Oncogene (2013), 1–15
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REVIEW
Senescence and aging: the critical roles of p53
A Rufini1, P Tucci1,2, I Celardo1 and G Melino1,3
p53 functions as a transcription factor involved in cell-cycle control, DNA repair, apoptosis and cellular stress responses. However,
besides inducing cell growth arrest and apoptosis, p53 activation also modulates cellular senescence and organismal aging.
Senescence is an irreversible cell-cycle arrest that has a crucial role both in aging and as a robust physiological antitumor response,
which counteracts oncogenic insults. Therefore, via the regulation of senescence, p53 contributes to tumor growth suppression,
in a manner strictly dependent by its expression and cellular context. In this review, we focus on the recent advances on the
contribution of p53 to cellular senescence and its implication for cancer therapy, and we will discuss p53’s impact on animal
lifespan. Moreover, we describe p53-mediated regulation of several physiological pathways that could mediate its role in both
senescence and aging.
Oncogene advance online publication, 18 February 2013; doi:10.1038/onc.2012.640
Keywords: senescence; aging; p53; mTOR; mitochondria; ROS
INTRODUCTION
Senescence represents a stress response in which cells withdraw
from the cell cycle and lose the capability to proliferate in
response to growth factors or mitogens.1,2 Senescent cells show
very distinctive changes in morphology, acquiring a typical flat
and enlarged shape and increase expression of recognized
biomarkers of senescence, including staining for b-galactosidase
at pH of 6.0 (senescence-associated-b-gal or SA-b-gal), decreased
replicative capacity, increased expression of p53, p21, p16 and
other cyclin-dependent kinase inhibitors, such as p27 and p15.
Finally, accumulation of transcriptionally inactive heterochromatic
structure (senescence-associated heterochromatic foci or SAHF)
has been reported, particularly in the promoters of E2F-target
genes.2–4
Initially thought to be a cell culture artifact, senescence has
been more recently observed in vivo in cancer lesions and during
physiological aging.3,5–10 Hence, recently, increasing interest has
focused on senescence as a novel approach in cancer therapy,
because of its inherent property to suppress cell proliferation,
senescence may protect against cancer onset.2 Intriguingly,
senescence is also intimately related to aging as both shared
ability to limit lifespan. The constant regeneration of somatic
tissues leads to accumulation of senescent cells, which limits
tissue renewal, perturbs normal tissue homeostasis and ultimately
elicits aging. Recent findings have established a causal role
between senescence and aging: selective killing of p16-positive
senescent cells in vivo ameliorates aging-related features in a
mouse model of progeroid syndrome.8,11
Senescence has been classically viewed as a state of permanent
growth arrest, during which cells are unable to re-enter the
cell cycle. Although this concept is still widely accepted, recent
studies have provided evidence that under certain conditions this
cellular status is reversible. In fact, stable suppression or even
more subtle changes in p53 expression in senescent fibroblasts
lead to rapid cell-cycle re-entry and immortalization, indicating
that both initiation and maintenance of senescence are p53
dependent,12–15 as discussed later in more detail. Regardless,
senescence is functional to both tumor suppression and
organismal aging, which means that p53 ability to regulate both
processes may heavily rely on its fundamental role in eliciting
cellular senescence.
MOLECULAR MECHANISMS IN SENESCENCE AND AGING
The senescence pathway can be triggered by multiple mechanisms. Originally, it was associated with replication exhaustion at
the end of the cellular lifespan, a process currently defined as
replicative senescence. Replicative senescence results from a
combination of events that include the progressive erosion of
telomeres during cell proliferation. This phenomenon can lead to
critically short telomeres that are sensed by the cells as doublestrand breaks. Double-strand breaks trigger the DNA damage
response (DDR), a signaling cascade centered around the ataxia
teleangectasia-mutated (ATM) kinase that activates p53 to elicit
cell-cycle arrest and to execute senescence.16–20 Ectopic
expression of telomerase, the enzyme responsible for telomere
stabilization, circumvents replicative senescence in human cells,21
and stabilization of telomeres is essential for tumor progression.
Telomeres are also important during aging. In humans, a positive
correlation between telomere length and longevity has been
suggested.20,22,23 In addition, a mouse model with depletion of
telomerase shows several signs of accelerated aging, including
anemia, kyphosis, osteoporosis, glucose intolerance, alopecia and
hair graying.24–26 Here, a crucial event points to a progressive loss
of the stem cell reservoir in these animals through induction
of apoptosis and senescence. These phenotypes correlate
with genomic instability and activation of p53,27 and genetic
ablation of p53 ameliorates symptoms in mice with critically short
telomeres.28 Thus telomere erosion links p53 to both senescence
and aging.
1
Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK; 2Department of Pharmaco-Biology, University of Calabria, Rende (CS), Italy and 3University of Rome
‘Tor Vergata’, Department of Experimental Medicine and Biochemical Sciences, and Biochemistry Laboratory, Istituto Dermopatico dell’Immacolata, Rome, Italy. Correspondence:
Dr G Melino, Medical Research Council, Toxicology Unit, Leicester University, Lancaster Road, Leicester LE1 9HN, UK.
