Anti-aging activity of the Ink4/Arf locus

Aging Cell

(2009)

8

, pp152–161 Doi: 10.1111/j.1474-9726.2009.00458.x

152

© 2009 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009

Blackwell Publishing Ltd

Anti-aging activity of the

Ink4/Arf

locus

Ander Matheu,

1,

† Antonio Maraver,

1

Manuel Collado,

1

Isabel Garcia-Cao,

1

Marta Cañamero,

2

Consuelo Borras,

3

Juana M. Flores,

4

Peter Klatt,

1

Jose Viña

3

and Manuel Serrano

1

1

Tumor Suppression Group

2

Comparative Pathology Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

3

Department of Physiology, University of Valencia, Valencia, Spain

4

Department of Animal Surgery and Medicine, Complutense University, Madrid, Spain

Summary

The proteins encoded by the

Ink4/Arf

locus, p16

Ink4a

, p19

Arf

and p15

Ink4b

are major tumour suppressors that opposeaberrant mitogenic signals. The expression levels of thelocus are progressively increased during aging andgenome-wide association studies have linked the locusto a number of aging-associated diseases and frailty inhumans. However, direct measurement of the global

impact of the

Ink4/Arf

locus on organismal aging andlongevity was lacking. In this work, we have examinedthe fertility, cancer susceptibility, aging and longevity ofmice genetically modified to carry one (

Ink4/Arf

-tg) ortwo (

Ink4/Arf

-tg/tg) intact additional copies of the locus.First, increased gene dosage of

Ink4/Arf

impairs the pro-duction of male germ cells, and in the case of

Ink4/Arf

-tg/tg mice results in a Sertoli cell-only-like syndrome anda complete absence of sperm. Regarding cancer, thereis a lower incidence of aging-associated cancer proportionalto the

Ink4/Arf

gene dosage. Interestingly, increased

Ink4/Arf

gene dosage resulted in lower scores in aging markersand in extended median longevity. The increased survivalwas also observed in cancer-free mice indicating that cancerprotection and delayed aging are separable activities of the

Ink4/Arf

locus. In contrast to these results, mice carryingone or two additional copies of the

p53

gene (

p53

-tg and

p53

-tg/tg) had a normal longevity despite their increasedcancer protection. We conclude that the

Ink4/Arf

locushas a global anti-aging effect, probably by favouringquiescence and preventing unnecessary proliferation.

Key words: anti-aging, antioxidant, ARF, cellular senescence,DNA damage, Ink4a, life-span studies, cancer, aging, tumorsuppression, p16Ink4a, p53

Introduction

The

Ink4a/Arf/Ink4b

locus, hereby abbreviated as

Ink4/Arf

,

encodes two members of the Ink4 family of cyclin-dependent

kinase inhibitors, p16

Ink4a

and p15

Ink4b

, and a completely

unrelated protein called p19

Arf

(Gil & Peters, 2006; Kim & Sharpless,

2006). The locus is among the most frequently inactivated loci

in human cancer, which reflects its central role in preventing

cancer development. The three products of the locus participate

in key cellular anti-proliferative responses, namely, senescence

and apoptosis. Proteins p16

Ink4a

and p15

Ink4b

inhibit the activity

of the Cdk4,6/cycD kinases promoting Rb-mediated proliferative

arrest; meanwhile, p19

Arf

inhibits the ubiquitin ligase Mdm2,

thus promoting p53 stabilization (Gil & Peters, 2006; Kim &

Sharpless, 2006). Current evidence indicates that mitogenic

over-stimulation of primary normal cells

in vitro

results in

upregulation of the locus and thereby decreased mitogenic

response and, eventually, upon maximal activation of the

locus, may result in apoptosis or senescence (Collado

et

al

.,

2007). At the organismal level, the

Ink4/Arf

locus is silent during

development and post-natal life, becoming progressively

expressed from adulthood until very old ages (Zindy

et

al

., 1997;

Krishnamurthy

et

al

., 2004; Ressler

et

al

., 2006).

Importantly, numerous independent genome-wide association

studies using large human cohorts have linked polymorphisms

in close vicinity to the

Ink4/Arf

locus with aging-associated

frailty and with a variety of aging-associated diseases, such as

myocardial infarction, type 2 diabetes and stroke [reviewed in

Sharpless & DePinho (2007), Melzer (2008); see also Matarin

et

al

. (2008)]. However, the functional significance of these

polymorphisms remains to be elucidated. A number of recent

studies have implicated p16

Ink4a

as an important negative regulator

of tissue regeneration upon acute damage in the haematopoietic

system, endocrine pancreas, skeletal muscle and fat (Janzen

et

al

., 2006; Krishnamurthy

et

al

., 2006; Molofsky

et

al

., 2006;

