Aging mice have increased chromosome instability that is exacerbated by elevated Mdm2 expression.

Abstract

Aging is thought to negatively affect multiple cellular processes including the ability to maintain chromosome stability. Chromosome instability (CIN) is a common property of cancer cells and may be a contributing factor to cellular transformation. The types of DNA aberrations that arise during aging before tumor development and that contribute to tumorigenesis are currently unclear. Mdm2, a key regulator of the p53 tumor suppressor and modulator of DNA break repair, is frequently overexpressed in malignancies and contributes to CIN. To determine the relationship between aging and CIN and the role of Mdm2, precancerous wild-type C57Bl/6 and littermate-matched Mdm2 transgenic mice at various ages were evaluated. Metaphase analyses of wild-type cells showed a direct correlation between age and increased chromosome and chromatid breaks, chromosome fusions and aneuploidy, but the frequency of polyploidy remained stable over time. Elevated levels of Mdm2 in precancerous mice increased both the numerical and the structural chromosomal abnormalities observed. Chromosome and chromatid breaks, chromosome fusions, aneuploidy and polyploidy were increased in older Mdm2 transgenic mice compared with wild-type littermates. Unexpectedly, chromosome fusions, aneuploidy and polyploidy rates in Mdm2 transgenic mice, but not chromosome and chromatid breaks, showed cooperation between Mdm2 overexpression and age. Notably, Mdm2 overexpression promoted gains in one or more chromosomes with age, while it did not affect the rate of chromosome loss. Therefore, aging increased specific forms of genomic instability, and elevated Mdm2 expression cooperated with aging to increase the likelihood of gaining certain chromosomal abnormalities of the kind thought to lead to cancer development.

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Aging mice have increased chromosome instability that is
exacerbated by elevated Mdm2 expression
Tamara Lushnikova1, Alyssa Bouska2, Jessica Odvody1, William D. Dupont3, and Christine
M. Eischen1,*
1 Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232
2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE
68198
3 Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, TN 37232
Abstract
Aging is thought to negatively affect multiple cellular processes, including the ability to maintain
chromosome stability. Chromosome instability (CIN) is a common property of cancer cells and
may be a contributing factor to cellular transformation. The types of DNA aberrations that arise
during aging prior to tumor development and that contribute to tumorigenesis are currently
unclear. Mdm2, a key regulator of the p53 tumor suppressor and modulator of DNA break repair,
is frequently overexpressed in malignancies and contributes to CIN. To determine the relationship
between aging and CIN and the role of Mdm2, pre-cancerous wild-type C57Bl/6 and littermate-
matched Mdm2 transgenic mice at various ages were evaluated. Metaphase analyses of wild-type
cells showed a direct correlation between age and increased chromosome and chromatid breaks,
chromosome fusions, and aneuploidy, but the frequency of polyploidy remained stable over time.
Elevated levels of Mdm2 in pre-cancerous mice increased both the numerical and the structural
chromosomal abnormalities observed. Chromosome and chromatid breaks, chromosome fusions,
aneuploidy, and polyploidy were increased in older Mdm2 transgenic mice compared to wild-type
littermates. Unexpectedly, chromosome fusions, aneuploidy, and polyploidy rates in Mdm2
transgenic mice, but not chromosome and chromatid breaks, showed cooperation between Mdm2
overexpression and age. Notably, Mdm2 overexpression promoted gains in one or more
chromosomes with age, while it did not affect the rate of chromosome loss. Therefore, aging
increased specific forms of genomic instability, and elevated Mdm2 expression cooperated with
aging to increase the likelihood of gaining certain chromosomal abnormalities of the kind thought
to lead to cancer development.
Keywords
aging; Mdm2; p53; chromosome instability; aneuploidy
INTRODUCTION
Genomic instability refers to the accumulation or acquisition of numerical and/or structural
abnormalities in chromosomes. It has long been observed that chromosome instability (CIN)
*Correspondence to: Christine M. Eischen, Vanderbilt University Medical Center, Department of Pathology, C3321 MCN, 1161 21st
Ave. South, Nashville, TN 37232-2561, Tel: (615) 322-3234, Fax: (615) 343-1633, christine.eischen@vanderbilt.edu.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Oncogene. 2011 November 17; 30(46): 4622–4631. doi:10.1038/onc.2011.172.