E-mail: [email protected]
Received 9 October 2012; revised 30 November 2012; accepted 7 December 2012
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Relevant for tumorigenesis, persistent oncogenic signaling is
another important trigger that activates a powerful senescence
response,29 known as oncogene-induced senescence (OIS). This
process, indeed, prevents cellular transformation. Oncogenic
H-Ras and, more generally, activation of oncogenes trigger
hyperproliferation. Enforced DNA replication results in DDR
followed by activation of senescence pathways, which must be
overcome for transformation to occur. This process fails in cells
that lack ATM activity or when cells cannot sense DNA damage or
transduce DDR signals to p53.29
A major determinant of senescence, at the molecular level, is
the intracellular accumulation of oxidative damage triggered by
reactive oxygen species (ROS).2,22,30–34 ROS are generally small,
short-lived and highly reactive molecules (for example, oxygen
anions, superoxide and hydroxyl radicals, and peroxides) formed
by partial reduction of oxygen, which, if not detoxified promptly
by antioxidant agents, can oxidize macromolecules and damage
organelles (Figure 1). Oxidation of DNA causes base modifications
(that is, mutations) leading to various pathologies in humans, such
as cancer, whereas oxidized proteins tend to form aggregates
resulting in diverse neurodegenerative pathologies. ROS are also
involved in aging as oxidative damage to various constituents of
the cell may limit lifespan.35 In this regard, as mitochondria are the
major source of ROS (Figure 1), a ‘mitochondrial free radical theory
of aging’ has been postulated, arguing that mitochondrialgenerated oxygen radicals cause widespread oxidative damage,
eventually resulting in aging.36,37 ROS enhance senescence and
aging, inducing toxicity, into a feed-forward cycle: ROS cause
Figure 1. Mitochondrial ROS. (a) A schematic model of ROS generation in the mitochondria. Superoxide (O2 ) generated by the respiratory
chain is mostly released to the matrix at complex I and the IMS at complex III. O2 can naturally dismute to hydrogen peroxide (H2O2) or is
enzymatically dismuted by matrix MnSOD or Cu/ZnSOD in the IMS or cytosol. H2O2 is detoxified in the matrix by catalase, the thioredoxin/
thioredoxin peroxidase system (TPx), or the glutathione/glutathione peroxidase system (GPx). Alternately, H2O2 can react with metal ions to
generate the highly reactive hydroxyl radical ( OH) via Fenton chemistry. O2 is not membrane permeable but can pass through ion channels
(solid lines), whereas H2O2 can pass freely through membranes. (b) ROS can have both endogenous or exogenous sources. The overall balance
in ROS cellular content is the result of ROS production and removal from specialized scavenging enzymes.
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damage to mitochondrial constituents, and, subsequently,
damaged mitochondria produce more ROS.35,38–40 The
senescence–ROS correlation has attracted great interest,
prompted by studies in several organisms, in which a negative
relationship between mitochondrial ROS production and lifespan
has been found.41,42 Moreover, senescence and aging are
associated with an increase in the levels of oxidative-damaged
proteins, lipids and DNA,43–45 consequently to ROS-mediated
damage to macromolecules such as proteins, nucleic acids and
lipids. Mitochondrial-generated ROS have also been involved in
OIS: Ras-driven senescence is associated with the accumulation of
dysfunctional mitochondria, a sharp rise in ROS and a drop in ATP
levels.46 Accordingly, chemical or genetic inhibition of the electron
transport chain suffices in inducing senescence in human
fibroblasts.46
Mechanistically, senescence relies on two main molecular
pathways: p53–p21 (discussed later) and p16INK4A-Rb. p16, a
cyclin/cdk inhibitor, prevents phosphorylation of Rb by cyclin/cdk
complexes. Hypophosphorylated Rb halts cell proliferation by
inhibitory binding to E2Fs transcription factors, thus preventing
them from stimulating transcription of genes involved in cellular
proliferation and DNA replication.17 In this context, p16-Rb
axis is pivotal to the establishment of cell-cycle arrest. During
OIS, suppression of Rb abolishes the establishment of a
proper senescent phenotype, but it is not sufficient to overcome
cell-cycle arrest; this depends on the concomitant p53-dependent
cell-cycle arrest.47
p53 IN SENESCENCE AND AGING
p53 is a tetrameric transcription factor heavily regulated by
posttranscriptional modifications.48–52 It is regarded as one of the
most powerful tumor suppressor genes owing to its ability to halt
cell proliferation and induce apoptosis and its activity is pivotal to
successful traditional chemotherapy, as many DNA-damageinducing drugs target tumors via p53-mediated apoptosis.49,53–55
Consequently, p53 is mutated or lost in the vast majority of
human cancers and considerable effort is focused on recovering
its function in anticancer therapy.56–61
p53 is clearly involved in cancer, but the existence of p53 in
short-living organisms that do not develop cancers, such as
flies and worms, suggests that tumor suppression is not its only
and, probably, original function. Indeed, recent studies have
shown that p53 influences development,62,63 reproduction,64
metabolism65 and longevity.
The first evidence linking p53 to aging arose from the analysis
of a mutant mouse model: in the attempt to develop a knock-in
(KI) of p53, Tyson and colleagues obtained an aberrant
serendipitous truncation of the N-terminal portion of the gene.
The truncated mutant proteins showed a robust constitutive p53
activity and the mutant mice presented an array of aging-related
features and severely reduced lifespan. In 2004, Scrable’s group
produced a transgenic mouse model overexpressing the truncated DNp53 or p44 isoform of p53.66,67 This mouse showed a
striking defect in growth with associated reduced lifespan and
accelerated aging. Interestingly, p44 overexpression resulted in
hyperactive p53 and increased IGF signaling, a master regulator of
aging.68 Recently, a KI mouse model of p53 was developed in
order to mimic constitutive phosphorylation (that is, activation) of
p53. This mouse model showed striking aging features, which
seemed to result from widespread apoptosis affecting the stem
cell compartments of several organs, hence compromising tissue
self-renewal.69 PUMA is a proapoptotic protein and a wellcharacterized p53 target that exerts a fundamental role in
induction of apoptosis and survival of stem cells: notably
depletion of PUMA in the context of p53 mutations rescued the
stem cell loss and ameliorated the aging phenotype.69 Thus,
widespread apoptosis of stem cells may underline p53-mediated
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aging phenotype, possibility in agreement with studies
highlighting an ‘aging’ process affecting stem cells.70 Although
the mechanisms underlining these phenotypes are still unclear,
these results led to the notion that excessive p53 activity
compromises healthy aging. On the other hand, whether lack or
reduced p53 activity affects lifespan has been difficult to assess,
owing to the severe tumor phenotype that accompanies loss of
p53.71 Nonetheless, recently developed in vivo models have shed
light on the issue. A fundamental residue in p53 is Serine 15 (Ser-15)
(ser-18 in mouse): phosphorylation of Ser-15 by ATM activates p53
in response to DNA damage. In 2006, Armata and colleagues
analyzed the phenotype of KI mice where Ser-18 of p53 was
replaced with non-phosphorylable alanine. These mice developed
signs of accelerated aging, indicating that physiological p53
activity may preserve tissues from aging-related damage.72,73 The
super-arf/p53 mouse model, developed by Serrano’s group,
provided an additional striking support to the anti-aging activity
of p53. These transgenic mice bear long genomic sequence of p53
and p19arf, allowing their increased expression (owing to
increased copy number, up to 4n), but maintaining endogenous
regulation (as the regulatory region of the loci are preserved). In
these circumstances, the authors noted an increase in lifespan and
an overall improvement of the aging-related health decline.74
Although p19arf may act independently of p53, it is worth
remembering that it does increase p53 activity preventing MDM2mediated p53 proteasomal degradation.75 Overall, these findings
suggest that loss of p53 is detrimental to aging.
