Amity University Rajasthan

Amity University Rajasthan

2009
SUBMITTED TO:
D0NE BY: Dr. Sandeep Shrivastva

Sunny Kataria
B.TECH (BIOTECH)
SEM – IIIrd
ROLL NO-24(AUR0821089)
AMITY INSTITUTE OF BIOTECHNOLOGY

Ageing: Complex network of genomic instability, Genome Maintenance

Pathways, cellular senescence and organismal aging- p53 is the central link.

Abstract
Aging can be defined as progressive age-related functional decline and increasing mortality over time. A large body of evidence
argues that DNA damage and mutations accumulate with age in mammals. Cells harboring mutations at defined loci have been
shown to increase with age in humans and mice. Different Genomic alterations accumulate in cells of different organs. It has
been proposed that DNA damage response activate p53 which through apoptosis and senescence leads to organismal aging.
There had been doubts over the hypothesis. In this paper I have investigated the evidence of what type of genomic instabilities
are known to accumulate with age and how they all converge to p53. I have also discussed how activation of p53 mediated
cellular senescence cause transcriptional changes observed in senescent cells. I have critically reviewed the link between various
genome maintenance pathways, genome instability, cellular senescence and organismal aging. In conclusion I have shown that
p53 lies central to pathways converging from various molecular and cellular level and diverging to age related phenotypes at
organismal level. The elucidation of such network models of aging might help elucidation of more complex networks, in order to
develop new drugs and technologies to help the elderly.
Introduction

Since the history of mankind aging has been an elusive mystery. Efforts have been made to decipher the mechanism
behind the rate of mortality and morbidity to increase with time of an organism. The most logical explanation is - aging
occurs due to genome alterations which activate p53 as tumor suppressor and which in turn cause cellular senescence
and effects gene transcription regulation or cause depletion of cellular reserves. Nothing in the universe is till eternity,
even at the quantum level information is exposed to random alterations, elementary particles decay, and symmetries
break with time. Moreover, the genome of organism is exposed to both endogenous and exogenous stresses, which
cause thousands of DNA lesions per cell per day. Although, the elegant DNA molecule has remarkable mechanisms to
repair these lesions but they are not perfect, perfect enough to restore the 100% of distorted information; it may make
errors and cause irreversible changes such as mutations and epimutations.

Some evolutionary theories of aging propose that the mechanism of natural selection selected mutations as an
advantage for reproductive success at young age, but it acts as a mechanism for degradation of soma at old age. One
such antagonistic gene is that encoding tumor suppressor p53. It is quite obvious that before reproductive maturity
most of the energy in the body will be utilized by soma to make the organism successfully reach the reproductive age.
Depending on how fast organism reproduces a balance should be maintained in distribution of energy between
maintenance of soma and germ line. Soma should be maintained in organisms like human beings whose reproductive
rate is slow to ensure that organisms survive long enough to produce maximum number of copies of its genome as
offspring. Moreover, in advanced organisms such as mice and human beings, apart from pathways that maintain
genome at molecular level extra reservoirs of stem and progenitor cells are provided by evolution. So it seems likely
that the availability of these reserves is significant to ensure longevity. In organism such as Nematodes it is well known
they lack such reserves and have life span of a few days. This gives us the first clue that somehow organismal aging,
cellular senescence and genomic instability must be linked by some common pathway. One such link is interaction
between caretaker and gatekeeper mechanism. Caretakers are those genes which encode proteins for DNA repair
pathways while gatekeepers are those which ensure any altered cells with genome should be eliminated to ensure the
longevity of multi-cellular organism as a whole. The best known example is a link between how NHEJ repair pathway
changes with age and activation of p53 which link to degradation of tissue by apoptosis of cellular reserves and by
increasing the number of senescent cells with age. p53 is a gatekeeper which promote longevity by providing resistance
to development of tumor, but it is only this p53 after the reproductive age which causes organism senescence.

Apart from interaction between gatekeepers and DNA repair the DNA repair can itself make errors which can lead to
Transcriptional changes over age by either sequence changes or can cause epimutations as a necessary side effect of
repair. Transcriptional changes could also occur due to change in methylation pattern and cytogenetic alterations as
we will see later. This would lead certain cells like senescent fibroblast to secrete different combination of protein such
as MMPs like collagenase and growth factors. This may explain why senescent fibroblast secretes chemicals that
degrade intracellular collagen matrix. It is important to mention here that transcriptional changes will not occur in
each and every cell but will occur randomly. Aging will therefore seem not to depend on instability at genomic level.
However on the other hand I would argue the rate with which random transcriptional noise will be manifested at the
organ level to cause tissue dysfunction predicts why the effect of genomic instability is successfully delayed until
reproductive maturity, why immunosenescence depend on the ratio of various types of T-cells rather than number of
cells, why different species have different life spans, why lifespan depend on rate of metabolism of species, why stem
cell reserves are so affective to give humans there maximum lifespan of 120 years.

If what we are assuming is true then the best known methods, Caloric restriction (CR) and Insulin/IGF signaling (IIS)
which increases both life span and health span must link between maintaining genome stability, inhibiting p53 and
maintaining heterochromatin in senescent cells to avoid transcriptional noise.
So, both transcriptional noise and interaction between p53 and caretakers seem to link with each other.

In this paper my central questions will be what is the evidence for the type of genomic instabilities that occur with age?
How caretaker DNA repair pathways are responsible for this instability? And how instability leads from cellular
senescent to organismal aging? In order to answer these I will review

1. What Changes occur in DNA repair mechanisms with age?

If we can find evidence that there is a decline in efficiency of repair pathway with age, or if it makes more errors with
age we can show its link to our hypothesis. In case if decline of efficiency there will be direct link between DNA damage
and activation of gatekeepers like p53. This will caused the decline in no of cells in tissues and its stem cell reserves.
While in case of repair becoming more error prone we could indirectly link that because of transcriptional changes
especially those in post-mitotic functional cells or the cells that became senescent there was a functional loss as well as
these cells secreted growth factors and extracellular matrix degrading factors which promoted cancer and malignancy
in other functionally intact cells and caused functional decline of its own tissue.

2. If cellular senescence and cell cycle is linked to aging and genome instability, is type of mutation accumulation
tissue specific?

If cellular senescence caused by p53 and thereafter changes occurring senescent cells has an important role in aging
then it should be that accumulation of genome alterations should be tissue specific.

3. What types of genome alterations accumulate with age if they do?

In this paper our mail goal is to see weather and which type of genomic alteration occurs with age and if all these
pathways affect by finally activating p53 and transcriptional noise. For this purpose I have discussed the evidence of
age related genomic alteration from cytogenetic changes and base sequence changes to epimutations. There role in
activation of p53 and transcriptional changes will help us explain aging as caused by pleiotropic effects of p53.

4. how longevity promoting CR links to hypothesis central to our paper?

 1. DNA DAMAGE: A LIKELY CAUSE OF AGING?

The prevailing view regarding causes of aging is that aging results from accumulation of
somatic damage. Damage to DNA can lead to cell cycle arrest, cell death or mutation.
The majority of Lesions and mutations do not kill the cell, but when accumulated in
sufficient numbers may lead to
1. deregulation of transcription patterns (Bahar et. al. 2006),
2. apoptosis when excessive damage accumulate and excessive apoptosis can
lead to depletion of stem cell reservoirs
3. increase in amount of senescent cells which would release more mitogens
increasing the risk of cancer with age, will accumulate more mutations and
lesions and would lead to decreased protein turnover rate and degradation of
extracellular proteins as well all these conditions will lead to reduced fitness and
ultimately the aging phenotype

It all begins from DNA damage as we will see throughout this paper. Although the rate
of DNA damage induction is very high(see table 1), steady-state levels of the most
frequently occurring lesions are only modest. Whereas absolute figures of tens of
thousands of lesions induced in cells each day suggest a very serious problem, it is
difficult to see how steady-state levels of a few lesions per 10 6 undamaged bases can
contribute to the adverse effects that develop during the course of the aging process. At
such low frequencies, with lesions continuously removed through DNA-repair pathways
and re-introduced, it would be unlikely for a lesion to reside in a gene or its regulatory
regions long enough to have adverse effects. It seems a reasonable conclusion that
DNA damage per se, even in the large absolute numbers of lesions induced in cells
each day, is unlikely to play a direct role in causing age-related functional impairment
and disease. However, DNA damage can have long-term adverse effects through
mutagenesis. Whereas extraordinarily efficient in resisting the avalanche of chemical
changes threatening the genome’s integrity at any given moment, DNA-damage
processing has not evolved to a level of perfection that would allow essentially no
errors. Errors are being made and while their frequency may be remarkably low
compared with the large numbers of lesions induced in our genome every day,
mutations may easily accumulate to a level where they might exert adverse effects. In
contrast to chemical changes in the DNA double helix, mutations are not recognized by
the cell’s DNA-damage-processing system and will be just as faithfully replicated as the
original, correct sequence. This is why mutations are irreversible and cannot be
removed, except through cell-elimination ‘repair’. The question that may next come in
mind is, what type of repair mechanism are at work, what mistakes can they make? And
if they make errors do mutations accumulate in different organs and tissues of
mammals, and, if so, what kind of mutations accumulate and can they have adverse
effects due to activation of p53?
Table 1: Number and type of lesions introduced per cell per day
source Lesion Estimated no. of Lesions induced per cell/day
SSBs 20000-40000
Spontaneous hydrolysis AP sites 10000
deamination 100-300
8-oxoG 27000
Oxidation
thymine glycol 270
N⁷- methylguanine 4000
methylation N³- methylguanine 600
N⁶- methylguanine 10 to 30
Glucose Glucose adducts 3
sun-exposure pyrimidine dimer 60000-80000
smoking PAHs 100-2000

Changes in DNA repair during aging

1.1 EVIDENCE FOR AGE-RELATED CHANGES IN DNA REPAIR FROM

THE STUDIES OF SOMATIC MUTATIONS

Accumulation of mutations with age has been studied extensively in mice and humans.
The early studies of mutations in the HPRT locus in cultured lymphocytes from young
and old individuals have shown accumulation of mutations with age in both humans and
mice (Finette et. al., 1994, Jones et. al., 1995, Dempsey et. al., 1993, Morley et. al.
1982, Morley et. al., 1998). The studies using transgenic mouse models allowed the
measurement of the mutation frequency in other mammalian tissues and loci. These
assays measure mutation frequency in chromosomally integrated LacZ (Vijg et. al.,
1997) or LacI (Kohler et. al., 2007, Stuart et. al., 2000) transgenes, which are rescued in
Escherichia coli and analyzed for mutations using betagalactosidase assay. Using these
mice, it was demonstrated that point mutations accumulate with age (Stuart et. al.,
2000, Vijg et. al., 2000 Dolle et. al., 1997 & 2000) and furthermore, the mutation rate is
higher in old animals (Stuart et. al., 2000). Not only did mutations accumulate but a
characteristic type of mutations, genomic rearrangements, appear in old individuals
(Vijg et. al., 2002, Ramsey et. al., 1995, Tucker et. al., 1999, Martin et. al., 1985).

Why does the rate of mutations increase with age and genomic rearrangements
appear? Multiple studies have shown a higher load of DNA damage in old organisms
(Chevanne et. al., 2003, Singh et. al., 2001, Sedelnikova et. al., 2004, d’Adda et. al.,
2003, Hamilton et. al., 2001). But why is there more damage? It is tempting to suggest
these changes are caused by DNA repair machinery becoming less efficient and more
error-prone with age. We will now discuss the studies, which directly measured DNA
repair efficiency in young and old

1.2 AGE-RELATED CHANGES IN MISMATCH REPAIR(MMR)

MMR removes mispaired bases resulting from replication errors,
recombination between imperfectly matched sequences and deamination of 5-methyl-
cytosine. DNA replication past a mismatched base pair would result in a point mutation.
The MMR system is also thought to play a role in repair of oxidative damage by
mechanisms that are not well understood (Skimmer et. al., 2005). Several lines of
evidence indicate the importance of the MMR system to the aging process. MMR is
essential for maintenance of repeated sequences, as mutations in MMR genes are
associated with a substantial destabilization of microsatellites (Karran et. al., 1996), and
microsatellite instability increases with aging in humans (Ben et. al., 2000, Krichevsky
et. al., 2004, Neri et. al., 2005). For example microsatellite instability can cause
heterochromatin alterations of telomeric DNA which can result in activation of p53 and
hence cause replicative senescence. The rate of MMR has been analyzed in; in vitro
aging human T cell clones (Annett et. al. 2005). Cells at different passages were treated
with mismatch-inducing agent and mismatch frequency was determined using a
modification of the alkaline comet assay. Results showed a decline in MMR with
increasing in vitro age. Thus, there is evidence of age-related alterations in MMR;
however, more studies are needed which would directly measure MMR capacity in
young and old individuals.

1.3 AGE-RELATED CHANGES IN BASE EXCISION REPAIR (BER)

Excision repair removes lesions that affect only one DNA strand, which permits excision
of the lesion and subsequent use of the complementary strand to fill the gap. BER
corrects small DNA alterations that do not distort the overall structure of DNA helix,
such as oxidized bases, or incorporation of uracil. Excision repair is critically important
for repairing base damage induced by reactive oxygen species. BER is classified into
two sub-pathways: short-patch BER; a mechanism whereby only 1 nucleotide is
replaced or long-patch BER; a mechanism whereby 2–13 nucleotides are replaced.
BER is initiated by DNA glycosylases, which cleave N-glycosylic bond of damaged
bases leaving apurinic/apyrimidinic site (AP site). The abasic site is then processed by
AP endonuclease (APE1) leaving a single-stranded gap. The gap is filled by DNA
polymerase b and ligated by DNA ligase (Wilson et. al., 2006, Almeida et. al., 2007).
The choice between short and long patch repair is currently under investigation. Various
factors are thought to influence this decision, including the type of lesion, the cell cycle
stage, and whether the cell is terminally differentiated or actively dividing.(Fortini et. al.,
2007). Although the choice was first thought to be species dependent but recent studies
cast some doubts.
A significant decrease in the mitochondrial incision activity of 8-oxoG DNA glycosylase,
uracil DNA glycosylase and the endonuclease III homolog was found in the brains of old
mice, whereas smaller changes were observed in nuclear incision activity (Chen 2002,
Wilson 2006, Imam 2006). Similarly, a decline in cleavage activity was observed in
mitochondrial, and to a lesser extent nuclear extracts from senescent human fibroblasts
(Shen et. al., 2003). A different in vitro assay measuring the repair of a synthetic DNA
substrate containing a single G:U mismatch showed a strong decline in BER activity in
brain, liver and germ cell nuclear extracts of old mice (Intano et. al., 2003). Germ cell
extracts from old mice were found to contain reduced levels of APE1, and
supplementation with purified recombinant enzyme restored the activity to the level of
young animals (Intano et al., 2002). Reduced abundance of DNA polymerase b in brain
extracts from mice and rats has been reported (Cabelof et al. 2002, Intano et al. 2003,
Krishna et al. 2005).

In addition to altered enzyme activity, the mechanism for age-related decline of BER
may lie in altered response to DNA damage. For example, the expression of DNA
polymerase b and AP endonuclease was induced by DNA damage in young mice, while
aged mice showed a lack of inducibility (Cabelof 2006). Furthermore, old mice and
senescent human fibroblasts were deficient in the translocation of oxoguanine DNA
glycosylase and AP endonuclease into both their nuclei and mitochondria (Szczesny
2003, Szczesny 2004). An intriguing finding showed that the efficiency of BER may be
sequence-specific with the promoters of the genes whose expression is down-regulated
with age being repaired poorly compared to genes that are not affected by age (Lu
2004).

The above studies indicate that increasing amount of senescent cells will be less
efficient in BER and hence will be more prone to oxidative damage. Even if decline was
itself responsible in causing senescence, then also the results indicate senescence will
promote more decline of this mechanism. This again indicates the relation between
transcriptional changes, genome maintenance, activation of p53 and aging.

1.4 AGE-RELATED CHANGES IN NUCLEOTIDE EXCISION REPAIR (NER)

NER removes short DNA oligonucleotides containing a damaged base (Hanawalt 2002). NER recognizes bulky lesions
caused by carcinogenic compounds, and covalent linkages between adjacent pyrimidines resulting from UV
exposure. NER is further classified into global genome repair (GG–NER) that occurs everywhere in the genome,
and transcription-coupled repair (TCR), which removes lesions in the transcribed strand of active genes. NER is a
multistep process involving multiple proteins (Hanawalt 2002). In GG–NER DNA damage is recognized by XPC–
HR23B complex, followed by damage verification by XPA. The DNA is unwound by XPB and XPD (ERCC3 and
ERCC2) helicases in complex with TFIIH basal transcription initiation factor, and the incision is made in the
damaged strand by XPF and XPG. The damaged strand is removed, and repair is completed by DNA polymerase
and DNA ligase. In the TCR pathway stalled RNA–PolII on the damaged DNA template is believed to initiate the
repair process, which requires the TCR-specific proteins CSB and CSA.
The relationship between NER and aging has long been studied by either evaluating repair during in vitro
senescence of human fibroblasts and lymphocytes, or in cells or tissues derived from donors of different age. Studies
using in vitro senescence yielded conflicting results. Hart et al, 1976 reported a reduction in unscheduled DNA
synthesis following DNA damage in late passage WI-38 human fibroblasts compared to early passage cells. While
no differences in the repair replication were found using the same cell line (Painter et. al., 1973). A more recent
study (Christiansen 2000) measured the kinetics of the disappearance of cyclobutane pyrimidines dimers (CPDs) from
genomic DNA in human fibroblasts and trabecular osteoblasts aged in vitro. In this method cells are treated with
UV, genomic DNA is extracted and incubated with T4 endonuclease V, which cleaves the DNA at pyrimidine
dimers. Then the DNA is cleaved with restriction enzymes, separated on an alkaline gel and the intensity of the
bands corresponding to specific genes is determined by Southern hybridization. Both actively transcribed and
inactive genes were analyzed, and no clear differences with age in the rate of CPD removal were found. Removal of
CPDs was also examined using in situ assay with CPD antibodies (Boyle 2005). This study showed reduced UV-
induced CPD removal in senescent compared to young fibroblasts.

Contrasting results between different studies may be explained by the sensitivity of cells to culture conditions such
as serum concentration, and differences in cell cycle distribution during treatment ( Al-Baker 2005). Furthermore,
replicative aging may vary between cell lines, depending on the cell donor. For example, a decline in NER with
increasing passages was documented for lymphocytes derived from two different donors but not in the lymphocytes
from the third donor (Annett 2004).

Studies of NER in cells or tissues from young and old individuals consistently showed a decline of NER capacity
with increasing age. Earlier works used unscheduled DNA synthesis (Vijg 1985) or a plasmid reactivation assay as a
measure of NER (Wei 1993, Grossman 1995, Moriwaki 1996). In the plasmid reactivation assay a plasmid containing
chloramphenicol acetyltransferase (CAT) is irradiated with UV to introduce DNA damage; plasmids are transfected
into host cells and percentage of CAT activity relative to undamaged control plasmid is measured. A large study
using human peripheral blood lymphocytes from 135 individuals aged 20 to 60 showed a decline of UV damage
repair (Wei 1993, Grossman 1995). The rate of decline was calculated as 0.63% per year, which amounts to ~25%
decrease over a 40-year period (Wei 1993, Grossman 1995). This analysis was further extended to show similar decline in
NER in skin fibroblasts (Moriwaki 1996). In addition to reduced plasmid reactivation, cells from older donors
introduced an increased number of mutations in the transfected plasmid. This suggests that not only the repair
becomes less efficient with age, but it also makes more errors.

A disadvantage of plasmid reactivation assay is that it does not differentiate between different types of
repair, and types of photoproducts. Other assays measured the rate of removal of specific photoproducts. A study
using T4 endonuclease V for detection of CPDs in hepatocytes of 6 and 24 months old rats showed that removal of
CPDs from two nontranscribed genes was 40% lower for cells isolated from old
rats than for cells isolated from young animals (Guo 1998). In contrast, the age-related decline of CPD removal was
less apparent in a transcribed gene, where only the rate of CPD removal was slower in old animals, but no difference
in the number of CPDs was found after 24 h (Guo 1998). By a similar assay, repair efficiency of telomeric DNA was
reported to be lower in fibroblasts isolated from older human donors (Kruk 1995). Thus, it appears that aging has a
greater effect on repair of nontranscribed genes.

Another method of detection of CPDs and (6–4) pyrimidone photoproducts (PP) became available with development
of CPD and (6–4) PP-specific antibodies (Mori 1991). DNA is isolated from cells after UV-irradiation and
hybridized with antibodies. In agreement with earlier results this method showed a decrease in removal of CPDs and
(6–4) PPs in dermal fibroblasts of older human donors ( Goukassian 2000). Decline in repair of (6-4) PPs was also
shown in round spermatids of 14 months old compared to 2 months old mice (Xu 2005). The method was further
developed to use PP-specific antibodies for immunohistochemistry, which allowed studying the removal of CPDs in
situ in human epidermis (Yamada 2006).

Skin at the upper arm of young and old volunteers was exposed to UVB, and biopsied at different time points after
irradiation. The biopsy material was either used for DNA extraction and probed with CPD antibodies or analyzed by
immunohistochemistry. CPDs were removed from epidermis at 4 days after irradiation in the young subjects, and
between 7 and 14 days in older subjects (Yamada 2006). Since the process of CPD removal is a combination of
NER and epidermal turnover, the CPDs are likely to be removed from aged skin after 7–14 days by turnover of the
epidermis. Thus, in situ studies further suggest that NER declines with age.

Another in situ study with human volunteers using a post-labeling method based on quantification of photoproducts
by HPLC found that photoproducts in UV-irradiated skin are induced at a higher frequency in old individuals (Xu
2000). The latter study, however, did not detect age-related differences in NER due to large individual variations in
the study population (Hemminki 2002).

1.5 AGE-RELATED CHANGES IN DOUBLE-STRAND BREAK (DSB) REPAIR

A DSB is the most lethal of all DNA lesions. If unrepaired, a DSB leads to loss of chromosome segments and
threatens the survival of the cell. Equally detrimental to the organism are misrepaired DSBs that destabilize the
genome and lead to genomic rearrangements. Genomic rearrangements become common in aging organisms (Dolle
1997, Vijg 2002 , Tucker 1999) ultimately leading to deregulation of transcription (Bahar 2006) and malignancies.

DSBs in DNA are repaired by two major mechanisms: homologous recombination (HR) and nonhomologous end
joining (NHEJ). During HR-mediated repair of DSB, the sister chromatid is used as a template to copy the missing
information into the broken locus. Repair by HR is mediated by Rad51 protein with the help of other members of
Rad52 epistasis group (Jackson 2002, Helleday 2003). Since sister chromatids are identical to each other, DNA
damage can be repaired faithfully with no genetic consequence.

In contrast, the NHEJ pathway simply fuses two broken ends with little or no regard for sequence homology. NHEJ
starts with binding of Ku70/Ku80 heterodimer to the broken DNA ends (Lieber 1999). Ku facilitates recruitment of
Artemis-DNA–PKcs complex, which processes the ends to prepare them for ligation (Lieber 2003). Next, the gaps
are filled by DNA polymerase of polX family, and covalently joined by XRCC4-DNA ligase IV complex. NHEJ is
rarely error-free leading to deletions or insertions of filler DNA (Gorbunova 1997, Sargent 1997, Seluanov 2004). DSBs
situated between two direct repeats can also be repaired by single-strand annealing (SSA), a highly mutagenic
recombinational mechanism in which the sequence between the repeats is deleted.

The first indication of age-related changes in DSB repair is the exponential increase in the incidence of cancer with
age (DePinho 2000), since cancer is associated with genome rearrangements and loss of heterozygosity (LOH).
Sequence analysis of genomic rearrangements in the LacZ locus in aged transgenic mice (Dolle 1997) and also in
the endogenous HPRT locus in human lymphocytes (Fuscoe 1994) did not reveal regions of extended homology at
the breakpoints, suggesting that the rearrangements may have resulted from erroneous NHEJ. Despite the
tremendous impact of DSB repair on aging process, age-related changes in DSB repair are currently understudied.
Early studies using variations of the comet assay documented age-related decline of the efficiency of rejoining of X-
ray-induced DNA breaks in normal human lymphocytes (Mayer 1989, Singh 1990). The methodology, however, did
not allow addressing a specific repair mechanism. Seluanov t. al., in 2004 has designed a sensitive assay for NHEJ. The
assay is based on a reporter cassette containing GFP gene interrupted by an intron, containing recognition sites for
the rare cutting endonuclease I-SceI. The GFP gene in the original construct is inactive, however, induction of DSBs
by I-SceI and subsequent repair restores GFP activity. Since NHEJ takes place within intronic sequences, GFP
activity is reconstituted even if repair is accompanied by deletions or insertions. The number of GFP positive cells
serves as a measure of NHEJ efficiency, and can be quantified by FACS.

1.5.1 Does cellular senescence cause decline in NHEJ?

In addition the fidelity of repair can be determined by sequencing the repair products. Using this assay to study
senescence-related changes of NHEJ in normal human fibroblasts, it was found that efficiency of NHEJ was reduced
up to 4.5-fold in pre-senescent and senescent relative to young cells. Strikingly, it was seen that end joining in old
cells was associated with extended deletions. These results indicate that end joining becomes less efficient and
more error-prone in senescent cells (Seluanov 2004). NHEJ in the brain of young and old rats has been analyzed
using in vitro plasmid rejoining assay. In this assay linearized plasmids are incubated with nuclear extract, and
plasmid concatamers resulting from NHEJ are quantified on a gel. This assay showed that NHEJ efficiency is
reduced in the brains of old rats ( Vyjayanti 2006 Ren 2004). NHEJ is also decreased in the brains of Alzheimer’s
disease patients compared to normal controls (Shackelford, 2006).

What is the molecular mechanism responsible for age related decline of NHEJ? The level of Ku, which is a protein
responsible for recognition and binding to DSBs, has been shown to decline markedly in the testis of aging rats (Um
2006), and lymphocytes from human donors of increasing age (Ju 2006). A 50% reduction in Ku70 and Ku80
protein levels in senescent human fibroblasts has been observed. Furthermore, intracellular distribution of Ku
differed between young and senescent cells. In young cells, Ku was distributed throughout the cytoplasm and the
nucleus, and translocated to the nucleus in response to γ-irradiation. It has been hypothesized that the cytoplasm of
young cells contains Ku that serves as a reserve that is recruited to the nucleus upon DNA damage. In contrast, in
senescent cells, all Ku was localized in the nucleus, so no additional Ku can be brought into the nucleus to repair
DNA damage.

Moreover, results suggest that nuclear Ku in senescent cells is unavailable for repair of new lesions (Seluanov
2007). Ku may be trapped by unrepairable DNA damage foci ( d’Adda 2003, Sedelnikova 2004) that accumulate in the
nuclei of senescent cells. Ku heterodimer forms a ring that is loaded onto the DNA strand (Walker 2001), and it is
unknown what takes Ku off the DNA. The release of Ku from DNA may be impaired in senescent cells.
Alternatively, Ku may remain at the damage sites and shortened telomeres, which cannot be repaired by DNA repair
machinery in senescent cells. Impaired nuclear targeting and DNA binding activities of Ku have been reported in
peripheral blood mononuclear cells of aged humans ( Doria 2004, Frasca 1999). Thus, decline of NHEJ with age may
be caused by reduced availability and altered regulation of Ku. Disruption of genes involved in DSB repair often
leads to premature aging phenotypes with notable examples being Werner syndrome, Ataxia telangiectasia, and
mice deficient for Ku80, DNA–PKcs and ERCC1 ( Gorbunova 2005). The exact mechanism by which these mutations
cause progeroid phenotype is unknown, and likely involves a combination of disregulation of transcription and an
increase of cellular senescence and apoptosis by activating p53. As DSB repair becomes less efficient during
normal aging the same pathways may contribute in a more subtle way into the onset of aged phenotype.

1.5.2 RELATIVE USAGE OF DSB REPAIR PATHWAYS WITH INCREASING AGE

As we discussed above DSBs can be repaired by three distinct pathways: mutagenic NHEJ and SSA, and
potentially non-mutagenic HR. Does the usage of DSB repair pathways change with age? This question was
addressed in Drosophila male germline (Preston 2006). The males carried a reporter cassette where DSBs were
generated by constitutively expressed I-SceI endonuclease. The cassette allowed differentiating repair events as
NHEJ, SSA or HR between two homologous chromosomes. HR in male germline has increased from 14% in young
flies to 60% in old ones, with a corresponding decrease in SSA and NHEJ.