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is a hallmark of cancer cells and is postulated to be required for tumorigenesis (Lengauer et
al., 1998, Negrini et al., 2010). Genomic changes, such as chromosome breaks,
translocations, genome rearrangements, aneuploidy, and telomere shortening have been
observed in aging organisms (Aubert and Lansdorp, 2008, Dolle and Vijg, 2002, Nisitani et
al., 1990, Tucker et al., 1999, Zietkiewicz et al., 2009). Characteristics of genetically
unstable cells include structural changes such as insertions, deletions, and translocations, as
well as changes in the number of chromosomes (aneuploidy or polyploidy). Cells can gain
or lose one or more chromosomes (aneuploidy) or gain part of or an entire genome
(polyploidy) (Ganem et al., 2007, Lengauer et al., 1998). Additionally, cells with genomic
instability frequently display increased chromosome or chromatid breaks, chromosomal
fusions, and centrosome amplification, which itself promotes CIN.
Mechanisms leading to CIN are diverse and incompletely understood. Unrepaired DNA
double-strand breaks or eroded telomeres can lead to CIN by serving as substrates for
chromosomal fusions and translocations (Morgan et al., 1998). Amplification of
centrosomes may lead to the missegregation of chromosomes and result in aneuploidy
(Ganem et al., 2007). The uncoupling of DNA replication and mitosis can result in
polyploidy, which is postulated to be a precursor to aneuploidy (Fujiwara et al., 2005,
Ganem et al., 2007, Thompson et al., 2010, Vitale et al., 2010). Whether this genomic
instability, commonly observed in cancer cells, precedes tumorigenesis or is a by-product of
transformation is not clear. However, mice with mutations in genes encoding proteins
involved in cell cycle control or DNA damage signaling or repair have increased CIN and
many spontaneously develop cancer (Garinis et al., 2008, Negrini et al., 2010). Therefore
maintaining genomic stability appears essential to limit tumorigenesis.
The oncogene Mdm2 is frequently amplified or overexpressed in many human and murine
cancers; approximately 10% of all human cancers harbor MDM2 amplifications (Marine and
Lozano, 2010, Rayburn et al., 2005). Elevated levels of Mdm2 are associated with increased
transformation in vitro and in vivo (Marine and Lozano, 2010). Mdm2 is an E3 ubiquitin
ligase that functions as a critical negative regulator of the tumor suppressor p53, and Mdm2
can also delay DNA repair through association with a DNA repair complex (Bouska and
Eischen, 2009, Marine and Lozano, 2010). Mdm2 interferes with the transcriptional activity
of p53 by binding to and ubiquitinating p53, targeting it for degradation by the proteosome
(Marine and Lozano, 2010). Inactivation of p53 function results in a loss of cell cycle
checkpoint control and polyploidy, aneuploidy, and tumorigenesis (Fujiwara et al., 2005,
Levine and Oren, 2009). Mdm2 also binds to Nbs1, a protein in the Mre11/Rad50/Nbs1
DNA repair complex, and inhibits DNA double-strand break repair (Alt et al., 2005, Bouska
et al., 2008). Cells with reduced levels of Nbs1 or that contain mutated Nbs1 have an altered
response to DNA damage and are delayed in repairing DNA damage (Difilippantonio et al.,
2005, Williams et al., 2002). Humans with mutations in NBS1 frequently develop
malignancies (Demuth and Digweed, 2007). Therefore Mdm2 regulates two proteins
essential for monitoring, signaling, and/or repairing DNA damage, which is thought to be a
contributing factor to tumorigenesis.
