Aging Cell

(2009)

8

, pp2–17 Doi: 10.1111/j.1474-9726.2008.00441.x

2

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

Blackwell Publishing Ltd

MSH2 deficiency abolishes the anticancer and pro-aging activity of short telomeres

Paula Martinez,

1

Irene Siegl-Cachedenier,

1

Juana M. Flores

2

and Maria A. Blasco

1

1

Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre, 28029 Madrid, Spain

2

Animal Surgery and Medicine Department, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

Summary

Mutations in the mismatch repair (MMR) pathway occurin human colorectal cancers with microsatellite instability.Mounting evidence suggests that cell-cycle arrest inresponse to a number of cellular stresses, includingtelomere shortening, is a potent anticancer barrier. Thetelomerase-deficient mouse model illustrates the anticancereffect of cell-cycle arrest provoked by short telomeres.Here, we describe a role for the MMR protein, MSH2, insignaling cell-cycle arrest in a p21/p53-dependent mannerin response to short telomeres in the context of telomerase-deficient mice. In particular, progressively shorter telomeresat successive generations of

MSH2

–/–

Terc

–/–-

mice did notsuppress cancer in these mice, indicating that MSH2deficiency abolishes the tumor suppressor activity of shorttelomeres. Interestingly, MSH2 deficiency preventeddegenerative pathologies in the gastrointestinal tractof

MSH2

–/–

Terc

–/–

mice concomitant with a rescue of pro-liferative defects. The abolishment of the anticancer andpro-aging effects of short telomeres provoked by MSH2abrogation was independent of changes in telomerelength. These results highlight a role for MSH2 in theorganismal response to dysfunctional telomeres, which inturn may be important in the pathobiology of humancancers bearing mutations in the MMR pathway.Key words: aging; cancer; mouse models; MMR; telomeres.

Introduction

The DNA mismatch repair (MMR) pathway maintains genomic

integrity by correcting DNA replication errors and preventing

recombination between divergent DNA sequences (reviewed in

Kunkel & Erie, 2005; Jiricny, 2006). At least three protein

heterodimers are involved in the initiation step of the eukaryotic

MMR process: MSH2/MSH6 (MutS

α

), MSH2/MSH3 (MutS

β

) and

MLH1/PMS2 (MutL

α

). Muts

α

binds both to base–base mismatches

and short insertion/deletion loops, while MutS

β

can only

recognize insertion/deletion loops containing up to 16 extra

nucleotides in one strand (Kunkel & Erie, 2005). MutL

α

possesses an intrinsic endonuclease activity, activated upon

mismatch recognition, that introduces incisions in the discon-

tinuous strand of the heteroduplex DNA, thereby generating

entry sites so exonucleases can digest unwound DNA (Kadyrov

et

al

., 2006). The resulting gap is filled by DNA polymerase III;

ligation of the remaining nick completes repair (Jiricny, 2006).

Furthermore,

in vitro

reconstitution of the MMR system showed

a requirement for EXO1 for both the 5

′

–3

′

and 3

′

–5

′

mismatch

corrections, suggesting that EXO1 is a downstream player of

the MMR pathway (Genschel

et

al

., 2000; Jiricny, 2006). In

support of this, mice deficient in EXO1 exhibited defects in the

MMR of DNA (Wei

et

al

., 2003).

Mutations in a subset of MMR genes, including

MSH2

,

MSH3

,

MSH6

,

MLH1

and

PMS2

, associate with human hereditary

non-polyposis colon cancer, stressing the crucial role of MMR

in the maintenance of genome integrity (Jiricny, 2006). Mice

deficient in different MMR genes, as well as in EXO1, show

an increase in both the spontaneous mutation rates and

susceptibility to develop cancer; the MSH2 knockout mouse having

the most severe cancer-prone phenotype (Baker

et

al

., 1995; de

Wind

et

al

., 1995; Prolla

et

al

., 1998; Wei

et

al

., 2003). A role

for MMR proteins in signaling DNA damage by direct involvement

of the p53 pathway has also been proposed (Duckett

et

al

., 1999;

Peters

et

al

., 2003; Luo

et

al

., 2004), which in turn could affect

tumorigenesis. Additionally, MMR factors activate ataxia tel-

angiectasia mutated (ATM) signaling in the S-phase checkpoint

response to DNA damage (Brown

et

al

., 2003).