In summary, the model that is emerging is an intensitybased model: physiological p53 activity prevents from cancer
and protects from aging, whereas unrestrained and excessive
p53 activation still protects from cancer, but is detrimental to
healthy aging.
Induction of p53 is pivotal for the establishment of senescence,
mainly following its activation by the DDR.76,77 Indeed, depletion
of p53 or abrogation of the upstream DDR signaling is sufficient to
impair OIS.29 Several p53-targets and regulators have been linked
to induction of senescence, including microRNAs,22,78,79 but the
molecular mechanisms are still elusive. One of the most wellestablished p53-target genes, CDKN1A/p21, has been proved to
be upregulated during replicative senescence.80–83 p21 has been
among the first identified downstream targets of p53, and it is an
essential mediator of p53-dependent cell-cycle arrest. p21depleted mouse embryonic fibroblasts are unable to undergo
p53-dependent G1 arrest after DNA damage.84 The obvious
dependency of p53 on p21 for the induction of cell-cycle arrest
and the established role of p21 as inhibitor of proliferation
suggest a crucial role for this gene in the induction of p53dependent senescence (Figure 2). Indeed, lack of p21 abrogates
senescence in several settings.85–87
Nonetheless, although p21 contributes to the growth arrest of
senescent cells, it is unlikely to be solely responsible for the
complex and paramount changes underpinning senescence and,
even more, aging. Moreover, p53 regulates a plethora of target
genes affecting several physiological and metabolic pathways, all
heavily involved in regulation of aging and establishment of
senescence.88 Here, we review several of these pathways and
discuss their potential implication in p53-induced senescence and
p53-regulated aging (Figure 2).
p53 and E2F7
In agreement with the idea that p21 is not sufficient to explain the
essential need for p53 in the establishment of senescence, two
recent papers have described E2F7 as a new p53 target involved
in cell-cycle arrest and senescence.47,89 In particular, Aksoi and
colleagues, in Scott Lowe’s laboratory, showed that E2F7 is
upregulated in a p53-dependent fashion during proliferative as
well as OIS. This gene is an atypical member of the E2F-family of
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Figure 2. Senescence regulation by p53. Several posttranslational modifications regulate p53 activity. Stimuli that activate the DDR, lead to
active p53 via ATM-mediated phosphorylation. Active p53 triggers expression of pro-senescence targets such as p21, responsible for G1 cellcycle arrest and E2F7, pivotal in repression of mitotic genes. In addition, p53 controls other pathways linked to aging, including ROS
generation and mTOR. In this regard, while steady-state levels of p53 are able to dampen ROS and limit oxidative damage, active p53 elevates
intracellular ROS, which participate in its proapoptotic and pro-senescent activities. Overall, the physiological role of p53-mediated regulation
of ROS in senescence and aging is still unclear.
transcription factors, as, unlike canonical E2Fs, it does not
heterodimerize with DP1 proteins,90,91 but binds DNA as a
monomer and promotes repression of several E2F target genes,
including E2F1. Moreover, many genes essential for mitosis, such
as cyclin A, cyclin B and cdc2/cdk1, are repressed in senescent
cells in a E2F7-dependent way. Hence, functionally, E2F7 arrests
cell-cycle progression at the mitotic phase. This may have some
important implications for tumorigenesis, explaining an apparent
conundrum. In fact, both p53 and Rb are necessary for a full
establishment of senescence. But, whereas p53-depleted cells are
immortalized and readily transformed by exogenous oncogenic
Ras alone, Rb-depleted cells, despite failing to undergo
proper senescent arrest, are not immortalized and expression of
Ras is not sufficient for their transformation and does not endow
them with tumorigenic capability.92 Aksoi and colleagues
demonstrate that p53-mediated upregulation of E2F7 is
potentially responsible for this difference. In fact, concomitant
inactivation of both Rb and E2F7 immortalizes cells and allows
Ras-mediated cellular transformation.47
p53 and mTOR
The kinase mechanistic target of rapamycin (mTOR), previously
known as mammalian TOR, is at the interface between growth and
starvation. When nutrients are available, mTOR is active and
promotes organism growth and anabolism. Conversely, in the case
of nutrient depletion, mTOR is promptly inactivated to favor
catabolism and growth arrest. Mechanistically, mTOR phosphorylates its substrates S6 kinase 1 and eIF4E-binding protein 1 to
regulate mRNA translation initiation and progression, thus
controlling the rate of protein synthesis.93 Hence, mTOR is
implicated in diseases showing growth deregulation and
metabolic compromise, such as cancer, diabetes and obesity94
and is a master regulator of senescence and aging in several
animal models, such as yeast,95–98 worms,99 flies100 and mice,101
where mTOR inhibition has been proved to prevent the
expression of some senescent markers.102 Overall, many findings
suggest that sustained mTOR signaling promotes cell and tissue
aging, fostering the idea that inhibition of mTOR may increase
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longevity. From yeast and Caenorhabditis elegans and up to mice
and primates, one of the most effective methods in prolonging
lifespan is caloric restriction (CR),103 achieved decreasing caloric
intake, without malnutrition. Importantly, mTOR is necessary for
the CR beneficial effect and CR fails to extend lifespan in
organisms where mTOR signaling has been reduced.104
Moreover, in Drosophila melanogaster, inhibition of mTOR during
CR results in selective increased translation of components of the
mitochondrial electron transport chain mediated by increased
activation of eIF4E-binding protein 1. This selective upregulation
leads to improved mitochondrial respiration, decreased ROS
production and results in reduced ROS-dependent senescence
and prolonged lifespan.105 Strikingly, the drug rapamycin, a
chemical inhibitor of mTOR, has been recently shown to prolong
lifespan in mammals.101
Active mTOR signaling promotes tumor growth and malignancy
and, to some extent, mTOR partners behave like oncogenes.106–110
Thus, even though it is normally related to cellular growth, mTOR
activity reinforces certain types of senescence,.111–113 In this
regard, it is of great interest that overexpression of the GTPase
protein mTOR-activator Ras homolog enriched in brain (Rheb)
triggers senescence in vivo and in vitro in an mTOR-dependent
fashion.111 Similar results were described upon in vivo
overexpression of the mTOR downstream target eIF4E.114
Moreover, the ability of mTOR to promote cell growth seems to
be pivotal to the establishment of senescence in cell-cyclearrested cells. Indeed, expression of p21 induces senescence when
mTOR is active, but it promotes quiescence when cells are serum
starved (that is, mTOR is inactive) or upon pharmacological
inhibition of mTOR by rapamycin.102,115 This may have important
implications in p53-induced senescence as detailed below.