Baker

et al

., 2008). It should be borne in mind that these experi-

mental systems probably involve acute mitogenic stimulation

and high levels of induction of p16

Ink4a

, and this may not be

predictive of the long-term impact of moderately increased

p16

Ink4a

on physiological regeneration. On the other hand,

studies from our laboratory and others, have implicated an

anti-aging function of p19

Arf

through its ability to reinforce

the activity of p53 thus favouring the elimination of damaged

cells (Matheu

et

al

., 2007; Baker

et

al

., 2008; Matheu

et

al

.,

2008). In this context, we consider of high relevance to

determine in a direct manner and under normal physiological

Correspondence

Manuel Serrano, Spanish National Cancer Research Centre (CNIO), 3 Melchor

Fernandez Almagro street, Madrid E-28029, Spain. Tel.: +34 91 732 8000;

fax: +34 91 732 8028; e-mail: [email protected]

†Present address: Division of Stem Cell Biology and Developmental Genetics,

MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

Accepted for publication

9 January, 2009

Anti-aging activity of the

Ink4/Arf

locus, A. Matheu

et al.


153

conditions the global impact of the

Ink4/Arf

locus on aging

and longevity.

Previous work from our laboratory has shown that a transgenic

allele of the entire locus,

Ink4a/Arf/Ink4b

, behaves similarly to

the endogenous allele (Matheu

et

al

., 2004). In particular, the

transgenic

Ink4/Arf

allele is minimally expressed in primary

embryonic cells but responds to mitogenic over-stimulation and

protects against neoplastic transformation by oncogenes. At

the organismal level, the transgenic allele results in a modest

increase (approximately 50%) in the expression levels of the

three genes,

Ink4a

,

Arf

and

Ink4b

, compared to the wild-type

situation, yet this translates into a significantly higher resistance

to chemically induced cancer, as well as, to aging-associated

spontaneous cancer (Matheu

et

al

., 2004; Matheu

et

al

., 2007).

Therefore, we regard this transgene as a suitable tool to evaluate

the effects of a modest and normally regulated increase in the

function of the locus. In the present work, we report the fertility,

cancer susceptibility, aging and longevity of mice carrying one

or two transgenic copies of the entire

Ink4a/Arf/Ink4b

locus.

Results

Male sterility in

Ink4/Arf

-tg/tg mice

Intercrosses between

Ink4/Arf

-tg mice (Matheu

et

al

., 2004)

were performed to generate the three genotypes of relevance

for this study, namely,

Ink4/Arf

-wt,

Ink4/Arf

-tg and

Ink4/Arf

-tg/

tg, all of them in a pure (> 99%) C57BL6 genetic background.

Therefore, the mice under study here can be considered to carry,

respectively, 2 (wt), 3 (tg) or 4 (tg/tg) gene doses of

Ink4a, Arf

and

Ink4b

. We have previously shown that

Ink4/Arf

-tg mice

have moderately increased levels of expression of

Ink4a

,

Arf

and

Ink4b

in a number of tissues at old ages (Matheu

et

al

., 2004).

In turn, as expected, doubly transgenic old

Ink4/Arf

-tg/tg mice

had even higher levels of expression of

Ink4a

and

Arf

in spleen

and liver compared to singly transgenic

Ink4/Arf

-tg mice (Sup-

plementary Fig. S1). The first noticeable phenotype of the

Ink4/Arf

-tg/tg mice was that the males were infertile, whereas the

females had no obvious fertility defects (Fig. 1A). We initially

considered the possibility that the insertion of the transgene into

the genome could have inactivated a gene specifically involved

in male fertility. To examine this, we generated mice homozygous

for the transgenic allele but lacking the endogenous alleles, i.e.

Ink4/Arf

-KO;tg/tg. Interestingly,

Ink4/Arf

-KO;tg/tg mice of both

sexes were fertile and yielded the same number of pups per

litter as wt mice (Fig. 1A). These results exclude the possibility

of an insertional mutagenic event responsible for the male

infertility and suggest a direct effect of the

Ink4/Arf

dosage on

male fertility. In support of the latter scenario, we observed a

reverse correlation between

Ink4/Arf

gene dosage and, both,

testis size and sperm count. In the case of

Ink4/Arf

-tg/tg mice,

their average testis weight was 18% compared to wt mice

(100%), and were totally devoided of spermatozoa (Fig. 1B

and C; see also below Fig. 2).

Ink4/Arf

-tg mice had intermediate

deficits in testis weight and sperm count compared to doubly

transgenic mice (Fig. 1B and C). These results point to a direct

impact of the

Ink4/Arf

gene on male fertility.