Can this finding be generalized to mammalian DNA repair? 4.5-fold decrease in NHEJ in senescent human
fibroblasts was in fact observed( Seluanov 2004), however, this was clearly not due to an increase in HR, since HR is
virtually absent in replicatively senescent cells (Seluanov 2007). Lack of HR in G0-arrested cells is not unexpected,
as HR is restricted to G2/M phase of cell cycle when a sister chromatid is available ( Saleh-Gohari 2004 Rothkamm 2003).
Potentially, a homologous chromosome can be used as a template in non-dividing cells; however, repair using a
homolog is three orders of magnitude less frequent than the use of a sister chromatid in mammalian cells (Johnson
2001, Stark 2003). This preference is explained by sister chromatids being in close proximity after DNA replication,
in contrast to homologs that are not paired in mitosis and may be harder to bring together. Expression of Rad51
protein is cell cycle-regulated (Yamamoto 1996, Chen 1997), and Rad51 becomes undetectable when cells enter
replicative senescence (Seluanov 2007). Therefore, the major pathway for repair of DSBs in mammalian G1/G0
cells is NHEJ (Rothkamm 2003 Saleh-Gohari 2004 Sonoda 2006). While senescent cells do not use HR, the situation is less
clear in an aging organism, which still contains dividing cells. However, several studies show that HR is more active
in embryological stem (ES) cells and in early embryonic development ( Orii 2006), compared to late developmental
stages and somatic cells. As the body ages, fewer cells divide, cell cycle slows down and more cells are in G1/G0
stage, therefore the usage of HR will further decrease.
Another important point is that HR tested by Preston et al., (2006) is HR between homologous chromosomes. In
mammalian cells mitotic recombination is rare and potentially dangerous process, as it is associated with Loss of
Heterozygosity (LOH) and cancer. Specific mechanisms controlled by Bloom protein (Luo 2000), the MMR system
(de Wind, 1995) and genetic divergence between homologs ( Shao, 2001) suppress mitotic recombination in mammalian
cells. LOH takes place in precancerous cells and is a major contributor to the genesis of tumor ( Shao, 2001). However,
LOH and cancer do occur with increased frequency with advancing age ( DePinho 2000, Grist 1992). Thus, the finding
of increased HR between homologs in aging flies may be compared to increased frequency of abnormal mitotic
recombination events in aging mammals. It is possible that mechanisms suppressing mitotic recombination start to
fail in aging cells, or that old individual contain more premalignant cells.

So until here we have seen that DNA repair mechanism do become less efficient with age and start making more
errors causing permanent mutations, genome rearrangements, LOH, and epimutations (discussed later). These
studies explains that why such few lesions accumulate with age. But the next questions that strike are that weather
mutations and other genome alteration do accumulate with age and to what extent? Except errors in repair are there
any other sources of Genome alterations? If yes how these alteration result in aging of organism?

Figure 1
Age-related changes in DNA repair and their consequences. Inefficient MMR leads to microsatellite instability, point mutations and
potentially, to increased frequency of LOH. Decline in efficiency and fidelity of BER and NER leads to point mutations. Less efficient
and more error-prone NHEJ results in point mutations and genomic rearrangements. As fewer cells are in G2 stage, the usage of sub pathways of
DSB repair (DSBR) may also change, where precise HR between sister chromatids declines, giving way to more mutagenic SSA and mitotic
recombination (MR) with homologous chromosome leading to genomic instability and LOH.

2. Mutations are tissue specific

Overall, there is evidence that NER, BER and NHEJ activity decline with age, albeit sometimes only
marginally and with some discrepancies between the different studies. The possible consequences of the
age-related decline in BER activity are still unclear. An age-related decrease in cell-proliferative activity
comes to mind as a possibility, which may require less BER activity than at a young age. Hanawalt, Tice
and Setlow, as well as some other researchers between 1980s and early 1990s, came to the conclusion that
there is no evidence for a drastic decline in DNA repair of DNA lesions during aging. This is not
surprising in view of the critical importance of genome maintenance for the cell. It should be noted that a
decline in DNA repair is not necessary to explain an accumulation of alterations in the somatic genome.
Since by nature genome-maintenance systems are imperfect, one would expect such alterations to
accumulate. However, in the presence of declining repair activities such an accumulation would be
expected to be exponential rather than linear.

If what we have discussed until now holds good in explaining the phenomena of aging, we should be able to see
evidence that type of mutations differ among tissues. One reason is that since type of repair pathway differ in mitotic
and post mitotic or senescent cells, there should be difference in type and quantity of mutations observed in different
organs and it will also differ in substructures of these organs.
In this section I will briefly review the observed tissue specific mutation accumulation in process of aging.

With the development of transgenic mouse models harboring chromosomally integrated reporter genes, it
became possible to directly test the hypothesis that somatic mutations in a neutral gene accumulate with
age in different organs and tissues. Using the lacI mouse model, Lee et al., 1994) were the first to
demonstrate an age-related increase in mutant frequency (expressed as the number of mutant reporter
genes as a function of the total number of recovered reporter genes) in spleen, from about 3 x10-5 in mice
of a few weeks old to (1–2) x 10-4 in 24-month-old animals. Shortly thereafter, Martijn Dollé
demonstrated age-related increases in the mutation frequency in some, but not all, organs in the mouse.
For example, Dollé demonstrated that mutation frequencies at a lacZ transgene increase with age in the
liver, from about 4 x10-5 in the young adults to about 15 x10-5 in old animals (about 30 months), whereas
such an increase was virtually absent in the brain (Dolle et. al., 1997). Also spleen, heart, and small intestine
showed increased mutation frequencies with age( Dolle et. al., 2000, Giese et. al., 2002) but not the testes(Martin
et. al., 2001). These organ-specific patterns of mutation accumulation at the lacZ transgene locus488 are
shown in Fig.2

Figure 2: Spontaneous lacZ mutant frequencies with age in

brain, testis, spleen, liver, heart, and small intestine of the
lacZ-plasmid transgenic mice. The lines represent hand-drawn
curves matching mean mutant frequencies at different age
groups. The survival curve of these mice, set to 100% at birth,
indicates 50% survival at 26.5 months of age. This was not
different from non-transgenic control animals of this strain.
( Vijg et. al., 2002)

The organ-specific mutation accumulation observed in

the lacZ-plasmid mice appeared to be very similar to that obtained by others. For example, Tetsuyo Ono
(Sendai, Japan) and co-workers used the original bacteriophage mouse model made by Jan Gossen also
observed an age-related increase of lacZ mutation frequency in liver, heart, and spleen, but virtually no
increase in brain and testes(Ono et. al., 2000) Also Stuart et al., (2000), analyzing the mutation frequency
in liver, brain, and bladder of lacI mice, observed a tissue-specific aging-related pattern, with mutations
accumulating in liver, more rapidly in bladder, but not in brain. Also using the LacI reporter mice, Hill et
al, (2005). analyzed spontaneous mutation frequency in middle to late adulthood of the mouse, at 10, 14,
17, 23, 25, and 30 months of age in adipose tissue, liver, cerebellum, and male germ cells. These authors
observed elevation of the mutation frequency with age in adipose tissue and liver, but not in cerebellum
or male germ cells, thereby extending and confirming the tissue specificity mentioned above. Mutation
accumulation in male germ cells at the LacI locus in the mouse was studied in the laboratory of Walter et
al. (1998). Interestingly, an initial decline in mutation frequency was observed during spermatogenesis,
with a subsequent increase in spermatogenic cells obtained from old mice. The mutation frequencies
eventually obtained in old mice were similar to the ones obtained in the aforementioned studies (at either
young or old age).Whereas several laboratories reported a generally lower spontaneous mutation
frequency in both brain and male germ cells, there seems to be consensus that in these tissues there is no
increase with age (or a very small one).Hence, it is possible that these investigators were able to
accurately measure lacI mutations at much lower levels than others, in which case the aging-related
increase escaped detection by other investigators.
Figure 3: A comparison of young and old
mice for the type of mutations found to
accumulate at the lacZ transgene locus.
Note that in the small intestine (SI) virtually
all such mutations are point mutations, but
not in liver (Li) or heart (He). Also note the
virtual lack of genome rearrangements in
brain (Br)..

Since bacteriophage models do not indicate a profound organ or tissue specificity with respect to the
spontaneous mutation spectra. By contrast, in the lacZ-plasmid model, with its approx. 20-fold smaller
concatamer, Martijn Dollé observed striking organ specificity with respect to the mutational spectra of the
old animals. Mutations in the lacZ reporter mouse model can be characterized by restriction digestion and
nucleotide sequencing of the positively selected lacZ-mutant plasmids recovered from a mouse tissue.
Mutations that do not alter the restriction pattern are point mutations—single base changes or very small
deletions. Mutations that cause changes in the restriction pattern are deletions and other types of
rearrangement. Only a few of these were deletions within the lacZ gene. Most of these mutations
appeared to involve genome rearrangements between the lacZ-plasmid cluster and the mouse
chromosomal DNA. As mentioned, such events can be physically characterized based on the small mouse
fragment that indicates the breakpoint in the mouse genome. Already, by simply separating mutations on
the basis of whether or not they changed the restriction-enzyme pattern in these two classes, striking
organ specificities were observed. For example, whereas in heart and liver both point mutations and
genome rearrangements were found to accumulate, in small intestine the age-related increase was entirely
due to point mutations(Dolle et al., 2000).

Further characterization of the point mutations by nucleotide sequencing of entire mutant lacZ genes
obtained from different organs at young and old age indicated that initially, at young age, the mutation
spectra were more or less the same for all organs. However, during aging they started to diverge
significantly, resulting at old age in drastic differences between organs, with a high frequency of GC to
AT base-pair substitutions at CpG sites in post-mitotic organs, such as brain and heart, and a more varied
pattern in the more proliferative tissues. Especially in brain and heart of old animals, GC to AT transition
mutations at CpG sites were dominant. Mutations are a typical consequence of the spontaneous
deamination of 5-methylcytosine. Further characterization of the genome rearrangements also required
nucleotide sequencing. Whereas some conclusions could be drawn about the nature of such mutations
based on the restriction patterns, characterization of the breakpoint in the mouse genome allowed their
identification as deletions, inversions, or translocations. Such physical mapping of 49 genome-
rearrangement mutations, mainly from heart and liver of young and old mice, indicated intrachromosomal
deletions or inversions, varying from smaller than 100 kb to 66 Mb, as well as translocation events. A
significant increase in the frequency of these genome rearrangements with age was found in heart and
liver, but not in brain. Assuming that the lacZ plasmid reporter locus is representative for the overall
genome, Martijn Dollé extrapolated the genome rearrangement frequencies from the 3-kb lacZ transgene
to the entire 6 x 109-bp diploid genome. The outcome was subsequently divided by a factor of two,
because only one of the two breakpoints needs to occur in a lacZ reporter gene. The results indicate that
up to 37 (in the aged heart) genome rearrangements can accumulate in an aged cell.(Dolle et al., 1995)

There are multiple explanations for organ specificity in the rate of mutation accumulation and the type of
mutations found during the course of the aging process. A most logical one is that the mutation frequency
is simply a function of organ-specific cell-proliferative activity. Indeed, it is reasonable to assume that
with higher proliferative activity there is a higher chance of replication error, one of the major
mechanisms of mutation generation. This could explain the relatively high mutation frequency in small
intestine, which increases about 5-fold during aging. It does not explain, however, the relatively low age
related increase in spleen, and the absence of an increase in testes; both these organs should contain
plenty of actively proliferating cells. If proliferative activity alone is important, then it is also not clear
why mutations accumulate in heart, a post-mitotic organ par excellence. To resolve at least some of these
issues, Ana Maria Garcia, investigated the possibility of intra-organ variation in the two tissues that were
most dissimilar in terms of mutation accumulation: the highly proliferative small intestine and the post-
mitotic brain. By separating the inner part or mucosa of the intestine, which mainly contains the rapidly
dividing epithelial cells, from the outer part or serosa, a layer of post-mitotic, smooth muscle cells, she
was able to directly compare the lacZ mutation frequency between proliferating and non-dividing cells in
this organ from 18-month-old mice. The results indicate significantly higher mutation frequencies in the
highly proliferative mucosa than in the serosa (Fig. 4).Most of the mutations in the mucosa were point
mutations, suggesting replication errors as their source. However, Ana Maria also observed a significant
increase in lacZ mutation frequency in the serosa, underscoring the fact that DNA replication is not
strictly necessary for mutations to accumulate with age.

Figure 4 More mutations have accumulated with age in the

mucosa than in the serosa of the small intestine from 18-
month-old mice. The excess mutations found to accumulate
in the mucosa were all point mutations (white).( Garcia, et
al)
 Figure 5: Mutation frequencies are higher in the
hypothalamus and hippocampus than in the whole brain.
Also the rate of age-related mutation accumulation is
significantly higher in these structures. Black bars, young
mice; white bars, 18-month-old mice; gray bars, 30-month-
old mice. (Garcia, et al)

With respect to the brain, Garcia was interested in the possibility that while in the overall organ mutations
were not (or only slowly) accumulating, certain sub-structures could nevertheless show increased
mutagenesis with age. Indeed, when subdividing different brain parts, she observed that in the
hypothalamus and hippocampus mutations were accumulating with age with the mutation frequency
higher than in the brain overall at all ages (Fig. 5). Therefore, factors other than DNA replication errors,
possibly closely related to the function of a particular organ or sub-structure, are likely to play a role in
determining the patterns of genomic instability as they arise during aging. Rita Busuttil, convincingly
demonstrated that mutations do not need replication for their induction. It all depends on the type of
mutation.

The results of above studies related to tissue specific mutation indicate that the Genome alterations occur
in both senescent and diving cells, however the type of alteration do depend on type whether cells are
mitotic or post mitotic. And hence, it is clear that both senescence and mutations compliment and
promote each other.

3. Types of Genome alterations that occur with age

Unlike DNA damage, which can be recognized and repaired through one of the pathways in the network
of genome-maintenance and -repair systems, DNA mutations due to errors in pathway, sequence changes
transpositions, cytogenetic changes, as well as epigenomic errors are irreversible. This makes them a true
molecular endpoint of the aging process. Below I will summarize the different types of genome alteration
that can occur in cells of animal tissues and the evidence that they accumulate with age in vivo, and
briefly discuss the likelihood that such changes could contribute to the aging phenotype.
3.1 CYTOGENETIC CHANGES
Cytogenetic changes are genomic alterations that can be visualized by light microscopy in cells, usually
during metaphase. They include chromosomal aberrations, abnormalities in the structure or number of
chromosomes, and extrachromosomal fragments, such as the extrachromosomal rDNA circles that
accumulate in yeast during replicative aging. A proportion of chromosomal aberrations is unstable and
gives rise to chromosome fragments without spindle-attachment organelles (kinetochores, centromeres).
These are termed acentric fragments. When the cell divides, some of these fragments are excluded from
the main daughter nuclei and form so-called micronuclei within the cytoplasm, either on their own or in
conjunction with other fragments.

Cytogenetic analysis provides evidence for an increasing level of genome alterations in a somatic tissue
during aging in vivo (Curtis et. al., 1963). Curtis and Crowley looked at the main cell type in the mouse
liver, parenchymal cells or hepatocytes, which normally do not divide. When inspecting parenchymal
cell metaphase plates after this so-called partial hepatectomy these workers found considerably higher
numbers of cells with abnormal chromosomes in old animals than in young animals (from about 10% of
the cells in 4–5-month-old mice to 75% in mice older than 12 months).

Later, such large structural changes in DNA—aneuploidy, translocations, and

dicentrics—were routinely observed to increase with donor age in white blood
cells of human individuals; that is, from about 2–4% of the cells carrying chromosome
abnormalities in young individuals to about six times more than this in the elderly. It is
conceivable that these chromosomal changes reflect changes in the hematopoietic
stem cells. The recent use of more advanced methods, such as chromosome painting,
has amply confirmed the increase in cytogenetic damage with age in lymphocytes from
both humans(Ramsey et al.,1995) and mice(tucker et al., 1999). Hence there is
currently universal consensus that the frequency of cytogenetic changes in both human
and mouse cells increases with age. In lymphocytes, which is still the only cell type
readily accessible for such determinations, the frequency of cells carrying one or more
of such abnormalities does not appear to rise much higher than approx. 10%.

However, it should be realized that even with the more recently emerged chromosome painting methods,
the smallest possible type of aberration that can still be detected is 2 million bp. Based on the early work
of Howard Curtis, the frequency of cytogenetic alterations may be higher in non-dividing cells, but in
the absence of independent confirmation of these results it is difficult to draw conclusions. However,
one type of chromosomal aberration—aneuploidy—has been recently found in post-mitotic tissues as
well. Aneuploidy is so widespread in tumors that there is probably no tumor that, upon analysis, does not
appear to depart from diploidy. Aneuploidy can have major adverse effects through so-called loss of
heterozygosity (LOH) due to the loss of one chromosome copy.

In the mouse embryo it has been demonstrated that over 30% of neural progenitor cells display
chromosomal aneuploidy, both loss and gain of chromosomes (Rehen et al., 2001) . This high frequency of
chromosomally aberrant neurons was also found in normal human brain using interphase fluorescence in
situ hybridization (or FISH) with probes for chromosome 21(Rehen et al., 2005). Chromosome 21-aneuploid
cells, including non-neuronal cells and neurons, were shown to constitute about 4% of the estimated 1
trillion cells in the human brain. Since only one autosome was analyzed it is likely that the overall
percentage of aneuploidy in the brain is much higher than that. This is in striking contrast with the
situation in human lymphocytes in which only 0.6% of the cells were chromosome 21-aneuploid. Routine
karyotyping in both human and mouse lymphocytes revealed aneuploidy in only about 3% of such cells.
Whereas it is not known whether the frequency of aneuploid cells in the brain increases with age, the high
numbers of aneuploid cells already in the brain of adults indicate that our genome is much less stable than
often thought.

A frequent consequence of aneuploidy is cell death. However, the widespread occurrence of aneuploidy
in tumor cells already indicates that cell death is by no means inevitable. Indeed, it has recently been
demonstrated that aneuploid neurons are functionally active and normally integrated in brain circuitry
(kingsbury et al.). This, together with the many other types of random genome alterations that have been
demonstrated in somatic cells, leads to genetic mosaicism in the cells and tissues of an individual
during development and aging. One may ask whether this genetic mosaicism contributes to individual
physiological and behavioral variation, for better or for worse. Thus far this question remains
unanswered, but a recent study provides some compelling evidence that large-scale genomic variation is
compatible with life and is more common than thought previously. For example, apart from the single-
nucleotide polymorphisms, of which there have been over 10 million, defined in the human genome,
large-scale copy-number polymorphism has been observed among individuals (Iafrate et al., 2004, Sebat et
al.,2004) and is thought to greatly contribute to genomic diversity and disease susceptibility (Feuk et al., 2005).
Whereas it is certainly possible that some random genomic alterations contribute positively to certain
complex physiological functions, such as those encoded in brain circuitry, it is reasonable to expect
adverse effects in most cases. This issue, as to how stochastic alterations can have a consistent effect on
function, is of major interest for the question that is central in this paper: how could random genomic
alterations lead to a series of consistent physiological changes that ultimately bring life to an end?

Observations in mice suggest that chromosomal aberrations in neuronal stem cells accumulate during
aging, eventually affecting almost all such cells. For most of the past century it was thought that the adult
brain was unable to generate new neurons, but that turned out to be wrong. (Reynolds et al., 2005) Stephen
Pruitt (Buffalo, NY, USA) and co-workers analyzed neurospheres from C57Bl/6_DBA/2 hybrid mice at
nine chromosome pairs for genomic areas of structural loss by making use of known single- nucleotide
polymorphisms between these two mouse strains ( Bailey et al., 2004). At such polymorphic loci, the loss
of one allele indicated LOH. It was found that whereas in 15 neurospheres from young mice no deletions
were detected, 16 out of 17 neurospheres from old animals displayed LOH on one or more chromosomes.
The results of this elegant study are especially interesting since they indicate the accumulation of
chromosomal mutations in stem or progenitor cells, a potential reservoir for cell-based therapies of aging-
related degeneration.

These studies again indicate that Transcriptional changes will occur with age. Cells with excessive
aneuploidy will lead to cell death or cancerous growth. Post-mitotic and senescent cells will be more
prone to cytogenetic changes. So, it is important to keep in mind this genomic alteration while designing
the model of aging.

3.2 Telomeric instability leads to senescence and apoptosis of mitotic cells

A genetic instability hotspot that has received a lot of attention is the telomere. Telomere shortening, due
to the loss of minisatellite repeat elements, has been observed to occur during repeated passaging of
human cells in culture, this causes replicative senescence and in the absence of p53 leads to crisis. This
latter stage is characterized by chromosomal aberrations, dominated by end-to-end fusions. This fusion is
prevented by activation of p53 since Telomeric end not bound to nuclear lamina are recognized as DSB
DNA damage p53 is activated in response which leads this cell to apoptosis or senescence. The
importance of telomerase in preventing this is underscored by the observation in the laboratories of
Woody Wright and Jerry Shay (both in Dallas, TX, USA) that its reconstitution prevents replicative
senescence and essentially immortalizes normal human cells (Bodnar et al., 1998).

Also in view of the important role of replicative senescence as an in vitro model of normal aging, this is
generally interpreted as strong evidence for telomerase as a major and perhaps the most important
genome-stability system in actively proliferating human cells. Its potential importance for aging in vivo is
underscored by the observation of premature aging of mice with an inactivated telomerase RNA
component and a number of functional studies.

In the genetic disorder dyskeratosis congenita, telomere shortening is accelerated, and patients have
premature onset of many age-related diseases and early death (Vulliamy et al., 2004). Is there any evidence
for telomere attrition in vivo to the extent that it can be considered as a major cause of aging, for example,
through increased replicative senescence? Telomere length can be measured as the mean length of
terminal restriction fragments, which involves a standard genomic Southern blot with terminal restriction
fragments detected by hybridization to a probe containing telomeric repeats. Whereas alternative methods
allowing the measurement of single telomeres were later developed (Lansdorp et al., 1996, Baird et al.,
2003), most data on telomere length during aging were obtained with analysis of terminal restriction
fragments.

Average telomere length in white blood cells and other tissues of humans have been measured by a
number of groups and found to decline from approx. 12.2 kb in a fetus to 7.2 kb in a 72-year-old (Butler
et al., 1998, Vaziri et al., 1993, Iwama 1998) . The greatest loss was found to occur at relatively
young age. No individuals have been found with a mean telomere length of less than 5 kb, even among
100-year olds. More recently, using a quantitative PCR method in a study of 143 normal unrelated
individuals over the age of 60 years, an association was found between telomere length in blood and
mortality(Cawthon et al., 2003). However, this was not confirmed in a later study involving 598 normal
individuals over 85 years(Martin 2005)

Whereas it is therefore questionable whether telomere attrition by itself has phenotypic consequences in
vivo, it should be kept in mind that average telomere length in blood is not the same as the average length
of telomeres in an individual cell or the shortest telomere of an individual chromosome. Hence, even if
average telomere length is not a predictor of survival, telomere loss could nevertheless be one of many
different forms of genomic instability contributing to normal aging, in this specific case, for example, by
resulting in an increased number of senescent cells.

3.3 mtDNA mutations occurs with age and cause DNA damage
The 16.5-kb mitochondrial genome has been implicated as a major target for somatic mutations during
aging(Taylor et. al., 2005). According to the mitochondrial theory of aging somatic mutations in mtDNA
accumulate with age as a consequence of the free radicals generated so close to their target. The resulting
degenerative changes in this important cell organelle are then expected to result in impaired function of
the respiratory chain, leading to increased free radical production and more mutations in mtDNA as well
as in nuclear DNA. This would lead to accelerated telomeric shorting and more 8-oxoG lesions in nuclear
DNA. More damage will definitely again result in functional loss, senescence and cell death by pathways
converging to p53. The logic of this vicious cycle has appealed greatly to gerontologists and explains the
importance assigned to the mtDNA as a target for age-related mutations. However, as we shall see,
recently obtained results shed some doubt on vicious-cycle ideas.

The main reasons to consider the mitochondrial genome as an important target for aging-related
spontaneous DNA mutations are its physical proximity to a major source of free radicals, the lack of
protective histone proteins, and the presumed absence of mtDNA-repair systems. However, this last point
turned out not to be entirely correct; several repair pathways have been described for mtDNA, most
notably an efficient BER system, and possibly also mismatch repair and recombinational repair (Larsen 2005).

Nevertheless, there is evidence for a relatively high susceptibility of mtDNA to oxidative DNA damage.
Apart from their increased vulnerability to DNA damage, mitochondria are the key organelles for
respiration through electron transfer and oxidative phosphorylation, arguably the most important cellular
function in eukaryotes.

Encoded by the mitochondrial genome are two rRNAs, 22 tRNAs, and genes encoding 13 mitochondrial
protein subunits. They are necessary to produce essential enzyme complexes involved in oxidative
phosphorylation. However, most mitochondrial components are encoded in the nuclear genome. We may
ask, what is the evidence that mutations in the mitochondrial genome accumulate to a level that starts
contributing to aging-related cellular degeneration and death?

Within human populations, the mitochondrial genome has a high rate of sequence divergence with
multiple polymorphic variants. This has led to its major role as a tool for examining human evolutionary
history. mtDNA is inherited exclusively through the mother and a number of human heritable diseases are
caused by specific mutations in the mitochondrial genome. These mutations typically impair oxidative
energy metabolism, causing such aging-related diseases as cardiomyopathy and neurodegeneration.
Somatic cells contain hundreds (epithelial cells) to hundreds of thousands (cardiomyocytes) of copies of
the mitochondrial genome per cell and disease-causing mutations are often present in only a fraction of
the total. This situation is called heteroplasmy. A mutation is called homoplasmic when it is carried by all
copies of the mitochondrial genome. Usually the clinical severity of the disease is correlated with the
proportion of the mutated mtDNA in the target tissues. There is a threshold above which a disease
phenotype becomes apparent (Rossignol 2003). This threshold may be 60% for deletions and as high as 90%
for point mutations. Similar to the situation with nuclear DNA mutations, deletions have a higher impact
because they affect a larger part of the mitochondrial genome. However, the possibility cannot be
excluded that physiological effects manifest at frequencies of deleted mitochondrial genome copies as
low as 20%.

For somatic mutations arising with age the situation is very similar to the mitochondriopathies. Somatic
mtDNA deletions increase with age in a tissue-specific manner (Arnheim 1992). In particular, muscle, heart,
and brain accumulate relatively high levels of deleted mtDNA. Intra-organ variation has been
demonstrated for the brain, with an accumulation in the cortex, but not in the cerebellum (Soong 1992). Hence,
this situation is highly reminiscent of what has been observed for nuclear DNA with the lacZ reporter
mice, in which similar striking inter- and intra-organ variation in mutation accumulation has been
observed. Also in this case, the question is whether or not the observed mutation frequencies can lead to
physiological consequences. For the nuclear genome this question cannot really be addressed at this stage,
but the mitochondrial genome is much smaller and relatively easily accessible due to its high copy
number per cell. Nevertheless, measuring mutations in the mitochondrial genome remains technically
challenging.

Before a mutation can have a phenotypic consequence, it must first clonally expand. There is now ample
evidence that mitochondrial mutations not only accumulate with age, but can also clonally expand to
reach levels that may be physiologically significant. Like so many studies aiming to detect and quantitate
low-frequency events, in this field one has to be extremely cautious in interpreting the work that has been
done. Most assays are based on PCR, which is notoriously prone to artifact. Some of the best work in
analyzing mtDNA mutations in aging is by Khrapko et al., (1999) , who developed a PCR-based method
to scan significant parts of mitochondrial genomes from single cells. Using this assay he demonstrated
that in human cardiomyocytes from old but not from young donors, mtDNA deletions occur at a
frequency of about one in 15 cells (Bodyak 2001). In such cells deleted mtDNA copies represented up to
65% of the total number of copies, although in most cells this fraction was much less. Excellent work in
this field has also been done by the groups of Doug Turnbull (Newcastle upon Tyne, UK) and Judd Aiken
(Madison, WI, USA). Turnbull’s group was the first to demonstrate mtDNA deletion mutations in muscle
fibers from normal elderly subjects with very low activity of cytochrome c oxidase (a marker of
respiratory-chain deficiency), suggestive of a mtDNA defect( Brierley 1998). Aiken’s group observed
clonality of deletion mutations in the mitochondrial genome in defective muscle fibers in aged rats (Cao 2001)
and rhesus monkeys(Gokey 2004) .Hence, in the case of muscle there is concrete evidence that mtDNA
deletions may actually cause defects in oxidative phosphorylation. Recent data from Khrapko’s
laboratory, using a novel single-molecule PCR approach, indicate high fractions of deleted mtDNA in
cytochrome c oxidase-deficient neurons in the substantia nigra of elderly humans (Kraytsberg 2006).