Nonetheless, this pro-senescence function of mTOR does not
apply universally: DNA-damage-induced senescence seems
refractory to mTOR inhibition,102 whereas in Ras-driven OIS
dampening of mTOR signaling has been reported116 (see
chapter p53 and autophagy). Moreover, mTOR promotes
senescence via autophagy inhibition, by decreasing lysosomal
degradation of intracellular components. Activated by nutrients,
mTOR inhibits autophagy, a process that may contribute
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to mitochondrial dysfunction ER stress and senescence.
Indeed, autophagy seems to be required for the senescence
response116–118 (Figure 2).
Recent evidence indicates that p53 can also prevent cell
growth, interacting with the mTOR pathway. Interestingly, p53
inhibits mTOR signaling through different ways.119 In fact, p53regulated sestrins repress mTOR activity directly.120 In addition,
p53 triggers expression of the AMP-activated protein kinase
(AMPK), which, in turn, inactivates mTOR.121,122 Finally, p53
upregulates PTEN, an inhibitor of the PI3K pathway, which is an
upstream-positive regulator of TOR.
Lately, mTOR regulation by p53 has been implicated in a
paradoxical antisenescence role of p53. Induction of p21 allows
the establishment of an irreversible senescent arrest. Nonetheless,
further accumulation of transcriptional-competent (but even
unphosphorylated) p53 triggers inhibition of mTOR and switches
cell status to a reversible cell-cycle arrest.14,123 Unfortunately, the
p53-target(s) responsible for this phenotype is yet unidentified,
but the ability of p53 to induce cell-cycle arrest and inhibiting
mTOR simultaneously could help explaining why moderate
increases of p53 activity protects from cancer and
simultaneously prolongs lifespan. In addition, these data support
our view that p53-mediated senescence is not simply an on–off
switch mediated by p21 induction, but it is a complex cellular
phenotype that can be fine tuned by regulation of several
additional targets and pathways.
p53 and autophagy
Autophagy is an evolutionary conserved self-eating mechanism by
which cellular cytoplasmic portions and organelles are delivered
to the lysosome for degradation. Degraded products are then
recycled for energy production or other metabolic processes,
which explains why autophagy is engaged in conditions of
nutrient deprivation.124 An additional role for the autophagic
process is the removal of damaged macromolecules and
dysfunctional mitochondria, avoiding the build-up of damage.
As such, autophagy is cytoprotective and can modulate aging and
influence cancer survival.125–127
The longevity pathways interact with the autophagic process to
regulate diverse cellular functions, including growth, differentiation, response to nutrient deprivation, oxidative stress, cell death,
as well as macromolecule and organelle turnover. Indeed,
mutations in genes that promote autophagy reduce lifespan in
C. elegans, D. melanogaster and yeast.125,128–131 Moreover, CR
induces autophagy via repression of the mTOR signaling, and
autophagy induction is essential for the anti-aging outcome of
reduced caloric intake.132,133
On the other hand, the role of autophagy in cancer is more
debatable.127,134–136 Robust engagement of autophagy in tumor
areas deprived of blood and nutrient supplies promotes survival of
cancer cells, suggesting an ‘oncogenic’ role for autophagy, and
several studies have proved that engagement of autophagy
protects cancer cell from chemotherapy.137–145 Conversely, allelic
disruption of some autophagic genes predisposes to tumor
development, indicating that autophagy may be required to
repress tumor onset.146–150 As far as senescence is concerned,
autophagy acts as an effector mechanism during OIS.116 In fact,
autophagy is engaged during OIS in a PI3k-mTOR-dependent
fashion and its inhibition delays the onset of the senescent
phenotype.116
p53 has a dual function in the control of autophagy: it can
either activate or repress autophagy.49,151–153 On the one hand,
nuclear p53 can induce autophagy through transcriptional
upregulation of targets such as AMPK, PTEN and sestrins that
activate autophagy mainly through inhibition of mTOR. Another
pivotal p53-target and positive regulator of autophagy is damageregulated autophagy modulator, which codes for a lysosomal
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protein. In response to p53 activation, damage-regulated
autophagy modulator is upregulated and elicits autophagy that
is necessary to mediate p53-dependent cell death. On the other
hand, cytoplasmic p53 represses autophagic flux through a
substantially unknown mechanism. Kroemer’s group showed
that loss of p53 activity can enhance autophagy and that
cytoplasmic, not nuclear, p53 is responsible for autophagy
inhibition. Importantly, in this context, inducers of autophagy,
such as starvation or rapamycin, induce degradation of p53 that is
necessary for autophagy induction.151 Although the physiological
implications of p53-regulated autophagy are unknown with
regard to senescence induction, there is evidence of their
involvement in the regulation of lifespan. Indeed, knockdown of
the C. elegans p53 ortholog Cep-1 increases lifespan, a phenotype
abrogated by inhibition of autophagy.154
p53 and ROS
As aforementioned, ROS or, more accurately, ROS-mediated
damage have been extensively implicated in the induction of
cellular senescence and in the onset of aging disorders. p53 shows
a Janus role, dictated by its dual capacity to inhibit or promote
senescence, by regulating ROS levels.14,42,155 Indeed, increasing
evidence suggests that transcriptional regulation of antioxidant
genes (including mitochondrial superoxide dismutase 2,
glutathione peroxidase 1 and mammalian sestrin homologs 1
and 2) accounts for p53’s ability to repress senescence by
dampening intracellular ROS levels.156–160 On the other hand, in
cells sensitive to p53-mediated apoptosis, DNA-damage-activated
p53 elicits a spike in intracellular ROS content, resulting in cell
death or senescence.42,155,161,162 The p53-dependent ROS
generation may well represent a crucial event for senescence
regulation, but its dual regulation of oxidative metabolism may
confer to p53 a double-edged role in the senescence process. As
far as aging is concerned, the free radical theory of aging states
that ROS-mediated damage has a direct detrimental effect on
animal well-being.38 Hence, p53 may counteract aging by
mitigating the oxidative burden, as suggested by reduced
oxidative damage in long-lived super-arf/p53 mice.74 However, it
is unclear whether induction of ROS by p53 in response to
stressors is involved in aging.