Histological analyses of testes from 2-month-old

Ink4/Arf

-tg/

tg mice revealed severe abnormalities in the testes. In particular,

atrophic seminiferous tubules lacking germ cells (Fig. 2). In

normal testes, the proliferative marker Ki67 is characteristically

present in spermatogonia, however, Ink4/Arf-tg/tg testes were

completely negative for Ki67, further confirming the absence

of germ cells in these mice (Fig. 2). In the case of singly transgenic

Ink4/Arf-tg mice, Ki67-positive cells were present albeit at lower

levels than in wt testes (Fig. 2). Importantly, spermatogonia were

clearly positive for p16Ink4a and p19Arf by immunohistochemistry

both in wt and in Ink4/Arf-tg testes, while Sertoli cells were

negative (Fig. 2). (Note that the p16 staining gives a non-specific

signal in the interstitial Leydig cells). Consistent with the fact

that Ink4/Arf-tg/tg testes are devoided of germ cells, these testes

also lacked p16Ink4a- or p19Arf-positive cells (Fig. 2). Finally, we used

Sox9, a well-established marker of Sertoli cells (Sekido & Lovell-

Badge, 2008), to determine the impact of the Ink4/Arf trans-

gene on this cell type. As shown in Fig. 2, all tubules in wt testes

showed the characteristic pattern of Sertoli cells interspersed with

spermatogonia; singly transgenic Ink4/Arf-tg testes had a mixture

of normal tubules together with others exclusively composed

by Sertoli cells; and, finally, more dramatically, the only cells present

in Ink4/Arf-tg/tg testes were Sox9-positive thus resembling the

so-called ‘Sertoli-cell-only syndrome’ (Fig. 2). It is important to

mention that similar phenotypes have been also observed in

Fig. 1 Male infertility in Ink4a/Arf-tg/tg mice. (A) The average litter size for crosses (n ≥ 5) between mice of the indicated sex and genotype. In the case of male Ink4/Arf-tg/tg mice, no pregnancies were obtained from a total of nine males. (B) Representative image of testis from mice (8-weeks-old) of the indicated genotypes (left). Weight of testes (n = 3 per genotype) (right). (C) Sperm count per epididymis from mice (8-week-old) of the indicated genotypes (n = 3 per genotype). Data are mean values ± SEM. Student’s t-test relative to wt: *P < 0.05; **P < 0.01; ***P < 0.001.

Anti-aging activity of the Ink4/Arf locus, A. Matheu et al.


154

mice with genetic alterations that upregulate the retinoblastoma

(Rb) tumour suppressor pathway (see Discussion).

Increased cancer resistance in Ink4/Arf-tg/tg mice

We have reported that a single transgenic copy of the Ink4/Arflocus confers significant cancer protection to Ink4/Arf-tg mice

compared to their wt counterparts (Matheu et al., 2004). We

wondered whether cancer protection by the Ink4/Arf locus

could be further increased in doubly transgenic Ink4/Arf-tg/tg

mice. Cohorts of wt, tg and tg/tg mice were observed during

their complete lifespan, sacrificed when moribund at old age,

and subjected to detailed necropsy and histopathological analysis.

Notably, almost half (48%) of the wt mice presented spontaneous

malignant tumours, and this incidence was decreased to 38%

in Ink4/Arf-tg mice (Fig. 3A). Remarkably, the presence of two

transgenic copies resulted in potent cancer protection decreasing

the incidence of aging-associated cancer to 10% in Ink4/Arf-tg/tg mice (Fig. 3A). These results demonstrate a correlation

between the gene dose of Ink4/Arf and resistance to aging-

associated cancer. Of relevance, these observations also indicate

that the transgenic locus is not subject to aging-associated

epigenetic silencing and that both transgenic copies remain active

at advanced ages yielding the expected phenotype of increased

cancer protection.

Increased longevity in Ink4/Arf-tg/tg mice

As previously described (Matheu et al., 2004), Ink4/Arf-tg mice

had a normal lifespan, not significantly different from wt mice

(log rank test, P = 0.09) (Fig. 3B). Remarkably, however, Ink4/Arf-tg/tg mice presented a significantly different survival curve

(logrank test, P = 0.001) with a more square shape. Median

survival was extended from 27.5 months to 31.7 months, i.e. an

extension of 15% (see detailed data in Supplementary Table S1).

To dissociate the possible contribution of decreased spontaneous

cancer to the extended lifespan, we eliminated from the previous

graph those mice that presented malignant tumours at their

time of death, thus focusing on the longevity of cancer-free

mice (Fig. 3C). Importantly, cancer-free Ink4/Arf-tg/tg mice still

Fig. 2 Depletion of spermatogonia in Ink4/Arf-tg/tg mice. Representative images of testis from mice (4- to 8-weeks-old, n = 5 per genotype) of the indicated genotypes analysed by immunohistochemistry to detect the indicated proteins. Note that the p16 staining gives a non-specific signal in the interstitial Leydig cells. In the tubules, germ cells are positive for p16Ink4a and p19Arf, whereas Sertoli cells are negative. Tubules from Ink4/Arf-tg/tg mice lack germ cells and therefore are negative for p16Ink4a and p19Arf; these tubules only contain Sertoli cells as indicated by the Sertoli cell marker Sox9.