Telomeres are protective structures at the ends of chromo-

somes that consist of tandem TTAGGG repeats bound to a

protein complex known as shelterin (Chan & Blackburn, 2002;

de Lange, 2005). Critical telomere shortening and loss of function

of telomere-binding proteins result in loss of telomere protection,

end-to-end chromosome fusions, and cell-cycle arrest or

apoptosis (van Steensel

et

al

., 1998; Goytisolo & Blasco, 2002;

de Lange, 2005). As a consequence, critical telomere shortening

in the context of the telomerase-deficient mouse model leads

to premature aging pathologies and reduced lifespan (Lee

et

al

.,

1998; Herrera

et

al

., 1999; Blasco, 2005). Telomere shortening is

also envisioned as a potent tumor suppressor mechanism (Gonzalez-

Suarez

et

al

., 2000; Blasco & Hahn, 2003; Blasco, 2005). Mice

lacking telomerase activity are resistant to cancer (Greenberg

et

al

., 1999; Gonzalez-Suarez

et

al

., 2000), with the only exception

of p53-deficient and TRF2-overexpressing genetic backgrounds

(Chin

et

al

., 1999; Artandi

et

al

., 2000; Blanco

et

al

., 2007).

Correspondence

Maria A. Blasco, Telomeres and Telomerase Group, Molecular Oncology

Program, Spanish National Cancer Centre, 28029 Madrid, Spain.

Tel.: +34 9 1732 8031; fax: +34 9 1732 8028; e-mail: mblasco@cnio.es

Paula Martinez and Irene Siegl-Cachedenier contributed equally to this work.

Accepted for publication

29 September 2008

MSH2 and signaling of short telomeres, P. Martinez

et al.

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

3

Telomerase activation, detected in a vast majority of human

cancers (Shay & Wright, 2006), is able to extend the lifespan of

cells in culture by maintaining telomeres (Bodnar

et

al

., 1998).

We have recently shown that PMS2 deficiency rescues

organismal survival and proliferative defects in telomerase-

deficient mice by attenuating p21-dependent cell cycle arrest

in response to short telomeres (Siegl-Cachedenier

et

al

., 2007).

These observations go in line with recent work from other

laboratories indicating that short telomeres provoke organismal

aging through a p21-dependent induction of cellular senescence

(Choudhury

et

al

., 2007; Feldser & Greider, 2007). Similarly,

previous findings in yeast also indicated that MMR deficiency

increases the survival of telomerase-deficient cells (Rizki &

Lundblad, 2001). MMR deficiency may rescue proliferative

defects in telomerase-deficient cells, this proposed mechanism

involves the activation of recombination-based telomere

elongation mechanisms associated with MMR deficiency the

so-called alternative lengthening of telomeres (ALT) (Rizki &

Lundblad, 2001; Bechter

et

al

., 2004). However, previous

observations from the study of PMS2- and MSH2-deficient mice

suggest that MMR defects do not lead to an increased telomere

length or telomere recombination (Campbell

et

al

., 2006; Siegl-

Cachedenier

et

al

., 2007).

Here, we demonstrate that, in the context of a telomerase-

deficient mouse background, MSH2 abrogation does not impact

on telomere length homeostasis. The work described here

strongly supports instead our previously proposed notion that

MMR pathway plays an important role in the cellular response

to telomere dysfunction by signaling a p21/p53-dependent cell-

cycle arrest in cells with critically short telomeres (Siegl-Cachedenier

et

al

., 2007). MSH2 deficiency was sufficient to abolish the

tumor suppressor effect of short telomeres, in marked contrast

to PMS2-deficiency, arguing that MSH2 is a more potent blocker

of the cell cycle in response to short telomeres than PMS2.

MSH2 deficiency is the third genetic scenario described to date

in which short telomeres fail to work as tumor suppressors,

along with p53 deficiency and TRF2 overexpression (Chin

et

al

.,

1999; Blanco

et

al

., 2007).