p53 and mitochondria
Mitochondria have been linked to aging, neurodegeneration163–165
and cancer.166 As stated, according to the free radical theory of
aging, ROS-mediated damage to cellular components is the
driving force behind aging.35 As mitochondria are the prime
source of ROS, they are as well the main targets of ROS-mediated
damage, a hypothesis known as ‘mitochondrial theory of aging’.
Impaired mitochondrial activity and the resulting imbalance in
oxidative and energetic metabolism can indeed severely affect
lifespan and negatively impact on aging.167 Study of telomerasedeficient animals has unveiled a link between p53, mitochondria
and aging. Telomerase maintains the stability of telomeres, the
nucleoprotein complexes responsible for the genomic integrity of
chromosomal ends. Eroded telomeric ends trigger widespread
DNA damage, which activates p53 and results in age-related
disorders.24,25 Importantly, depletion of p53 ameliorates the agerelated degeneration in telomerase-deficient animals, partially
abolishing p53-mediated cell death.28 Intriguingly, upon telomere
dysfunction, active p53 represses expression of peroxisome
proliferator-activated receptor gamma, coactivator 1 alpha and
beta (PGC-1a/b). PGC proteins regulate mitochondrial physiology
and energetic metabolism (Figure 3). Their repression decreases
mitochondrial biogenesis, reduces oxygen consumption and
increases ROS levels.168 These findings bridge DNA damage,
mitochondria and aging, and prove that p53 regulation of
mitochondrial respiration is likely to affect animal longevity.
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Figure 3. p53 regulates mitochondrial function. Mitochondria are pivotal regulators of aging and accumulating evidences link them to cellular
senescence. In unstressed cells, p53 sustains mitochondrial respiration promoting the transcription of the nuclear-encoded mitochondrial
protein synthesis of cytochrome c oxidase 2 (a). Conversely, stress-activated p53 (for example, consequently to telomere erosion) represses
PGC1a, a positive regulator of mitochondrial function and biogenesis, resulting in impaired mitochondrial respiration and bioenergetic, which
has been directly implicated in p53-mediated regulation of animal lifespan (b). In addition, potentially, p53 could affect mitochondria
indirectly, through regulation of autophagy. Autophagy of mitochondria, or mitophagy, is essential for the removal of damaged organelles
and, hence, the preservation of an efficient pool of healthy mitochondria. Nuclear p53 activates autophagy, whereas cytoplasmic p53 inhibits
it. Whether this regulations affect mitochondria and, generally, their physiological implication in aging and senescence still awaits
investigation (c,d).
Intriguingly, basal p53 activity is necessary for maintenance of
mitochondrial function. Indeed, p53 promotes the expression of
synthesis of cytochrome c oxidase 2, a component of the complex
IV of the electron transport chain. p53 null tissues and cells have
reduced complex IV activity resulting in impaired oxygen
consumption. However, whether this has a role in aging or
senescence has not been investigated yet.
Intriguingly, mTOR signaling has been reported to sustain
respiration in human cells,169 whereas autophagy of mitochondria,
or mitophagy, removes damage organelles and helps maintain a
healthy pool of mitochondria.170,171 Hence, the ability of p53 to
inhibit both mTOR and its dual regulation of autophagy may well
be implicated in regulation of mitochondrial function during
senescence or aging, a possibility that needs further investigation.
p53 and sirtuins
The crosstalk between p53 and Sirt1 represents a crucial point of
regulation of p53 signaling, implicated in many biological
processes such as senescence. Sirt1 belongs to a family of
evolutionary conserved NAD þ -dependent protein deacetylase,
classified as class III histone deacetylase, able to deacetylate target
histone and non-histone proteins, and thus participates in the
regulation of chromatin structure and of DNA accessibility for
processing and repair, as well as in transcriptional control
networks via deacetylation of transcription factors and cofactors.58,172–179 SIRT1 is necessary for the establishment of
senescence,180,181 and SIRT1 is strongly downregulated in
senescent cells. A major substrate for SIRT1 is p53, and the
deacetylation of p53 regulates cell cycle, cellular senescence and
stress resistance in various cell types. Deacetylation inhibits p53’s
ability to transcriptionally activate some, but not all, target
genes—including those involved in apoptosis, proliferation, ROS
production and presumably also senescence.58,182,183 Following
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DNA damage, SIRT1 relocalizes from its constitutive loci to sites of
DNA damage where it promotes DNA repair and hence genomic
stability. Thus, both SIRT1 and p53 are chromatin/DNA responders
that help maintain genomic stability and are coordinated so that
SIRT1 favors repair and survival, while p53 elicits programmed
removal of overly damaged cells via apoptosis. The presence of a
chronic DDR (as may be seen in cancer cells), which is linked to the
induction of senescence, can directly increase p53 acetylation
by promoting the interaction with the acetyl transferases
CBP/p300.184 Acetylation of p53 is also seen to be important
during Ras-induced or replicative senescence, where it is
antagonized by SIRT1.180,181,185,186 In keeping with this, cells
harboring p53 with acetyl-mimicking mutations of the last seven
lysine residues have an accelerated entry into senescence and are
very resistant to senescence bypass,187 although the cell-cycle
arrest response in these cells remains normal. Conversely,
mutations that abolish acetylation of the lysine residues located
in the DNA-binding domain fails to establish replicative as well as
OIS.188 Thus, these data strongly suggest that deacetylation of p53
by Sirt1 impedes the induction of senescence. Whether this is
relevant in tumor formation is unclear. Indeed, SIRT1-mediated
repression of p53 activity supports the idea that SIRT1 could be
oncogenic. However, several mouse models proved that SIRT1 acts
as a tumor suppressor reducing cancer incidence even in p53
heterozygous mice.189–191 Thus, the physiological meaning of
SIRT1-mediated deacetylation of p53 remains to be elucidated.