155

presented a clear increase in median lifespan compared to the

corresponding wt mice (Fig. 3C and Supplementary Table S1).

This suggests that the Ink4/Arf transgene has an impact on

longevity that cannot be attributed to its cancer protection

activity. Of note, similar to Ink4/Arf/p53-tg mice (Matheu et al.,2007), Ink4/Arf-tg/tg mice showed no extension in the maximum

lifespan (Fig. 3B), suggesting the existence of aging-inducing

mechanisms that are not mitigated by the Ink4/Arf locus.

Together with cancer, kidney lesions, mostly consisting in

glomerulonephritis with chronic interstitial nephritis, constitute

the most frequent pathology in aged C57BL6 mice affecting

more than 50% of the wt mice (Hayashi et al., 1988; Lipman

et al., 1998; Matheu et al., 2007). Interestingly, old Ink4/Arf-tg/tg

showed a significantly lower incidence of kidney lesions com-

pared to wt mice (Fig. 3D).

Finally, to evaluate whether the longevity effect of the Ink4/Arf locus was exclusive of this tumour suppressor or was a

general property associated to anti-cancer genes, we performed

a similar longevity analysis in mice with increased gene dosage

of p53. Before, we had reported that p53-tg mice, carrying one

additional transgenic copy of p53, are significantly protected

from cancer compared to wt mice (Garcia-Cao et al., 2002).

Interestingly, a single p53-tg allele had no impact on normal

aging or on aging driven by short telomeres (Garcia-Cao et al.,2002; Garcia-Cao et al., 2006), but in combination with the

Ink4/Arf transgenic allele (Ink4/Arf/p53-tg mice) it resulted in an

increased median longevity (Matheu et al., 2007). Considering

that the longevity effect that we are reporting here is only clearly

noticeable in doubly transgenic Ink4/Arf-tg/tg, we considered

of importance to perform a longevity study in an equivalent

Fig. 3 Enhanced cancer protection and extended longevity in Ink4/Arf-tg/tg mice. (A) Spontaneous cancer incidence in old mice of the indicated genotypes. Mice were sacrificed when they showed overt signs of poor health, such as reduced activity or dramatic weight loss, and analysed for malignant lesions by histology. Statistical significance vs. wt mice was calculated using the Fisher’s Exact test: #P < 0.1; ***P < 0.001. (B) Survival curves of cohorts of mice with the indicated Ink4/Arf genotype. Statistical significance of the Kaplan-Meier curves was assessed vs. wt mice using the logrank test: tg mice, P = 0.1; tg/tg mice, P = 0.001. (C) Cancer-free survival curves. The survival data used for the curves in (B) were filtered to exclude those animals that presented malignant tumours at the time of death. Logrank test vs. wt mice: tg mice, P = 0.1; tg/tg mice, P = 0.05. (D) Incidence of kidney lesions in old moribund mice. Most kidney lesions correspond to glomerulonephritis with chronic interstitial nephritis. Statistical analysis was done as in (A). For the wt cohort: a total of 111 mice were scored for survival, 95 of them were analysed by necropsy and histopathology, 49 were free of tumours and the rest, 46, had tumours at the time of death. For the tg cohort: a total of 54 mice were scored for survival, 40 were analysed, 25 were free of tumours and 15 had tumours at the time of death. For the tg/tg cohort: a total of 25 mice were scored, 19 were analysed, 17 were free of tumours and 2 presented tumours. (E) Survival curves of cohorts of mice with the indicated p53 genotype (P > 0.5 in all comparisons).



156

cohort of doubly transgenic p53-tg/tg mice (as before, all cohorts

are in a pure C57BL6 genetic background). Interestingly,

p53-tg/tg mice had the same longevity profile as p53-tg and

wt mice (Fig. 3E). It should be noted that these results do not

negate an anti-aging effect of p53, but only indicate that

increasing p53 by itself, as in p53-tg/tg mice, is not sufficient

to impact longevity. In summary, we conclude that the Ink4/Arf locus possesses an intrinsic capacity to increase longevity

that is independent of its cancer protection activity.

Delayed aging in Ink4/Arf-tg/tg mice

We wondered whether the increased median longevity could

be correlated with a delayed onset of aging. For this, we

examined various biomarkers of aging in old mice of the three

relevant genotypes. In particular, we examined aging markers

previously validated by others and by us (Sedelnikova et al.,2004; Herbig et al., 2006; Matheu et al., 2007), such as the

neuromuscular coordination assay, hair regrowth, and DNA

damage signalling in the liver, the latter measured as the

percentage of cells positive for γH2AX or 53BP1 (Fig. 4A–D). In

all these assays, old Ink4/Arf-tg/tg performed better than

wt mice; and in the case of hair regeneration and DNA

damage accumulation, Ink4/Arf-tg/tg mice also performed

better than singly transgenic mice (Fig. 4A–D). Together, these

observations suggest that the Ink4/Arf locus has the capacity

to delay aging.