Several studies have linked Mdm2 overexpression to genomic instability through both p53-
dependent and p53-independent mechanisms. Overexpression of human Mdm2 in murine
fibroblasts that contain wild-type p53 resulted in aneuploidy and centrosome amplification
(Carroll et al., 1999). Recently, loss of p53 was demonstrated to be important for the
survival and proliferation of aneuploid cells (Fujiwara et al., 2005, Thompson et al., 2010,
Vitale et al., 2010). Mammary epithelial cells from mammary specific-Mdm2 transgenic
mice have increased polyploidy, likely due to endoreduplication, and this occurred
irrespective of p53 status (Lundgren et al., 1997). Additionally, B-cells from juvenile Mdm2
transgenic mice, where Mdm2 expression was driven from its native promoter, have
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increased chromosome and chromatid breaks and aneuploidy compared to B-cells from
wild-type mice (Wang et al., 2008). Furthermore, through interaction with Nbs1, Mdm2
overexpression in wild-type or p53-null mouse embryonic fibroblasts inhibited DNA
double-strand break repair, leading to genomic instability and transformation (Alt et al.,
2005, Bouska et al., 2008). Thus, elevated expression of Mdm2 is linked to genomic
instability, but whether elevated levels of Mdm2 affect chromosomal stability as a mammal
ages is unknown. Therefore, we performed an in depth study of the effect of aging alone and
together with Mdm2 overexpression on genome stability. We show that specific
chromosomal alterations linked to chromosomal instability increase with age, and elevated
levels of Mdm2 lead to a greater amount of chromosomal instability that accumulated with
age prior to tumor development. The increased numerical and structural chromosome
abnormalities in Mdm2 transgenic mice likely contribute to their increased predisposition to
cancer.
RESULTS
Chromosome instability increases as mice age
To determine the specific effects of aging on overall genome stability and the contribution
of increased Mdm2 levels to this process, approximately 3,600 metaphase spreads of
splenocytes from 72 littermate-matched pre-cancerous wild-type C57Bl/6 and Mdm2
hemizygous transgenic mice 2–14 months old were analyzed for chromosomal alterations.
(Mice older than 14 months were not evaluated due to the emergence of cancer in the Mdm2
hemizygous transgenic mice.) The number of chromosomal aberrations, including
chromosome and chromatid breaks, chromosome fusions, and metaphases with an abnormal
number of chromosomes, were quantified in approximately 50 metaphases from each
mouse. There was a small, gradual increase in the total number of chromosomal
abnormalities in wild-type mice as they aged (Fig. 1A and 1B). In contrast, Mdm2 transgenic
mice had a marked increase in CIN with increasing age (p<0.0005). The frequency of
chromosomal abnormalities in wild-type and Mdm2 transgenic mice were similar at 2
months of age, but at 14 months old the number of chromosomal abnormalities in Mdm2
transgenic mice was twice that in wild-type mice (Fig. 1B). Therefore, as mice age, the
frequency of CIN increases, and this is significantly augmented by elevated levels of Mdm2.
Increased chromosome and chromatid breaks in aging mice
To identify the specific chromosomal alterations that are correlated with aging, and to
examine the effects of Mdm2 overexpression on these alterations, we first evaluated
chromatid and chromosome breaks from metaphase spreads of splenocytes from pre-
cancerous wild-type C57Bl/6 mice and Mdm2 transgenic littermates. Both chromatid and
chromosome breaks were detected in wild-type and Mdm2 transgenic mice with
chromosome breaks dominating in both genotypes (Fig. 2A–2C). The number of metaphases
with breaks gradually increased with age for both wild-type and Mdm2 transgenic mice (Fig.
2D). As we previously reported for splenocytes from mice 3 weeks old (Wang et al., 2008),
at 2 months old, a higher percentage of metaphases from Mdm2 transgenic mice had
chromosome and chromatid breaks compared to metaphases from wild-type littermates. The
mean number of metaphases with DNA breaks was significantly higher for Mdm2 transgenic
mice compared to wild-type mice at all ages evaluated (p=0.001) (Fig. 2D). Moreover, tri-
radial chromosomes, which lead to DNA breaks, were detected only in Mdm2 transgenic
cells (Fig. 2E). Surprisingly, the rate of increase of DNA breaks with age was statistically
similar between wild-type and Mdm2 transgenic cells (p=0.49). Therefore, although Mdm2
overexpression increased the frequency of chromosome and chromatid breaks at every age
evaluated, Mdm2 overexpression did not accelerate the rate at which breaks occurred with
age.