SIRT1 was initially identified as a ‘longevity’ gene in
C. elegans,192 yeast193 and Drosophila,194 findings that spurred
research in mammalian models. The idea that SIRT1 is necessary to
prolong animal lifespan has been heavily questioned.195
Nonetheless, mice-overexpressing SIRT1 have a reduced
incidence of age-related metabolic disorders, including diabetes,
liver steatosis and196 cancer. In other words, it is now evident that
SIRT1 is not necessary to live longer, but to live healthier.
& 2013 Macmillan Publishers Limited
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Currently, it is unclear whether p53 is required for the metabolic
function of SIRT1.
p53-INDUCED SENESCENCE IN CANCER THERAPY
The cancer cell phenotype is characterized by sustained proliferative signaling, alteration of cellular homeostasis and metabolism.197,198 Senescence is a robust physiological antitumor
response that is engaged by tissues to counteract oncogenic
insults. Accumulating evidence of its involvement in the onset and
therapeutic response in humans has spurred considerable efforts
towards its therapeutic exploitation.176,199 Indeed, therapyinduced senescence is an emerging appealing approach to halt
tumor growth; several agents are reported to induce senescence
by acting on different pathways, as demonstrated in vitro and in
human tumors and in tumor models (Table 1). The DDR entails
senescence as an anticancer mechanism; indeed, many drugs
induce senescence following DNA damage. In OIS, the genetic
lesions that initiate tumorigenesis (for example, RAS overactivation) promote senescence at the early stage of cellular transformation. For transformation to occur, additional genetic
modifications are necessary to overcome senescence.17,77,82,200,201
Therefore, designing innovative therapeutic approaches to trigger
tumor regression re-activating senescence programs and effectors
is appealing. In this regard, modulation of p53 activity may be
appropriate.56,57,202–207 Recent findings have demonstrated that
reactivation of p53 in tumors elicits a robust tumor regression
mediated by induction of senescence.203,204 These studies
boosted the long-standing efforts to develop drugs able to
reactivate
p53
in
tumors-bearing
null
or
mutant
p53.59,73,186,205,208–211 Nonetheless, the efficiency of p53mediated tumor clearance is also stage-specific and dependent
on the overall ‘oncogenic’ burden. Indeed, using a mouse model
of K-Ras-driven lung cancer, two different research groups
demonstrated that p53 reactivation is efficient only in advanced
cancers characterized by sustain Ras-Raf-Mef-Erk signaling. In
addition, in the same system, p53 tumor suppressor activity does
rely on co-expression of p19ARF. Although these results cast doubt
on the potential therapeutic benefit of p53 restoration in cancer
therapy, they also unveil an interesting parallelism between p53
tumor suppressive function and regulation of aging. Indeed, in the
super-arf/p53 mouse model, increased expression of p19 is
essential to prolong lifespan, whereas increased dosage of p53
alone does not suffice.212,213 These findings highlight the
existence of a p19–p53 axis, where ARF expression seems to be
necessary to fully engage p53 activity.
The phytoalexin resveratrol is known to possess a variety of
cancer-preventive, therapeutic and chemosensitizing properties. It
has been reported that chronic treatment with resveratrol in a
subapoptotic concentration induces senescence-like growth arrest
in tumor cells (Table 1). Resveratrol has proved to act by increasing
the level of ROS and induce a p21–p53-dependent senescence.214
This anticancer property of resveratrol is particularly intriguing on
the light of its debated role in regulating aging and sirtuin
function. Because of the debate on the issue, we refer to other
reviews for details,125,215 but in summary, there is evidence that
resveratrol may improve healthy aging, especially by
counteracting obesity and diabetes, and that this could be
mediated, at least partially, by activation of Sirt1 and induction
of autophagy. Hence, resveratrol is able to suppress tumor growth,
while improving organismal metabolism.
A promising target for senescence induction in cancer cells is
the enzyme telomerase. Findings have demonstrated that short
telomeres induce senescence, limiting tumor suppression.216,217
The evidence that senescence induced by telomere shortening is
an in vivo tumor suppression process comes from studies in
mTERC / mice, in which shortened telomeres decrease
tumorigenesis when block of apoptosis is due to p53-mutant
& 2013 Macmillan Publishers Limited
Table 1. Table reports anticancer drugs currently undergoing clinical
trials (source www.clinicaltrial.gov), which induce cellular senescence
in cancer cell lines and tumors
Agent
Mechanism
p53
Status
Resveratrol
Hydroxyurea
Mitoxantrone
Cyclophosphamide þ doxorubicin
þ 5-fluorouracil
Carboplatin þ docetaxel
Diaziquone/AZQ
VO-OHpic
MLN4924
ROS
ROS
DNA damage
DNA damage
þ
þ
þ/
ND
DNA damage
DNA damage
PTEN
Cul1 SCF subunit
inhibitor
Aurora kinase A
inhibitor
KIF11
DNA damage
DNA damage
HDAC inhibitor
DNA damage
þ/
þ/
þ
þ/
MLN8054
K858
Irotecan
Etoposide
MS-275
Cetuximab þ radiation þ EGFR
inhibitor
Pemetrexed
GSK690693
Everolimus
Folate
antimetabolite
AKT inhibitor
mTOR inhibitor
þ
þ/
þ
þ/
þ/
þ/
þ/
þ/
þ
Abbreviations: mTOR, mechanistic target of rapamycin; ND, not determined; ROS, reactive oxygen species; WT, wild type. ‘p53 status’ indicates
the p53 form expressed in cells in which the studies have been conducted:
‘ þ ’ denotes p53 WT-expressing cells, ‘ ‘’ denotes p53 null- or mutantexpressing cells, ‘ þ / ’ denotes that studies have been conducted in both
p53 WT- and null-/mutant-expressing cells.