Antioxidant defences in Ink4/Arf-tg/tg mice

Recent evidence has demonstrated that p53, upon exposure

to low intensity stress, such as it might happen during aging,

moderately activates anti-oxidant genes, most prominently

sestrins Sesn1 and Sesn2 (Sablina et al., 2005). Previously, we have

reported that Ink4/Arf/p53-tg mice have higher hepatic

expression of Sesn1 and Sesn2, and we have also shown that

the livers from these mice have a higher global anti-oxidant

potential as reflected by their higher levels of reduced-glutathione

(GSH) (Matheu et al., 2007). Importantly, we recapitulated the

same observations in old (≥ 24 months) Ink4/Arf-tg/tg mice

both regarding the expression of Sesn1 and Sesn2 (Fig. 5A), as

well as, with regard to GSH levels (Fig. 5B). Also, the levels of

reactive oxygen species in the spleens of Ink4/Arf-tg/tg mice

were lower than in Ink4/Arf-tg mice (Supplementary Fig. S2).

Together, these findings suggest that Ink4/Arf-tg/tg mice are

protected against oxidative damage through an increased

expression of anti-oxidant genes probably mediated by p53.

Discussion

Here, we have addressed the global impact of the entire Ink4a/Arf/Ink4b locus (abbreviated as Ink4/Arf) on fertility, cancer,

aging and longevity.

The Ink4/Arf locus is strongly upregulated during human and

mouse aging (Zindy et al., 1997; Krishnamurthy et al., 2004;

Fig. 4 Biomarkers of aging in Ink4/Arf-tg/tg mice. (A) Neuromuscular coordination assay, also known as tightrope test, of old (> 20 months) mice (wt, n = 23; tg, n = 26; tg/tg, n = 12) of the indicated genotypes. Statistical significance vs. wt mice was calculated using the Fisher’s Exact test: *P < 0.05. (B) Hair re-growth capacity of aged (≥ 24 months) mice (wt male, n = 5; wt female, n = 6; tg male, n = 3; tg female, n = 7; tg/tg male, n = 4; tg/tg female, n = 4). Hair regrowth was scored 15 days after plucking as explained in Methods. Statistical significance vs. wt mice was calculated using the Student’s t-test: #P = 0.06). (C and D) Aging-associated accumulation of γH2AX-positive cells (C) and 53BP1-positive cells (D) in the liver. Cryosections from aged (≥ 24 months) mice (n = 3–4 per genotype) were analysed for the percentage of γH2AX positive nuclei by confocal microscopy. Data are mean values ± SEM, Student’s t-test is relative to wt: ***P < 0.001.



157

Ressler et al., 2006), and it has been genetically linked to numerous

aging-associated human diseases including general organismal

frailty [reviewed in Sharpless & DePinho (2007), Melzer (2008);

see also Matarin et al. (2008)]. However, these observations do

not address whether the expression of the Ink4/Arf locus pro-

motes or protects from aging. Work with genetically modified

mice has generated a wealth of valuable information on the role

of the Ink4/Arf locus in cancer and stem cell biology, but has

not directly examined the impact of the locus on aging. It is

interesting to note that the effects of a transgenic allele con-

stitutively overexpressing p16Ink4a (from 2- to 20-fold depending

on the tissue), were minimal or absent in the pancreatic islets

and in the neural stem cells of old mice (Krishnamurthy et al.,2006; Molofsky et al., 2006). Regarding p19Arf, we and others

have concluded that it has an anti-aging effect (Matheu et al.,2007; Baker et al., 2008). Considering these precedents, we

regard of relevance to study directly the global impact of the

Ink4/Arf locus on aging and longevity.

The first obvious phenotype that we have noticed in the

Ink4/Arf-tg/tg mice is the complete sterility of the males. This

prompted us to analyze the testis and sperm production of these

mice, observing a clear negative impact of the Ink4/Arf-tg allele

that results in progressively smaller testes and sperm counts in

singly transgenic and doubly transgenic mice compared to wt

mice. Histologically, Ink4/Arf-tg/tg mice were completely

devoided of spermatogonia and only presented Sertoli cells in

the testicular tubules. A number of evidences point to p16Ink4a

and p15Ink4b, but not p19Arf, as responsible for this phenotype.