For point mutations the situation is more complicated because such mutations are more difficult to detect.
Using a similar single-cell PCR approach, Khrapko’s group sequenced about 5% of the mitochondrial
genome in a number of cells from tissues as diverse as buccal epithelium and heart muscle obtained from
donors of different ages(Nekhaeva 2002). They discovered that many cells contained high proportions
(varying from about 30 to 100%) of clonal mutant mtDNA expanded from single initial mutant mtDNA
molecules. Virtually all expanded mutations were observed in tissues from elderly. They concluded that
in an aged human body, there is on average approximately one expanded mtDNA point mutation per cell.

Rather than post-mitotic cells, such as cardiomyocytes or muscle fibers, Turnbull’s group analyzed
intestinal colonic crypts, clonal populations of cells derived from one or two stem cells, for mtDNA
mutations(Taylor 2003). They sequenced the entire mitochondrial genome from individual colonic crypts
obtained by laser micro-dissection from elderly individuals. Pathogenic point mutations were detected in
most but not all of the cytochrome c oxidase-deficient crypts. Since the frequency of such crypts
increased with age these findings support a more general role of mtDNA mutations, including point
mutations, in aging.

3.4 Mutations due to transposons

The families of repeat elements that are such a major component of the mammalian genome are also
recognized mutational hot spots. Some of these repeat families plays an important role in the regulation of
gene-expression patterns. As early as 1969, Roy Britten (Corona del Mar, CA, USA) and Eric Davidson
(Pasadena, CA, USA) proposed that networks of dispersed DNA repeats, thereby focusing on
transposons, coordinate the expression of batteries of genes involved in development. There is evidence
that both the mobile genetic elements (mainly retrotransposons) and simple, tandem repeat loci are
unstable. Such instability would allow rapid evolutionary change at the level of gene regulation rather
than through protein structural alterations. Whereas, as yet, evidence has emerged for networks of
miRNAs that act as additional layers of gene regulation there are no consistent data pointing towards a
role of the main repeat families in the genome; that is, retrotransposons and tandem repeats.

Nevertheless, it is conceivable that age-related instability of these sequences has adverse effects on
normal patterns of gene regulation. There are two major families of retrotransposons, termed LINES and
SINES, with L1 and Alu, respectively, as their main representatives in humans. L1 repeats, the only
active human mobile element, encode the machinery to move both itself and Alu, which is much shorter
and does not contain an open reading frame. The capability of such elements to retrotranspose has been
demonstrated in human cell lines(Kimberland et al., 1999). The recently evidence for both expression and
retrotransposition of L1 elements in mouse neural progenitor cells in the brain as well as in such cells in
vitro was obtained. There was some evidence that L1 insertion occurred preferentially in or near genes
with an effect on differentiation of the neural progenitor cells. It has not been possible to accurately
determine the frequency of the insertion events, which may vary from one in 10 to one in 1000 cells.
Hence, there is ambiguous evidence that they contribute significantly to somatic instability and nothing is
known about a possible increase in transposition activity with age(Johnson et al., 2002) . The authors
suggested that the observed L1 transposition could positively contribute to brain plasticity, it is more
likely that such events would contribute to the random accumulation of mutations in the genome to
possibly exert adverse effects. Indeed, overexpression of L1 proteins leads to toxicity (Bourc’his et al., 2004)
and L1 is able to induce a p53-dependent apoptotic response (Haoudi 2004). Moreover, L1 is preferentially
expressed in the germ line. Its lower overall expression level in somatic cells may be a consequence of
methylation((Bourc’his et al., 2004).

3.5 Epigenetic mutations - Changes in DNA modification, conformation and

nuclear architecture

Thus far, most studies of genome alterations in aging have focused on DNA damage (chemical changes)
or mutations (sequence changes).However, more and more attention is now focused on another source of
phenotypic variation that is not based on variations in the base sequence of DNA. Such so-called
epigenetic variation is heritable information encoded by modifications of genome and chromatin
components that affect gene expression. The structural basis for epigenetic variation involves DNA
methylation or histone-modification patterns. Epigenetic information is only partially stable and destined
to change during development and in carrying out essential somatic functions. Hence, it would not be
surprising if stochastic changes in the epigenome turned out to be more relevant as a potential cause of
aging than DNA sequence changes.

Epigenetic patterns of transcription control have now been conclusively shown to be essential in the
regulation of gene expression. After embryogenesis, when epigenetic patterns are erased and reset,
development and differentiation are largely facilitated by epigenetic programs. Random changes in these
patterns are genuine mutations and likely to have adverse effects, similar to changes in DNA sequence.
Called epimutations by Robin Holliday (Sydney, Australia) ( Holliday, et al., 1991), they have now been
demonstrated to be critically important as causal factors in cancer (Feinberg 2006).

3.5.1 Heterochromatin reorganizes in response to DNA damage:

Why does Sir protein redistribution occur during ageing? The answer may come from the
growing appreciation of the importance of chromatin in maintaining genomic stability and
facilitating DNA repair. During the DNA-repair process, chromatin needs to be unpacked and
reassembled, which has an important influence on the rate and type of repair. One possibility is
that the relocalization of Sir proteins is an active defence process that the cell initiates to
stabilize its DNA. In 1999, four studies showed that a single DNA break is sufficient to illicit a
DNA-damage-checkpoint response that releases Sir proteins from mating-type loci and
telomeres and relocalize them to the DNA break, possibly to facilitate the repair process.
Indeed, genomic DNA from SIR2 mutants was shown to be more susceptible to cutting by an
endogenously expressed EcoRI endonuclease25. However, no defect in DNA end-joining was
observed using a plasmid-based assay26, a discrepancy that may be explained by the lack of
chromatin on plasmid DNA. This finding fits with the observation of Tyler and colleagues, who
found that Sir2 and other histone deacetylases modify the chromatin that surrounds the break
site in a temporally coordinated manner, which appears to be a prerequisite of efficient repair.
We refer to this process as the relocalization of chromatin-modifying factors (RCM) response.
The damage-mediated relocalization of Sir proteins appears to have little effect on long-term
genomic silencing patterns in young yeast cells. However, the accumulation of DNA damage
and increased rDNA instability in old yeast cells eventually leads to a chronic RCM response,
alterations in silencing and irreversible genomic changes. A role for DNA damage in age-related
genomic instability has also been reported for loci other than the rDNA. DNA-break-induced loss
of heterozygosity at artificially generated heterozygous loci was found to increase dramatically
with age and, depending on the locus, was aggravated in the absence of Sir2, again indicating a
protective role for heterochromatin. Both increased susceptibility to DNA damage and an
accumulation of defective DNA-repair enzymes might explain how ageing can promote this
global genomic instability.

3.5.2 Lessons from human progeroid syndromes:

WS, HGPS and ataxia telangiectasia (AT) are rare genetic premature ageing disorders that
demonstrate both the dramatic consequences of defects in nuclear architecture and the diverse
sets of genes that are involved in its maintenance.WS is characterized by a genome that is
highly unstable owing to the lack of functional RecQ helicase. HGPS shows similarities to WS
but proceeds more rapidly. One known cause of HGPS is a single base change in the LMNA
gene, which encodes lamin A, an essential structural component of the nuclear membrane. The
mutant LMNA gene generates a truncated splice variant that disturbs the structure of the
nuclear membrane and causes large changes in the nuclear architecture. Nuclei from patients
with HGPS are characterized by a dysmorphic shape and a loss of heterochromatin-related
proteins that are associated with the nuclear membrane, such as heterochromatin protein-1
(HP1), as well as altered histone-modification patterns that reflect a general disturbance in silent
heterochromatin (FIG. 6).

Interference with aberrant LMNA splicing can reverse the structural defects that are typical for
HGPS in cell culture, which demonstrates a direct causal relationship between the LMNA gene
and HGPS. The truncated LMNA splice variant has also been found in naturally old humans,
which implicates changes in lamin A in the normal ageing process. A conserved role for lamin A
in the ageing process is consistent with the recent finding that neuronal cells in the nematode
Caenorhabditis elegans also show changes in the nuclear architecture in aged animals, and a
loss of function mutation in the worm orthologue of lamin A causes a decrease in life
expectancy. Like WS and HGPS, AT is characterized by a defective nuclear architecture,
progressive neurological degeneration, growth retardation, genomic instability and premature
ageing. Patients with AT have an inherited defect in the AT mutated (ATM) gene, which
encodes a protein kinase that initiates the DNA-repair cascade. DNA repair and ATM in
particular seem to be required for telomere maintenance, and defective ATM can disturb the
interactions between telomeres and the nuclear matrix. The contribution of this effect on AT
pathology remains unclear and is complicated by the range of defects that are observed in
ATM-defective cells. The yeast ATM orthologue Tel1 has also been linked to telomere
maintenance, which further suggests that pathways that are involved in the maintenance of
nuclear architecture are highly conserved.

3.5.3 Chromatin remodeling

Changes in nuclear architecture do not appear to be restricted to defects in the structural

components of the nucleus. An age-related loss of epigenetic silencing at certain repetitive
elements was reported almost 20 years ago. Shen et al. recently reported a possible
mechanistic link between mammalian ageing and changes in heterochromatin. Chromatin
remodeling may occur in two ways either by histone modification or by changes in methylation
patterns. Older individuals show altered activity in their histone-modifying enzymes, which
causes a loss of perinuclear heterochromatin and concomitant changes in gene expression. An
important function of heterochromatin is the suppression of recombination. This would suggest
an age-related loss of heterochromatin, which is in keeping with reported changes in
nucleosome spacing with age and an altered sensitivity to nucleases (Kanungo et al., 1994).
Interestingly, David Sinclair (Boston, MA, USA) and co-workers recently demonstrated that in
mice SIRT1 is bound to repetitive DNA and to promoters of a number of genes. In the brain,
such binding was found to decrease with the age of the mouse. In mouse ES cells SIRT1 was
released from repetitive DNA in response to genotoxic stress in the form of treatment with
hydrogen peroxide.

Numerous other epigenetic changes in nuclear architecture and gene expression have
been associated with ageing. More than a decade ago, Imai and colleagues showed that
collagenase, a gene associated with cellular ageing, is differentially regulated during
cellular senescence. This effect appears to be due to changes in the subnuclear
localization of the collagenase gene as cells undergo senescence. In young cells, the
collagenase gene is repressed by the transcription factor OCT1. A considerable proportion of
OCT1 was found in the heterochromatic nuclear periphery, where it co-localized with lamin B, a
component of the nuclear membrane. This interaction was abrogated in senescent cells and,
concomitantly, collagenase repression was lost. On the basis of these findings, the authors
proposed a model of age-associated heterochromatin reorganization that would account for
such transcriptional changes in a global manner.

This idea gained support from recent studies of cellular senescence, most notably by Lowe and
colleagues, who found that senescence is associated with an overall increase in non-
pericentromeric, facultative heterochromatin domains, known as senescence-associated
heterochromatin foci (SAHFs; FIG. 5). SAHFs form repressive chromatin structures that can be
found at, but are not limited to, promoter regions of certain cell-cycle regulators, in particular
target promoters of the cell-cycle regulator E2F. Since E2F represses p53 hence this again links
aging to p53, the central component in aging phenomena and hints the link between
collagenase activity and senescence associated with p53.. Although the repression of cell-cycle
regulators is an important function of SAHFs during cellular senescence, the frequency and
distribution of these foci suggests a much broader impact of SAHFs. This notion is further
supported by the finding that the formation of SAHFs appears to rely on the recruitment of
proteins from promyelocytic leukemia nuclear bodies, which have been implicated in numerous
cellular processes including transcriptional regulation, apoptosis and cellular defence in
response to stress (most notably to DNA damage).

A connection between senescence-associated heterochromatin formation and mammalian

ageing has recently been made using baboon skin fibroblasts. Tissue from older individuals
accumulates cells containing heterochromatic foci that are reminiscent of SAHFs in senescent
cells. These foci were found in >15% of the total cell population in aged tissues, which suggests
that a significant fraction of aged tissue may be expressing markers of senescence and
undergoing large-scale heterochromatic changes. Importantly, the emergence of
heterochromatin foci occurred simultaneously with telomere shortening, which points to a shift
from stable, perinuclear heterochromatin to induced, or facultative, heterochromatin. These
studies raise the intriguing possibility that the age-associated loss of genomic silencing detected
in previous studies may be linked to, or caused by, the formation of SAHF-like heterochromatic
foci, a phenomenon reminiscent of the RCM response that occurs in yeast in response to DNA
damage and ageing.

However, it is important to keep in mind that cellular senescence — although it is a likely

contributor to cancer and organismal ageing — does not equal the complex physiological
processes that, together, define what is called ageing. The relevance of the aforementioned
findings to the functional decline of higher organisms remains to be elucidated.

Figure 6 | Comparison of age-related

changes in nuclear architecture
between yeast and mammalian cells. a
| Schematic of accelerated ageing (top)
and normal ageing (bottom) in a
replicating yeast nucleus. In young yeast
cells, telomeres, mating-type loci (MATa
and MATand ribosomal DNA (rDNA;
orange) are silenced by silent
information regulator-2 (Sir2)-containing
complexes (purple circles). In wild-type
yeast, homologous recombination at the
highly repetitive rDNA locus generates
extrachromosomal rDNA circles (ERCs)
during cell division. Lack of the DNA
helicase Sgs1 causes genomic instability
at the rDNA and leads to increased ERC
formation, accelerated changes in
nuclear architecture and premature
ageing23 (top). Sites of DNA damage
and both ERCs and rDNA recruit
components of the Sir2- silencing
complex, causing a loss of silencing at
telomeres and mating-type loci (green
represents areas of transcriptional
derepression). b | Changes in nuclear
architecture of human cells in a model of
accelerated ageing (top) and normal
ageing (bottom). Young cells show
dense, transcriptionally inaccessible
perinuclear heterochromatin surrounding
less densely packed, transcriptionally active euchromatin. Grey circles represent sites of facultative heterochromatin and blue
ovals depict constitutive, perinuclear heterochromatin (BOX 2). In Hutchinson–Gilford progeria syndrome (HGPS), a defect in
the nuclear lamina component lamin A leads to an accelerated loss of pericentromeric heterochromatin and concomitant changes
in nuclear architecture that are accompanied by transcriptional deregulation13,58,59 (green areas). Similar changes have been
observed during normal ageing. Cellular stress can cause the formation of repressed senescence-associated heterochromatin foci
(SAHFs)12.

3.5.3.1 Changes in methylation patterns occur with age can affect by causing chromatin
remodeling

DNA methylation acts synergistically with histone deacetylation to repress transcription. Methylation
patterns are intricately related to changes in histone-modification patterns. Together these epigenomic
control systems are two sides of the same coin: chromatin remodeling Epigenomic alterations are now
also increasingly recognized as part of aging and its associated pathologic phenotypes( Richardson et al., 2003,
Bandyopadhyay et al., 2003). Early evidence indicated a general demethylation in most vertebrate tissues with
aging(Wilson et al., 1987).

In mitotically active tissue, it is possible that this reflects some deficiency in maintenance remethylation
as this normally occurs after DNA replication. Such a general demethylation during aging may
desuppress silenced retrotransposons, such as L1, to increase genome instability as indicated earlier(see
above). L1 retrotransposons can again activate p53 and cause organismal aging. Genome-wide
demethylation has been suggested to be a step in carcinogenesis, based on the results of a study
suggesting that defects in DNA methylation might contribute to the genomic instability of some
colorectal tumor cell lines ((Lengauer et al., 1997) .Evidence that supports this notion comes from another
study in which elevated mutation rates were found at both the endogenous Hprt gene and an integrated
viral thymidine kinase (tk) transgene in mouse embryonic stem cells nullizygous for the major DNA
methyltransferase (Dnmt1) gene. Gene deletions were the predominant mutations at both loci. The major
cause of the observed tk deletions was either mitotic recombination or chromosomal loss accompanied by
duplication of the remaining chromosome (Chen et al., 1998). These results suggest that the role of methylation
may include the suppression of genomic instability. This may be especially important in repeat-rich
genomic regions, to prevent illegitimate recombinations.
.

There are now numerous reports in the literature on changes in methylation status of individual genes
during aging(van Remmen et al., 1995). However, the functional relevance of these changes is largely unknown.
This situation is different for cancer, in which hypermethylation has been associated with the silencing of
genes involved in cell-cycle regulation, tumor-cell invasion, DNA repair, apoptosis, and cell
signaling(Feinberg et al., 2006). The target for such hypermethylation events are the CpG islands located in
gene promoter regions. Methylation of CpG islands is strongly correlated with transcriptional
suppression. CpG islands are generally unmethylated in normal somatic cells, the changes in chromatin
structure resulting from hypermethylation would effectively silence transcription.

After comparing more than 2000 loci in T lymphocytes isolated from newborn, middle-aged, and elderly
people, the conclusion was that 29 loci (approx. 1%) changed methylation status with age: 23 cases of
hypermethylation and six cases of hypomethylation. Most of these methylation changes happened before
middle age. Interestingly, the same CpG islands affected in T cells also changed with age in other tissues,
suggesting that methylation instability is locus-specific. Interestingly, in T cells and various tissues from
aging mice no methylation changes were observed.

There is other evidence pointing in the same direction. In a recent study, global and locus-specific
epigenetic differences between identical twins were reported to arise with age (Fraga 2005). In this study,
involving 80 twins varying in age from 3 to 74 years, a variety of epigenomic markers, including X-
chromosome inactivation, total genomic 5-methylcytosine content, sequence-specific DNA methylation
(by bisulfite genomic sequencing), and global methylation status (by RLGS), were assessed in blood in
comparison with measures of gene expression (using microarrays and quantitative real-time PCR). The
results indicate that while these monozygotic twins are epigenetically indistinguishable early in life, at
older age they start to exhibit remarkable differences in overall content and genomic distribution of 5-
methylcytosine DNA and histone acetylation, affecting their gene-expression profiles.

Evidence for a relaxation of epigenetic control of gene expression during aging has been reported by
multiple authors, with the stability of X inactivation and genomic imprinting the obvious targets of study.
In the mouse an approx. 50-fold age-related increase in liver cells that had re-activated the gene ornithine
transcarbamoylase on the inactive X chromosome has been observed. This was ascribed to an age-related
reduction in DNA methylation (Wareham, et al., 1987)

Age-associated loss of epigenetic repression of X-linked genes has been analyzed quantitatively for the
gene Atp7a in various tissues of the same X–autosomal translocation mouse model( Bennett-Baker et al.,
2003). The results indicated a mean organ-specific relaxation of gene repression in the oldest group of
mice of up to 2.2% (this is the fraction of transcripts from the inactive X relative to its active sister
chromosome).Using the same quantitative methodology, the Igf2 gene, imprinted in one mouse strain but
not in another, was shown to increase relaxation of its epigenomic repression at old age by up to 6.7%.

In humans, the situation may be different. Indeed, in a study of the X-linked HPRT locus in 41 women
heterozygous for mutations at this locus (causing Lesch–Nyhan syndrome), an increase with age in HPRT
+
skin fibroblast clones over the expected 50% (as a consequence of random X inactivation) was not
found(Migeon 1988). Further studies showed that the silent locus does not detectably re-activate
spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor
of methylation. Robin Holliday proposed that age-related reactivation of a silent X-linked gene may be
many times higher in mouse cells than in human cells as part of the much tighter genetic control systems
in long-lived versus short-lived animals. Hence, this could be another example of increased genome
maintenance capacity of humans as compared to mice.

On the other hand, it would be in conflict with the aforementioned RLGS results indicating substantially
more alterations in global methylation status in humans compared with mice. Suffice it to say that also in
this case more, extended data-sets are needed to draw definite conclusions. Decreased epigenomic control
during aging would be in keeping with the so-called dysdifferentiation theory of aging proposed by
Richard Cutler (Phoenix, AZ, USA). According to this hypothesis the primary process of aging is the
time-dependent drifting away of cells from their proper state of differentiation. Some experimental
support for the hypothesis had already been obtained earlier in the laboratory of Cutler himself in the
form of increased hybridization of globin gene probes to RNA extracted from brain and liver of old
versus young mice. This was well before the emergence of PCR, and even before the age of Northern-blot
analysis.

3.5.3.2 Chromatin remodeling is also side effect of DNA damage repair mechanisms
In yeast, DNA damage induces an RCM response that disrupts heterochromatin and alters gene
expression. A wealth of literature has recently implicated chromatin-remodeling enzymes in the
DNA-repair process in yeast and other more complex organisms. DNA double-strand break
(DSB) repair involves the recruitment of histone modifiers to the repair site, together with other
repair complex components such as Ku70/80 and DNA ligase IV. DSBs trigger the DNA-
damage sensor kinases ATM, ATR or DNA-PK, which phosphorylate the surrounding histones
H2A and H2AX in yeast and mammals, respectively.

The extent of this modification can reach into mega-bases, potentially affecting the epigenetic
regulation of several genes. In yeast, chromatin-remodeling factors that are involved in DNA
repair, such as histone acetyltransferases (HATs) and histone deacetylases (HDACs) as well as
histone methyltransferases, are then recruited to the break site. Although the precise role for
these chromatin-remodeling complexes during DNA repair is not fully understood, it is presumed
that they dictate the type of repair and facilitate the repair process by changing the chromatin
composition around the site of damage. In yeast, HATs (such as Esa1) and HDACs (in
particular Sin3, Rpd3 and the sirtuin Hst1) are part of chromatin remodeling complexes that
promote an ordered and dynamic progression of histone modifications over the time-course of
DSB repair.

Importantly, these proteins are not specialized DNA-repair enzymes but, rather, chromatin-
modifying enzymes that have several functions outside DNA repair, including gene silencing.
Recruitment of such factors to sites of damage may therefore be accompanied by a loss of
function at their original sites, as is the case for the Sir complex. Although a role for histone
deacetylation during DNA repair has not been demonstrated in mammalian cells, there is
accumulating evidence for chromatin remodeling besides H2AX phosphorylation. For example,
histone methylation has been directly linked to the recruitment of DNA-repair factors in human
cells. Specifically, methylation of Lys79 on histone H3 recruits the p53-binding protein-1
(53BP1). Recruitment of these factors is thought to tether the DNA-repair complex to the site of
damage. Importantly, this mechanism appears to be evolutionarily conserved between yeast
and mammals because the fission yeast 53BP1 homologue Crb2 is also recruited to DSBs, a
process that requires methylation of Lys20 on H4. The fact that 53BP1 can also be found in the
DNA-damage-associated heterochromatin foci of aged monkey fibroblasts further corroborates
the idea that histone modifiers may have a crucial role during the DNA-damage response in
mammals and points towards DNA damage as an inducer of global changes in chromatin
architecture.

The formation of transient heterochromatic foci around sites of DNA damage may explain how
DNA damage might directly mediate gene repression. Consistent with this notion, many of the
gene-expression changes that are observed in aged individuals occur in a stochastic fashion, as
does most DNA damage. It is also conceivable that certain genomic regions are more prone to
damage than others, which could explain some of the predictable, coregulated changes that are
observed between aged individuals of the same species. Indeed, DNA breaks occur more
commonly in certain euchromatic, active regions of the genome. Furthermore, fragile sites on
chromosomes that are prone to breakage are well documented in mammals. It will be
interesting to investigate whether these sites of preferential DNA damage correlate with loci that
become deregulated with age.

Although it is conceivable how DNA damage might lead to gene repression, it is less obvious
how age-related stress and DNA damage could account for gene activation. This question is
particularly important to address because the fraction of genes that are significantly up-
regulated with age roughly equals or even exceeds the fraction that is down-regulated.
Moreover, transcript levels overall appear to be increased in old animals. One explanation may
be that DNA damage interferes with the expression of transcriptional repressors, leading
indirectly to an induction of sets of target genes. However, the diversity of genes that show
increased expression with age indicates that other processes may also be at work.
‘epigenetic balance hypothesis’ model propose, age-related gene-expression changes are
manifestations of the redistribution of chromatin modifiers from one genomic locus to another.
The model also encompasses the idea that DNA damage mediates chromatin remodeling and
changes in nuclear architecture that occur over a lifetime, which fits with evidence that oxidative
stress and DNA damage can accelerate the ageing process. Based on the observations of Tyler
and colleagues28, it is plausible that chromatin modifications during DNA repair are never fully
restored to their pre-damaged state, resulting in progressive alterations in both chromatin-
modification patterns and gene expression28.

The consequences of the redistribution of chromatin modifying enzymes would be twofold.

Previously silent regions may become transcriptionally active, leading to ectopic gene
expression and, possibly, destabilization of previously heterochromatic repetitive DNA. By
contrast, genes may become repressed near sites of DNA damage through remodeling
processes that are similar to the formation of SAHFs. According to the model, nuclear structure
and organization is progressively and inexorably altered over time, resulting in the functional
decline of cells and tissues. The model is consistent with both the stochastic changes in gene
expression as described by Vijg and colleagues49 and the reproducible tissue specific
transcriptional changes that occur as organism age, which will be dictated by the original
architecture of a given tissue.

Moreover, epigenomic status needs maintenance during and after DNA transactions. Errors
during this process may be inevitable, similar to the DNA-sequence mutations that occur after
the repair of chemical damage in DNA. However, it is also possible that epimutations are
consequences of DNA-sequence changes. Since the local structure of DNA is nucleotide
sequence-dependent, mutations can be expected to influence the chromatin, thereby causing
gene-expressional changes. The same is true for DNA damage, which has been demonstrated
to cause changes in nucleosome positioning(Mann et. al., 1997). DNA methylation and DNA–
histone and DNA–non-histone protein interactions together are thought to organize the genome
into transcriptionally active and inactive zones. DNA damage and mutations can disturb this
pattern of regulation, thereby causing normally silenced alleles to be expressed and vice versa.
It is therefore possible that age-related changes in DNA methylation patterns and histone
protein changes are secondary rather than primary events in the aging process.

However, this discussion sends us back to the same link we started from DNA repair make
errors and cause methylation pattern changes, DNA repair itself modify chromatin and cause
cellular senescence by activation of p53 or may cause gene transcriptional noise and promote
later activation of senescence and cause tissue dysfunction.

4. CR increase life-span by activating the pathways that are involved in

Genome Maintenance and also leads to repression of p53.
 4.1 Pathways of CR
Sensing the conditions of CR seems to occur at two different levels: the nutrient level and the energy level (Fig.). At the nutrient
level, the main pathways involved include the endocrine insulin/IGF-1 pathway (Dilova et al. 2007) and the nutrient-responsive
kinases target of rapamycin (TOR), cyclic AMP-dependent kinase (PKA), and Sch9/AKT. From the energetic standpoint, the two
main cellular events during CR are a decrease in the ratios of NADH/NAD+ and ATP/ADP. This oxidative and hypo-energetic
state of the cell is believed to trigger a response through the activation of different pathways, the best known of which involve the
NAD+-dependent deacetylases called Sirtuins and the AMP-dependent kinase (AMPK). The elements involved do not work
independently (Fig. 6). In fact, as we shall see, they seem to synergize for fine-tuning the regulation of cellular responses.

Fig 7: Main pathways associated with

response to CR. Metabolic fluctuations
signal through a nutrient and energy
level. A decrease
in the nutrient levels down-regulates the
IIS pathway and the nutrient-dependent
kinases TOR, PKA, and AKT, which in
turn up-regulate stress response
organizers such as the transcription
factors NF-kB and FOXOs. In parallel,
the cell’s energetic imbalance produced
by CR upregulates the NAD+-
dependent family of Sirtuins and the
nutrient-dependent kinase AMPK,
which in turn activate and modulate the
stress response. Sirtuins are also
involved in the activation of the LKB1
kinase. Overall, as is shown in the
bottom part of the figure, survival and life span increase is activated through the activation of several processes (in blue) and inhibition of others
(in green).