Results

The lifespan of mice lacking MSH2 does not decrease with successive generations of Terc deficiency

To address the role of MSH2 in the organismal response to short

telomeres, we obtained increasing generations (G1–G3) of

mice doubly deficient in telomerase and MSH2 in a C57BL6

genetic background, G1–G3

Terc

–/–

/MSH2

–/–

mice, as well as the

corresponding G1–G3

Terc

–/–

littermate controls (Experimental

procedures), and monitored the lifespan of the different mouse

cohorts. As shown in Fig. 1, successive generations of G1–G3

Terc

–/–

mice showed a progressive decrease in both the median

survival and the mean lifespan compared to wild-type controls,

in agreement with previous findings reporting a premature loss

of mice survival associated with short telomeres (Herrera

et

al

.,

1999; García-Cao

et

al

., 2006; see also Supporting Fig. S1a for

Kaplan–Meier survival curves and Supporting Table S1 for

complete statistical analysis). Single mutant

MSH2

–/–

mice also

showed a marked reduction in the median survival and the

mean lifespan due to an early onset of cancer (Figs 1a,b, 3;

Supporting Fig. S1), in agreement with a previous report

(Reitmair

et

al

., 1996). Unexpectedly, the simultaneous lack of

telomerase and MSH2 in G1–G3

MSH2

–/–

/Terc

–/–

mice did not

result in a further reduction of the median survival and the mean

lifespan of these mice compared to the MSH2-deficient controls

(Fig. 1a,b; Supporting Fig. S1). We indeed observed a statistically

Fig. 1 MSH2 deficiency attenuates loss of organismal survival associated to short telomeres. (a) Median survival from Kaplan–Meier plots of mice of the indicated genotypes (see Supporting Fig. S1 for full Kaplan–Meier plots). Statistical comparisons using the log rank test are shown. (b) Mean lifespan of mice of the indicated genotypes. Error bars are shown. Gray bars, MSH2+/+ mice; black bars, MSH2–/– mice. Statistical comparisons using the Student’s t-test are shown. For statistical significance comparisons between mouse generations, see Supporting Table S1. n, number of mice per genotype used in the analysis.

MSH2 and signaling of short telomeres, P. Martinez

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

4

significant increase in the median survival when comparing

G2 and G3

MSH2

–/–

/Terc

–/–

mice to the MSH2-deficient

Terc

+/+

controls (Fig. 1a,b; Supporting Fig. S1; Supporting Table S1 for

complete statistical significance analysis). These results suggest

that telomerase deficiency partially ameliorates the MSH2-

associated loss of organismal survival.

MSH2 abrogation rescues aging pathologies associated to short telomeres

We performed next a thorough histopathological analysis to

determine the cause of death of the different mouse cohorts

(Experimental procedures). The most frequent and life-

threatening degenerative lesions associated to short telomeres in

G1–G3 Terc−/– mice are the atrophies of the intestinal epithelia;

they contribute to the early mortality of these mice (Herrera

et al., 1999; García-Cao et al., 2006; Siegl-Cachedenier et al.,2007). As illustrated in Fig. 2a, the age of onset of these lesions

progressively anticipates with increasing Terc–/– generations (see

also Fig. 2b,c for a comparison between mouse cohorts of a

similar age range at their time of death, as well as for some

representative images of the lesions). These lesions, however,

were largely absent in both wild-type and single MSH2-deficient

mice at the time of their death (Fig. 2a–c). Strikingly, the per-

centage of G1–G3 MSH2–/–/Terc–/– mice showing intestinal

degenerative lesions was dramatically reduced compared to

their G1–G3 Terc–/– controls (p < 0.001 for all comparisons)

(Fig. 2a–c). Furthermore, when we considered the whole set of

degenerative lesions in the different organs of these mice,

we observed a similar effect (Supporting Fig. S2). When we

analyzed mouse cohorts that were ≤ 60 weeks old at the time

of their death, we obtained similar results, ruling out an age-

dependent effect in the protection from degenerative

pathologies associated to MSH2 deficiency (Fig. 2b).

Indeed, very few animals deficient in MSH2 display intestinal

atrophy regardless of Terc–/– generation status (G1, G2 or G3),

while late-generation Terc–/– exhibit an early onset and high

incidence of intestinal atrophy (Fig. 2b). Finally, the rescue of

degenerative pathologies in the G1–G3 MSH2–/–/Terc–/– mice

correlated with the observed attenuation of the low body

weight phenotype in mice with dysfunctional telomeres (data

not shown). Altogether, these results indicate that MSH2

deficiency rescues degenerative pathologies associated with

increasing generations of telomerase-deficient mice.