First, male sterility has not been reported for a variety of genetic

modifications that upregulate p53 (Garcia-Cao et al., 2002;

Tyner et al., 2002; Matheu et al., 2004; Mendrysa et al.,2006; Matheu et al., 2007), although there is one exception

(Maier et al., 2004). In contrast, impaired spermatogenesis has

been reported for numerous mice carrying genetic modifications

that result in inhibition of the Cdk4/Cdk2/E2f1 pathway, which

is a downstream target of the Ink4 proteins through inhibition

of Cdk4 (Gil & Peters, 2006; Kim & Sharpless, 2006). In particular,

severe or complete absence of spermatogonia has been reported

for Cdk4-null mice (Rane et al., 1999; Tsutsui et al., 1999; Mettus

& Rane, 2003), Cdk2-null mice (Berthet et al., 2003; Ortega

et al., 2003), Skp2-null mice (due to their abnormally high levels

of the Cdk inhibitor p27Kip2) (Fotovati et al., 2006), and E2f1-

null mice (Field et al., 1996; Yamasaki et al., 1996). Together,

we conclude that male germ cells are particularly sensitive to

the inhibition of the cell cycle by the Ink4/Arf locus, probably

due to the effect of the p16Ink4a and p15Ink4b proteins, rather

than to the effect of p19Arf.

Regarding cancer susceptibility, as anticipated from our

previous data with singly transgenic Ink4/Arf-tg mice (Matheu

et al., 2004; Matheu et al., 2007), doubly transgenic mice

were even less susceptible to aging-associated cancer, which

corroborates the well-established cancer protection activity of

the Ink4/Arf locus. The most interesting data came from the

longevity curves of these mice because there was a clear and

statistically significant increase in median longevity (+15%). The

survival curve of the Ink4/Arf-tg/tg mice had a more squared

shape than in the case of wt mice (Fig. 3B). For example, at

2 years of age, approximately 25% of the wt mice have died

whereas none of the Ink4/Arf-tg/tg mice has died at this age;

also, despite retaining the same maximal lifespan, only 5% of

wt mice reached 33 months of age, whereas 40% of the Ink4/Arf-tg/tg mice were alive at this age (Fig. 3B). The lack of impact

on maximum lifespan is suggestive of additional aging processes

that are not mitigated by the Ink4/Arf locus, one of them likely

being telomere shortening (Tomas-Loba et al., 2008).

At a first approximation, the increased longevity of the Ink4/Arf transgenic mice could be due to a lower incidence of cancer.

However, this explanation was not supported by the longevity

curves of the cancer-free mice, which still presented a clear

increase in longevity. Moreover, despite the potent effect of the

transgenic p53 allele on cancer protection (Garcia-Cao et al.,2002; Matheu et al., 2007; Tomas-Loba et al., 2008), mice p53-

tg and p53-tg/tg had longevity curves indistinguishable from the

control wt cohort, further implying that death by cancer has a

small contribution to longevity in C57BL6 laboratory mice. This

observation does not negate a role of p53 in aging, but it

reinforces the idea that the Ink4/Arf locus possesses a potent

anti-aging activity that is separable from its anti-cancer effect.

Fig. 5 Increased anti-oxidant protection in Ink4/Arf-tg/tg mice. (A) Expression of antioxidant genes in vivo. Liver samples from aged (≥ 24 months) mice (n = 3 per genotype) were analysed for the expression of the antioxidant genes sestrin 1 (Sesn1) and sestrin 2 (Sesn2) by real-time quantitative RT-PCR. PCR data were normalized to β-actin expression and are expressed relative to gene expression levels in wt tissues. (B) Levels of reduced glutathione (GSH) in the liver of aged (≥ 24 months) mice (wt, n = 8; tg, n = 7; tg/tg, n = 6). GSH was measured by high-performance liquid chromatography. Data correspond to the mean ± SEM. Statistical significance vs. wt was calculated using the Student’s t-test: #P = 0.02; *P < 0.05).



158

The analysis of pathologies in moribund aged mice showed

a significant difference in the incidence of kidney lesions, which

is the most common aging-associated pathology, together with

cancer, in C57BL6 mice (Hayashi et al., 1989; Lipman et al.,1998; Matheu et al., 2007). Doubly transgenic Ink4/Arf-tg/tg

mice presented an incidence of kidney lesions (26%) that was

less than half the incidence observed in the control wt mice (60%).

We do not know at present whether this is a direct effect of

the Ink4/Arf locus on renal biology or whether this is secondary

to other physiological processes, but this beneficial effect on renal

function could certainly contribute to the increased longevity of

Ink4/Arf transgenic mice. In this context it is interesting to mention

the case of mice lacking Skp2, and hence overexpressing the

Cdk2 inhibitor p27Kip2. These mice present a lower proliferative

response upon severe kidney damage compared to wt mice

and, interestingly, this decreased compensatory proliferation

is indeed protective and helps to preserve renal function in the

face of damage (Suzuki et al., 2007). Based on these data, we

speculate that a lower proliferative response in doubly transgenic

Ink4/Arf-tg/tg mice may protect renal function at old ages.