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Increased chromosome fusion in cells from Mdm2 transgenic mice
Unrepaired DNA breaks can serve as substrates for genomic rearrangements (Morgan et al.,
1998), which have been found to accumulate in aging mice (Dolle and Vijg, 2002). Since
aging wild-type and Mdm2 transgenic mice have elevated levels of DNA breaks, we
evaluated metaphases for chromosomal fusions from both genotypes of mice at 2–14 months
of age. Although fusions were rare, they were identified in both wild-type and Mdm2
transgenic mice (Fig. 3A–3C). Most (66%) of the fusions identified for both genotypes were
centromeric-centromeric fusions also known as Robertsonian translocations (Fig. 3A & 3B),
which are known to occur in mice (Garagna et al., 2001). Centromeric-telomeric fusions
accounted for a quarter of the fusions detected (Fig. 3C), and telomeric-telomeric fusions
were rare for both genotypes and accounted for less than 10% of the fusions identified. At 2
months of age, wild-type and Mdm2 transgenic mice had a similar low number of
metaphases with fused chromosomes, and both genotypes showed an increase in metaphases
with fused chromosomes as the mice aged (Fig. 3D). However, Mdm2 transgenic mice had a
greater percentage of metaphases with fused chromosomes than wild-type mice with the
greatest difference detected at older ages (p=0.05) (Fig. 3E). Other deleterious chromosomal
fusion abnormalities were only observed in cells from Mdm2 transgenic mice. Specifically,
three chromosomes fused centromere to telomere and a break in one of the fused
chromosomes were detected in a metaphase from a 10 month old Mdm2 transgenic mouse
(Fig. 3F). Additionally, a very rare fusion, a ring chromosome, was observed in cells from
Mdm2 transgenic mice as young as 5 months old and in 14 month old mice (Fig. 3G).
Therefore, fused chromosomes were more common in Mdm2 transgenic mice. The
prevalence of these abnormalities increased with age in both wild-type and Mdm2 transgenic
mice, but the rate of increase was greater for Mdm2 transgenic mice, suggesting an
interaction between Mdm2 and age on chromosomal fusions. Moreover, severe structurally
abnormal chromosomes were only detected in pre-cancerous cells in Mdm2 transgenics and
not in wild-type cells.
Increased polyploid DNA content in cells from aging Mdm2 transgenic mice
A cell becomes polyploid by gaining whole sets of chromosomes or an entire genome
through a variety of incompletely understood mechanisms (Ganem et al., 2007, Lengauer et
al., 1998). The number of chromosomes from metaphases of splenocytes from wild-type and
Mdm2 transgenic mice were quantified to determine the frequency of polyploid cells (near
triploid, triploid, near tetraploid, and tetraploid DNA content). Polyploid metaphases (40
chromosomes is normal) were detected in both wild-type and Mdm2 transgenic mice (Fig.
4A–4C). At two months of age, the number of metaphases with 50–80 chromosomes was
similar for wild-type and Mdm2 transgenic littermates (Fig. 4C & 4D). As the mice aged, the
number of metaphases with 50–80 chromosomes significantly increased in Mdm2 transgenic
mice (p=0.04), but remained relatively constant for wild-type mice (Fig. 4D). Furthermore,
Mdm2 transgenic mice also had cells that contained more than 4N DNA (Fig. 4E). Our
results indicate that age had little effect on the development of polyploidy in wild-type mice,
but elevated levels of Mdm2 significantly increased the rate of emergence of polyploid cells
in mice over 14 months. Thus, there was a greater likelihood of gaining partial or whole
genomes as mice aged, if Mdm2 was overexpressed.
