
manner to the mammary gland, p53 plays a crucial role in
cell cycle arrest in the regenerating livers of the Aurora-A
transgenic mice.
Using linkage analysis and haplotype mapping of interspecific mouse crosses in a search for low-penetrance susceptibility genes for cancer predisposition, Ewart-Toland et al.
(34) identified Aurora-A gene as a candidate skin tumor oncogenic gene. The authors showed that a common genetic
variant in Aurora-A, 91T→A (Phe31Ile), is preferentially amplified in tumor cells and is associated with aneuploidy and
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686 Li et al.
chromosome instability. The Ile31 allele is a high-risk allele
that induces more rapid cell growth of rat cells and increased
xenograft tumorigenicity in nude mice than the Phe31 low-risk
allele (34). In another Aurora-A association analysis for risk of
cancer development in humans, Ewart-Toland et al. (35)
showed that Aurora-A is a general cancer susceptibility gene
for multiple tumor types with low penetrance. The results from
the Aurora-A tumorigenesis study using the transgenic mouse
approach showed that conditional expression of Aurora-A led
to hyperplastic focus formation in the mammary glands but no
malignant tumors were found over a long period of follow-up
(17). In another transgenic study, the Aurora-A protein was directly expressed in the mammary gland using the mouse mammary tumor virus promoter. This study revealed that
overexpression of Aurora-A could cause mammary tumor formation in ∼40% of transgenic mice at ∼20 months of age (18).
In the present study, we overexpressed the human Aurora-A
cDNA, which was obtained from HuH-7 hepatoma cells by reverse transcription-PCR (Phe31; Supplementary Fig. S6), in
mouse liver and showed that Aurora-A overexpression could
cause low incidence (3.8%) of hepatic tumor formation but
not malignant hepatocellular carcinoma; this was also at long
latency in the transgenic mice. Our study in liver is consistent
with the previous studies and supports Aurora-A as a low-penetrance susceptibility gene in both human and mouse for tumor
formation, which may modify cancer risk and thereby accelerate cancer formation by cooperation with additional factors,
such as p53 deficiency.
Materials and Methods
Generation of Aurora-A Transgenic Mice
The PEPCKex vector contains the human PEPCK promoter,
a 0.3-kb ApoAI intron, and polyadenylate signal of human
growth hormone gene as previously described (36). The
PEPCK-Aurora-A transgenic construct was generated by inserting the human Aurora-A cDNA into the PEPCKex plasmid.
The human Aurora-A cDNA used in this study was cloned from
HuH-7 hepatoma cell line by reverse transcription-PCR; the
amino acid sequence is shown in Supplementary Fig. S6. The
Aurora-A transgenic mice were generated by pronucleus microinjection of FVB/N fertilized eggs (37). The transgenic mice
were bred in a specific pathogen-free facility. The genotypes
of the mice were determined by PCR and/or Southern analysis
of genomic DNA isolated from mouse tails (38). To detect the
Aurora-A transgene, the 515-bp DNA fragment was amplified
by PCR using the primers 5′-GGGTAAAGGAAAGTTTGGTAATG-3′ and 5′-CCACCTTCTCATCATGCATCC-3′. The
transgenic copy numbers were estimated by comparing the relative intensity of the transgene with the copy number controls
using Southern blot hybridization. We mixed different amounts
of transgenic DNA equal to 1, 5, 10, 25, and 50 copies of the
transgene per diploid mouse genome with liver genomic DNA
isolated from wild-type mice to mimic the transgenic mouse
genome. For example, to mimic that there is one copy of a 6kb transgene present in the mouse genome, we mix 10 μg of
liver genomic DNA with 20 pg of the 6-kb transgenic DNA
fragment.
Collection of Fetal and Postnatal Livers
FVB/N wild-type females were bred with wild-type males
for collection of embryos at different embryonic stages. Virginal
plugs were checked in the morning before 10:00 a.m. Sampling of the embryos was carried out between 11:00 a.m. and
1:00 p.m. to obtain fetal livers at embryonic stage E11.5 to
E18.5 (vaginal plug = embryonic stage E0.5). Postnatal day
1 and day 7 livers were also obtained from FVB/N inbred
mice (37).