R172P expression.218 Furthermore, the DDR activated by telomere
dysfunction induces the ATM/ATR and Chk1/Chk2 activation,
which consequently phosphorylates and stabilizes p53.29
Evidence that deletion of key regulators of senescence, for
example, p53, p27, PRAK or Arf, induces tumor progression and
senescence block, connect the loss of senescence to tumor
transformation.82,219–222 Eighty to ninety percentage of human
cancers seem to be associated with unlimited proliferation due to
activation of telomerase.223,224 Therefore, the inhibition of
telomerase could be a promising therapeutic target for cancer,
because telomere shortening induces senescence in cancer cells21
and this kind of approach could offer the additional advantage to
specifically target cancer cells, characterized by telomerase
expression, unlike normal cells.
A FAMILY MATTER: p63 AND p73 IN SENESCENCE AND AGING
Two others p53 homologs of p53 have been characterized over
the past two decades: p63 and p73.225 Like p53, both proteins
contain three domains: a N-terminal transactivation domain, a
DNA-binding domain and an oligomerization domain responsible
for tetramerization. In addition, the use of different promoters and
alternative splicing results in the expression of multiple isoforms.
Briefly, alternative splicing at the 30 -end of the primary transcript
originates three isoforms in p63 and at least seven in p73, some of
which contain an additional C-terminal protein/protein interaction
domain, the sterile alpha motif, absent in p53.225 Although several
studies have attempted to dissect specific functions of these
proteins, at present clear-cut roles for these different variants have
not been attributed. As mentioned, the use of alternative
promoters generates two additional N-terminal variants. An
upstream promoter transcribed longer, transcriptionally
competent isoforms containing a transactivation domain (TAp63
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Mechanisms of p53-mediated regulation
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and TAp73), whereas an internal downstream promoter originates
shorter isoforms that lack the transactivation domain (DNp63 and
DNp73) and are thought to act as dominant negatives.226
Both TAp63 and TAp73 are activated in response to DNA
damage by the non-receptor tyrosine kinase c-Abl227–232 and act
as proapoptotic molecules.233,234 Although they are involved in
cancer and chemotherapy response,226,235 p73 has a major role in
regulation of inflammation236,237 and brain development through
several mechanisms,238,239 including preservation of neural stem
cells240–242 and, importantly, its depletion predisposes to agerelated neurodegeneration in mouse models.243–245 In addition,
TAp73 has been involved in the preservation of genomic stability
and fertility, and is important for accurate mitotic and meiotic
division.64,246–248 p63’s role in cancer onset and metastatic spread
has been widely investigated,249–251 but it also has a fundamental
role in epithelial development and maintenance of the epithelial
stem cell reservoir252–258 and protects the female germ line
against DNA damage.259 Nonetheless, like their sibling p53, both
genes have a role in senescence and aging. Indeed, the recent
development of several N-terminal selective-knockout (KO) mouse
models has helped understand the involvement of these isoforms
in complex biological processes, such as senescence and aging.
p63 null mice (that is, lacking all isoforms) show a very severe
phenotype and die shortly after birth,260,261 although long-term
analysis of heterozygous mice allowed detection of premature
aging.262 These findings were strengthened by the development
of inducible TAp63-KO mice.263 In this setting, depletion of p63 in
the epithelial compartment was sufficient to accelerate aging,
which correlated with accumulation of senescence markers in vivo
and in isolated keratinocytes. In particular, the establishment of
the senescent status relies on PML, a known mediator of
senescence.263–265 Further insights into p63 regulation of the
aging process were provided by development of TAp63 isoformspecific KO. Flores and colleagues showed that absence of TAp63
has severe effects and results in skin ulceration, premature aging
and reduced lifespan. Interestingly, this correlates with strong
cellular senescence triggered by genomic instability, which is
responsible for the loss of the epithelial stem cell repertoire.266,267
Although these findings suggest that lack of p63 induces
senescence, other reports have shown that TAp63 mediates the
induction of OIS in keratinocytes, similarly and independently of
p53.268 Hence, it appears that p63, especially its transcriptionally
competent isoforms, share a Janus role with p53: their activation
in response to oncogenic stress is necessary to halt transformation
via senescence, but their absence compromises the stem cell
reservoir and promotes aging. Nonetheless, to fully address the
role of p63 in senescence and aging, it is necessary to take into
account the activity of the N-terminal truncated proteins. Indeed
DNp63 isoforms are by far the most abundant isoforms expressed
in epithelial cells. They have been consistently reported to support
the maintenance of the stem cell compartment of the skin and to
antagonize the induction of replicative senescence.255,269
Moreover, DNp63 downregulation by oncogenic K-Ras is
necessary for establishment of OIS and tumor prevention in
keratinocytes.270 Unfortunately, it is still unclear to what extent
these isoforms affect aging, and data on isoform-specific KO are
eagerly awaited.
p73 has been linked to OIS and in fact expression of DNp73 has
been reported to bypass Ras-induced senescence allowing cellular
transformation to occur.271 Moreover, oncogenic Ras promotes a
switch from TAp73 to DNp73 expression to sustain transformation.
Indeed, transformed mouse fibroblasts, lacking DNp73, fails to
form tumors in nude mice because of ensuing senescence.244
Recently, by mean of isoform-specific KO models,247 we have
demonstrated that mice lacking TAp73 show an aggravation of
several aging-related parameters (hunchback, cataract, alopecia
and skin thinning among others).34 In addition, fibroblasts isolated
from null embryos were more susceptible to oxidative stress and
Oncogene (2013), 1 – 15
underwent senescence at higher rate than wild-type counterparts.
In the quest for a mechanism underlining the observed
phenotypes, we showed that TAp73 regulates expression of a
cytochrome c oxidase 4 subunit 1 (Cox4i1), a protein essential for
assembly of fully functional mitochondrial complex 4.
Consequently, lack of TAp73 impaired activity of the complex 4
of the electron transport chain and decreased oxygen
consumption both in vitro and in vivo, similarly to depletion of
p53. This resulted in reduced ATP cellular content and increased
cellular ROS and oxygen sensitivity. Hence, we proposed that the
mitochondrial and metabolic dysfunction triggered by depletion
of TAp73 underlined the accelerated aging in KO animals.34
In summary, regulation of senescence and aging are shared
functions of the p53-family members.