In addition to the above-mentioned pathological analyses, we

have examined a number of aging-associated markers that

allow to evaluate the progression of aging in particular organs

or systems. Specifically, aged transgenic mice performed better

than wt mice in the tightrope assay that measures neuromuscular

coordination, and in the hair re-growth assay. Finally, we examined

the presence of molecular markers of aging-associated DNA

damage response (Sedelnikova et al., 2004; Herbig et al., 2006;

Matheu et al., 2007). When compared to wt mice, the livers of

aged Ink4/Arf-tg/tg had a lower percentage of γH2AX and

53BP1 positive cells. These data on aging biomarkers, together

with the increased longevity and the lower incidence of

aging-associated kidney lesions, indicate that the Ink4/Arf locus

contributes to maintain tissular fitness at old ages.

When considering the mechanisms that could explain the

anti-aging activity of the locus, it is best to consider p19Arf

separately from the Ink4 proteins. In the case of p19Arf, current

evidences, based on the combination with an extra gene copy

of p53 or with a hypomorphic mutation in BubR1, suggest that

p19Arf possesses anti-aging activity (Matheu et al., 2007; Baker

et al., 2008). The anti-aging activity of p19Arf is likely mediated

by the stabilization of p53, which upregulates the basal expression

of anti-oxidant genes and eliminates irreversibly damaged cells

(Vousden & Lane, 2007). In support of this, we have observed

increased basal levels of the anti-oxidant p53 targets Sestrinsand of GSH (reduced-glutathione). However, as shown here,

increased p53 has not the same effect on aging as increased

Ink4/Arf, despite similar quantitative protection from aging-

associated cancer (Matheu et al., 2007). This suggests that

p19Arf-mediated activation of p53 conveys an anti-aging activity

that is not achieved by simply increasing p53 gene dosage and

this probably reflects the ability of the Ink4/Arf locus to respond

to mitogenic over-stimulation.

Regarding p16Ink4a, we envision two opposite scenarios: First,

Ink4a could be a pro-aging gene whose negative effects

on aging are surmounted by the anti-aging activity of Arf.However, as discussed above, current data on transgenic mice

overexpressing Ink4a have not found significant effects on stem

cell numbers or tissue proliferation at advanced ages (Krishna-

murthy et al., 2006; Molofsky et al., 2006). Alternatively, we

favour the possibility that Ink4a and Ink4b are indeed anti-aging

genes. We propose that the mechanism to explain the anti-

aging activity of Ink4a,b is precisely their capacity to decrease

proliferation. There are examples in support of the concept that

low proliferation rates are beneficial at advanced ages or for

the long-term viability of stem cell pools. First, calorie restriction,

the gold standard of anti-aging interventions, decreases global

proliferation rates [see, for example Varady et al. (2008)]. Also,

low levels of the growth factor IGF1 are characteristically

associated to aging and play an anti-aging role (Garinis et al.2008). Finally, ablation of p53 or its target p21Cip1 results in loss

of quiescence and depletion of long-term haematopoietic stem

cells and neural stem cells at advanced ages (Cheng et al., 2000;

Kippin et al., 2005; Liu et al., 2009).

It is conceivable that the upregulation of the Ink4a/Arf locus

during aging reflects a protective response elicited by an

aberrant or unbalanced mitogenic milieu. In the case of Ink4a,b,

their anti-aging activity may be due to their ability to oppose

mitogenic over-stimulation, thus avoiding unnecessary exhaustion

of proliferative potential, particularly in progenitor and stem cells.

In the case of Arf, its anti-aging activity is probably mediated

by p53, including its anti-oxidant effect and, also, its ability to

decrease proliferation rates. Although the proposed mechanisms

are conceptually similar to the mechanisms that explain the

anti-cancer activity of the Ink4/Arf locus, it is important to note

that they are quantitatively different: progressive upregulation

of the locus during aging likely results in slower proliferation rates;

whereas acute upregulation of the locus during oncogenesis

results in complete cessation of proliferation. Future work will be

necessary to dissect the detailed anti-aging mechanisms involved.

Experimental procedures

Mice

Mice were housed at the pathogen-free barrier area of the

Spanish National Cancer Research Center (CNIO), Madrid. Mice

were observed on a daily basis and sacrificed when they showed

signs of morbidity or overt tumours, in accordance to the

Guidelines for Humane Endpoints for Animals Used in BiomedicalResearch. Doubly transgenic Ink4/Arf-tg/tg mice were generated

by intercrossing singly transgenic mice Ink4/Arf-tg (Matheu

et al., 2004). The genetic background of all the mice used in

this study is > 99% C57BL6 (after at least seven backcrosses

with pure C57BL6).

Tightrope test and hair regrowth assay

For the tightrope test, also known as neuromuscular coordina-

tion assay, mice were placed on a bar of circular section (60 cm



159

long and 1.5 cm diameter) and the test was considered

successful when a mouse did not fall during a period of 60 s

in at least one trial out of five consecutive trials. For the hair

re-growth assay, dorsal hair was removed by plucking from a

square of approximately 1.5 cm × 1.5 cm. Hair re-growth was

scored 2 weeks later based on digital photographs and a semi-

quantitative assessment using an arbitrary scale from 1 to 4 (four

being complete hair regeneration). Scoring was done blindly by

two investigators who obtained essentially identical scores.