4.2 CR and chromatin maintenance

The evidence suggests that the biological effects of CR are closely related to chromatin function. In fact, given the
parallels between aging and loss of chromatin integrity, one of the models for explaining the delaying effect of CR
on aging is based on increased genomic stability (Fig.7 Heydari et al. 2007). This has been shown clearly in yeast where, Sirtuin
Sir2p has been linked to aging control through the modulation of chromatin structure (Guarente et al., 2000). The consequences
of CR on chromatin historically have been interpreted as antagonistic to the well-known effects produced by ROS in chromatin
during oxidative stress. While this is partially true, it is becoming increasingly clear that ROS are not the only consideration in
this process.

Current data suggest that at least part of the CR effect on life span could be elicited through a long-term activation of protective
machinery under low-intensity stressors (e.g., activation of detoxifying machinery, cell cycle progression, DNA repair, and
inhibition of apoptosis and senescence) (Masoro et al., 2005). This would entail a tighter and more efficient global control on the
cell. This theory, known as the Hormesis theory, was proposed by Masoro in 1998 (Masoro et al., 1998), and is currently under
study (Masoro et al., 2005). The evidence suggests that responses to CR involving chromatin are twofold: mediating cellular
adaptation to changes in metabolism through the control of gene expression, and promoting the protection of genome integrity
and chromatin structure (Fig. 6). The former occurs not only in the nucleus but also in mitochondrial DNA. CR induces major
changes in gene expression patterns, which in mammals are apparently highly tissue-specific (Fu et al. 2006). The genes targeted
can be classified generally as those encoding proteins involved in:
(1) Metabolic pathways,
(2) Stress responses,
(3) DNA damage repair,
(4) Chromatin structure regulation,
(5) Detoxification, and
(6) Mitochondria-related processes.

The mechanisms that mediate these changes include modulation of the activity of certain key transcription factors that regulate a
specific set of genes (e.g., FOXO transcription factors) (Guarente and Picard et al., 2005). Also implicit to the CR response is
gene inactivation, which seems to occur via two different mechanisms: regulation of individual genes by modulation of specific
transcription factors, and heterochromatinization of large parts of the genome that might include many genes, as has been
observed in the case of nucleolar rDNA with either TOR inhibition or yeast Sir2p activation (Guarente et al., 2000; Tsang et al.,
and Zheng et al., 2007). The former may occur through changes in DNA-binding affinity, enzymatic activity, or cellular
localization of key transcription factors such as stress-responsive transcription factors, or via recruitment of silencing machinery
(e.g., histone deacetylases [HDACs] or methyltransferases). CR leads to protection of genome integrity and preservation of
chromatin structure from yeast to mammals (Heydari et al. 2007). This effect is closely related to that described above, since
part of the machinery involved in genomic protection depends on appropriate gene expression. This protection occurs via two
series of mechanisms. The first encompasses activation of DNA repair (Cabelof et al. 2003), ROS detoxification machinery (Xia
et al. 1995), and increased fidelity of DNA replication (Srivastava et al. 1993). The second comprises direct modulation of
chromatin structure, which has positive effects on DNA recombination, DNA replication, and cell cycle progression, among other
processes (Vaquero et al., 2009).

CR increases cellular resistance to oxidative stress

by reducing the accumulation of both nuclear and
mitochondrial DNA damage produced by ROS
(Fig. 7). However, the level of DNA
protection in each cell compartment is not
equivalent among different tissues (Stuart et
al. 2004). CR increases DNA repair activity
and reverses the decreased levels of such
activity observed in aged mice (Heydari et al.
2007). The DNA repair pathways up-regulated
by CR are the three main mechanisms
involving ssDNA, NER (Guo et al. 1998),
BER (Cabelof et al. 2003), S-phase-dependent
mismatch repair (Tsao et al. 2002), and the
most common form of DSB repair, NHEJ
(Um et al. 2003). Interestingly, CR not only
increases expression of the DNA repair
machinery (Heydari et al. 2007), but also
amplifies the activity and fidelity of critical
enzymes such as DNA polymerases a and b
(Cabelof et al. 2003). The effects of CR on
NHEJ are tissue-specific, being most
pronounced in kidneys and lungs, but less
evident in testes or liver. These effects seem to
occur via altered levels of the NHEJ
modulation complex Ku70/86 (Um et al.
2003)

Figure 8. CR response on chromatin. CR has an

effect on chromatin at three different levels—
chromatin structure, gene expression (up-regulation of certain genes and down-regulation of others as indicated by the arrows), and DNA repair
—which in general produce an increase in genome stability and can explain, at least partially, the life span increase effect of CR

Several groups of factors have been postulated as being mediators of the CR response on chromatin. The candidates with the best
credentials for such a role are the Sir2 NAD +-dependent deacetylases known as Sirtuins that have emerged recently as major
players in signaling to chromatin during responses to CR (Guarente and Picard 2005; Saunders and Verdin 2007; Vaquero et al.
2007b). Another interesting case is that of TOR. In addition to its signaling pathway, TOR participates directly in the regulation
of nucleolar chromatin. Additionally, given the strong relationship between CR and the NAD +/NADH levels, it is likely that
other chromatin related factors whose activity is strongly influenced by the redox state of the cell might be involved in CR
responses.
 4.3 Calorie restriction counters gene-expression changes and helps in genome
maintenance and interaction with p53

We have seen above in epigenetic mutations hat how sirtuins affect chromatin remodeling this leaves no doubt that sirtuins are
involved in changes in regulating genetic expressions. Moreover i n higher organisms such as worms and flies, sirtuins
may exert lifespan-extending effects via modulation of factors involved in IIS. In mammals, indirect evidence points
to potential roles for sirtuins in regulating longevity; most studies have focused on SIRT1. SIRT1 possesses
multiple metabolic functions that might indicate a beneficial role in longevity: these include increasing cellular
stress resistance and suppressing apoptosis via deacetylation of FOXO proteins, p53, and E2F1 [Luo 2001, Vaziri
2001, Langley 2002, Alcendor 2004, Greer 2005 ,Chen 2005 , Alcendor 2007, 69]; and decreasing white adipose
tissue formation (Picard 2004). SIRT1 also plays a protective role in model systems of neurodegeneration (Kim
2007).

The mammalian homologue of the yeast Sir2 gene (Sir2α in mouse [Imai 2000] and SIRT1 in human [Frye t
al., 1999]) inhibits the activity of p53 by removing the acetyl-groups from C-terminal lysine. p53 deacetylation
decreases the apoptotic and transcriptional functions of p53 in response to both DNA damage and oxidative
stress [Luo et al., 2001, Vaziri et al., 2001]. Thus, SIRT1- and p53-mediated pathways converge, providing
yet another mechanism whereby modulation of p53 activity affects life span.

5. DNA damage, stem cells and differentiated somatic tissues

Cancer is a major age-related disease in mice, humans and many other mammals ( Balducci et al., 2005). In the absence
of cancer, aging is mostly characterized by tissue atrophy and degeneration (Gessert et al., 2002). Most mammalian
tissues can be described as being comprised of two major cellular components: stem or progenitor cells, which are
responsible for regenerative capacity or repair after injury, and differentiated somatic cells, responsible for adult
stem cell support and specialized tissue/organ functions. Based on this classification, two major mechanisms can
account for tissue degeneration associated with age: loss of stem cell pool division potential (loss of regenerative
capacity) and loss of differentiated somatic cell function, which directly leads to loss of organ function. Loss of
differentiated somatic cell function can additionally indirectly affect adult stem and progenitor cells by altering the
tissue microenvironment that is essential for stem cell support (the stem cell niche). In general, loss of stem cell pool
division potential can occur through multiple mechanisms including stem cell senescence, death or dysfunction of
the niche. One specific mechanism that can account for the loss of both stem cell and differentiated somatic cell
function is the gradual accumulation of persistent DNA damage.

Persistent DNA damage and its erroneous resolution include telomeric dysfunction ( Sedelnikova et al., 2004, Herbig et al.,
2006, Jeyapalan et al., 2007) and somatic mutations (Vijg et al., 2002), both of which increase with age; both also have been
proposed to contribute to the loss of stem and differentiated somatic cell function with age (Bahar et al., 2006,
Carlson et al., 2007 ). DNA damage accumulation in stem cells has been detected in mice and clearly contributes to
the attrition of stem cell division potential during aging ( Rossi et al.,2007). Thus, it is likely that DNA damage
contributes to aging by limiting stem cell division potential and by also interfering with somatic tissue functions,
including stem cell niches. p53 signaling networks that link lesions in the DNA to the activation of DNA damage
checkpoints, which in turn activate effectors of critical cell fate decisions is cause of loss of stem cells.

6. p53 activity and ensuing cellular outcomes: to be or not to be

Since the beginning of this paper we have been discussing how various pathways that may be
involved in aging begins from DNA damage and its repair and finally converge to p53 or
functional decline through transcriptional noise. It is from this point from where various
phenotypes that show up at level of organs and organism diverge. Below I will review briefly the
fates of a cell after 53 is activated and then I finally discuss these studies we have reviewed in
the paper with various models of p53 mutants.

Activation of p53 through the DNA Damage Repair (DDR) usually leads to either proper repair of the lesion or
elimination of the damaged cell from the proliferative cell pool. How stringent must this mechanism be to prevent
pathological loss of cells and how does it impact organ homeostasis? The steps outlined below are general scenarios,
based in some cases on indirect evidence and in all cases highly dependent on the cell, tissue and physiological
context. Once a cell has been damaged and the DDR and p53 are activated (Figure 8), a complex signaling network
is engaged to result in a long-term cell fate decision. In normal mammalian cells, there are four such options. In all
cases, initial processing of the DNA lesions begins immediately, while activation of cell cycle checkpoints by
CHK1, CHK2 and p53 lead to transient cell growth arrest (Bakkenist et al., 2004). While initial processing of the
lesions continues, p53 physically localizes to sites of DNA damage to promote repair (Al Rashid et al., 2005) and
simultaneously stimulates the transcription of direct effectors of the cell growth arrest (e.g. the cyclin-dependent
kinase inhibitor p21) as well as effectors required for efficient DNA repair of complex lesions that require longer
processing (e.g. GADD45) (Guillouf et al.,1995).

Once these early steps are in place, there are several potential cellular outcomes, most of which are heavily
influenced by the cell type as well as the severity of the DNA lesions (Figure 8).

(i) Transient arrest and proper repair: when DNA damage is not severe; most of the time the result is
proper repair of the lesions and recovery of the damaged cell with few, if any, consequences. Clearly
this cellular outcome in which p53 promotes transient arrest and repair, is most favorable for the cell
and organism.

(ii) Defective repair: alternatively, the repair attempt may fail. If the cell survives and does not arrest
growth permanently (cell fate decisions 3 and 4 below), the result can be mutations, including
chromosomal aberrations. This scenario, promotes the development of cancer.

(iii) Apoptosis: when the damage is very severe, p53 transcriptionally upregulates effectors of apoptosis
such as proteins of the BH3-only family (PUMA, NOXA and BAX), and downregulates repressors of
apoptosis such as BCL-2 and SURVIVIN ( Oren et al., 2003). Newly expressed BH3-only proteins
translocate to the mitochondria and promote an apoptotic cascade starting at the mitochondrial
membrane (Danial et al., 2004). Moreover, p53 itself can translocate to the mitochondria and promote the
mitochondrial apoptotic cascade by interfering directly with anti-apoptotic BCL-2 family members
(Murphy et al., 2004). Apoptosis is an important mechanism for eliminating supernumerary cells during
embryonic development, and a potent tumor suppressor mechanism in neonatal and adult organisms.
Because apoptosis irreversibly removes cells from tissues, this cell fate decision can also deplete
tissues of stem or progenitor cells and contribute to organ degeneration (Danial et al., 2004).

(iv) Cellular senescence: cellular outcomes following DNA damage are highly dependent on cell type and
physiological context (Danial et al., 2004). While lymphoid lineages usually undergo apoptosis following
severe DNA damage (Lowe et al., 1993), stromal and some epithelial lineages undergo senescence (Di
Leonardo 1994). Senescence is a complex genetic program and a final cell fate decision that
establishes permanent cell growth arrest. The senescence response also causes dramatic alterations in
cellular metabolism including the secretion of proteins that can modify the tissue microenvironment
(Campisi et al., 2005). In most cell types, activation of p53 is crucial for initiating the senescence
response following DNA damage. In some cells, p53 is also important for maintaining the senescence
growth arrest. In others, p53 is required only to establish the senescence growth arrest, which
subsequently becomes irreversible and p53-independent ( Beausejour et al., 2003). Senescent cells have
been shown to accumulate with age in vivo (Kishi et al., 2004, Melk et al., 2003, Dimri et al., 1995).
Because they have lost the ability to proliferate, senescent cells can no longer participate in tissue
renewal and repair; thus, senescence can deplete both stem (Wang et al., 2006, Janzen et al., 2006,
Molofsky et al., 2006) and stroma (Herbig et al., 2006, Jeyapalan et al., 2007) cell pools. Moreover,
 because senescent cells persist, they have the ability to alter the tissue microenvironment, and can
therefore also promote the degeneration of organs and stem cell niches ( Carlson et al., 2007,Campisi et al.,
2005). Finally, senescent cells secrete factors such as matrix metalloproteinase-3 (MMP-3), which
favors extra-cellular matrix remodeling, promotes defects in epithelial cell differentiation and
stimulates cancer cell growth (Campisi 2005, Parrinello 2005, Liu 2007).

These four cellular outcomes and their potential impact on tissue homeostasis are summarized in Figure 8 and are all
orchestrated by the p53 signaling network. There is now little doubt that activation of tumor suppression
mechanisms such as apoptosis and senescence suppress the development of cancer and therefore promote longevity.
In contrast, loss of p53 leads to a dramatic reduction in DNA damage-induced cell fate decisions (Lowe et al., 1993,
Ruley et al., 1993, Lotem et al., 1993, Christophorou et al., 2005, Christophorou et al., 2006, Komarova et al., 2004)
and this deficit shortens life span in mice and humans due to early cancer development. In light of the cellular
outcome model above, the reasons for this are easily understood. Defects in p53-dependent cell fate decisions such
as apoptosis and senescence can promote the survival of cells with potentially severe genotoxic damage, leading to
excessive mutation accumulation and neoplastic transformation.

While it is clear that reduced p53 activity shortens life span by promoting cancer, the model above paradoxically
predict that loss of p53 should also prevent permanent cell fate decisions such as apoptosis and senescence that
promote tissue and organ degeneration. Moreover, if p53 does promote tissue degeneration associated with aging,
increased p53 activity should promote aging phenotypes further and do so despite a reduced incidence of cancer.
Some of these possibilities can and have been tested in mice.

Figure 8. The impact of p53-mediated cell fate decisions on tissue homeostasis and organismal longevity: In the presence of severe DNA damage,
effectors triggered by p53 cause transient cell growth arrest, apoptosis or senescence, which in turn promote tissue atrophy and organismal aging.
In contrast, loss of p53 function prevents critical cell fate decisions and dramatically favors cancer.

7. Discussions
It had been since long proposed that Genomic instability leads to aging phenotype. However
there had always been missing links in approving any proper theory of aging. Through above
reviews I have tried to fill in most of the missing links.

It is quite obvious that any genome is essential component of cells to maintain life alteration will
lead them to initiate repair processes. DNA damage lesions due to various endogenous and
exogenous stresses are the starting point of phenomena of aging. However there is no evidence
in support of any effective amount accumulation of lesions as the repair mechanisms are always
working in our cells. There are many mechanisms that have evolved to ensure an organism live
long enough to produce maximum copies of its genome as offspring and hence these lesions
are removed and kept below threshold level before they can cause adverse effects. In broad
category we can divide these mechanisms as caretakers and gatekeepers.

The ‘caretaker genes’ normally prevent DNA damage and genomic instability (Campisi et al.,
2003). These genes, which include genes involved in DNA repair pathways, may maintain
health and enhance longevity by increasing the stability of the genome, reducing the risk of
cancer and postponing ageing (Burkle et al., 2000). In contrast, deficiency of these genes
causes a number of progeroid syndromes, including Werner, Rothmund–Thompson, Cockayne
and trichothiodystrophy. These patients frequently have an increased incidence of cancer and
exhibit striking features of premature ageing. The ‘gatekeeper genes’, which include genes
responsible for cell death and cell cycle arrest, include the classic tumour suppressor
genes(Campisi et al., 2003). The relationship between the gatekeeper genes, tumour
suppressors and the ageing process is less clear than in the case of genes involved in DNA
repair and maintenance .(Hasty et al., 2003)

Gatekeepers mediate tumor suppressor mechanisms and are frequently inactivated by

mutations or epigenetic silencing in a variety of neoplasms. Humans and mice that carry germ
line mutations in one allele of a tumor suppressor have an increased susceptibility to cancer.
Since loss of a copy of a tumor suppressor predisposes to cancer, an increase in copy number
or in the activity of a tumour suppressor might be expected to better protect against tumor
development. Although an increase in tumor suppressor activity can diminish the risk of
developing cancer, it has been proposed that it may simultaneously accelerate age-related loss
of tissue cellularity. Enhancing tumor suppressor function could compromise proliferation of
stem cells, enhance apoptosis or increase senescence, thereby driving the ageing process. p53
is a classic tumor suppressor that is mutated in the majority of human cancers. p53 functions by
halting cellular proliferation in response to a variety of cellular stresses, including DNA damage,
hypoxia and activated oncogenes. p53 effectively limits the proliferation of damaged cells and
hence protects against malignancy. Specific stress signals result in posttranslational
modifications of p53 and this induces activation of distinct transcriptional programmes and
outcomes in the cell. p53 activation can effectively inhibit cellular proliferation by inducing cell
cycle arrest, apoptosis or senescence. In addition, a variety of negative and positive feedback
loops is activated which communicate with signal transduction pathways, modulating the p53
response. Thus, a network of pathways converge upon p53 to tightly regulate its activity (Harris
et al., 2005).

There is sufficient evidence that all pathways of caretakers or DNA repair, specifically, MMR,
excision repair and DSB repair, become less efficient with age leading to accumulation of
mutations. Much less is known about the causes of this deterioration. Several studies point out
age-related decrease in expression of DNA repair enzymes or their activities. Why do levels of
DNA repair enzymes decline with age, and why are the entire DNA repair systems affected?
Since DNA repair and DNA damage response are tightly controlled processes it is tempting to
speculate that DNA damage response becomes less efficient or somewhat deregulated with
age. Less efficient DNA repair will cause transcriptional changes through mutations while it
slower speed will respond by activating more p53 mediated apoptotic pathway. Even beyond
DNA damage response, it is a general notion that old organisms become more sensitive to
stress, hence stress responses may be altered. Recently a connection was made between the
stress response elicited by DNA damage and insulin/IGF1 signaling. As stress signaling
becomes disregulated with age it may in turn affect DNA repair. Do the observed changes in
DNA repair activities with age contribute to the onset of aging? It may be difficult to infer cause–
effect relationship from the studies of normal aging, however, mouse knockout studies can
provide answers. Defects in DNA maintenance may lead to accelerated aging. Notable
examples are mice deficient in NHEJ or mice deficient in TCR. It has to be mentioned that not
all DNA repair mutants show progeroid phenotypes, and it appears that only mutations in
particular pathways affect lifespan. However, as most premature aging syndromes are caused
by mutations in DNA repair genes it is reasonable to assume therefore that normal aging is
caused, in part by the decline in DNA repair capacity.

The overall sequence of events can be as follows gradually impair the function of genes
involved in stress response and DNA repair. DNA repair becomes less efficient and more error-
prone leading to cascading accumulation of DNA damage and mutations, which further
exacerbate age-related physiological decline. Accumulation of mutations may contribute to
aging by affecting essential genes and disturbing transcription patterns. In addition, unrepaired
DNA damage may contribute to aging by increasing apoptosis. A shift in the balance between
cell death and cell renewal may lead to exhaustion of the stem cell pool, loss of tissue cellularity
and functional decline. Accelerated aging due to loss of stem cells has been observed in mice
with overactive p53. Mice with mutated DNA polymerase g lacking the proofreading activity
exhibit accelerated aging due to increased apoptosis, and remarkably several NER mutants
undergo premature aging due to increased apoptosis, which is not associated with an increase
in mutation frequency. Furthermore, DNA damage and mutation may perturb chromatin
structure and cause epigenetic changes.

I next asked the question that if aging is complex network of pathways involved in genome
maintenance, errors made during its maintenance and gatekeepers. The way Genome
instability is manifested in various tissues and organs should have diversity. That is mutations
should be tissue specific. There should be different patterns of observed genome alterations in
different organs. One reason for such an assumption is that various organs differ in cell
proliferative activity since senescent and quiescent cells manifest p53 there should have
occurred transcriptional changes due to various factors such sequence changes and
epimutations. This would also mean that DNA repair mechanisms will also be affected
differently in post mitotic and mitotic cells. One may easily guess that this will cause different
type of mutations and genome alterations in different organs to accumulate.

In fact I got somewhat similar answer to my assumptions. Indeed first clue was that mutation
frequency differed in different organs. While liver, bladder, spleen, adipose tissue, small
intestine showed high increase of mutation frequency with age, increase in mutation frequency
in brain was negligible. But however, I next asked that is it cause of brain being a post mitotic
organ. But perhaps this is not the correct direction to move in. Good amount of increase in
mutation frequency have been observed in heart, a post mitotic organ par excellence. Also,
germline did not show any good increase in mutation frequency, so it was clear only proliferative
activity was not enough to explain organ specific mutation frequencies.

Next I asked is there any difference in mutation spectra within different organs? The answer is
perhaps yes. For e.g. while heart and live showed both point mutation and genomic
rearrangements mutations in small intestine were only point mutations. Mutations spectra was
similar at younger ages while it diverged exponentially with age in different organs. In brain and
heart high frequency of GC to AT transition mutation at CpG sites was dominant. This made the
picture much more clear that there is indeed a difference in mutation spectra with different type
of cell lines, and it differed in quiescent , senescent , post mitotic and mitotic cells. Later, it was
also shown that even intra organ variation exist in mutation spectra(see above)

Cytogenetic changes appear to accumulate in almost every cell type, but alterations were much
higher in post mitotic cells especially aneuploidy. For e.g. mice parenchymal cells of liver which
exist in quiescent state showed 75% chromosomal abnormalities at old age as compared to
10% in young age. in the overall population of neurons in human brain 3-4% were found to be
aneuploid while only 0.6% of all lymphocytes were aneuploid.

My next question was what are other hot spots of genomic instabilities and what types of
instabilities do occur and if they are in some way related to functional decline either by p53
activation or following increase in transcriptional noise. First I tried to figure out if accumulation
of cytogenetic changes can lead to adverse effect of aging. However, as mentioned above
cytogenetic alteration mostly accumulated more after activation of p53 mediated cell cycle
arrest. It was obvious that this was not primary but secondary cause of age related phenotype. It
is well known that aneuploidy can loss of heterozygosity which would lead to functional decline.

Telomeres, p53 and mTR-/- mice The next attraction was definitely telomeres as it is well
known that telomere shortening causes replicative senescence. However doubt in considering
it as a cause of organismal aging was due to long telomeres of mouse genome. If its shortening
was to be considered as a primary cause then mouse should out live humans which is too
absurd to be a fact. However, knock out mouse model with defects in telomerase enzyme
components lead to premature aging phenotypes in mice and dyskeratosis congenita, which
has aspects of premature aging, in humans (Marrone et al., 2005). Dysfunctional telomeres are
recognized by the DNA repair machinery as DNA-DSBs and thus activate the DDR and p53
(Rodier et al., 2005, d’Adda et al., 2003, von Zglinick et al., 2005). Mice deficient in the
telomerase catalytic subunit (Terc) (mTR-/- mice) are prone to telomere erosion in all tissues,
and late generation animals have a decreased life span and show decreased body weight,
increased skin lesions and delayed wound healing (Blasco et al., 1997, Rudolph 1999).
Because mice have long telomeres, early generation mTR-/- mice are phenotypically normal
and have a normal life span. Importantly, some of the premature aging phenotypes observed in
later generation mTR-/- mice could be rescued by p53 deficiency. This result indicates that, at
least in a telomerase-deficient background, short telomeres activate p53, which contributes to
premature aging (Chin et al., 1999).
mtDNA Mutations and p53 The next hot spot to accelerate genome instability is mtDNA
mutations. Being one the first molecules exposed to ROS mtDNA is hot spot of mutations.
Mutations in mitochondrial genome will lead to reduced efficiency of electron transport enzymes
leading to accelerate production of ROS, this will definitely pose a threat on other cellular
components and nuclear DNA. ROS causes accelerated shortening of telomeric DNA and
hence directly or indirectly may activate p53. A pathogenic mtDNA mutation causes respiratory
chain deficiency only if the fraction of mutated mtDNA exceeds a certain threshold level. These
mutations often undergo apparently random mitotic segregation and the levels of normal and
mutated mtDNA can vary considerably between cells of the same tissue. In human ageing,
segregation of somatic mtDNA mutations leads to mosaic respiratory chain deficiency in a
variety of tissues, such as brain, heart and skeletal muscle. A similar pattern of mutation
segregation with mosaic respiratory chain deficiency is seen in patients with mitochondrial
disease syndromes caused by inherited pathogenic mtDNA mutations. Dufour et al., (2008)
experimentally addressed the role of mosaic respiratory chain deficiency in ageing and
mitochondrial disease in mouse with a mixture of normal and respiratory chain-deficient neurons
in cerebral cortex and reported that a low proportion (>20%) of respiratory chain-deficient
neurons in the forebrain are sufficient to cause symptoms, whereas premature death of the
animal occurs only if the proportion is high (>60-80%). They also showed that respiratory chain-
deficient neurons induce death of normal neurons by a trans-neuronal degeneration
mechanism. These studies and those discussed before shows that mtDNA damage as a cause
of aging could not be overlooked. However yet again this pathway converges to p53 mediated
apoptosis and cellular senescence, however there are some doubts regarding viscous cycle.

The next type of genomic instability which occurred with age was retrotransposition however
there are only few evidences in its support. But with evidence regarding change in methylation
pattern instability of L1 retrotransposons should not be underestimated as it can cause genomic
instability and is well known to directly activate p53. There is emerging evidence of
retrotransposons instability in age related neurodegenerative diseases.

Finally, I questioned how epimutations can contribute to process of aging. And to my surprise it
was well linked with CR, one of the most successful tools in aging research. The story begins
from human segmental progerias such as WS and HGPS. WS occurs due to defect in RecQ
helicase, while HGPS is characterized by mutation in LMNA gene which encodes Lamin A
protein. truncated LMNA splice variants are also found in normal aging old homo sapiens.
Mutated Lamin A cause loss of heterochromatin with unstable telomeric ends and alters histone
modification pattern. This again cause activation of p53 by either direct exposure of telomeric
heterochromatin or by changes in methylation patter freeing L1 retrotransposons. Secondly, this
modification of heterochromatin leads to increased transcriptional noise in the organs. Also in
another segmental progeria known as AT, the aging phenotype is caused by mutation in ATM
gene which is responsible for detection of DNA DSBs and initiating NHEJ repair pathway. ATM
recruits Protein Kinase which maintains telomeric stability by mediating interaction between
telomeric DNA and nuclear matrix. Mutated ATM will lead to more accumulation of DSBs and
unstable telomeric ends both responsible for activation of p53.

Sirtuins which are activated during CR protect the heterochromatin. As any DNA break is
detected sir proteins on telomeric repeat elements are relocalized to DNA break where it causes
chromatin remodeling to facilitate DNA repair. This can cause the previous site more prone to
DNA damage as well. SIRT1 bound to telomeric DNA and mating type loci is found to decrease
with age.

Moreover, we know cells undergone p53 mediated senescence release MMPs such as
collagenase which degrade the extracellular matrix and lead to tissue dysfunction as well as
promote cancer by increasing chances of malignancy. It was found that such a swap in
collagenase activity is mediated as a result of chromatin remodeling after p53 mediated
senescence have been initiated. In young cell OCT1 is found in heterochromatic nebular
periphery localized with Lamin B. While, in old senescent cells the OCT1 localization is
inhabited by formations of SHAFs. SHAFs are known to be associated with inhibition of E2F,
which is cause and which is effect is however unclear at present. But SHAFs causes
delocalization of OCT1 transcription factor at promoter of collagenase gene and hence cause
activation of collagenase. Therefore it is now quite clear that why activation of p53 and increase
of number of such senescent cells with respect number of intact cells can cause age related
phenotypes.