MSH2 abrogation largely abolishes the tumor suppressor activity of short telomeres

Premature death of MSH2-deficient mice was associated to an

early onset of malignant tumors (Fig. 3a), in agreement with the

findings of previous reports (Reitmair et al., 1995; de Wind

et al., 1995). In particular, MSH2–/– mice presented a very high

incidence of lymphoma, which affected 67% of the mouse

cohort compared to less than 40% of wild-type controls bearing

this tumor at the time of their death. To rule out an age-

dependent effect, we analyzed tumor incidence in mouse

cohorts that were ≤ 60 weeks old at the time of their death

(Fig. 3b). In particular, MSH2–/– mice die of tumors and the same

trend is observed in different generations MSH2–/–Terc–/– mice

within the same age range (Fig. 3b). In marked contrast, single

Terc-deficient mice displayed a progressively decreased cancer

incidence with increasing mice generations (Fig. 3a,b), which is

in agreement with the tumor suppressor activity of short

telomeres and telomerase deficiency when in a p53-proficient

genetic background (reviewed in Gonzalez-Suarez et al.,2000; Blasco & Hahn, 2003). Indeed, when we compared

age-matched mice from 60 to 100 weeks old, we observed that

50% of wild-type mice (n = 10) developed cancer whereas none

of the G2 MSH2+/+Terc–/– mice (n = 24) showed malignant

tumors at the time of their death. Strikingly, the tumor suppressor

activity of short telomeres was completely abolished in successive

generations of Terc–/–/MSH2–/– mice, with 82% of G3 Terc–/–/MSH2–/– mice succumbing to malignant tumors compared to

less than 20% of G3 Terc–/– controls (Fig. 3b). Again, the age

at the time of death of these two mouse cohorts was very similar

(Figs 1a,b and 3a), ruling out an age-dependent effect on cancer

incidence in these mice. The fact that a higher percentage of

G3 Terc–/–/MSH2–/– mice developed tumors compared to wild-type

animals (82% and 67%, respectively) suggests that telomere

dysfunction may cooperate with Msh2 deficiency to induce

tumor formation (Fig. 3b). In summary, mice harboring a MSH2deletion showed a high incidence of cancer regardless of the

Terc genotype, indicating that the tumor suppressor effect of

telomerase deficiency and short telomeres was fully blocked by

the MSH2 null mutation.

Of note, these results are in marked contrast to those

previously described for G1–G3 Terc–/–/PMS2–/– mice, which lack

a different MMR component: the PMS2 protein (Siegl-Cachedenier

et al., 2007). In particular, progressive telomere shortening in

G1–G3 Terc–/–/PMS2–/– mice effectively protected against PMS2-

induced cancer. This marked difference in cancer behavior

between PMS2- and MSH2-deficient mice suggests that MSH2

deletion has a stronger tumorigenic effect than PMS2 deficiency

(see Discussion). Finally, the fact that short telomeres cannot

suppress the cancer-prone phenotype associated with MSH2

deficiency explains that the median lifespan and survival of

MSH2–/–/Terc–/– mice are not prolonged compared to Terc–/–

cohorts despite their marked reduction in degenerative

pathologies. Therefore, while MSH2 null mutation could

rescue the premature aging phenotypes of Terc–/– mice,

lack of telomerase activity, however, could not exert its

well-known tumor suppressor effect in the context of MSH2

deficiency.

MSH2 abrogation rescues cell-cycle arrest but not apoptosis provoked by short telomeres

Decreased survival of late-generation Terc-deficient mice has

been previously shown to be the consequence of a decreased

tissue renewal capacity, which in turn is thought to be determined

MSH2 and signaling of short telomeres, P. Martinez et al.

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5

by the balance between apoptosis and proliferation in tissues

with an elevated cell turnover, such as the gastrointestinal

tract (Lee et al., 1998; Herrera et al., 1999). As mentioned

above, the gastrointestinal tract was the most frequently

affected organ in single G1–G3 Terc–/– mouse cohorts (Fig. 2;

Supporting Fig. S2). To address a putative role for MSH2 in

signaling apoptosis in response to short telomeres, we measured

apoptosis levels in the small intestine epithelium (crypt area)