PH and Liver Regeneration
In this study, all PH experiments were done using male mice
at 9 to 10 wk of age; age-matched transgenic and wild-type
mice with the same inbred background (i.e., FVB/N) were used
for comparison. They were treated under the regulations of the
“Guide for the Care and Use of Laboratory Animals” (39).
About 70% of the liver, including the median and left lateral
lobes, was removed after they had been anesthetized with
50 mg/kg ketamine i.p. (40, 41). There was a mortality rate
of 15% to 20% recorded during the 7 d of observation period
after the surgery; this mortality rate is similar in the wild-type
mice and Aurora-A transgenic mice with or without p53 knockout background. Regenerated liver tissues were harvested from
five to nine surviving mice for each time point in each group of
mice.
Trp53 (p53) Knockout Mice
The p53 knockout mice were obtained from JAX (BALB/c
congenic strain). The p53 heterozygous mice were backcrossed
to FVB/N inbred mice for five generations to obtain FVB/N
congenic strain (∼97% FVB/N background). Allele-specific
PCR was used to distinguish the wild-type and knockout p53
alleles (42).
RNA Analysis
Total RNA was isolated using Trizol Reagent (Life Technologies). Northern blot and slot blot hybridization were done
as described previously (38). The cDNA probe for mouse
Aurora-A (NM_011497, from base 1208 to 1721) was generated by reverse transcription-PCR. The Cyclin B1 cDNA (RIKEN clone H3076D10, NCBI BG078426) was obtained from
the NIA mouse 15K cDNA library. The cDNA probes for
Cyclin D1, Pcna, Cdk2, and Cdk1 were prepared as previously described (36). The hybridization signal was standardized
against the intensity of the 28S rRNA for all of the RNA
samples.
Western Blot and IHC Analyses
Western blot was done as described previously (43) and detected using Visualizer kit (Upstate). The following antibodies
were used: Aurora-A (recognizes both human and mouse Aurora-A proteins; BD Biosciences), p53 (BD Biosciences),
Hsp70 (BD Biosciences), and β-tubulin (Upstate). IHC staining
of Aurora-A protein was done using paraffin-embedded liver
sections (3 μm). Liver sections were soaked in 10 mmol/L sodium citrate of antigen retrieval buffer (pH 6.0), placed in a microwave oven (650 W; Sunpentown), and irradiated for 5 min
at superpower and 15 min at defrosting power. The sections
were then incubated with primary antibody against Aurora-A
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Aurora-A Overexpression in Mouse Liver
(1:50; BD Biosciences) at 4°C for 18 to 24 h and visualized by
the Chemicon IHC Select System.
DNA Synthesis and Mitotic Index
The number of S-phase cells was estimated by BrdUrd
incorporation into nuclei. Mice were injected with BrdUrd
(100 mg/kg; Sigma) i.p. 2 h before sacrifice. The IHC analysis
was done on paraffin-embedded liver sections using antiBrdUrd antibodies (1:100; DAKO) and LSAB kit (DakoCytomation). For the mitotic index, the numbers of hepatocytes with
mitotic figures showing visibly condensed chromosomes were
counted.
Liver Histology
The regenerating livers were collected, fixed with formalin,
and embedded in paraffin. Serial liver sections were subjected
to H&E and periodic acid-Schiff reaction staining (44). Fat accumulation was shown by Oil Red O staining of cryostat frozen
sections.
Statistical Analysis
The results are presented as mean ± SD from at least three
independent experiments. Differences among multiple groups
were analyzed by one-way ANOVA (Statistical Package for
the Social Sciences 14.0 statistical software). Comparisons between two groups were done using a Student's t test. A P value
of <0.05 was considered significant.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
mitotic entry and G2 checkpoint in mammalian cells. Genes Cells 2002;7:
1173–82.
12. Bischoff JR, Anderson L, Zhu Y, et al. A homologue of Drosophila aurora
kinase is oncogenic and amplified in human colorectal cancers. EMBO J 1998;17:
3052–65.