Also Zmpste24 is a metalloproteinase that participates in the maturation of lamin A. Mice models deficient in
Zmpste24 and lamin A, exhibit many features of premature aging (Sullivan 1999, Pendas 2002, Bergo 2002).
Microarray analysis of transcriptional alterations in tissues from Zmpste24-/- mice showed an upregulation of
several p53 target genes (Varela et al., 2005). Although the levels of p53 itself were unchanged, it was proposed that
a stress response triggered by nuclear envelope abnormalities in Zmpste24-/- mice activated p53. When compared to
wild-type cells, fibroblasts from Zmpste24-/- mice showed a p53-dependent decrease in proliferative capacity and
premature cellular senescence. Crossing Zmpste24-/- mice to p53-/- mice further tested the possibility that the
progeroid symptoms observed in Zmpste24-/- mice were due to hyperactive p53. Indeed, loss of p53 in the
Zmpste24-/- background partially rescued the aging phenotype characteristic of Zmpste24 deficiency, markedly
increasing body weight and life span. Thus the premature aging syndrome caused by alterations in nuclear lamina
structure triggers activation of p53, possibly through increased DNA damage and DDR signaling (Scaffidi 2006, Liu
2005), which contributes to the premature aging of Zmpste24-/- mice.

I also showed in the review that chromatin remodeling is caused as a side effect if DNA repair and methylation
pattern which also cause this remodeling can be caused as mutations arising due to DNA repair. Also, indicated by
Zmpste24-/- mouse model it become clears that both genome maintenance pathways and activation of p53 are
strongly linked to aging phenotype.

Till now we have discussed what causes cellular senescence and we saw all pathways converge to p53 but what
pathways diverge from this point to cause organismal aging. As discussed many times in this paper cellular
senescence is a genetic programme of essentially irreversible cell cycle arrest that blocks the cell’s response to
proliferative stimuli and growth factors (Lowe et al., 2004). Senescent cells are found in the tissues of elderly
patients. Therefore, it has been suggested that senescence through p53 mediated contributes to organismal
ageing (Dimri et al., 1995). All our discussion till now has also lead to same predictions. I next asked what is
so special about senescent cells that they cause such adverse affect. We just have discussed the relation
between chromatin remodeling, p53 activated senescent cell and MMPs. Apart from this, Senescent cells have
a large, flat morphology, are metabolically active, resistant to apoptosis and display high senescence-
associated β-galactosidase (SA-β-gal) activity. Cellular senescence inhibits cell division, thereby preventing
immortalization of cultured cells and limiting malignant transformation (Campisi et al., 2005). Activated
oncogenes and oxidative damage can trigger cellular senescence via activation of the tumour suppressor p53.
Upon p53 activation, cyclin-dependent kinase (CDK) inhibitors p21 Cip/Waf and p16Ink4a, as well as the negative
modulator of Mdm2 activity, p14Arf (p19Arf in mice), are induced [Brown et al., 1997, Lundberg 2000 ]. p53
activation is crucial for establishing and maintaining the senescent phenotype and high p16Ink4a expression can
render this growth arrest irreversible (Beausejour et al., 2003). In addition to increased expression of p53 and CDK
inhibitors, senescence is accompanied by up-regulation of genes encoding a variety of secreted growth factors,
matrix remodelling enzymes and inflammatory cytokines. This ‘secretary phenotype’ (Campisi et al., 2005) disrupts
the normal structure and function of tissues. Therefore, senescent cells could contribute to age-related
pathology by inducing or promoting chronic tissue remodelling and local inflammation, which compromises
tissue integrity and function. This secretary phenotype can also promote proliferation, invasion and malignant
transformation of neighboring pre-malignant cells ( Krtolica et al., 2001), suggesting that senescence could promote
tumorigenesis at older age. Interestingly, p53 keeps the senescent secretory phenotype in check (Campisi et al
). Campisi and colleagues demonstrate that p53 deficiency in senescent cells reverses the growth arrest in cells
that express low levels of p16 Ink4a, but these proliferating cells had an exaggerated secretary phenotype. These
data indicate that modulation of p53 influences the tissue microenvironment, suggesting a mechanism whereby
p53 affects both tumorigenesis and tissue homeostasis.

Although senescence has been proposed as a mechanism that contributes to ageing, in vivo evidence for a
correlation between senescence and ageing has been lacking. Recently, it was discovered that deficiency of
p63, a member of the p53 family, induces a programme of cellular senescence that leads to accelerated ageing
in vivo [Keyes et al., 1999]. p63 heterozygous mutant mice have reduced lifespan and develop a number of features
of age-related decline. In this study, p63 ablation in cultured primary keratinocytes was found to evoke
cellular senescence. In vivo, Cre-mediated ablation of p63 during development arrests epithelial
morphogenesis concomitant with increased expression of senescence markers SA-β-gal, p16Ink4a and
promyelocytic leukemia (PML) protein. In the adult, enforced p63 deficiency in stratified epithelia caused
cellular senescence and accelerated ageing. Interestingly, age-associated features appeared to occur globally in
this mouse model, supporting the idea that senescent cells can ‘communicate’ to cause a systemic loss of tissue
homeostasis.

The finding that p63 deficiency induces a programme of cellular senescence that leads to accelerated ageing at
the level of the whole animal provides evidence that senescence contributes to the ageing process and defines
p63 as a novel mediator of both cellular senescence and organismal ageing. Importantly, p53 negatively
regulates certain isoforms of p63 [ Ratovitski et al., 2001]. Therefore, enhanced p53 activity, e.g. in response to UV-
or gamma-irradiation, compromises p63 expression, supporting the idea that p53-mediated p63 deficiency
contributes to both cellular senescence and ageing. The hypothesis that the balance between the tumour
suppressive- and age-promoting functions of p53 is intriguing and places p53 at a nodal point between cancer
and ageing. However, the role of p53 in ageing is still controversial.
Figure 10. Pathways involving p53. A number of pathways converge on, and modulate, p53 activity and various cellular
outcomes

The signaling pathways leading from DNA damage to p53 activation have been extensively investigated, and
provide intriguing insights into how the activity of a tumor suppressor can promote lifespan by preventing cancer
and promoting genome maintenance, while paradoxically reducing lifespan through promotion of cell fate decisions
that can result in tissue degeneration.

Figure 11. DNA damage response signaling pathways leading to p53 activation: DNA damage activates PIKK (ATM, ATR and DNA-PK), which
leads to activation of checkpoint kinases (CHK1, CHK2) and p53. Activated p53 integrates the output of the DDR signaling network and triggers
various cell fate decisions.

For either BER or NER, it is believed that recruitment of the ATR/ATRIP (ATM and Rad3-related kinase/ATR-
interacting protein) complex to an ssDNA intermediate in the repair process promotes initiation of the DDR. The
DDR subsequently entails activation of the checkpoint kinase CHK1 and ultimately p53 activation (Bakkenist 2004,
Zou 2003, Cortez 2006). When the BER or NER pathways are deficient, the lesion likely converts into a DNA-DSB
and initiates the DDR by activating the ATM (ataxia telangiectasia mutated) kinase (Marini 2006, Jiang 2006)
(Figure). Replication stress during S-phase can create ssDNA, which either promotes ATR activation as described
above or progresses to DNA-DSB (Zou 2003, Cortez 2006). Examples of replication stress include lesions that
retard or interrupt replication fork progression, and the inappropriate firing of replicons, such as that caused by the
hyper-proliferation induced by certain oncogenes (Di Micco 2006, Bartkova 2006).

DNA-DSBs are caused by many stimuli including ssDNA breaks on opposite DNA strands, replication fork
collapses, high energy radiation or dysfunctional telomeres, and are dangerous and potentially lethal lesions ( Bassing
2004). DNA-DSBs require prompt repair, which can be achieved through two independent but not mutually exclusive
mechanisms: error prone non-homologous end joining (NHEJ) or relatively error-free homologous recombination
(HR) (Bassing 2004). In response to DNA-DSBs both ATM and DNA-PK (DNA-dependent protein kinase) are
activated. Activation of DNA-PK can lead to p53 phosphorylation on serine 15 and 37 ( Pluquet 2001), but mostly
promotes repair of the DNA-DSB through NHEJ (Bakkenist 2004). On the other hand, activation of ATM leads to
direct phosphorylation of p53 on serine 15 and 37 (Pluquet 2001), as well as phosphorylation and activation of another
checkpoint kinase (CHK2), which also phosphorylates p53 on serine 20.

It should be noted that while the pathways described above converge on p53, all three of the phosphoinositide 3-
kinase-related kinases (PIKK)— ATM, ATR and DNA-PK—as well as the CHK1 and CHK2 kinases—can
phosphorylate a multitude of downstream substrates with important functions in DNA repair and cell cycle arrest
(Bakkenist 2004). One example of this feedback on repair from the DDR includes the phosphorylation of the histone
variant H2AX at sites of DNA-DSBs by all three PIKKs. Phosphorylated H2AX (gamma-H2AX) is essential for
stabilizing DNA repair complexes and promoting efficient repair (Celeste 2002). Nevertheless, p53 remains a key
target of the DDR pathway. DDR-mediated multisite p53 phosphorylations reduces binding of the H/MDM2
(human/mouse double minute 2) ubiquitin transferase to the p53 molecule, which in turn allows replacement of
ubiquitin moieties by acetylation, resulting in p53 stabilization and full activation (Lavin 2006).

Upon activation, depending on the severity of the stress signals, the pattern of post-transcriptional modifications and
the cellular context,
1) p53 acts as a potent transcriptional activator or repressor,
2) can translocate to the mitochondria to induce apoptosis
3) and can localize directly to sites of DNA damage and promote proper repair ( Oren 2003, Ho 2003, Al Rashid 2005,
38. Murphy 2004, Marchenko 2000)

p53 mutant models

We have been discussing that various pathways in aging will lead to activation of p53 but before coming to any
conclusion I want to discuss the various experiments with p53 mutant models. Although a physiological role for p53
in aging is controversial, studies with different mouse models indicate a delicate balance between the tumor
suppressive and age promoting functions of p53. Here, we discuss several of these mouse models (summarized in
Table below). Note that when referring to altered aging phenotypes in mouse models, in general, premature aging
refers to phenotypes that occur very early (weeks) after birth, whereas accelerated aging refers to phenotypes that
take longer to develop (months/years), but that would normally take much longer to develop in control animals.

models that alter p53 activity

∗ p53 activity is increased in cooler tissues (e.g. skin) and is decreased in warmer tissues which are prone to tumour development.
∗∗ p53 activity is increased as measured by increase in expression of some p53 target genes, such as p21 and GADD45. p53 activity is decreased
as
well in the same mouse model, as measured by the decrease in PTEN, a p53 target gene, and by increase in IGF-R1, which is normally repressed
by wild-type p53.

Ku80 null mice.

Ku80 is a DNA repair protein that associates with Ku70 to comprise the DNA-binding component of DNA-PK
(Chechlacz 2001). Ku80 null mice exhibit accelerated aging phenotypes, including skin atrophy, osteopenia,
hepatocellular degeneration and shortened life span (Vogel 1999). A lack of NHEJ and therefore increased DNA
damage load could account for the accelerated aging phenotype. In support of this hypothesis, mouse embryo
fibroblasts (MEFs) derived from Ku80-/- mice clearly underwent p53-dependent premature replicative senescence.
A requirement for p53 was illustrated by the limited proliferative capacity of Ku80-/- cells compared to Ku80-/-
p53+/- fibroblasts (Lim 2000). Thus, p53 deficiency rescues the premature replicative senescence of Ku80-/- MEFs.
If, as suggested, increased DNA damage and increased p53 activity is responsible for the accelerated aging
phenotype of Ku80-/- mice, crossing these animals with p53-deficient mice might rescue the accelerated aging.
Interestingly, mice deficient in both Ku80 and p53 were much more cancer-prone than mice that were deficient only
in p53 (Lim et al., 2000). The increase in cancer incidence was large enough to obscure any beneficial effect that
loss of p53 might have conferred on the accelerated aging observed in Ku80-deficient mice. Clearly, loss of p53
cannot rescue accelerated aging phenotypes if cancer incidence is dramatically increased.

Telomerase-deficient mice.
Telomerase is a key enzyme that maintains telomere length in stem cells and other proliferating cells. Defects in
telomerase enzyme components lead to telomere dysfunction, resulting in premature aging phenotypes in mice and
dyskeratosis congenita, which has aspects of premature aging, in humans ( Marrone et al., 2005). Dysfunctional
telomeres are recognized by the DNA repair machinery as DNA-DSBs and thus activate the DDR and p53 (Rodier
et al., 2005, d’Adda et al., 2003, von Zglinick et al., 2005). Mice deficient in the telomerase catalytic subunit (Terc)
(mTR-/- mice) are prone to telomere erosion in all tissues, and late generation animals have a decreased life span and
show decreased body weight, increased skin lesions and delayed wound healing ( Blasco et al., 1997, Rudolph 1999 ).
Because mice have long telomeres, early generation mTR-/- mice are phenotypically normal and have a normal life
span. Importantly, some of the premature aging phenotypes observed in later generation mTR-/- mice could be
rescued by p53 deficiency.

This result indicates that, at least in a telomerase-deficient background, short telomeres activate p53, which
contributes to premature aging (Chin 1999).
Zmpste24 protease null mice.
Zmpste24 is a metalloproteinase that participates in the maturation of lamin A. Lamin A is an essential component
of the nuclear envelope, defects in which can cause premature aging phenotypes in humans and mice (Sullivan 1999,
Pendas 2002, Bergo 2002). Some humans with progeroid syndromes have been shown to carry mutations in the
genes encoding Lamin A as well as ZMPSTE24 (Chen 2003, Eriksson 2003). Not surprisingly, mice deficient in
Zmpste24 and lamin A exhibited many features of premature aging (Sullivan 1999, Pendas 2002, Bergo 2002).
Microarray analysis of transcriptional alterations in tissues from Zmpste24-/- mice showed an upregulation of
several p53 target genes (Varela 2005).

Although the levels of p53 itself were unchanged, it was proposed that a stress response triggered by nuclear
envelope abnormalities in Zmpste24-/- mice activated p53. When compared to wild-type cells, fibroblasts from
Zmpste24-/- mice showed a p53-dependent decrease in proliferative capacity and premature cellular senescence.
Crossing Zmpste24-/- mice to p53-/- mice further tested the possibility that the progeroid symptoms observed in
Zmpste24-/- mice were due to hyperactive p53. Indeed, loss of p53 in the Zmpste24-/- background partially rescued
the aging phenotype characteristic of Zmpste24 deficiency, markedly increasing body weight and life span.

Thus the premature aging syndrome caused by alterations in nuclear lamina structure triggers activation of p53,
possibly through increased DNA damage and DDR signaling (Scaffidi 2006, Liu 2005), which contributes to the
premature aging of Zmpste24_/_ mice.

BRCA1 hypomorphic mutant mice.

BRCA1 is a tumor suppressor protein that acts both as a checkpoint protein and DNA damage repair protein (Zhang
2005). About 98% of embryos carrying, a hypomorphic mutation in Brca1 (BRCA1-11/-11), die between days 12 to
18 of gestation. Importantly, haploid loss of p53 completely suppressed this embryonic lethality, allowing Brca1 -11/-
11 mice to survive to adulthood (Xu 2001). In those adult animals, Brca1-11/-11 p53+/- females developed tumors
whereas males exhibited accelerated aging phenotypes.

These findings suggest that genomic instability and DNA damage due to BRCA1 deficiency lead to a rapid cancer
phenotype in females. They also reveal a scenario in which increased DNA damage in cancer spared males may
result in a remaining hyperactive p53 allele, which could promote accelerated aging (Cao 2003). Recent findings
show that indeed, the ATM-Chk2-p53 DDR pathway is activated upon BRCA1 deficiency in mice (Cao 2006).
Consistent with an important role for p53 in the DDR, complete or haploid loss of ATM or Chk2 rescued the Brca1
deficiency-associated embryonic lethality and accelerated aging in adult mutant mice.

These findings are consistent with a model in which BRCA1 deficiency impairs proper DNA damage repair, which
in turn promote p53 hyperactivity through the DDR components ATM and Chk2 (see Figure 9). In cancer spared
animals, absence of ATM or Chk2 prevents the hyperactivity of p53 caused by BRCA1 deficiency, hence these mice
are rescued from accelerated aging. Mouse models that affect p53 directly

Short isoforms of p53 in mice: The p53+/m mice The first evidence for a direct role for p53 in promoting
aging phenotypes came from Tyner et al., who generated mice in which a spontaneous recombination event deleted
a stretch of DNA upstream of the mouse p53 gene that included exons 1–6 of the p53 coding sequence. The mutant
p53 allele (m allele) contained exons 7–11 and was presumed to be under the transcriptional control of a promoter
from an upstream gene.

Experiments with MEFs isolated from p53+/m mice indicated that the m allele product could enhance the stability and
the trans-activation activity of p53. Consistent with an increase in p53 activity, p53+/m mice were strikingly cancer
resistant at 18 months of age compared to similarly aged p53 +/+ mice. Surprisingly, however, the cancer resistant
p53+/m mice had a 20–30% shorter life span. Moreover, the mutant mice exhibited several signs of accelerated aging
including tissue atrophy (in the skin, skeletal muscle, liver and lymphoid organs). Thus, enhanced p53 activity could
drive aging phenotypes at the cost of tumor suppression, at least in these mutant mice.
It was suggested that the accelerated aging was caused by an impaired ability of stem cells to produce adequate
numbers of progenitor and mature differentiated cells. A recent analysis of hematopoietic stem cell dynamics in
p53+/m mice confirmed that, compared to wild-type mice, these mice exhibits a reduced number of proliferating
hematopoietic stem cells with age. Haploid insufficiency for p53 (p53+/-) partially rescued the reduced hematopoietic
stem cell proliferative potential, presumably due to diminished p53 activity.

While the phenotypes of p53+/m mice suggest that increased p53 activity promotes tumor suppression at the cost of
aging, there are two major caveats to consider. First, due to the low expression level of the m allele in p53+/m tissues,
no m-derived protein could be detected. Second, and more important, the unknown stretch of DNA upstream of the
p53 gene that was deleted in the m allele has recently been characterized and shown to include 24 upstream genes
in addition to the p53 truncation (Gentry 2005). Absence of one or more of these 24 genes can in principle, give rise
to the accelerated aging and cancer resistance of the p53+/m mice.

Short isoforms of p53 in mice: the P+/+ mice.

Subsequent studies not only confirmed the idea that p53 status can modulate organismal aging but removed some of
the ambiguities present in the above studies (6). Maier et al. created transgenic mice that over-expressed p44 (a short
naturally occurring p53 isoform that lacks the main trans-activation domain) (P+/+ mice). By 4 months of age, P+/+
mice showed selected signs of premature aging. By 1 year of age, most of the P+/+ mice had died, whereas the non-
transgenic mice were still alive and healthy.

P+/+ mice had a very low incidence of cancer, suggesting that p44 overexpression increased wild-type p53 activity.
Indeed, the over-expressed p44 short isoform modulated p53 functions by trans-activation, resulting in enhanced
expression(p21, Mdm2 and IGFBP-3) or repression(Gadd45 and PTEN) of certain p53 target genes [Maier 2004]
, suggesting that p53 function is compromised for a subset of target genes.

The premature ageing phenotype of P+/+ mice was shown to be due at least in part to hyper-activation of the
signaling cascade initiated by IGF, as measured by increased phosphorylated Akt and Forkhead (FKHR)
(Figure ). Mouse models with reduced expression of IGF-1 [ Holzenberger 2003] and insulin [Bluher 2003] receptors
have a significant increase in life span. In the P+/+ mouse model, it was proposed that the increase in IGF
signaling is responsible for the age related phenotypes observed. p53 controls the level of IGF-1 receptor
[Werner 1996] and also controls both the level (by direct promoter trans-activation [ Stambolic 2001]) and the
activity of the dual lipid–protein phosphatase, PTEN, which affects the IGF signaling by dephosphorylation of
phosphatityl-inositol triphosphate (PIP3).
PTEN expression is repressed in some tissues of P+/+ mice, suggesting that a decrease in p53 function, rather
than enhanced function, leads to the activation of the IGF signaling pathway and premature ageing. It was also
seen that in older P+/+ mice expression of the IGF-1 receptor (IGF-1R) was elevated in tissues that contain cells
that proliferate throughout life (testis and spleen), but not in tissues containing post mitotic cells, such as the
brain. In addition, mouse embryonic fibroblasts transfected with increasing amounts of p44 cDNA expressed
higher levels of IGF-1R protein relative to mock controls. Since p53 regulates the level of IGF-1R expression
by inhibiting its transcription [Werner 1996], one could conclude that a compromise, rather than an enhanced
p53 activity, contributes to the premature ageing phenotype of P+/+ mice (see Figure ). Future studies are
needed to test whether the ageing phenotype in P+/+ mice is due to modulation of IGF independent pathways
and to determine whether the age-associated features can be genetically rescued by decreased insulin signaling.

The common underlying characteristic of the p53+/m, pL53 and P+/+ mouse models is the presence of a
modified p53 allele that likely alters wild-type p53 function. Both the p53+/m and the P+/+ mouse models
involve expression of N-terminally truncated p53 isoforms and enhanced p53 activity, and the age associated
phenotype in these models was shown to be dependent upon the presence of the wild-type p53 allele [Tyner
2002, Maier 2004]. The recent discovery that the p53 locus has dual promoters and gives rise to a number of p53
isoforms [Bourdon 2005], including those with N-terminal truncations similar to p44, suggests that the age
associated phenotypes in P+/+ and p53+/m mice might be due to an altered balance of p53 isoform expression.
Indeed, isoforms that lack the N-terminus of p53 have been shown to alter the preference of full length p53 for
specific promoters [Bourdon 2005], suggesting a mechanism whereby N-terminally truncated isoforms of p53
enhance the function of wild-type p53. This suggests that altered p53 function and not simply an overall
increase in p53 function contributes to the ageing phenotype observed in the p53+/m and P+/+ mouse models. It
would be interesting to determine whether the insulin-signaling pathway that is responsible for the age-
associated phenotype of P+/+ mice is affected in the p53+/m and pL53 mouse models as well.

Figure 12. IGF signaling is modulated by p53. Abnormal p53 activity in P+/+ mice leads to accelerated ageing by affecting the
IGF signaling pathway. In wild-type mice (left), p53 modulates and maintains the normal cellular proliferation via IGF and p21.
In P+/+ mice (right), p53 activity responsible for modulation of the IGF signaling pathway is decreased, resulting in an overall
increase in this signal transduction pathway. In contrast, p53-mediated expression of p21 is increased, limiting cellular
proliferation. This altered p53 activity leads to accelerated ageing at the level of the whole organism. The increase and decrease
in level/activity is depicted by an increase or decrease in border thickness, respectively

The pL53 mouse model

This mutant p53 transgenic mouse, pL53, was originally developed by Bernstein and colleagues. The pL53
strain contains approximately 20 copies of a temperature sensitive mutant transgene of p53 (ala135val) that is
expressed at high levels. These mice have age associated phenotypes such as osteoporosis, delayed wound
healing, reduced hair growth and a decrease in subcutaneous fat [ Tyner 2002]. In contrast to p53+/m mice which
are protected from developing tumors, pL53 mice have a modest increase in tumour incidence relative to
controls. The enhanced tumour incidence in this model is consistent with the prediction that the mutant p53
transgene functions in a dominant negative fashion to inhibit wild-type p53 function. However, this also
presents an apparent paradox with the accelerated ageing phenotype. Indeed, Tyner and colleagues propose
that the temperature-sensitive pL53 protein mice acts as an oncoprotein in internal tissues (where the
temperature is elevated) and as a wild-type protein in the cooler tissues (i.e. skin) where enhanced p53 activity
inhibits proliferation and impairs wound healing [ Tyner 2002]. It would be interesting to establish whether p53
transcriptional activity varies between internal and external tissues of pL53 mice.

The super-p53 mice:

Though, Loss-of-function p53 mouse models have been used extensively for cancer research, the generation of
gain-of-function models for p53 has been impeded by the difficulties in reproducing the transcriptional
regulation of the endogenous gene. Early attempts to express p53 transgenes resulted in high expression of p53
protein in particular cell types that led to atrophy of the targeted organ [ Allemand 1999 Godley 1995 Nakamura 2009]
consistent with the effect of over-expressing p53 in Drosophila [Bauer 2005]. In 2002, Garcia-Cao et. al.
generated a bacterial artificial chromosome (BAC) transgenic p53 mouse model that carried extra copies of the
entire p53 locus [Garcia-Cao 2002]. In this mouse model, p53 expression is regulated by the same cis-acting
control regions as the endogenous p53 allele. These ‘super p53’ mice exhibited an increased DNA damage
response and were shown to be tumor-resistant, demonstrating that enhanced p53 is tumour-suppressive in
vivo. Mice with extra copies of p53 were clearly tumour-resistant, but did this enhanced tumour suppression
accelerate the ageing process?

No — ‘super p53’ mice aged normally as measured by life span, skin thickness, lordokyphosis and
osteoporosis. These mice have a normal basal level of p53 despite an increase in copy number. Upon whole
body irradiation, super-53 mice showed elevated levels of p21 mRNA in all tissues and increased apoptosis in the
thymus, compared to irradiated control mice. These effects were further enhanced in animals carrying a second
copy of the BAC-p53 transgene. The fact that ‘super p53’ mice have normal basal level of p53 (and p19 Arf)
compared to controls, explains the lack of correlation between the increase in p53-mediated tumour
suppression and an accelerated ageing phenotype.

Indeed, it was hypothesized that the basal level of p53 would have to be elevated in order to accelerate ageing,
therefore, the presence of a chronic stress signal is required to activate p53 and induce premature ageing in this
model. In rodents, expression of p19Arf increases with age [Krishnamurthy 2004], which suggests that cells with
chronic p53 activity might accumulate. More recently, it was shown that the double super-p53/p19ARF mice can
effectively eliminate cells harboring age-associated persistent DNA damage markers in the absence of any
premature or accelerated aging phenotypes. In fact, the superp53/ p19ARF animals were cancer-free, showed
increased resistance to oxidative stress and displayed general signs of delayed aging (Matheu 2007). The authors
suggested that increased, but properly regulated p53 activity; provide both stress resistance and tumor suppression
activity. It remains to be seen if super-p53 or super-p53/p19ARF mice display accelerated aging phenotypes when
under other types of chronic stress.

However, the accelerated aging shown by telomerase-null mice was not further accelerated when these mice were
crossed with super-53 mice ( Garcia-Cao, 2006). It was observed that cells harboring DNA damage due to defective
telomeres were more efficiently eliminated in super-p53 mice as compared to wild-type mice, although p53 did not
have any effect on telomere-driven aging. The authors suggested that the decrease in damaged cells was not of a
sufficient magnitude to delay the pathologies associated with aging in mice.

The hypomorphic Mdm2 mice:

A number of tumors that retain wild-type p53 have compromised p53 function due to amplification of Mdm2.
Mdm2 binds the transcriptional activation domain of p53 and prevents its interaction with the transcriptional
machinery. Mdm2 also facilitates the nuclear export of p53, and ubiquitinates p53 and promotes its degradation by
the proteasome. Therefore, one way of modulating p53 function is to interfere with Mdm2 expression or
activity. Mdm2-deficient mice are embryonic lethal; deletion of p53 rescues this lethality, indicating that very high
levels of p53 are incompatible with embryonic development (Jones 1995, Montes 1995).

In 2004, Bond and colleagues reported a single nucleotide polymorphism (SNP309) in the promoter of the
human Mdm2 gene that influences its level of expression [Bond 2004]. Cells homozygous for the GG
polymorphism have enhanced expression of Mdm2 that accelerates tumour formation. In contrast, cells
homozygous for the TT allele have normal Mdm2 expression and are considered to be wild-type for p53
activity. It showed that the GG allele in its homozygous state is able to decrease the latency of tumour
development in Li–Fraumeni patients who carry a germ line mutation in p53. From this body of evidence, one
could conclude that altering the balance between p53 and its upstream mediator Mdm2 has deep implications
in controlling tumorigenesis and possibly longevity.