Fig. 2 MSH2 deficiency rescues degenerative pathologies in Terc–/– mice. (a) Intestinal degenerative lesions in of G1–G3 MSH2–/–/Terc–/–, wild-type, MSH2–/– and G1–G3 Terc–/– control mice at their time of death. Each circle or square represents a mouse. Open circles, MSH2+/+ mice without lesions; closed circles, MSH2+/+ mice with lesions; open squares, MSH2–/– mice without lesions; closed squares, MSH2–/– mice with lesions. The black and gray lines represent the mean lifespan of mice with lesions and without lesions, respectively. (b) Percentage of mice of the indicated genotypes showing mild, moderate or severe intestinal atrophy at the time of death. Only mice that were ≤ 60 weeks old were included in the analysis (see red dotted line in (a)) n, number of mice of each genotype. Statistical comparisons between genotypes using the chi-squared test are shown. (c) Representative images of degenerative lesions of the large intestine. Lesions were classified as mild, moderate or severe lesions according to the pathological findings. (i,v) Sections of healthy intestine. (ii,vi) Mild intestinal lesions were characterized by focal atrophy (black arrows), loss of mucosal glands and few inflammatory cells in the lamina propia (white arrows). (iii,vii) Moderate intestinal lesions showed diffuse atrophy of the mucosal glands with focal hyperplasia and cysts in some crypts (yellow arrow heads). Some ulcers and a moderate cellular infiltrate of lymphocytes, plasma cells and macrophages are present in lamina propia and submucosa (white arrows). (iv,viii) Severe intestinal lesions are characterized by severe atrophy (black arrows) and focal regenerative hyperplasia of glands (yellow arrow heads). The intestine is ulcerated and has marked infiltration of mixed inflammatory cells (white arrows) in the lamina propia, submucosa and muscular layer with a narrow intestinal lumen. Chronic peritonitis could be present.

MSH2 and signaling of short telomeres, P. Martinez et al.

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6

using two independent techniques: caspase-3 immunohisto-

chemistry and TUNEL staining (Fig. 4a,b,d) (Experimental

procedures). As expected, G1–G3 Terc-deficient mice showed

increased apoptosis in the gastrointestinal tract compared to

wild-type controls (Fig. 4a,b,d; see Supporting Table S2 for

complete statistical significance analysis). Interestingly, the

frequency of apoptotic cells was indistinguishable between G1–

G3 Terc–/–/MSH2–/– mice and their corresponding single G1–G2

Terc–/– controls (Fig. 4a,b,d; see Supporting Table S2 for complete

statistical significance analysis), arguing that the rescue of

degenerative pathologies associated to MSH2 abrogation is not

due to decreased apoptosis in Terc–/–/MSH2-–/– mice compared

to the single Terc–/– counterparts.

We determined next the proliferative index of the gastro-

intestinal tract epithelium in mice of the different genotypes

(crypt area) by performing immunohistochemistry with the Ki67

mitotic marker (Experimental procedures). Successive generations

of telomerase-deficient mice presented a lower percentage

of proliferating cells in the gastrointestinal tract compared

to wild-type controls, in agreement with their higher incidence

of intestinal degenerative pathologies (Fig. 4c,d; see Supporting

Table S2 for complete statistical significance analysis). Single

MSH2-deficient mice showed significantly higher proliferation

rates compared to wild-type mice (Fig. 4c,d; see Supporting

Table S2 for complete statistical significance analysis).

Importantly, MSH2 abrogation in G1–G3 Terc–/–/MSH2–/– mice

significantly rescued proliferative defects in the gastrointestinal

tract epithelium of these mice compared to single G1–G3 Terc-

deficient controls (p ≤ 0.0001 for all comparisons; Fig 4c,d;

see Supporting Table S2 for complete statistical significance

analysis). This was particularly striking in the case of G3 Terc–/–/MSH2–/– mice, where 80% of the gastrointestinal tract epithelium

cells were positive for Ki67 compared to less than 40% in the

single G3 Terc–/– controls. Altogether, these results indicate that

MSH2 deficiency resumes proliferation of cells from Terc–/–/MSH2–/– mice, resulting in a rescue of degenerative pathologies

in these mice.

MSH2 deficiency does not prevent telomere shortening in Terc–/– mice

Given the well-known role of MMR as a repressor of recombi-

nation processes (Modrich, 1994; Jiricny, 2006), we next addressed

whether MSH2 deficiency could be rescuing telomere length and

telomere capping defects associated to Terc deficiency by increasing

recombination between telomeric sequences. To address this,

we first measured telomere length directly on small intestine

sections using quantitative fluorescence in situ hybridization

(Q-FISH; Experimental procedures). Single mutant Terc–/– and

double mutant Terc–/–/MSH2–/– mice showed similar shortening

of telomeres and frequency of critically short telomeres associated

to successive mouse generations (G2–G3) in the gastrointestinal

tract epithelium (Fig. 5a; see Supporting Fig. S3 and Table S3 for

statistical analysis), indicating that the rescue of gastrointestinal

tract degenerative pathologies in G1–G3 Terc–/–/MSH2–/– mice

is not associated to a rescue of short telomeres in these mice.