Elevated rates of aneuploidy in aging Mdm2 transgenic mice
Polyploidy is postulated to be a precursor to aneuploidy, which is a loss or gain of whole
chromosomes and can result from chromosome missegregation during mitosis (Fujiwara et
al., 2005, Ganem et al., 2007, Storchova and Kuffer, 2008, Thompson et al., 2010, Vitale et
al., 2010). The number of chromosomes in metaphases of splenocytes from wild-type and
Mdm2 transgenic littermates of various ages were counted to assess aneuploidy. Metaphases
with greater than or less than the normal number of 40 chromosomes were detected in both
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wild-type and Mdm2 transgenic mice (Fig. 5A & 5B), but the frequency beyond 2 months of
age was different for each (Fig. 5C & 5D). As wild-type mice aged, they showed a small but
steady, significant increase in the number of metaphases that were aneuploid (Fig. 5D,
p=0.04). Notably, aging Mdm2 transgenic mice had a large significant, non-linear increase
in aneuploid cells such that the mean number of aneuploid cells in mice 14 months old was
more than twice that in mice 2 months old (p<0.0005) (Fig. 5D). Therefore, Mdm2
overexpression appeared to accelerate the rate of aneuploidy development with age
compared to wild-type mice.
To assess aneuploidy more in depth, metaphases that lost chromosomes were distinguished
from metaphases that gained chromosomes. As mice aged, both wild-type and Mdm2
transgenic mice had increased numbers of metaphases with less than 40 chromosomes
(p=0.003), doubling the mean frequency between 2 months and 14 months of age (Fig. 6A
& 6B). Unexpectedly, wild-type and Mdm2 transgenic mice had a similar number of
metaphases with less than 40 chromosomes at all ages evaluated. Consequently, both
genotypes of mice showed a similar rate of increase in metaphases with less than 40
chromosomes over 14 months (p=0.88) (Fig. 6B). In contrast, the rate of increase in
metaphases with more than 40 chromosomes over 14 months was greater in Mdm2
transgenic mice than wild-type mice (Fig. 6C & 6D). At 2 months of age there were slightly
more metaphases from Mdm2 transgenic mice that had greater than 40 chromosomes
compared to wild-type mice. However, as the mice aged, the number of metaphases with
greater than 40 chromosomes slightly increased in wild-type mice, but this increase was not
significant (p=0.34). In contrast, the number of metaphases with greater than 40
chromosomes profoundly increased with age in Mdm2 transgenic mice and was statistically
significant (p=0.003) (Fig. 6D). Mdm2 transgenic mice had triple the mean number of
metaphases with greater than 40 chromosomes at 14 months old compared to at 2 months of
age. These data indicate that as wild-type and Mdm2 transgenic mice age, both gains and
losses of chromosomes increased. However, Mdm2 overexpression only resulted in an
exacerbation of the rate of gains in chromosomes and not in the loss of chromosomes.
DISCUSSION
Genomic instability is characterized by chromosomal alterations, including changes in the
number of chromosomes (aneuploidy or polyploidy) and/or in the structure of chromosomes
(breaks, fusion, insertions, deletions, and translocations) (Lengauer et al., 1998, Negrini et
al., 2010). Limited published studies suggest that genome instability increases with age in
mammals (Aubert and Lansdorp, 2008, Nisitani et al., 1990, Tucker et al., 1999, Zietkiewicz
et al., 2009). The incidence of cancer increases with age, and cancer cells commonly have
genome instability, but the contribution chromosomal aberrations make to the development
of malignancies is incompletely understood. This study shows that specific numerical and
structural chromosomal changes increased with age in wild-type C57Bl/6 mice and were
exacerbated with Mdm2 overexpression. Importantly, all chromosomal alterations in both
wild-type and Mdm2 transgenic mice occurred prior to overt tumor development, indicating
genome instability precedes tumorigenesis and likely contributes to the development of
tumors. Moreover, Mdm2 transgenic mice had greatly increased and more severe
chromosomal alterations than wild-type mice that correlated to age; they also develop cancer
significantly earlier in life than wild-type mice, which may never develop a malignancy.
Therefore, our data from approximately 3,600 metaphases from 72 littermate-matched mice
from 2 to 14 months old provide new insight into aging and the types of chromosomal
alterations associated with it that may lead to tumorigenesis.
Polyploidy is considered a prerequisite to aneuploidy (Fujiwara et al., 2005, Ganem et al.,
2007, Storchova and Kuffer, 2008, Thompson et al., 2010, Vitale et al., 2010), which did
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