Pharmacological inhibitors of Mdm2 that interfere with its ability to mediate p53 degradation are currently
being tested as potential anti-cancer therapies. For example, intraperitoneal injection of the small molecule
RITA (reactivation of p53 and induction of tumour cell apoptosis), a drug that dissociates p53 from Mdm2,
inhibits human tumour xenografts in immune-compromised mice by 90% [ Issaeva 2004]. Apart from inhibiting
Mdm2 binding, RITA reduces the binding of p53 to p300, iASPP and Parc, but not to BCLx L. It has been
shown that p300 affects p53 by both facilitating and inhibiting its function [Gu 1997, Grossman 2003].
Inhibition of iASPP and Parc binding might facilitate the growth-suppressive effect of RITA. Enhanced iASPP
expression, as found in some breast tumors, might confer resistance to drug-induced apoptosis [ Bergamaschi 2003].
Parc, on the other hand, has been implicated in sequestering p53 in the cytoplasm, where it is non-functional
[Nikolaev 2003]. Other inhibitors of Mdm2 are the Nutlins. Nutlin-3 is a compound that dissociates Mdm2 from
p53 and activates p53-mediated apoptosis [Vassilev 2004]. Mice treated orally with Nutlin-3 daily displayed a
90% decrease in tumour growth of human xenografts of the osteosarcoma cell line SJSA-1, relative to controls,
suggesting that inhibition of Mdm2 provides a potent mechanism for anti-cancer therapy [ Vassilev 2004].

Similarly, acute activation of p53 in adult mice lacking Mdm2 but carrying an inducible p53 protein was
incompatible with survival (Ringshausen 2006). Mendrysa et al.(2006) generated mice that express low levels of Mdm2
(mdm2 puro/-7-12 mice) owing to the presence of one hypomorphic (puro) and one null allele (-7–12). These mice
expressed 30% of the normal level of Mdm2 ( Mendrysa 2006, Mendrysa and Perry et al., 2006). As expected, these mice also
showed increased basal levels of p53 and, upon activation, increased expression of p53 target genes as well as
increased apoptosis in the small intestine. mdm2 puro/-7–12 mice were also highly resistant to cancer, presumably as a
consequence of increased p53 activity. Despite having increased p53 activity, these mice had a normal life span, and
showed no signs of accelerated aging.

Garcia-Cao and colleagues hypothesized that p53 requires a stress signal to accelerate ageing. This idea is
consistent with the fact that the increased p53 activity in mdm2puro/-7–12 mice does not correlate with an
increase in phosphorylation of serine 18 (serine 15 in humans) [Mendrysa 2006], which is the major
phosphorylation site in stress-induced activation. The lack of the full spectrum of accelerated ageing features
in mice with increased p53 activity due to reduced levels of Mdm2 suggests that an increase in p53 function
alone is not sufficient to trigger ageing. As in the case of the ‘super p53’ mice, p53 is presumably under
normal control in these Mdm2-compromised models, a situation that is distinct from the altered expression of
the p53+/m, pL53 and P+/+ mouse models.

p66Shc null mice

p66Shc is an adaptor protein important in the signaling response to reactive oxygen species [ Luzi 2000]. Cells that
lack this protein have increased resistance to apoptosis by UV irradiation or hydrogen peroxide treatment.
Consistent with the established role of p66 Shc in cultured cells, p66Shc-deficient mice have an extended life
span [Migliaccio 1999]. Very interestingly, the p53 apoptotic response in these mice is compromised,
suggesting that decreased p53 function is associated with increased longevity. It will be interesting to
determine whether enhanced p53 function of ‘super p53’ mice can suppress the life span extending effect of
p66Shc deficiency and to assess whether p53+/m and P+/+ mice function similarly.

Aging and human p53 polymorphism at amino acid 72

Taken together, the mouse models support the idea that p53 is a potent tumor suppressor that can, under some
circumstances, also promote aging. Insights into the role of p53 in aging are also derived from studies of human
populations. Human p53 has a Pro/Arg polymorphism at amino acid residue 72. Humans carrying one or two copies
of the Pro or Arg p53 form have been studied for cancer susceptibility, mortality and survival. The results support a
role for p53 in regulating life span in humans. A recent systematic literature survey done by van Heemst et al.
showed that humans with the Pro/Pro genotype had a higher risk of developing cancer compared to the Arg/Arg
genotype (Bonafe 2004). This finding agrees with a previous finding that p53Arg is a more potent inducer of
apoptosis than the p53Pro form (Dumont 2003, van Heemst 2005). A prospective study done by the same group in
1226 subjects aged 85 or over showed that the Pro/Pro carriers had a 41% increased survival, despite a 2.54-fold
increase in cancer mortality, compared to the Arg/Arg carriers(Bonafe 2004). The authors suggested that in older
survivors, p53Arg protected against cancer more efficiently than p53Pro but at the cost of a diminished life span.
In another instance, Orsted et al. studied a large Danish population of 9219 participants aged 20–95 years for
longevity, survival after cancer diagnosis, and risk of cancer (Orsted 2007). The authors found that overall 12-year
survival was higher for Pro/Pro carriers compared to Arg/Arg carriers. Moreover, although Pro/Pro carriers had the
same cancer risk as Arg/Arg carriers, the survival of Pro/Pro individuals after cancer or a life threatening disease
was higher compared to Arg/Arg individuals. The authors suggested that the increased longevity of Pro/Pro
individuals after a cancer diagnosis or other life threatening disease might also be due to an increase in survival.

Again, carriers of a form of p53 that is a less potent inducer of apoptosis had increased survival after stress,
suggesting that high p53 activity could be detrimental in humans. While limited in scope, correlations about this
naturally occurring polymorphism and its impact on aging corroborates to some extent the data obtained from mouse
models described above.

Decrease in neuronal p53 activity in flies prolongs life span

Bauer et al., 2005 reported that inhibition of p53 specifically in neurons of the fly increases life span. In this
study, two different altered forms of Dmp53 were expressed in neurons, using the tissue-specific UAS–GAL4
system. The first of these dominant-negative p53 proteins was a C-terminal fragment that lacks the DNA
binding domain. The second dominant-negative form was a 259H mutant that carries a point mutation in the
DNA binding domain and is not capable of binding DNA. These two mutated forms of p53 can form tetramers
with wild-type Dmp53 but cannot bind DNA. Neuronal expression of these dominant-negative versions of p53
increased the life span of the flies, suggesting that decreased p53 function enhances longevity. In contrast to
the life span extension caused by neural specific expression of these dominant-negative forms of p53, life
span was significantly reduced when these dominant-negative proteins were expressed in non-neural
tissues, such as muscle and the fat body. This result was consistent with the finding that flies globally
deficient for Dmp53 (Dmp53-null flies) have a shortened life span, and is consistent with the idea that p53
deficiency in non-neuronal tissues reduces longevity.

Interestingly, the authors found that increasing wild-type Dmp53 activity in neurons during development was
lethal in flies and that over-expression of wild-type p53 in adult neurons did not compromise lifespan. The
authors argue that Dmp53 activation during fly development is required for removing or inactivating damaged
cells before they undergo division and cause extensive dysfunction. This hypothesis for Dmp53’s crucial role
in development explains why reducing Dmp53 function during Drosophila development and also in non-
neuronal adult tissue compromises life span. Taken together, these findings indicate that neural-specific
inhibition and activation of p53 function significantly extends and reduces life span, respectively.

In addition to finding that p53 regulates longevity; it was demonstrated that the increase in life span in flies in
which the neuronal activity of Dmp53 was reduced is associated with life span extension by caloric restriction.
Indeed, flies in which Dmp53 activity was reduced did not have an additional increase in life span when
subjected to caloric restriction. These results raise the possibility that a decrease in p53 activity is one of the
mediators of the caloric restriction/Sir2 life span-extending pathway and are consistent with the hypothesis that
excessive activity of p53 compromises tissue homeostasis and thereby contributes to ageing in vivo. In support
of this hypothesis, Sidransky and colleagues determined that p53+/m mice had reduced expression of Sirt1 that
correlated with their reduced longevity [ Sommer 2006]. Future experiments should address whether Sirt1 is also
compromised in P+/+ mice.

Conclusion

In this paper I discussed the various type of genomic instability that occur with age. I
focused on decline efficiency of DNA repair mechanism with age and discussed how
they will lead to activation of p53 to cause organismal aging from the most fundamental
molecular level. We saw NER and NHEJ decline with age We saw that mutation spectra
is tissue specific because of difference is cell proliferative activities to some extent. This
again meant that p53 mediated cellular senescence also differently guided the path of
mutation in various different organs. We saw that in senescent cells epimutations cause
transcriptional changes which lead to secretion of MMPs and Growth factors and promote
cancer and other age related phenotypes in nearby tissues. We also saw CR increase lifespan by inhibiting p53
and maintaining heterochromatin as well as efficient DNA repair.

The tumor suppressor p53 protects the genome by promoting the repair of potentially carcinogenic lesions in the
DNA, thereby preventing mutations. In addition, p53 eliminates or arrests the proliferation of damaged or mutant
cells by the processes of apoptosis and cellular senescence. However, there is mounting evidence that apoptosis and
cellular senescence can lead to aging phenotypes, possibly by depletion of important stem cell and other stromal cell
pools. Thus, integration of DNA damage signaling by p53 has been optimized to balance the beneficial effects of
tumor suppression against the detrimental effects of tissue degeneration. Finally by studying various p53 affected
models it became clear that Genomic instability do increase with age and it cause age related phenotype by
activation of p53. All genome maintenance pathways converge on p53 as a central node and age related
phenotypes diverge from this node. It became very crucial to maintain the proper balance between various
isoforms of p53 to ensure a good healthspan and life span.

The intricate web of pathways that converge upon p53 and the extensive feedback loops that modulate p53
function present an unparalleled challenge in unraveling the role of this tumour suppressor in the ageing
process. Nonetheless, the variety of in vivo models of altered p53 function clearly implicates p53 as a
modulator of ageing in these systems. Furthermore, the evidence that polymorphisms in human p53 affect life
span convincingly demonstrate that these models provide insight into the process of ageing that is likely
relevant to ageing in humans. Future studies should help to delineate the myriad of molecular mechanisms
whereby p53 regulates ageing and may eventually help to design ways to enhance tissue function and maintain
youthfulness, thereby increasing the quality of life. . I conclude that p53 lies at the central node between aging,
Genome maintenance pathways, cellular senescence and organismal aging.
References
Aguilaniu H, Gustafsson L, Rigoulet M, Nystrom T. Asymmetric Inheritance of Oxidatively Damaged Proteins During
Cytokinesis. Science 2003;299:1751. [PubMed: 12610228]

Al Rashid, S.T., Dellaire,G., Cuddihy,A., Jalali,F., Vaid,M., Coackley,C., Folkard,M., Xu,Y., Chen,B.P. et al. (2005) Evidence for the direct
binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res., 65, 10810–10821.

Al-Baker, E.A., Oshin, M., Hutchison, C.J. and Kill, I.R. (2005) Analysis of UV-induced damage and repair in young and senescent human
dermal fibroblasts using the comet assay. Mech. Ageing Dev., 126, 664–672.

Alcendor RR, Gao S, Zhai P, et al. Sirt1 regulates aging and resistance to oxidative stress in the heart. Circ Res 2007;100:1512–
21. [PubMed: 17446436]

Alcendor RR, Kirshenbaum LA, Imai S, Vatner SF, Sadoshima J. Silent information regulator 2alpha, a longevity factor and
class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes. Circ Res 2004;95:971–80.
[PubMed: 15486319]

Allemand I, Anglo A, Jeantet AY, Cerutti I, May E. Testicular wild-type p53 expression in transgenic mice induces spermiogenesis alterations
ranging from differentiation defects to apoptosis. Oncogene 1999;18(47):6521–6530.

Almeida, K.H. and Sobol, R.W. (2007) A unified view of base excision repair: lesion-dependent protein complexes regulated by post-
translational modification. DNA Repair (Amst), 6, 695–711.

Al-Regaiey KA, Masternak MM, Bonkowski M, Sun L, Bartke A. Long-lived growth hormone receptor knockout mice:
interaction of reduced insulin-like growth factor i/insulin signaling and caloric restriction. Endocrinology 2005;146:851–60.
[PubMed: 15498882]

Ames, B.N.DNA damage from micronutrient deficiencies is likely to be a major cause of cancer.Mutat. Res. 475, 7–20. 2001.

Annett, K., Duggan, O., Freeburn, R., Hyland, P., Pawelec, G. and Barnett, Y. (2005) An investigation of DNA mismatch repair capacity under
normal culture conditions and under conditions of supra-physiological challenge in human CD4+T cell clones from donors of different ages. Exp.
Gerontol., 40, 976–981.

Annett, K., Hyland, P., Duggan, O., Barnett, C. and Barnett, Y. (2004) An investigation of DNA excision repair capacity in human CD4+ T cell
clones as a function of age in vitro. Exp. Gerontol., 39, 491–498.

Arnheim, N. & Cortopassi, G. Deleterious mitochondrial DNA mutations accumulate in aging human tissues.Mutat.Res. 275,
157–167. 1992.

Astrom SU, Okamura SM, Rine J. Yeast cell-type regulation of DNA repair. Nature 1999;397:310. [PubMed: 9950423]
Atamna, H., Cheung,I. and Ames,B.N. (2000) A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.
Proc. Natl Acad. Sci. USA, 97, 686–691.

Bahar, R., Hartmann, C.H., Rodriguez, K.A., Denny, A.D., Busuttil, R.A., Dollé, M.E. T. et al. Increased cell-to-cell variation in
gene expression in aging mouse heart.Nature 441, 1011–1014. 2006.

Bahar, R., Hartmann, C.H., Rodriguez, K.A., Denny, A.D., Busuttil, R.A., Dolle, M.E., Calder, R.B., Chisholm, G.B., Pollock, B.H. et al. (2006)
Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature, 441, 1011–1014.

Bahar, R., Hartmann, C.H., Rodriguez, K.A., Denny, A.D., Busuttil, R.A., Dolle, M.E., Calder, R.B., Chisholm, G.B., Pollock, B.H. et al. (2006)
Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature, 441, 1011–1014.

Bailey, K.J., Maslov, A.Y., & Pruitt, S.C. Accumulation of mutations and somatic selection in aging neural stem/progenitor cells.
Aging Cell 3, 391–397. 2004.

Baird, D.M., Rowson, J.,Wynford-Thomas, D., & Kipling, D. Extensive allelic variation and ultrashort telomeres in senescent
human cells.Nat. Genet. 33, 203–207. 2003.

Bakkenist, C.J. and Kastan,M.B. (2004) Initiating cellular stress responses. Cell, 118, 9–17.
Balaban, R.S., Nemoto,S. and Finkel,T. (2005) Mitochondria, oxidants, and aging. Cell, 120, 483–495.

Balducci, L. and Ershler, W.B. (2005) Cancer and ageing: a nexus at several levels. Nat. Rev., 5, 655–662.

Bandyopadhyay, D. & Medrano, E.E. The emerging role of epigenetics in cellular and organismal aging. Exp. Gerontol. 38,
1299–1307. 2003.

Barja G. Free radicals and aging. Trends Neurosci 2004;27:595–600. [PubMed: 15374670]

Bartkova, J., Rezaei,N., Liontos,M., Karakaidos,P., Kletsas,D., Issaeva,N., Vassiliou,L.V., Kolettas,E., Niforou,K. et al. (2006)
Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature, 444, 633–637.

Bassing, C.H. and Alt,F.W. (2004) The cellular response to general and programmed DNA double strand breaks. DNA Repair (Amst), 3, 781–
796.

Bauer JH, Poon PC, Glatt-Deeley H, Abrams JM, Helfand SL. Neuronal expression of p53 dominant-negative proteins in adult Drosophila
melanogaster extends life span. Curr Biol 2005;15(22):2063–2068.

Baur JA, Pearson KJ, Price NL, et al. Resveratrol improves health and survival of mice on a highcalorie diet. Nature
2006;444:337–42. [PubMed: 17086191]

Beard BC, Wilson SH, Smerdon MJ. Suppressed catalytic activity of base excision repair enzymes on rotationally positioned
uracil in nucleosomes. Proc Natl Acad Sci U S A 2003;100:7465–70. [PubMed: 12799467]

Beausejour, C.M., Krtolica,A., Galimi,F., Narita,M., Lowe,S.W., Yaswen,P. and Campisi,J. (2003) Reversal of human cellular senescence: roles
of the p53 and p16 pathways. EMBO J., 22, 4212–4222.

Ben Yehuda, A., Globerson,A., Krichevsky, S., Bar On, H., Kidron, M., Friedlander, Y., Friedman, G. and Ben Yehuda, D. (2000) Ageing and
the mismatch repair system. Mech. Ageing Dev., 121, 173–179.

Bennett-Baker, P.E., Wilkowski, J., & Burke, D.T. Age-associated activation of epigenetically repressed genes in the mouse.
Genetics 165, 2055–2062. 2003

Bensaad, K., Tsuruta,A., Selak,M.A., Vidal,M.N., Nakano,K., Bartrons,R., Gottlieb,E. and Vousden,K.H. (2006) TIGAR, a p53- inducible
regulator of glycolysis and apoptosis. Cell, 126, 107–120.

Bergamaschi D, Samuels Y, O’Neil NJ, Trigiante G, Crook T, Hsieh JK, et al. iASPP oncoprotein is a key inhibitor of p53 conserved from worm
to human. Nat Genet 2003;33(2):162–167.

Bergo, M.O., Gavino,B., Ross,J., Schmidt,W.K., Hong,C., Kendall,L.V., Mohr,A., Meta,M., Genant,H. et al. (2002) Zmpste24 deficiency in mice
causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect. Proc. Natl Acad. Sci.USA, 99, 13049–13054.

Blasco,M.A., Lee,H.W., Hande,M.P., Samper,E., Lansdorp,P.M., DePinho,R.A. and Greider,C.W. (1997) Telomere shortening and tumor
formation by mouse cells lacking telomerase RNA. Cell, 91, 25–34.

Bluher M, Kahn BB, Kahn CR. Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 2003;299(5606):572–574.

Bodnar, A.G., Ouellette, M., Frolkis, M.,Holt, S.E., Chiu, C.P.,Morin, G.B. et al. Extension of life-span by introduction of
telomerase into normal human cells. Science 279, 349–352. 1998.

Bodyak, N.D., Nekhaeva, E.,Wei, J.Y., & Khrapko, K. Quantification and sequencing of somatic deleted mtDNA in single cells:
evidence for partially duplicated mtDNA in aged human tissues. Hum. Mol. Genet. 10, 17–24. 2001.

Bokov, A., Chaudhuri,A. and Richardson,A. (2004) The role of oxidative damage and stress in aging. Mech. Ageing Dev., 125, 811–826.

Bonafe,M., Salvioli,S., Barbi,C., Trapassi,C., Tocco,F., Storci,G., Invidia,L., Vannini,I., Rossi,M. et al. (2004) The different apoptotic potential
of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death. Cell Death Differ., 11, 962–973.

Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC, et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53
tumour suppressor pathway and accelerates tumour formation in humans. Cell 2004;119(5):591–602.

Bordone L, Motta MC, Picard F, et al. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biol
2006;4:e31. [PubMed: 16366736]

Bouffler, S.D., Bridges, B.A., Cooper, D.N., Dubrova, Y., McMillan, T.J., Thacker, J. et al. Assessing radiation-associated
mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers. Radiat.Res. 165, 249–268. 2006.
Bourc’his, D. & Bestor, T.H. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L.Nature
431, 96–99. 2004.

Bourdon,J.C., Fernandes,K., Murray-Zmijewski,F., Liu,G., Diot,A., Xirodimas,D.P., Saville,M.K. and Lane,D.P. (2005) p53 isoforms can
regulate p53 transcriptional activity. Genes Dev., 19, 2122–2137.

Boyle, J., Kill, I.R. and Parris, C.N. (2005) Heterogeneity of dimer excision in young and senescent human dermal fibroblasts. Aging Cell, 4,
247–255.

Brachmann CB, Sherman JM, Devine SE, Cameron EE, Pillus L, Boeke JD. The SIR2 gene family, conserved from bacteria to
humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev 1995;9:2888–902. [PubMed:
7498786]

Brierley, E.J., Johnson, M.A., Lightowlers, R.N., James, O.F., & Turnbull, D.M. Role of mitochondrial DNA mutations in human
aging: implications for the central nervous system and muscle. Ann.Neurol. 43, 217–223. 1998.
Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science
1997;277(5327):831–834.

Burkle A. Poly(ADP-ribosyl)ation: a posttranslational protein modification linked with genome protection and mammalian longevity.
Biogerontology 2000;1(1):41–46.

Butler, M.G., Tilburt, J., DeVries, A., Muralidhar, B., Aue, G., Hedges, L. et al. Comparison of chromosome telomere integrity
in multiple tissues from subjects at different ages. Cancer Genet. Cytogenet. 105, 138–144. 1998.

Cabelof DC, Yanamadala S, Raffoul JJ, Guo Z, Soofi A, Heydari AR. 2003. Caloric restriction promotes genomic stability by induction of base
excision repair and reversal of its agerelated decline. DNA Repair (Amst) 2: 295–307.

Cabelof, D.C., Raffoul, J.J., Ge, Y., Van Remmen, H., Matherly, L.H. and Heydari, A.R. (2006) Age-related loss of the DNA repair response
following exposure to oxidative stress. J. Gerontol. A Biol. Sci. Med. Sci., 61, 427–434.

Cabelof, D.C., Raffoul, J.J., Yanamadala, S., Ganir, C., Guo, Z. and Heydari, A.R. (2002) Attenuation of DNA polymerase beta dependent base
excision repair and increased DMS-induced mutagenicity in aged mice. Mutat. Res., 500, 135–145.

Campisi J. Cancer and ageing: rival demons? Nat Rev Cancer 2003;3(5):339–349.

Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005;120:513–22.
[PubMed: 15734683]

Cao, L., Kim,S., Xiao,C., Wang,R.H., Coumoul,X., Wang,X., Li,W.M., Xu,X.L., De Soto,J.A. et al. (2006) ATM-Chk2-p53 activation prevents
tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency. EMBO J., 25, 2167–2177.

Cao, L., Li,W., Kim,S., Brodie,S.G. and Deng,C.X. (2003) Senescence, aging, and malignant transformation mediated by p53 in mice lacking the
Brca1 full-length isoform. Genes Dev., 17, 201–213.

Cao, Z., Wanagat, J., McKiernan, S.H., & Aiken, J.M. Mitochondrial DNA deletion mutations are concomitant with ragged red
regions of individual, aged muscle fibers: analysis by laser-capture micro-dissection. Nucleic Acids Res. 29, 4502–4508. 2001.

Carlson, M.E. and Conboy,I.M. (2007) Loss of stem cell regenerative capacity within aged niches. Aging Cell, 6, 371–382.

Cawthon, R.M., Smith, K.R., O’Brien, E., Sivatchenko, A., & Kerber, R.A. Association between telomere length in blood and
mortality in people aged 60 years or older. Lancet 361, 393–395. 2003.

Celeste, A., Petersen, S., Romanienko, P.J., Fernandez-Capetillo, O, Chen, H.T., Sedelnikova, O.A., Reina-San-Martin, B.,
Coppola, V., Meffre, E. et al. . Genomic instability in mice lacking histone (2002) H2AX. Science (New York, N.Y.), 296, 922–927

Celic I, Masumoto H, Griffith WP, Meluh P, Cotter RJ, Boeke JD, Verreault A. The sirtuins hst3 and Hst4p preserve genome
integrity by controlling histone h3 lysine 56 deacetylation. Curr Biol 2006;16:1280–9. [PubMed: 16815704]

Chechlacz, M., Vemuri,M.C. and Naegele,J.R. (2001) Role of DNA dependent protein kinase in neuronal survival. J. Neurochem., 78, 141–154.

Chen D, Steele AD, Lindquist S, Guarente L. Increase in activity during calorie restriction requires Sirt1. Science 2005;310:1641.
[PubMed: 16339438]

Chen WY, Wang DH, Yen RC, Luo J, Gu W, Baylin SB. Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-
dependent DNA-damage responses. Cell 2005;123:437–48. [PubMed: 16269335]
Chen, D., Cao, G., Hastings, T., Feng, Y., Pei, W., O’Horo, C. and Chen, J. (2002) Age-dependent decline of DNA repair activity for oxidative
lesions in rat brain mitochondria. J. Neurochem., 81, 1273–1284.

Chen, F., Nastasi,A., Shen,Z., Brenneman,M., Crissman,H. and Chen,D.J. (1997) Cell cycle-dependent protein expression of mammalian
homologs of yeast DNA double-strand break repair genes Rad51 and Rad52. Mutat. Res., 384, 205–211.

Chen, L., Lee,L., Kudlow,B.A., Dos Santos,H.G., Sletvold,O., Shafeghati,Y., Botha,E.G., Garg,A., Hanson,N.B. et al. (2003) LMNA mutations
in atypical Werner’s syndrome. Lancet, 362, 440–445.

Chen, R.Z., Pettersson, U., Beard, C., Jackson-Grusby, L., & Jaenisch, R. DNA hypomethylation leads to elevated mutation
rates.Nature 395, 89–93. 1998.

Cheng H, Mostoslavsky R, Saito Si, et al. Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient
mice. Proc Natl Acad Sci USA 2003;100:10794–9. [PubMed: 12960381]

Chevanne, M., Caldini,R., Tombaccini,D., Mocali,A., Gori,G. and Paoletti,F. (2003) Comparative levels of DNA breaks and sensitivity to
oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians. Biogerontology, 4, 97–104.

Chin,L., Artandi,S.E., Shen,Q., Tam,A., Lee,S.L., Gottlieb,G.J., Greider,C.W. and DePinho,R.A. (1999) p53 deficiency rescues the adverse
effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell, 97, 527–538.

Christiansen, M., Stevnsner, T., Bohr, V.A., Clark, B.F. and Rattan, S.I. (2000) Gene-specific DNA repair of pyrimidine dimers does not decline
during cellular aging in vitro. Exp. Cell Res., 256, 308–314.

Christophorou, M.A., Martin-Zanca,D., Soucek,L., Lawlor,E.R., Brown-Swigart,L., Verschuren,E.W. and Evan,G.I. (2005) Temporal dissection
of p53 function in vitro and in vivo. Nat. Genet., 37, 718–726.

Christophorou, M.A., Ringshausen,I., Finch,A.J., Swigart,L.B. and Evan,G.I. (2006) The pathological response to DNA damage does not
contribute to p53-mediated tumour suppression. Nature, 443, 214–217.

Chua KF, Mostoslavsky R, Lombard DB, et al. Mammalian SIRT1 limits replicative life span in response to chronic genotoxic
stress. Cell Metab 2005;2:67–76. [PubMed: 16054100]

Clark, S.J., Harrison, J., Paul, C.L., & Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22,
2990–2997. 1994.

Cohen HY, Miller C, Bitterman KJ, et al. Calorie restriction promotes mammalian cell survival by inducing the SIRT1
deacetylase. Science 2004;305:390–2. [PubMed: 15205477]

Coolbaugh-Murphy, M.I., Xu, J., Ramagli, L.S., Brown, B.W., & Siciliano, M.J.Microsatellite instability (MSI) increases with
age in normal somatic cells.Mech.Ageing Dev. 126, 1051–1059. 2005.

Cortez,D. (2005) Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling. Genes Dev., 19, 1007–1012.

Curtis, H. & Crowley, C. Chromosome aberrations in liver cells in relation to the somatic mutation theory of aging.Radiat.Res.
19, 337–344. 1963.

d’Adda di Fagagna,F., Reaper,P.M., Clay-Farrace,L., Fiegler,H., Carr,P., Von Zglinicki,T., Saretzki,G., Carter,N.P. and Jackson, S.P. (2003) A
DNA damage checkpoint response in telomere-initiated senescence. Nature, 426, 194–198.

d’Adda di Fagagna,F., Reaper,P.M., Clay-Farrace,L., Fiegler,H., Carr,P., Von Zglinicki,T., Saretzki,G., Carter,N.P. and Jackson,S.P. (2003) A
DNA damage checkpoint response in telomere-initiated senescence. Nature, 426, 194–198.

Danial, N.N. and Korsmeyer,S.J. (2004) Cell death: critical control points. Cell, 116, 205–219.

Dasgupta B, Milbrandt J. Resveratrol stimulates AMP kinase activity in neurons. Proc Natl Acad Sci U S A 2007;104:7217–22.
[PubMed: 17438283]

de Boer,J., Andressoo,J.O., de Wit,J., Huijmans,J., Beems,R.B., van Steeg,H., Weeda,G., van der Horst,G.T., van Leeuwen,W. et al. (2002)
Premature aging in mice deficient in DNA repair and transcription. Science (New York, N.Y.), 296, 1276–1279.

de Wind, N., Dekker,M., Berns,A., Radman,M. and te Riele,H. (1995) Inactivation of the mouse Msh2 gene results in mismatch repair
deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell, 82, 321–330.