Sperm count

For each mouse (8-week-old), the cauda epididymidis and vas

deferens from each side were harvested separately. The sperm-

containing fluid was squeezed out of the vas and the cauda was

cut into pieces to extract the remaining sperm. The sperm fluid and

the pieces of cauda were suspended in 1 mL of DMEM containing

25 mM Hepes and incubated during 10 min at room temperature

to allow the sperm to swim out. The total number of spermatozoa

was counted with a haematocytometer for each cauda/vas and

the average between the two was obtained for each mouse.

Histological and immunohistochemical methods

For hematoxylin and eosin staining, testes were fixed overnight

in Bouin´s fixative. For immunohistochemistry, testes were fixed

first in formalin for 4 hours and then overnight in diluted Bouin´s

fixative. After fixation, testes were stored in 70% ethanol at

4 °C until processed. Tissues were dehydrated in increasing con-

centration of ethanol, embedded in paraffin wax, and sectioned

at a thickness of 5 μM. Sections were stained with haematoxylin

and eosin or processed for inmunohistochemical analysis with

the following antibodies: anti-Ink4a (M156, Santacruz), anti-Arf

(5-C3–1, SantaCruz), Ki67 (310QD, Master Diagnostica) and

anti-Sox9 (AB5535, Chemicon).

Determination of DNA damage in tissues

DNA damage was assessed by confocal immunofluorescence

against 53BP1 (from Novus Biologicals) or γH2AX (antibody

clone JBW301 from Upstate Biotechnology) on cryosections as

previously described (Garcia-Cao et al., 2006).

Quantitative RT-PCR

Total RNA was extracted from liver with Trizol (Invitrogen,

Carlsbad, CA). Reverse transcription was performed using

random priming and Superscript Reverse Transcriptase (Life

Technologies), according to the manufacturer’s guidelines.

Quantitative real-time PCR was performed using DNA Master

SYBR Green I mix (Applied Biosystems) in an ABI PRISM 7700

thermocycler (Applied Biosystems, Carlsbad, CA). Variations in

input RNA were corrected by substracting the number of PCR

cycles obtained for β-actin. The primers used were: Sesn1: 5′-CCA GGT AGG AAC ACT GAT GC-3′ and 5′-GTC TGG ATA

ACA TCA CAT TAG-3′; Sesn2, 5′-CTC ACA GCT GGT CTG TGT G-

3′ and 5′-CCT CCG TGT GGC AAT ACC-3′; Ink4a, 5′-AAC TCT

TTC GGT CGT ACC CC-3′ and 5′-GCG TGC TTG AGC TGA AGC

TA-3′; Arf 5′-GCC GCA CCG GAA TCC T-3′ and 5′-TTG AGC

AGA AGA GCT GCT ACG T-3′; actin, 5′-GGC ACC ACA CCT

TCT ACA ATG-3′ and 5′-GTG GTG GTG AAG CTG TAG CC-3′.

Determination of reduced glutathione

Reduced glutathione (GSH) was determined by high-performance

liquid chromatography (HPLC) as described (Asensi et al., 1994).

Acknowledgments

We are indebted to Maribel Muñoz for excellent mouse colony

management and Marta Riffo for help with sperm extraction.

A. Matheu was supported by a predoctoral fellowship from the

Spanish Ministry of Education and Science (MEC). A. Maraver

is funded by the ‘Juan de la Cierva’ Program (MEC) and MC by

the ‘Ramon y Cajal’ Program (MEC). Work at the laboratory of

MS is funded by the CNIO and by grants from the MEC (SAF2005-

03018 and OncoBIO-CONSOLIDER), from the Government of

Madrid (GsSTEM), from the European Union (INTACT and

PROTEOMAGE), and from the ‘Marcelino Botin’ Foundation.

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Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Fig. S1 Expression of Ink4a and Arf in old transgenic mice.

(A) Spleen histological sections from aged (≥ 24 months) mice were

analysed for the expression of p19Arf by immunohistochemistry.

Quantifications indicate average ± SE of positive cells. (B) Liver

samples from aged (≥ 24 months) tg (n = 5) or tg/tg (n = 5) mice



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were analysed for the expression of Ink4a and Arf by quantitative

real-time PCR. PCR data were normalized to β-actin expression

and are expressed relative to gene expression levels in tg tissues

(mean ± SEM.).

Fig. S2 Reactive oxygen species (ROS) in old transgenic mice.

Splenocytes from aged (≥ 24 months) tg (n = 2) or tg/tg (n = 2)

samples were analysed for ROS levels by FACS analysis using

DCF. Data are mean values ± SEM. Normalized to ROS levels in

tg splenocytes, which were set to 100%.

Table S1 Delayed aging of Ink4/Arf transgenic mice.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.

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Anti-aging activity of the Ink4/Arf locus

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