13. Zhou H, Kuang J, Zhong L, et al. Tumour amplified kinase STK15/BTAK
induces centrosome amplification, aneuploidy and transformation. Nat Genet
1998;20:189–93.
14. Chen SS, Chang PC, Cheng YW, Tang FM, Lin YS. Suppression of the
STK15 oncogenic activity requires a transactivation-independent p53 function.
EMBO J 2002;21:4491–9.
15. Katayama H, Sasai K, Kawai H, et al. Phosphorylation by aurora kinase A
induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet 2004;
36:55–62.
16. Liu Q, Kaneko S, Yang L, et al. Aurora-A abrogation of p53 DNA binding
and transactivation activity by phosphorylation of serine 215. J Biol Chem 2004;
279:52175–82.
17. Zhang D, Hirota T, Marumoto T, et al. Cre-loxP-controlled periodic AuroraA overexpression induces mitotic abnormalities and hyperplasia in mammary
glands of mouse models. Oncogene 2004;23:8720–30.
18. Wang X, Zhou YX, Qiao W, et al. Overexpression of aurora kinase A in
mouse mammary epithelium induces genetic instability preceding mammary
tumor formation. Oncogene 2006;25:7148–58.
19. Smith MW, Yue ZN, Geiss GK, et al. Identification of novel tumor markers
in hepatitis C virus-associated hepatocellular carcinoma. Cancer Res 2003;63:
859–64.
20. Jeng YM, Peng SY, Lin CY, Hsu HC. Overexpression and amplification of
Aurora-A in hepatocellular carcinoma. Clin Cancer Res 2004;10:2065–71.
21. Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology 2006;43:
S45–53.
22. Leu JI, Crissey MA, Craig LE, Taub R. Impaired hepatocyte DNA
synthetic response posthepatectomy in insulin-like growth factor binding
protein 1-deficient mice with defects in C/EBP β and mitogen-activated
protein kinase/extracellular signal-regulated kinase regulation. Mol Cell Biol
2003;23:1251–9.
23. Li W, Liang X, Kellendonk C, Poli V, Taub R. STAT3 contributes to the
mitogenic response of hepatocytes during liver regeneration. J Biol Chem
2002;277:28411–7.
We thank Yi-Ru Chen, Huei-Jane Chen, and Shu-Pei Wu for technical assistance.
24. Greenbaum LE, Li W, Cressman DE, et al. CCAAT enhancer-binding protein
β is required for normal hepatocyte proliferation in mice after partial hepatectomy. J Clin Invest 1998;102:996–1007.
References
25. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth by
tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor
necrosis factor receptor. Proc Natl Acad Sci U S A 1997;94:1441–6.
Acknowledgments
1. Marumoto T, Zhang D, Saya H. Aurora-A—a guardian of poles. Nat Rev
Cancer 2005;5:42–50.
2. Giet R, Petretti C, Prigent C. Aurora kinases, aneuploidy and cancer, a coincidence or a real link? Trends Cell Biol 2005;15:241–50.
26. Fukuda T, Mishina Y, Walker MP, DiAugustine RP. Conditional transgenic
system for mouse aurora a kinase: degradation by the ubiquitin proteasome
pathway controls the level of the transgenic protein. Mol Cell Biol 2005;25:
5270–81.
3. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA. Human TPX2
is required for targeting Aurora-A kinase to the spindle. J Cell Biol 2002;158:
617–23.
27. Tanaka M, Ueda A, Kanamori H, et al. Cell-cycle-dependent regulation of
human aurora A transcription is mediated by periodic repression of E4TF1. J Biol
Chem 2002;277:10719–26.
4. Hirota T, Kunitoku N, Sasayama T, et al. Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells.
Cell 2003;114:585–98.
28. Hoque A, Carter J, Xia W, et al. Loss of aurora A/STK15/BTAK overexpression correlates with transition of in situ to invasive ductal carcinoma of the breast.
Cancer Epidemiol Biomarkers Prev 2003;12:1518–22.
5. Hutterer A, Berdnik D, Wirtz-Peitz F, Zigman M, Schleiffer A, Knoblich JA.
Mitotic activation of the kinase Aurora-A requires its binding partner Bora. Dev
Cell 2006;11:147–57.