Dempsey, J.L., Pfeiffer, M. and Morley, A.A. (1993) Effect of dietary restriction on in vivo somatic mutation in mice. Mutat. Res., 291, 141–145.
DePinho, R.A. (2000) The age of cancer. Nature, 408, 248–254.

Di Leonardo, A., Linke,S.P., Clarkin,K. and Wahl,G.M. (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term
induction of Cip1 in normal human fibroblasts. Genes Dev., 8, 2540–2551.

Di Micco, R., Fumagalli,M., Cicalese,A., Piccinin,S., Gasparini,P., Luise,C., Schurra,C., Garre,M., Nuciforo,P.G. et al. (2006)
ncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature, 444, 638–642.

Dilova I, Easlon E, Lin SJ. 2007. Calorie restriction and the nutrient sensing signaling pathways. Cell Mol Life Sci 64: 752–767.

Dimri, G.P., Lee,X., Basile,G., Acosta,M., Scott,G., Roskelley,C., Medrano,E.E., Linskens,M., Rubelj,I. et al. (1995) A biomarker that identifies
senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA, 92, 9363–9367.

Dolle, M.E.T., Giese,H., Hopkins,C.L., Martus,H.-J., Hausdorf,J.M. and Vijg,J. (1997) Rapid accumulation of genome rearrangements in liver
but not in brain of old mice. Nat. Genet., 17, 431–434.

Dolle, M.E.T., Snyder,W.K., Gossen,J.A., Lohman,P.H.M. and Vijg,J. (2000) Distinct spectra of somatic mutations accumulated with age in
mouse heart and small intestine. Proc. Natl Acad. Sci. USA, 97, 8403–8408.

Donehower, L.A., Harvey,M., Slagle,B.L., McArthur,M.J., Montgomery,C.A.Jr, Butel,J.S. and Bradley,A. (1992) Mice deficient for p53 are
developmentally normal but susceptible to spontaneous tumours. Nature, 356, 215–221.

Doria, G., Barattini, P., Scarpaci,S., Puel,A., Guidi,L. and Frasca,D. (2004) Role of immune responsiveness and DNA repair capacity genes in
ageing. Ageing Res. Rev., 3, 143–151.

Dubrova, Y.E., Jeffreys, A.J., & Malashenko, A.M. Mouse minisatellite mutations induced by ionizing radiation.Nat. Genet. 5,
92–94. 1993.

Dufour E, Terzioglu M, Sterky FH, Sörensen L, Galter D, Olson L, Wilbertz J, Larsson NG

(2008) Age-associated mosaic respiratory chain deficiency causes trans-neuronal degeneration.
Hum Mol Genet. 2008 May 15;17(10):1418-26.

Dumont,P., Leu,J.I., Della Pietra,A.C.III, George,D.L. and Murphy,M. (2003) The codon 72 polymorphic variants of p53 have markedly different
apoptotic potential. Nat. Genet., 33, 357–365.

Eriksson, M., Brown,W.T., Gordon,L.B., Glynn,M.W., Singer,J., Scott,L., Erdos,M.R., Robbins,C.M., Moses,T.Y. et al. (2003)
Recurrent de novo point mutations in lamin A cause Hutchinson- Gilford progeria syndrome. Nature, 423, 293–298.

Fabrizio P, Gattazzo C, Battistella L, Wei M, Cheng C, McGrew K, Longo VD. Sir2 blocks extreme life-span extension. Cell
2005;123:655–67. [PubMed: 16286010]

Feinberg, A.P., Ohlsson, R., & Henikoff, S. The epigenetic progenitor origin of human cancer. Nat. Rev. Genet. 7, 21–33. 2006.

Fenech,M.& Morley, A.A. The effect of donor age on spontaneous and induced micronuclei.Mutat.Res. 148, 99–105. 1985.

Feuk, L., Marshall, C.R., Wintle, R.F., & Scherer, S.W. Structural variants: changing the landscape of chromosomes and design
of disease studies.Hum.Mol. Genet. 15 (suppl 1), R57–R66. 2006.

Finette, B.A., Sullivan, L.M., O’Neill, J.P., Nicklas, J.A., Vacek, P.M. and Albertini, R.J. (1994) Determination of hprt mutant frequencies in T-
lymphocytes from a healthy pediatric population: statistical comparison between newborn, children and adult mutant frequencies, cloning
efficiency and age. Mutat. Res., 308, 223–231.

Fortini P, Dogliotti E (April 2007). "Base damage and single-strand break repair: mechanisms and
functional significance of short- and long-patch repair subpathways". DNA Repair (Amst.) 6 (4): 398–409.
doi:10.1016/j.dnarep.2006.10.008. PMID 17129767. 

Fraga, M.F., Ballestar, E., Paz, M.F., Ropero, S., Setien, F., Ballestar,M.L. et al. Epigenetic differences arise during the lifetime
of monozygotic twins. Proc.Natl.Acad. Sci. USA 102, 10604–10609. 2005.

Frasca, D., Barattini,P., Tirindelli,D., Guidi,L., Bartoloni,C., Errani,A., Costanzo,M., Tricerri,A., Pierelli,L. et al. (1999) Effect of age on DNA
binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC). Exp. Gerontol., 34, 645–658.
Frojdo S, Cozzone D, Vidal H, Pirola L. Resveratrol is a class IA phosphoinositide 3-kinase inhibitor. Biochem J. 2007epub
ahead of print Frye RA. Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize
NAD and may have protein ADP-ribosyltransferase activity. Biochem Biophys Res Commun 1999;260(1):273–279.

Fu C, Hickey M, Morrison M, McCarter R, Han ES. 2006. Tissue specific and non-specific changes in gene expression by aging and by early
stage CR. Mech Ageing Dev 127: 905–916.

Fuscoe, J.C., Zimmerman, L.J., Harrington-Brock, K. and Moore, M.M. (1994) Deletion mutations in the hprt gene of T-lymphocytes as a
biomarker for genomic rearrangements important in human cancers. Carcinogenesis, 15, 1463–1466.

Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado LM, Klatt P, Flores JM, et al. ‘Super p53’ mice exhibit enhanced DNA damage
response, are tumour resistant and age normally. EMBO J 2002;21(22):6225–6235.

Garcia-Cao, I., Garcia-Cao,M., Tomas-Loba,A., Martin- Caballero,J., Flores,J.M., Klatt,P., Blasco,M.A. and Serrano,M.(2006) Increased p53
activity does not accelerate telomere-driven ageing. EMBO Rep., 7, 546–552.

Garcia-Salcedo JA, Gijon P, Nolan DP, Tebabi P, Pays E. A chromosomal SIR2 homologue with both histone NAD-dependent
ADP-ribosyltransferase and deacetylase activities is involved in DNA repair in Trypanosoma brucei. Embo J 2003;22:5851–62.
[PubMed: 14592982]

Gentry,A. and Venkatachalam,S. (2005) Complicating the role of p53 in aging. Aging Cell, 4, 157–160.

Gerhart-Hines Z, Rodgers JT, Bare O, et al. Metabolic control of muscle mitochondrial function and fatty acid oxidation through
SIRT1/PGC-1alpha. Embo J 2007;26:1913–23. [PubMed: 17347648]

Godley LA, Kopp JB, Eckhaus M, Paglino JJ, Owens J, Varmus HE. Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric
bud and possess small kidneys. Genes Dev 1996;10(7):836–850.

Gokey, N.G., Cao, Z., Pak, J.W., Lee, D., McKiernan, S.H., McKenzie, D. et al. Molecular analyses of mtDNA deletion
mutations in microdissected skeletal muscle fibers from aged rhesus monkeys. Aging Cell 3, 319–326. 2004.

Gorbunova, V. and Levy, A.A. (1997) Nonhomologous DNA end joining in plant cells is associated with deletions and filler DNA insertions.
Nucleic Acids Res., 25, 4650–4657.

Gorbunova, V. and Seluanov,A. (2005) Making ends meet in old age: DSB repair and aging. Mech. Ageing Dev., 126, 621–628.

Goukassian, D., Gad, F., Yaar, M., Eller, M.S., Nehal, U.S. and Gilchrest, B.A. (2000) Mechanisms and implications of the age associated
decrease in DNA repair capacity. FASEB J., 14, 1325–1334.

Green, D.R. and Chipuk,J.E. (2006) p53 and metabolism: inside the TIGAR. Cell, 126, 30–32.

Greer EL, Brunet A. FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene
2005;24:7410–25. [PubMed: 16288288]

Grist, S.A., McCarron,M., Kutlaca,A., Turner,D.R. and Morley,A.A. (1992) In vivo human somatic mutation: frequency and spectrum with age.
Mutat. Res., 266, 189–196.

Grossman, L. and Wei,Q. (1995) DNA repair and epidemiology of basal cell carcinoma. Clin. Chem., 41, 1854–1863.

Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997;90(4):595–606.

Guarente L, Picard F. 2005. Calorie restriction—The SIR2 connection. Cell 120: 473–482.

Guarente L. 2000. Sir2 links chromatin silencing, metabolism, and aging. Genes & Dev 14: 1021–1026.

Guillouf, C., Grana,X., Selvakumaran,M., De Luca,A., Giordano,A., Hoffman,B. and Liebermann,D.A. (1995) Dissection of the genetic
programs of p53-mediated G1 growth arrest and apoptosis: blocking p53-induced apoptosis unmasks G1 arrest. Blood, 85, 2691–2698.

Guo Z, Heydari A, Richardson A. 1998. Nucleotide excision repair of actively transcribed versus nontranscribed DNA in rat hepatocytes: Effect
of age and dietary restriction. Exp Cell Res 245: 228–238.

Guo, Z., Heydari, A. and Richardson, A. (1998) Nucleotide excision repair of actively transcribed versus nontranscribed DNA in rat
hepatocytes: effect of age and dietary restriction. Exp. Cell Res., 245, 228–238.

Haigis MC, Mostoslavsky R, Haigis KM, et al. SIRT4 Regulates Glutamate Dehydrogenase and Insulin Secretion in Pancreatic
Beta Cells. Cell 2006;126:941–54. [PubMed: 16959573]
Hamilton, M.L., Van Remmen,H., Drake,J.A., Yang,H., Guo,Z.M., Kewitt,K., Walter,C.A. and Richardson,A. (2001) Does oxidative damage to
DNA increase with age? Proc. Natl Acad. Sci. USA, 98, 10469–10474.

Hanawalt, P.C. (2002) Sub-pathways of nucleotide excision repair and their regulation. Oncogene, 21, 8949–8956.

Haoudi, A., Semmes, O.J.,Mason, J.M., & Cannon, R.E. Retrotransposition-competent human LINE-1 induces apoptosis in
cancer cells with intact p53. J. Biomed. Biotechnol. 2004, 185–194. 2004.

Harris SL, Levine AJ. The p53 pathway: positive and negative feedback loops. Oncogene 2005;24(17):2899–2908.

Hart, R.W. and Setlow, R.B. (1976) DNA repair in late-passage human cells. Mech. Ageing Dev., 5, 67–77.

Hasan S, El-Andaloussi N, Hardeland U, et al. Acetylation regulates the DNA end-trimming activity of DNA polymerase beta.
Molecular cell 2002;10:1213–22. [PubMed: 12453427]

Hasty P, Campisi J, Hoeijmakers J, van Steeg H, Vijg J. Ageing and genome maintenance: lessons from the mouse? Science
2003;299(5611):1355–1359.

Helleday, T. (2003) Pathways for mitotic homologous recombination in mammalian cells. Mutat. Res., 532, 103–115.

Hemminki, K. and Snellman, E. (2002) How fast are UV-dimers repaired in human skin DNA in situ? J. Invest. Ermatol. 119, 699; discussion
700–692.

Herbig, U., Ferreira,M., Condel,L., Carey,D. and Sedivy,J.M. (2006) Cellular senescence in aging primates. Science (New York, N.Y.), 311,
1257.

Heydari AR, Unnikrishnan A, Lucente LV, Richardson A. 2007. Caloric restriction and genomic stability. Nucleic Acids Res 35: 7485–7496.

Ho, J. and Benchimol,S. (2003) Transcriptional repression mediated by the p53 tumour suppressor. Cell Death Differ., 10, 404–408.

Holliday, R.Mutations and epimutations in mammalian cells.Mutat.Res. 250, 351–363. 1991.

Holmes SG, Rose AB, Steuerle K, Saez E, Sayegh S, Lee YM, Broach JR. Hyperactivation of the silencing proteins, Sir2p and
Sir3p, causes chromosome loss. Genetics 1997;145:605–14. [PubMed: 9055071]

Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in
mice. Nature 2003;421(6919):182–187.

Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan.
Nature 2003;425:191–6. [PubMed: 12939617]

Iafrate, A.J., Feuk, L., Rivera, M.N., Listewnik, M.L., Donahoe, P.K., Qi, Y. et al. Detection of large-scale variation in the
human genome.Nat. Genet. 36, 949–951. 2004

Imai S, Armstrong CM, Kaeberlein M, Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone
deacetylase. Nature 2000;403(6771):795–800.

Imam, S.Z., Karahalil, B., Hogue, B.A., Souza-Pinto, N.C. and Bohr, V.A. (2006) Mitochondrial and nuclear DNA-repair capacity of various
brain regions in mouse is altered in an age-dependent manner. Neuro biol. Aging, 27, 1129–1136.

Intano, G.W., Cho, E.J., McMahan, C.A. and Walter, C.A. (2003) Age-related base excision repair activity in mouse brain and liver nuclear
extracts. J. Gerontol. A Biol. Sci. Med. Sci., 58, 205–211.

Intano, G.W., McMahan, C.A., McCarrey, J.R., Walter, R.B., McKenna, A.E., Matsumoto, Y., MacInnes, M.A., Chen, D.J. and Walter, C.A.
(2002) Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice. Mol. Cell.
Biol., 22, 2410–2418.

Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, et al. Small molecule RITA binds to p53, blocks p53–HDM-2 interaction
and activates p53 function in tumours. Nat Med 2004;10(12):1321–1328.

Iwama, H., Ohyashiki, K., Ohyashiki, J.H., Hayashi, S., Yahata, N., Ando, K. et al. Telomeric length and telomerase activity vary
with age in peripheral blood cells obtained from normal individuals. Hum. Genet. 102, 397–402. 1998.

Jackson, S.P. (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis, 23, 687–696.

Janzen, V., Forkert,R., Fleming,H.E., Saito,Y., Waring,M.T., Dombkowski,D.M., Cheng,T., DePinho,R.A., Sharpless,N.E. et al. (2006) Stem-cell
ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature, 443, 421–426.
Jeffreys, A.J.,Neumann, R., & Wilson,V. Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism
for studying variation and mutation by single molecule analysis. Cell 60, 473–485. 1990.

Jeyapalan, J.C., Ferreira,M., Sedivy,J.M. and Herbig,U. (2007) Accumulation of senescent cells in mitotic tissue of aging primates. Mech. Ageing
Dev., 128, 36–44.

Jiang, G. and Sancar,A. (2006) Recruitment of DNA damage checkpoint proteins to damage in transcribed and nontranscribed sequences. Mol.
Cell. Biol., 26, 39–49.

Johnson, R.D. and Jasin,M. (2001) Double-strand-break-induced homologous recombination in mammalian cells. Biochem. Soc. Trans., 29, 196–
201.

Johnson,T.E.,Henderson, S.,Murakami, S., de Castro, E., de Castro, S.H.,Cypser, J. et al. Longevity genes J. Inherit.Metab.Dis.
25, 197–206. 2002.

Jones, I.M., Thomas, C.B., Tucker, B., Thompson, C.L., Pleshanov, P., Vorobtsova, I. and Moore, D.H. II (1995) Impact of age and environment
on somatic mutation at the hprt gene of T lymphocytes in humans. Mutat. Res., 338, 129–139.

Jones, S.N., Roe,A.E., Donehower,L.A. and Bradley,A. (1995) Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature,
378, 206–208.

Ju, Y.J., Lee,K.H., Park,J.E., Yi,Y.S., Yun,M.Y., Ham,Y.H., Kim,T.J., Choi,H.M., Han,G.J. et al. (2006) Decreased expression of DNA repair
proteins Ku70 and Mre11 is associated with aging and may contribute to the cellular senescence. Exp. Mol. Med., 38, 686–693.

Kaeberlein M, Andalis AA, Liszt GB, Fink GR, Guarente L. Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-
independent mechanism. Genetics 2004;166:1661–72. [PubMed: 15126388]

Kaeberlein M, McVey M, Guarente L. The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by
two different mechanisms. Genes Dev 1999;13:2570–80. [PubMed: 10521401]

Kanungo, M.S. Genes and Aging.Cambridge University Press,New York, 1994.

Karran, P. (1996) Microsatellite instability and DNA mismatch repair in human cancer. Semin. Cancer Biol., 7, 15–24.

Kennedy BK, Steffen KK, Kaeberlein M. 2007. Ruminations on dietary restriction and aging. Cell Mol Life Sci 64: 1323–1328.

Keyes WM, Wu Y, Vogel H, Guo X, Lowe SW, Mills AA. p63 deficiency activates a programme of cellular senescence and leads to accelerated
ageing. Genes Dev 2005;19(17):1986–1999.

Khrapko, K., Bodyak, N., Thilly, W.G., van Orsouw, N.J., Zhang, X., Coller, H.A. et al. Cell-by-cell scanning of whole
mitochondrial genomes in aged human heart reveals a significant fraction of myocytes with clonally expanded deletions.Nucleic
Acids Res. 27, 2434–2441. 1999.

Kim D, Nguyen MD, Dobbin MM, et al. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s
disease and amyotrophic lateral sclerosis. Embo J 2007;26:3169–79. [PubMed: 17581637]

Kimberland, M.L., Divoky, V., Prchal, J., Schwahn, U., Berger, W., & Kazazian, H.H., Jr. Full-length human L1 insertions retain
the capacity for high frequency retrotransposition in cultured cells. Hum. Mol. Genet. 8, 1557–1560. 1999.

Kingsbury, M.A., Friedman, B., McConnell, M.J., Rehen, S.K., Yang, A.H., Kaushal, D. et al. Aneuploid neurons are
functionally active and integrated into brain circuitry. Proc. Natl. Acad. Sci. USA 102, 6143–6147. 2005.

Kishi, S. (2004) Functional aging and gradual senescence in zebrafish. Ann. NY Acad. Sci., 1019, 521–526.

Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Sorge, J.A., Putman, D.L. and Short, J.M. (1991) Spectra of spontaneous and
mutagen-induced mutations in the LacI gene in transgenic mice. Proc. Natl Acad. Sci. USA, 88, 7958–7962.

Komarova, E.A., Kondratov,R.V., Wang,K., Christov,K., Golovkina,T.V., Goldblum,J.R. and Gudkov,A.V. (2004) Dual effect of p53 on
radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice. Oncogene, 23, 3265–3271.

Kraytsberg, Y., Kudryavtseva, E., McKee, A.C., Geula, C., Kowall, N.W., & Khrapko, K. Mitochondrial DNA deletions are
abundant and cause functional impairment in aged human substantia nigra neurons. Nat. Genet. 38, 518–520. 2006.

Krichevsky S. Pawelec, G., Gural, A., Effros, R.B., Globerson,A., Yehuda, D.B. and Yehuda, A.B. (2004) Age related microsatellite instability in
T cells from healthy individuals. Exp. Gerontol., 39, 507–515.
Krishna, T.H., Mahipal, S., Sudhakar, A., Sugimoto, H., Kalluri, R. and Rao, K.S. (2005) Reduced DNA gap repair in aging rat neuronal extracts
and its restoration by DNA polymerase beta and DNA-ligase. J. Neurochem., 92, 818–823.

Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest
2004;114(9):1299–1307.

Krontiris, T.G.Minisatellites and human disease. Science 269, 1682–1683. 1995.

Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J. Senescent fibroblasts promote epithelial cell growth and tumourigenesis: a link
between cancer and aging. Proc Natl Acad Sci USA 2001;98(21):12072–12077.

Kruk, P.A., Rampino, N.J. and Bohr, V.A. (1995) DNA damage and repair in telomeres: relation to aging. Proc. Natl Acad. Sci. USA, 92, 258–
262.

Kuro-o,M. (2001) Disease model: human aging. Trends Mol. Med., 7, 179–181.

Lagouge M, Argmann C, Gerhart-Hines Z, et al. Resveratrol improves mitochondrial function and protects against metabolic
disease by activating SIRT1 and PGC-1alpha. Cell 2006;127:1109–22. [PubMed: 17112576]

Lamming DW, Latorre-Esteves M, Medvedik O, et al. HST2 mediates SIR2-independent life-span extension by calorie
restriction. Science 2005;309:1861–4. [PubMed: 16051752]

Langley E, Pearson M, Faretta M, et al. Human SIR2 deacetylates p53 and antagonizes PML/p53- induced cellular senescence.
Embo J 2002;21:2383–96. [PubMed: 12006491]

Lansdorp, P.M., Verwoerd, N.P., van de Rijke, F.M., Dragowska, V., Little, M.T., Dirks, R.W. et al. Heterogeneity in telomere
length of human chromosomes.Hum.Mol. Genet. 5, 685–691. 1996.

Larsen, N.B., Rasmussen, M., & Rasmussen, L.J. Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5,
89–108. 2005.

Lasko, D., Cavenee,W. and Nordenskjold,M. (1991) Loss of constitutional heterozygosity in human cancer. Annu. Rev. Genet., 25, 281–314.

Lavin, M.F. and Gueven, N. (2006) The complexity of p53 stabilization and activation. Cell Death Differ., 13, 941–950.

Lee SE, Paques F, Sylvan J, Haber JE. Role of yeast SIR genes and mating type in directing DNA double-strand breaks to
homologous and non-homologous repair paths. Curr Biol 1999;9:767–70. [PubMed: 10421582]

Lemieux ME, Yang X, Jardine K, et al. The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in
mammals. Mech Ageing Dev 2005;126:1097–105. [PubMed: 15964060]

Lengauer, C., Kinzler, K.W., & Vogelstein, B. DNA methylation and genetic instability in colorectal cancer cells.
Proc.Natl.Acad. Sci. USA 94, 2545–2550. 1997.

Lieber, M.R. (1999) The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in
multicellular eukaryotes. Genes Cells, 4, 77–85.

Lieber,M.R., Ma,Y., Pannicke,U. and Schwarz,K. (2003) Mechanism and regulation of human non-homologous DNA endjoining. Nat. Rev. Mol.
Cell Biol., 4, 712–720.

Lim,D.S., Vogel,H., Willerford,D.M., Sands,A.T., Platt,K.A. and Hasty,P. (2000) Analysis of ku80-mutant mice and cells withdeficient levels of
p53. Mol. Cell. Biol., 20, 3772–3780.

Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.
Science 2000;289(5487):2126–2128.

Liu, D. and Hornsby, P.J. (2007) Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase
secretion. Cancer Res., 67, 3117–3126.

Liu, L.,Wylie, R.C.,Andrews, L.G., & Tollefsbol, T.O.Aging, cancer and nutrition: the DNA methylation
connection.Mech.Ageing Dev. 124, 989–998. 2003.

Liu,B., Wang,J., Chan,K.M., Tjia,W.M., Deng,W., Guan,X., Huang,J.D., Li,K.M., Chau,P.Y. et al. (2005) Genomic instability in laminopathy-
based premature aging. Nat. Med., 11, 780–785.
Longo VD, Kennedy BK. Sirtuins in aging and age-related disease. Cell 2006;126:257–68. [PubMed: 16873059]

Lotem, J. and Sachs,L. (1993) Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some
agents. Blood, 82, 1092–1096.

Lowe SW, Cepero E, Evan G. Intrinsic tumour suppression. Nature 2004;432(7015):307–315.

Lowe, S.W., Schmitt,E.M., Smith,S.W., Osborne,B.A. and Jacks,T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes.
Nature, 362, 847–849.

Lu, T., Pan, Y., Kao ,S.Y., Li, C., Kohane, I., Chan, J. and Yankner, B.A. (2004) Gene regulation and DNA damage in the ageing human brain.
Nature, 429, 883–891.

Lundberg AS, Hahn WC, Gupta P, Weinberg RA. Genes involved in senescence and immortalization. Curr Opin Cell Biol 2000;12(6):705–709.
Luo J, Nikolaev AY, Imai S, Chen D, Su F, Shiloh A, et al. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell
2001;107(2):137–148.

Luo J, Nikolaev AY, Imai S, et al. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 2001;107:137–
48. [PubMed: 11672522]

Luo, G., Santoro,I.M., McDaniel,L.D., Nishijima,I., Mills,M., Youssoufian,H., Vogel,H., Schultz,R.A. and Bradley,A. (2000) Cancer
predisposition caused by elevated mitotic recombination in Bloom mice. Nat. Genet., 26, 424–429.

Luzi L, Confalonieri S, Di Fiore PP, Pelicci PG. Evolution of Shc functions from nematode to human. Curr Opin Genet Dev 2000;10(6):668–
674.

Ma W, Stafford LJ, Li D, Luo J, Li X, Ning G, Liu M. 2007. GCIP/CCNDBP1, a helix–loop–helix protein, suppresses tumorigenesis. J Cell
Biochem 100: 1376–1386.

Maas NL, Miller KM, DeFazio LG, Toczyski DP. Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3
and Hst4. Molecular cell 2006;23:109–19. [PubMed: 16818235]

Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, et al. Modulation of mammalian life span by the short isoform of p53. Genes
Dev 2004;18(3):306–319.

Malkin, D., Li,F.P., Strong,L.C., Fraumeni,J.F.Jr, Nelson,C.E., Kim,D.H., Kassel,J., Gryka,M.A., Bischoff,F.Z. et al. (1990) Germ line p53
mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science (New York, N.Y.), 250, 1233–1238.
Mallette, F.A., Gaumont-Leclerc,M.F. and Ferbeyre,G. (2007) The DNA damage signaling pathway is a critical mediator of oncogeneinduced
senescence. Genes Dev., 21, 43–48.

Mann, D.B., Springer,D.L., & Smerdon, M.J.DNA damage can alter the stability of nucleosomes: effects are dependent on
damage type. Proc.Natl.Acad. Sci. USA 94, 2215–2220. 1997.

Marchenko, N.D., Zaika, A. and Moll, U.M. (2000) Death signal induced localization of p53 protein to mitochondria. A potential role in
apoptotic signaling. J. Biol. Chem., 275, 16202–16212.

Marini, F., Nardo,T., Giannattasio,M., Minuzzo,M., Stefanini,M., Plevani,P. and Falconi,M.M. (2006) DNA nucleotide excision repair-dependent
signaling to checkpoint activation. Proc. Natl Acad. Sci. USA, 103, 17325–17330.

Marrone, A., Walne,A. and Dokal,I. (2005) Dyskeratosis congenita: telomerase, telomeres and anticipation. Curr. Opin. Genet. Dev., 15, 249–
257.

Martin SG, Laroche T, Suka N, Grunstein M, Gasser SM. Relocalization of telomeric Ku and SIR proteins in response to DNA
strand breaks in yeast. Cell 1999;97:621–33. [PubMed: 10367891]

Martin, G.M., Smith,A.C., Ketterer,D.J., Ogburn,C.E. and Disteche,C.M. (1985) Increased chromosomal aberrations in first metaphases of cells
isolated from the kidneys of aged mice. Isr. J. Med. Sci., 21, 296–301.

Martin-Ruiz, C.M.,Gussekloo, J., van Heemst,D., von Zglinicki,T.,& Westendorp, R.G.Telomere length in white blood cells is
not associated with morbidity or mortality in the oldest old: a population-based study.Aging Cell 4, 287–290. 2005.

Masoro EJ. 1998. Hormesis and the antiaging action of dietary restriction. Exp Gerontol 33: 61–66.

Masoro EJ. 2005. Overview of caloric restriction and ageing. Mech Ageing Dev 126: 913–922.

Matheu,A., Maraver,A., Klatt,P., Flores,I., Garcia-Cao,I., Borras,C., Flores,J.M., Vina,J., Blasco,M.A. et al. (2007) Delayed ageing through
damage protection by the Arf/p53 pathway. Nature, 448, 375–379.
Matoba, S., Kang,J.G., Patino,W.D., Wragg,A., Boehm,M., Gavrilova,O., Hurley,P.J., Bunz,F. and Hwang,P.M. (2006) p53 regulates
mitochondrial respiration. Science (New York, N.Y.), 312, 1650–1653.