CONCLUDING REMARKS
The molecular mechanisms underlying the senescence pathway
are becoming increasingly topical owing to its role in tumor
suppression, giving great relevance for its potential exploitation in
cancer therapy. Although these molecular mechanisms are only
partially elucidated and are currently under intense investigation,
it is evident that p53 has a key role in its regulation. Indeed, p53
can modulate senescence at different levels. Surprisingly, p53
seems to show a dual effect, promoting or in same case inhibiting
the senescence program. This dual effect of p53 is still unclear; a
possible explanation can be the dependence on the degree and
type of stress or the cellular milieu where p53 is active. Indeed,
mild stress can induce p53 to repair the cell and activate
antioxidant mechanisms, while more severe stress leads p53 to
induce apoptosis and senescence, via ROS generation. In the
context of cancer therapy, the ability of p53 to regulate
senescence is emerging as a promising and alternative way to
eliminate cancerous cells, because p53-signaling pathways can be
manipulated at several steps to stimulate senescence. Beyond its
antitumor ability, senescence is emerging as a casual factor in
aging.11 Presently, while it is clear that modulation of p53 activity
affects lifespan, the contribution of senescence to this function
needs further investigation. In a recent mouse model of
hyperactive p53 and consequent aggravation of aging disorders,
the concomitant removal of the proapoptotic p53 target PUMA
suffices to rescue the stem cell repertoire and increase the lifespan
of mutant mice. This suggests that apoptosis may be critical to
p53-induced aging. But, on the other hand, it does not exclude an
additional role for senescence. In particular, apoptosis can explain
the defect in tissues subject to extended self-renewal (for
example, bone marrow or gut), but it is unclear whether it could
explain the aging-related degeneration that accompanies
quiescent tissues (such as liver or brain).272 In this context,
senescence may have a more critical role, which would be worth
investigating.
Mechanistically, we have proposed several mechanisms that are
at the crossroads between senescence and aging: ROS scavenging
and generation, mitochondrial function, mTOR signaling and
autophagy. Importantly, these biological processes are intimately
linked: mTOR positively regulates respiration in mammalian
cells169,273 and renders cancer cells addicted to mitochondrial
activity.274 Moreover, it inhibits autophagy, and autophagy of
mitochondria (a process known as mitophagy) is essential to
remove dysfunctional mitochondria, whose accumulation would
result in increased ROS production and reduced energy efficiency.
Activation of autophagy depends on ROS, while autophagy
dampens intracellular ROS build-up.38,275–277 Intriguingly, SIRT1mediated deacetylation of PGC-1a is required for its activation of
mitochondrial biogenesis and gluconeogenesis,278 thus
antagonizing p53-negative regulation of PGC-1a. In other words,
p53 is controlling a network of tightly connected biological
processes that impinge on senescence and aging. Future efforts
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9
Figure 4. The unified theory of aging and the p53 family. Recent findings suggest that p53 regulates mitochondrial function in both stress and
unstressed conditions. In unstressed cells, it regulates expression of synthesis of cytochrome c oxidase 2 (SCO2) and help sustaining and
oxidative energy production. In response to stress, such as telomere erosion, p53 is activated in response to DNA damage and directly repress
the expression of PGC1a/b protein, causing reduction in mitochondrial mass and function and increasing ROS content. Similar results were
described for p73: deletion of the TA isoforms worsen aging in mice and results in increased cellular ROS levels, oxidative damage and
senescence triggered by mitochondrial dysfunction. In this case, the target responsible, at least partially, for the described phenotype is
cytochrome c oxidase 4 subunit 1 Cox4i1. Unfortunately, data are still missing with regards to p63 and it is not clear whether, upon stress,
activated p63 and p73 are able to regulate mitochondrial function similarly to p53. Further studies aim to investigate these possibilities are
eagerly awaited.
are necessary to fully address how p53 regulates these processes,
how they interrelate in the context of p53 regulation and their
overall relevance to cancer and aging.
Finally, a growing body of evidence points to mitochondria as
crucial factors in aging and senescence. Indeed, dysfunctional
mitochondrial function accompanies OIS, and several mouse
model of dysfunctional mitochondria show worsening of the
aging phenotype.168,279–285 Strikingly, overexpression of the
scavenging enzyme catalase exerts negligible effects on
longevity if targeted to the peroxisomes or the nuclei, but
extends median lifespan by 20% when targeted to the
mitochondria,286 and a recent study demonstrated that this
genetic manipulation prevents the age-associated decline in
mitochondrial activity.281 Our recent data on TAp73-selective KO
and the findings of Ronald DePinho’ s Laboratory on the p53PGC1a axis both support a model whereby depletion or
hyperactivation of the p53 family members leads to aging
through impaired mitochondrial function and metabolic
compromise (Figure 4).272 These findings also explain the aging
decline of quiescent organs (such as liver) that do not depend on
a strong stem cell pool (such as the haematopoietic system or
skin) and thereby are less susceptible to apoptosis mediated by
DNA damage and p53 activation. Future studies to thoroughly
address the mitochondrial function and metabolic profile of longlived mouse models such as super-arf/p53 or short-lived p53knock-in mice are desirable. Similarly, these recent studies urge
immediate work to investigate whether p63 shares with its
siblings the ability to regulate oxidative metabolism, making it
definitively a family matter. Intriguingly, a recent paper
demonstrated that TAp63 upregulates expression of SIRT1 and
AMPKa2 (one of the subunit of AMPK). Although the authors do
& 2013 Macmillan Publishers Limited
not investigate mitochondrial function in detail, they demonstrate
that TAp63 null mice have reduced activity of SIRT1 and AMPK
in vivo, which renders these animals extremely susceptible to
obesity and insulin resistance following high-fat diet regimen.287
Although further studies are necessary, it is becoming increasingly
evident that regulation of energetic metabolism is a main function
of the p53 family of genes with essential consequences on animal
lifespan and well-being.
ABBREVIATIONS
DDR, DNA damage response; ROS, reactive oxygen species.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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