29. Sieber O, Heinimann K, Tomlinson I. Genomic stability and tumorigenesis.
Semin Cancer Biol 2005;15:61–6.
6. Honda K, Mihara H, Kato Y, et al. Degradation of human Aurora2 protein
kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway.
Oncogene 2000;19:2812–9.
7. Castro A, Arlot-Bonnemains Y, Vigneron S, Labbe JC, Prigent C, Lorca T.
APC/Fizzy-related targets Aurora-A kinase for proteolysis. EMBO Rep 2002;3:
457–62.
8. Lim SK, Gopalan G. Antizyme1 mediates AURKAIP1-dependent degradation
of Aurora-A. Oncogene 2007;26:6593–603.
9. Meraldi P, Honda R, Nigg EA. Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53−/− cells. EMBO J 2002;
21:483–92.
10. Anand S, Penrhyn-Lowe S, Venkitaraman AR. AURORA-A amplification
overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol.
Cancer Cell 2003;3:51–62.
11. Marumoto T, Hirota T, Morisaki T, et al. Roles of aurora-A kinase in
30. Kops GJ, Weaver BA, Cleveland DW. On the road to cancer: aneuploidy and
the mitotic checkpoint. Nat Rev Cancer 2005;5:773–85.
31. Andreassen PR, Lohez OD, Lacroix FB, Margolis RL. Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol Biol
Cell 2001;12:1315–28.
32. Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic checkpoint following spindle disruption. Mol Cell Biol 1998;18:1055–64.
33. Khan SH, Wahl GM. p53 and pRb prevent rereplication in response to microtubule inhibitors by mediating a reversible G1 arrest. Cancer Res 1998;58:
396–401.
34. Ewart-Toland A, Briassouli P, de Koning JP, et al. Identification of Stk6/
STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and
human. Nat Genet 2003;34:403–12.
35. Ewart-Toland A, Dai Q, Gao YT, et al. Aurora-A/STK15 T+91A is a general
low penetrance cancer susceptibility gene: a meta-analysis of multiple cancer
types. Carcinogenesis 2005;26:1368–73.
Mol Cancer Res 2009;7(5). May 2009
Downloaded from mcr.aacrjournals.org on June 14, 2017. © 2009 American Association for Cancer Research.
687
Published OnlineFirst May 12, 2009; DOI: 10.1158/1541-7786.MCR-08-0483
688 Li et al.
36. Li CC, Chou CK, Wang MH, Tsai TF. Overexpression of ABIN-2, a negative
regulator of NF-κB, delays liver regeneration in the ABIN-2 transgenic mice.
Biochem Biophys Res Commun 2006;342:300–9.
37. Hogan B, Beddington R, Costantini F, Lacy E. Manipulating the mouse embryo: a laboratory manual. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor
Laboratory Press; 1994.
38. Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed.
Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2001.
39. National Research Council. Guide for the care and use of laboratory animals.
Washington (DC): National Academy Press; 1996.
40. Higgins GM, Anderson RM. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 1931;12:186–202.
41. Greene AK, Puder M. Partial hepatectomy in the mouse: technique and perioperative management. J Invest Surg 2003;16:99–102.
42. Jacks T, Remington L, Williams BO, et al. Tumor spectrum analysis in p53mutant mice. Curr Biol 1994;4:1–7.
43. Harlow E, Lane D. Using antibodies: a laboratory manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1999.
44. Young B, Heath JW. Wheater's functional histology: a text and colour atlas.
4th ed. Churchill Livingstone: Elsevier Science Limited; 2002.
Mol Cancer Res 2009;7(5). May 2009
Downloaded from mcr.aacrjournals.org on June 14, 2017. © 2009 American Association for Cancer Research.
Published OnlineFirst May 12, 2009; DOI: 10.1158/1541-7786.MCR-08-0483
Aurora-A Overexpression in Mouse Liver Causes
p53-Dependent Premitotic Arrest during Liver Regeneration
Chao-Chin Li, Hui-Yi Chu, Chu-Wen Yang, et al.
Mol Cancer Res 2009;7:678-688. Published OnlineFirst May 12, 2009.
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