Mayer, P.J., Lange, C.S., Bradley, M.O. and Nichols, W.W. (1989) Age-dependent decline in rejoining of X-ray-induced DNA doublestrand
breaks in normal human lymphocytes. Mutat. Res., 219, 95–100.

McBurney MW, Yang X, Jardine K, et al. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis.
Mol Cell Biol 2003;23:38–54. [PubMed: 12482959]

Melk, A., Kittikowit,W., Sandhu,I., Halloran,K.M., Grimm,P., Schmidt,B.M. and Halloran,P.F. (2003) Cell senescence in rat kidneys in vivo
increases with growth and age despite lack of telomere shortening. Kidney Int., 63, 2134–2143.

Mendrysa SM, O’Leary KA, McElwee MK, Michalowski J, Eisenman RN, Powell DA, et al. Tumour suppression and normal aging in mice with
constitutively high p53 activity. Genes Dev 2006;20(1):16–21.

Mendrysa, S.M., O’Leary,K.A., McElwee,M.K., Michalowski,J., Eisenman,R.N., Powell,D.A. and Perry,M.E. (2006) Tumor suppression and
normal aging in mice with constitutively high p53 activity. Genes Dev., 20, 16–21.

Mendrysa,S.M. and Perry,M.E. (2006) Tumor suppression by p53 without accelerated aging: just enough of a good thing? Cell cycle
(Georgetown, Tex.), 5, 714–717.

Migeon, B.R., Axelman, J., & Beggs, A.H. Effect of ageing on reactivation of the human X-linked HPRT locus.Nature 335, 93–
96. 1988.

Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, et al. The p66shc adaptor protein controls oxidative stress response and life
span in mammals. Nature 1999;402(6759):309–313.

Mills KD, Sinclair DA, Guarente L. MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-
strand breaks. Cell 1999;97:609–20. [PubMed: 10367890]

Moazed D. Enzymatic activities of Sir2 and chromatin silencing. Current opinion in cell biology 2001;13:232–8. [PubMed:
11248558]

Molofsky, A.V., Slutsky,S.G., Joseph,N.M., He,S., Pardal,R., Krishnamurthy,J., Sharpless,N.E. and Morrison,S.J. (2006) Increasing
p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature, 443, 448–452.

Monckton,D.G.Using robots to find needles.Mech.Ageing Dev. 126, 1046–1050. 2005.

Montes de Oca Luna,R., Wagner,D.S. and Lozano,G. (1995) Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53.
Nature, 378, 203–206.

Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M. and Nikaido, O. (1991) Simultaneous establishment of monoclonal antibodies specific
for either cyclobutane pyrimidine dimer or (6-4) photoproduct from the same mouse immunized with ultraviolet irradiated DNA. Photochem.
Photobiol. 54, 225–232.

Moriwaki, S.-I., Ray, S., Tarone, R.E., Kraemer, K.H. and Grossman, L. (1996) The effect of donor age on the processing of UV-damaged DNA
by cultured human cells: reduced DNA repair capacity and increased mutability. Mutat. Res., 364, 117–123.

Morley, A. (1998) Somatic mutation and aging. Ann. N. Y. Acad. Sci., 854, 20–22.

Morley, A.A., Cox, S. and Holliday, R. (1982) Human lymphocytes resistant to 6-thioguanine increase with age. Mech. Ageing Dev., 19, 21–26.
Mostoslavsky R, Chua KF, Lombard DB, et al. Genomic instability and aging-like phenotype in the absence of mammalian
SIRT6. Cell 2006;124:315–29. [PubMed: 16439206]

Moynihan KA, Grimm AA, Plueger MM, et al. Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-
stimulated insulin secretion in mice. Cell Metab 2005;2:105–17. [PubMed: 16098828]

Murphy, M.E., Leu,J.I. and George,D.L. (2004) p53 moves to mitochondria: a turn on the path to apoptosis. Cell cycle (Georgetown, Tex.), 3,
836–839.

Nakamura T, Pichel JG, Williams-Simons L, Westphal H. An apoptotic defect in lens differentiation caused by human p53 is rescued by a
mutant allele. Proc Natl Acad Sci USA 1995;92(13):6142–6146.

Nekhaeva, E., Bodyak, N.D., Kraytsberg, Y., McGrath, S.B., Van Orsouw, N.J., Pluzhnikov, A. et al. Clonally expanded mtDNA
point mutations are abundant in individual cells of human tissues. Proc. Natl.Acad. Sci. USA 99,
5521–5526. 2002.
Neri, S., Gardini, A., Facchini, A., Olivieri, F., Franceschi, C., Ravaglia , G. and Mariani, E. (2005) Mismatch repair system and aging:
microsatellite instability in peripheral blood cells from differently aged participants. J. Gerontol. A Biol. Sci. Med. sci., 60, 285–292.

Nikolaev AY, Li M, Puskas N, Qin J, Gu W. Parc: a cytoplasmic anchor for p53. Cell 2003;112(1):29–40.

Nilsen H, Lindahl T, Verreault A. DNA base excision repair of uracil residues in reconstituted nucleosome core particles. Embo J
2002;21:5943–52. [PubMed: 12411511]

Oren, M. (2003) Decision making by p53: life, death and cancer. Cell Death Differ., 10, 431–442.

Orii, K.E., Lee,Y., Kondo,N. and McKinnon,P.J. (2006) Selective utilization of nonhomologous end-joining and homologousrecombination DNA
repair pathways during nervous system development. Proc. Natl Acad. Sci. USA, 103, 10017–10022.

Orsted,D.D., Bojesen,S.E., Tybjaerg-Hansen,A. and Nordestgaard,B.G. (2007) Tumor suppressor p53 Arg72Pro polymorphism and longevity,
cancer survival, and risk of cancer in the general population. J. Exp. Med., 204, 1295–1301.

Painter, R.B., Clarkson, J.M. and Young, B.R. (1973) Letter: ultraviolet- induced repair replication in aging diploid human cells (WI-38). Radiat.
Res., 56, 560–564.

Parrinello, S., Coppe, J.P., Krtolica, A. and Campisi, J. (2005) Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter
epithelial cell metalloproteinase secretion. Cancer Res., 67, 3117–3126.differentiation. J. Cell Sci., 118, 485–496.

Pendas, A.M., Zhou,Z., Cadinanos,J., Freije,J.M., Wang,J., Hultenby,K., Astudillo,A., Wernerson,A., Rodriguez,F. et al. (2002) Defective
prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice. Nat. Genet., 31, 94–99.

Picard F, Kurtev M, Chung N, et al. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature
2004;429:771–6. [PubMed: 15175761]

Pluquet, O. and Hainaut,P. (2001) Genotoxic and non-genotoxic pathways of p53 induction. Cancer Lett., 174, 1–15.

Preston, C.R., Flores, C. and Engels, W.R. (2006) Age-dependent usage of double-strand-break repair pathways. Curr. Biol., 16, 2009–2015.

Pruitt K, Zinn RL, Ohm JE, et al. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA
hypermethylation. PLoS Genet 2006;2:e40. [PubMed: 16596166]

Ramsey, M.J., Moore,D.H., Briner,J.F., Lee,D.A., Olsen,L.A., Senft,J.R. and Tucker,J.D. (1995) The effects of age and lifestyle factors on the
accumulation of cytogenetic damage as measured by chromosome painting. Mutat. Res., 338, 95–106.

Ramsey, M.J.,Moore, D.H., 2nd, Briner, J.F., Lee, D.A., Olsen, L., Senft, J.R. et al. The effects of age and lifestyle factors on the
accumulation of cytogenetic damage as measured by chromosome painting. Mutat.Res. 338, 95–106. 1995.

Ratovitski EA, Patturajan M, Hibi K, Trink B, Yamaguchi K, Sidransky D. p53 associates with and targets delta Np63 into a protein degradation
pathway. Proc Natl Acad Sci USA 2001;98(4):1817–1822.

Rehen, S.K., McConnell, M.J., Kaushal, D., Kingsbury, M.A., Yang, A.H., & Chun, J. Chromosomal variation in neurons of the
developing and adult mammalian nervous system. Proc.Natl.Acad. Sci.USA 98, 13361–13366. 2001.

Rehen, S.K., Yung, Y.C., McCreight, M.P., Kaushal, D., Yang, A.H., Almeida, B.S. et al. Constitutional aneuploidy in the
normal human brain. J.Neurosci. 25, 2176–2180. 2005.

Ren, K. and de Ortiz, S.P. (2002) Non-homologous DNA end joining in the mature rat brain. J. Neurochem., 80, 949–959.
Reynolds, B.A. & Rietze, R.L. Neural stem cells and neurospheres—re-evaluating the relationship. Nat. Methods 2, 333–336.
2005.

Richardson, B. Impact of aging on DNA methylation.Ageing Res.Rev. 2, 245–261. 2003.

Rincon M, Rudin E, Barzilai N. The insulin/IGF-1 signaling in mammals and its relevance to human longevity. Exp Gerontol
2005;40:873–7. [PubMed: 16168602]

Ringshausen, I., O’Shea,C.C., Finch,A.J., Swigart,L.B. and Evan,G.I. (2006) Mdm2 is critically and continuously required to suppress lethal p53
activity in vivo. Cancer Cell, 10, 501–514.

Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a
complex of PGC-1alpha and SIRT1. Nature 2005;434:113–8. [PubMed: 15744310]
Rodier,F., Kim,S.H., Nijjar,T., Yaswen,P. and Campisi,J. (2005) Cancer and aging: the importance of telomeres in genome maintenance. Int J.
Biochem. Cell Boil., 37, 977–990.

Rogina B, Helfand SL. Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc Natl Acad Sci U S
A 2004;101:15998–6003. [PubMed: 15520384]

Rossi, D.J., Bryder,D., Seita,J., Nussenzweig,A., Hoeijmakers,J. and Weissman,I.L. (2007) Deficiencies in DNA damage repair limit the function
of haematopoietic stem cells with age. Nature, 447, 725–729.

Rossignol, R., Faustin, B., Rocher, C., Malgat, M., Mazat, J.P., & Letellier, T. Mitochondrial threshold effects. Biochem. J. 370,
751–762. 2003.

Rothkamm, K., Kruger, I., Thompson, L.H. and Lobrich, M. ( 2003) Pathways of DNA double-strand break repair during the mammalian cell
cycle. Mol. Cell. Biol., 23, 5706–5715.

Rudolph,K.L., Chang,S., Lee,H.W., Blasco,M., Gottlieb,G.J., Greider,C. and DePinho,R.A. (1999) Longevity, stress response, and cancer in
aging telomerase-deficient mice. Cell, 96, 701–712.

Ruley, H.E., Lowe,S.W., Jacks,T. and Housman,D.E. (1993) p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell, 74,
957–967.

Sablina, A.A., Budanov,A.V., Ilyinskaya,G.V., Agapova,L.S., Kravchenko,J.E. and Chumakov,P.M. (2005) The antioxidant function of the p53
tumor suppressor. Nat. Med., 11, 1306–1313.

Saleh-Gohari, N. and Helleday, T. (2004) Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S
phase of the cell cycle in human cells. Nucleic Acids Res., 32, 3683–3688.

Sargent, R.G., Branneman,M.A. and Wilson,J.H. (1997) Repair of site-specific double-strand breaks in mammalian chromosome by homologous
and illegitimate recombination. Mol. Cell. Biol., 17, 267–277.

Saunders LR, Verdin E. 2007. Sirtuins: Critical regulators at the crossroads between cancer and aging. Oncogene 26: 5489–5504.

Scaffidi,P. and Misteli,T. (2006) Lamin A-dependent nuclear defects in human aging. Science (New York, N.Y.), 312, 1059–1063.

Sebat, J., Lakshmi, B., Troge, J., Alexander, J., Young, J., Lundin, P. et al. Large-scale copy number polymorphism in the human
genome. Science 305, 525–528. 2004.

Sedelnikova, O.A., Horikawa,I., Zimonjic,D.B., Popescu,N.C., Bonner,W.M. and Barrett,J.C. (2004) Senescing human cells and ageing mice
accumulate DNA lesions with unrepairable doublestrand breaks. Nat. Cell Biol., 6, 168–170.

Seluanov, A., Mittelman,D., Pereira-Smith,O.M., Wilson,J.H. and Gorbunova,V. (2004) DNA end joining becomes less efficient and more error-
prone during cellular senescence. Proc. Natl Acad. Sci. USA, 101, 7624–7629.

Seluanov,A., Danek,J., Hause,N. and Gorbunova,V. (2007) Changes in the level and distribution of Ku proteins during cellular senescence. DNA
Repair.

Shackelford, D.A. (2006) DNA end joining activity is reduced in Alzheimer’s disease. Neurobiol. Aging, 27, 596–605.

Shao, C., Stambrook,P.J. and Tischfield,J.A. (2001) Mitotic recombination is suppressed by chromosomal divergence in hybrids of
distantly related mouse strains. Nat. Genet., 28, 169–172.

Shen, G.P., Galick, H., Inoue, M. and Wallace, S.S. (2003) Decline of nuclear and mitochondrial oxidative base excision repair activity in late
passage human diploid fibroblasts. DNA Repair (Amst), 2, 673–693.

Shi T, Wang F, Stieren E, Tong Q. SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and
thermogenesis in brown adipocytes. The Journal of biological chemistry 2005;280:13560–7. [PubMed: 15653680]

Sinclair DA, Guarente L. Extrachromosomal rDNA circles--a cause of aging in yeast. Cell 1997;91:1033–42. [PubMed:
9428525]

Singh, N.P., Danner, D.B., Tice, R.R., Brant, L. and Schneider, E.L. (1990) DNA damage and repair with age in individual human lymphocytes.
Mutat. Res., 237, 123–130.

Singh, N.P., Ogburn,C.E., Wolf,N.S., van Belle,G. and Martin,G.M. (2001) DNA double-strand breaks in mouse kidney cells with age.
Biogerontology, 2, 261–270.

Skinner, A.M. and Turker, M.S. (2005) Oxidative mutagenesis, mismatch repair, and aging. Sci. Aging Knowledge Environ., 2005, re3.
Slagboom, P.E., de Leeuw,W.J., & Vijg, J. Messenger RNA levels and methylation patterns of GAPDH and beta-actin genes in
rat liver, spleen and brain in relation to aging.Mech. Ageing Dev. 53, 243–257. 1990.

Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63. [PubMed: 8658196]

Sommer M, Poliak N, Upadhyay S, Ratovitski E, Nelkin B, Donehower L, et al. DNp63alpha over-expression induces downregulation of Sirt1
and an accelerated aging phenotype in the mouse. Cell Cycle 2006;5(17):2005–2011.

Sonoda, E., Hochegger,H., Saberi,A., Taniguchi,Y. and Takeda,S. (2006) Differential usage of non-homologous end-joining and homologous
recombination in double strand break repair. DNA Repair (Amst), 5, 1021–1029.

Soong,N.W.,Hinton,D.R.,Cortopassi, G., & Arnheim, N.Mosaicism for a specific somatic mitochondrial DNA mutation in adult
human brain.Nat. Genet. 2, 318–323. 1992.

Srivastava VK, Miller S, Schroeder MD, Hart RW, Busbee D. 1993. Age-related changes in expression and activity of DNA polymerase a: Some
effects of dietary restriction. Mutat Res 295: 265–280.

Stambolic V, MacPherson D, Sas D, Lin Y, Snow B, Jang Y, et al. Regulation of PTEN transcription by p53 . Mol Cell 2001;8(2):317–325.

Stark, J.M. and Jasin,M. (2003) Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks. Mol. Cell.
Biol., 23, 733–743.

Strehler, B.L. Genetic instability as the primary cause of human aging. Exp.Gerontol. 21, 283–319. 1986.

Stuart JA, Karahalil B, Hogue BA, Souza-Pinto NC, Bohr VA. 2004. Mitochondrial and nuclear DNA base excision repair are affected
differently by caloric restriction. FASEB J 18: 595– 597.

Stuart, G.R. and Glickman,B.W. (2000) Through a glass, darkly: reflections of mutation from lacI transgenic mice. Genetics, 155, 1359–1367.

Stuart, G.R., Oda, Y., de Boer, J.G. and Glickman, B.W. (2000) Mutation frequency and specificity with age in liver, bladder and brain of LacI
transgenic mice. Genetics, 154, 1291–1300.

Suh,Y., Lee,K.A., Kim,W.H., Han,B.G., Vijg,J. and Park,S.C. (2002) Aging alters the apoptotic response to genotoxic stress. Nat. Med., 8, 3–4.

Sullivan, T., Escalante-Alcalde,D., Bhatt,H., Anver,M., Bhat,N., Nagashima,K., Stewart,C.L. and Burke,B. (1999) Loss of A-type lamin
expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell. Biol., 147, 913–920.

Szczesny, B., Bhakat, K.K., Mitra, S. and Boldogh, I. (2004) Age-dependent modulation of DNA repair enzymes by covalent modification and
sub-cellular distribution. Mech. Ageing Dev., 125, 755–765.

Szczesny, B., Hazra, T.K., Papaconstantinou, J., Mitra, S. and Boldogh, I. (2003) Age-dependent deficiency in import of mitochondrial DNA
glycosylases required for repair of oxidatively damaged bases. Proc. Natl Acad. Sci. USA, 100, 10670–10675.
Taylor, R.W. & Turnbull, D.M. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 6, 389–402. 2005.
Taylor, R.W., Barron, M.J., Borthwick, G.M., Gospel, A., Chinnery, P.F., Samuels, D.C. et al. Mitochondrial DNA mutations in
human colonic crypt stem cells. J. Clin. Invest. 112, 1351–1360. 2003.

Thomas, S. & Mukherjee, A.B. A longitudinal study of human age-related ribosomal RNA gene activity as detected by silver-
stained NORs.Mech.Ageing Dev. 92, 101–109. 1996

Tissenbaum HA, Guarente L. Increased dosage of a sir–2 gene extends lifespan in Caenorhabditis elegans. Nature 2001;410:227–
30. [PubMed: 11242085]

Tra, J.K.T., Lu, Q., Kuick, R., Hanash, S., & Richardson, B. Infrequent occurrence of age-dependent changes in CpG island
methylation as detected by restriction landmark genome scanning.Mech.Ageing Dev. 123, 1487–1503. 2002.

Tsang CK, Li H, Zheng XS. 2007a. Nutrient starvation promotes condensin loading to maintain rDNA stability. EMBO J 26: 448–458.

Tsang CK, Wei Y, Zheng XF. 2007b. Compacting DNA during the interphase: Condensin maintains rDNA integrity. Cell Cycle 6: 2213–2218.

Tsang CK, Zheng XF. 2007. TOR-in(g) the nucleus. Cell Cycle 6: 25–29.

Tsao JL, Dudley S, Kwok B, Nickel AE, Laird PW, Siegmund KD, Liskay RM, Shibata D. 2002. Diet, cancer and aging in DNA mismatch repair
deficient mice. Carcinogenesis 23: 1807–1810.

Tsuchiya M, Dang N, Kerr EO, et al. Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in
yeast. Aging Cell 2006;5:505–14. [PubMed: 17129213]
Tsukamoto Y, Kato J, Ikeda H. Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae.
Nature 1997;388:900–3. [PubMed: 9278054]

Tucker, J.D., Spruill, M.D., Ramsey, M.J., Director, A.D., & Nath, J. Frequency of spontaneous chromosome aberrations in
mice: effects of age.Mutat.Res. 425, 135–141. 1999.

Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, et al. p53 mutant mice that display early ageingassociated
phenotypes. Nature 2002;415(6867):45–53.

Um JH, Kim SJ, Kim DW, Ha MY, Jang JH, Chung BS, Kang CD, Kim SH. 2003. Tissue-specific changes of DNA repair protein Ku and
mtHSP70 in aging rats and their retardation by caloric restriction. Mech Ageing Dev 124: 967–975.

Valenzano DR, Terzibasi E, Genade T, Cattaneo A, Domenici L, Cellerino A. Resveratrol prolongs lifespan and retards the onset
of age-related markers in a short-lived vertebrate. Curr Biol 2006;16:296–300. [PubMed: 16461283]

van Heemst,D., Mooijaart,S.P., Beekman,M., Schreuder,J., de Craen,A.J., Brandt,B.W., Slagboom,P.E. and Westendorp,R.G. (2005) Variation in
the human TP53 gene affects old age survival and cancer mortality. Exp. Gerontol., 40, 11–15.

van Remmen, H.,Ward,W.F., Sabia, R.V., & Richardson, A. Gene expression and protein degradation. In Handbook of
Physiology—Aging, pp. 171–234 (ed. Masoro, E.). Oxford University Press, New York, 1995.

Vaquero A, Scher M, Erdjument-Bromage H, Tempst P, Serrano L, Reinberg D. 2007a. SIRT1 regulates the histone methyltransferase SUV39H1
during heterochromatin formation. Nature 450: 440–444.

Vaquero A, Sternglanz R, Reinberg D. 2007b. NAD+-dependent deacetylation of H4 lysine 16 by class III HDACs. Oncogene 26: 5505–5520.

Vaquero A. 2009. The conserved role of sirtuins in chromatin regulation. Int J Dev Biol 53: 303–322.

Varela, I., Cadinanos,J., Pendas,A.M., Gutierrez-Fernandez, A., Folgueras,A.R., Sanchez,L.M., Zhou,Z., Rodriguez,F.J., Stewart,C.L. et al.
(2005) Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation. Nature,437, 564–568.

Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by smallmoleculeantagonists of
MDM2. Science 2004;303(5659):844–848.

Vaziri H, Dessain SK, Ng Eaton E, et al. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 2001;107:149–59.
[PubMed: 11672523]

Vaziri, H., Schachter, F.,Uchida, I.,Wei, L.,Zhu, X., Effros, R. et al. Loss of telomeric DNA during aging of normal and trisomy
21 human lymphocytes.Am J.Hum. Genet. 52, 661–667. 1993.

Vijg, J. (2000) Somatic mutations and aging: a re-evaluation. Mutat. Res., 447, 117–135.

Vijg, J. and Dolle,M.E.T. (2002) Large genome rearrangements as a primary cause of aging. Mech. Ageing Dev., 123, 907–915.

Vijg, J., Dolle, M.E.T., Martus, H.-J, and Boerrigter, M.E.T.I. (1997). Transgenic mouse models for studying mutations in vivo: applications in
aging research. Mech. Ageing Dev., 98, 189–202.

Vijg, J., Mullaart, E., Lohman, P.H. and Knook, D.L. (1985) UV-induced unscheduled DNA synthesis in fibroblasts of aging inbred rats. Mutat.
Res., 146, 197–204.

Villeponteau, B. The heterochromatin loss model of aging. Exp. Gerontol. 32, 383–394. 1997.

Viswanathan M, Kim SK, Berdichevsky A, Guarente L. A role for SIR-2.1 regulation of ER stress response genes in determining
C. elegans life span. Developmental cell 2005;9:605–15. [PubMed: 16256736]

Vogel,H., Lim,D.S., Karsenty,G., Finegold,M. and Hasty,P. (1999) Deletion of Ku86 causes early onset of senescence in mice. Proc. Natl Acad.
Sci. USA, 96, 10770–10775.

von Zglinicki,T., Saretzki,G., Ladhoff,J., d’Adda di Fagagna,F. and Jackson,S.P. (2005) Human cell senescence as a DNA damage response.
Mech. Ageing Dev., 126, 111–117.

Vulliamy, T.,Marrone, A., Szydlo, R.,Walne, A.,Mason, P.J.,& Dokal, I. Disease anticipation is associated with progressive
telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Nat. Genet. 36, 447–449. 2004.

Vyjayanti, V.N. and Rao, K.S. (2006) DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons. Neurosci. Lett., 393,
18–22.
Walker, J.R., Corpina,R.A. and Goldberg,J. (2001) Structure of the Ku heterodimer bound to DNA and its implications for doublestrand break
repair. Nature, 412, 607–614.

Wang C, Chen L, Hou X, et al. Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage. Nat Cell Biol
2006;8:1025–31. [PubMed: 16892051]

Wang F, Nguyen M, Qin FX, Tong Q. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction. Aging
Cell 2007;6:505–14. [PubMed: 17521387]

Wang Y, Tissenbaum HA. Overlapping and distinct functions for a Caenorhabditis elegans SIR2 and DAF-16/FOXO. Mech
Ageing Dev 2006;127:48–56. [PubMed: 16280150]

Wang, Y., Schulte,B.A., LaRue,A.C., Ogawa,M. and Zhou,D. (2006) Total body irradiation selectively induces murine hematopoietic stem cell
senescence. Blood, 107, 358–366.

Wareham, K.A., Lyon, M.F., Glenister, P.H., & Williams, E.D. Age related reactivation of an X-linked gene.Nature 327, 725–
727. 1987.

Wei, Q., Matanoski,G.M., Farmer,E.R., Hedayati,M.A. and Grossman,L. (1993) DNA repair and aging in
basal cell carcinoma: a molecular epidemiology study. Proc. Natl Acad. Sci. USA, 90, 1614–1618.
Weitao T, Budd M, Campbell JL. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability.
Mutation research 2003;532:157–72. [PubMed: 14643435]

Werner H, Karnieli E, Rauscher FJ, LeRoith D. Wild-type and mutant p53 differentially regulate transcription of the insulinlike growth factor I
receptor gene. Proc Natl Acad Sci USA 1996;93(16):8318–8323.

Wilson, D.M., III and Bohr, V.A. (2006) The mechanics of base excision repair, and its relationship to aging and disease. DNA Repair (Amst).

Wilson, V.L., Smith, R.A., Ma, S., & Cutler, R.G. Genomic 5-methyldeoxycytidine decreases with age. J. Biol. Chem. 262,
9948–9951. 1987.

Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay
ageing in metazoans. Nature 2004;430:686–9. [PubMed: 15254550]

Wu, C., Miloslavskaya,I., Demontis,S., Maestro,R. and Galaktionov,K. (2004) Regulation of cellular response to oncogenic and oxidative stress
by Seladin-1. Nature, 432, 640–645.

Xia E, Rao G, Van Remmen H, Heydari AR, Richardson A. 1995. Activities of antioxidant enzymes in various tissues of male Fischer 344 rats
are altered by food restriction. J Nutr 125: 195–201.

Xu, G., Snellman, E., Bykov, V.J., Jansen, C.T. and Hemminki, K. (2000) Effect of age on the formation and repair of UV photoproducts in
human skin in situ. Mutat. Res., 459, 195–202.

Xu, G., Spivak, G., Mitchell, D.L., Mori, T., McCarrey, J.R., McMahan, C.A., Walter, R.B., Hanawalt ,P.C. and Walter, C.A. (2005) Nucleotide
excision repair activity varies among murine spermatogenic cell types. Biol. Reprod., 73, 123–130.

Xu, X., Qiao, W., Linke, S.P., Cao, L., Li, W.M., Furth, P.A., Harris, C.C. and Deng, C.X. (2001) Genetic interactions between tumor
suppressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis. Nat. Genet., 28, 266–271.

Yamada, M., Udono, M.U., Hori, M., Hirose, R., Sato, S., Mori, T. and Nikaido, O. (2006) Aged human skin removes UVB-induced pyrimidine
dimers from the epidermis more slowly than younger adult skin in vivo. Arch. Dermatol. Res., 297, 294–302.

Yamamoto, A., Taki,T., Yagi,H., Habu,T., Yoshida,K., Yoshimura,Y., Yamamoto,K., Matsushiro,A., Nishimune,Y. et al. (1996) Cell cycle-
dependent expression of the mouse Rad51 gene in proliferating cells. Mol. Gen. Genet., 251, 1–12.

Yuan Z, Zhang X, Sengupta N, Lane WS, Seto E. SIRT1 regulates the function of the Nijmegen breakage syndrome protein.
Molecular cell 2007;27:149–62. [PubMed: 17612497]

Zhang J. Resveratrol inhibits insulin responses in a SirT1-independent pathway. Biochem J 2006;397:519–27. [PubMed:
16626303]

Zhang,J. and Powell,S.N. (2005) The role of the BRCA1 tumor suppressor in DNA double-strand break repair. Mol. Cancer Res., 3, 531–539.
Zou, L. and Elledge,S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science (New York, N.Y.), 300,
1542–1548.