To obtain more robust and extensive data of telomere length

frequencies on individual chromosomes, we next determined

telomere length by Q-FISH on metaphase spreads from primary

(passage 3) littermate mouse embryonic fibroblasts (MEF) of the

Fig. 3 MSH2 abrogation abolishes the tumor suppression effect of Terc deficiency. (a) Incidence of malignant tumors (lymphoma and carcinoma) in increasing generations of MSH2–/–/Terc–/–, wild-type, MSH2–/– and G1–G3 Terc–/– control mice at their time of death. Open circles, MSH2+/+ mice without tumors; closed circles, MSH2+/+ mice with tumors; open squares, MSH2–/– mice without tumors; closed squares, MSH2–/– mice with tumors. The black and gray lines represent the mean lifespan of mice with and without tumors, respectively. (b) Percentage of mice of the indicated genotype showing lymphoma and carcinoma tumors at their time of death. Only mice that were ≤ 60 weeks old were included in the analysis (see red dotted line in part a). n, number of mice of each genotype. Statistical comparisons between genotypes using the chi-squared test are shown.

MSH2 and signaling of short telomeres, P. Martinez et al.

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different genotypes (Experimental procedures). Again, we

found a similar rate of telomere loss with increasing generations

(G1–G3) of single Terc–/– and double Terc–/–/MSH2–/– MEFs

(Fig. 5b; see Supporting Table S4 for complete statistical analysis).

Finally, we confirmed the Q-FISH results obtained in MEF by

using a Southern-based ‘terminal restriction fragment’ (TRF)

analysis (Supporting Fig. S4). Altogether, these results indicate

that MSH2 deficiency does not impact on telomere length

regulation.

MSH2 deficiency does not rescue telomere-associated chromosomal instability in Terc-deficient mice

Next, we performed cytogenetic analysis of primary (passage 3)

MEFs derived from G1 to G3 single Terc–/– and double mutant

Terc–/–/MSH2–/– mice using telomere Q-FISH on metaphase

spreads to determine the frequency of telomere-associated

chromosomal aberrations in these mice. In agreement with

progressive telomere shortening associated to telomerase

deficiency, we detected similarly increased or even higher

frequencies of chromosome ends lacking detectable TTAGGG

signal (‘signal free’ ends) in G1–G3 Terc–/–/MSH2–/– MEFs compared

to littermate G1–G3 Terc–/– MEFs (Fig. 5c,d). In agreement with

these findings, chromosome and chromatid fusions were

significantly increased both in single late-generation G2–G3

Terc–/– and in double mutant late-generation G2–G3 Terc–/–/MSH2–/– MEFs compared to wild-type controls (Fig. 6a).

Furthermore, as expected from the known role of MSH2 in

maintaining chromosomal stability, we found a higher frequency

of fusion events in late-generation G2–G3 Terc–/–/MSH2–/– MEFs

compared to single G2–G3 Terc–/– controls, although the

differences did not reach statistical significance (Fig. 6a).

Interestingly, breaks and fragments were similarly increased in

single G2–G3 Terc–/– and doubly deficient G2–G3 Terc–/–/MSH2–/–

cells (Fig. 6b), suggesting that telomere shortening may lead

these types of aberrations. Of notice, MSH2 abrogation per se

in single MSH2-deficient cells also resulted in increased frequencies

of breaks and fragments (Fig. 6b), most likely due to the known

role of MSH2 in the repair of replicative errors and in the

regulation of mitotic recombination processes (Jiricny, 2006).

Fig. 4 MSH2 deficiency rescues proliferation defects but not apoptosis in Terc−/– mice. (a) Percentage of caspase-3-positive cells in small intestine sections of mice of the indicated genotype. (b) Percentage of TUNEL-positive cells in small intestine sections of mice of the indicated genotype. (c) Percentage of Ki67-positive cells in small intestine sections of mice of the indicated genotype. The mice analyzed were age-matched ranging from 20 to 50 weeks old. n, number of mice analyzed per genotype. Bars represent standard error. The total number of cells scored for each genotype is shown in brackets. Gray bars, MSH2+/+ mice; black bars, MSH2–/– mice. Statistical comparisons between genotypes using the chi-squared test are shown. For statistical comparisons between generations, see Supporting Table S2. (d) Representative images of small intestine sections of mice of the indicated genotype stained for caspase-3, TUNEL and Ki67. Caspase-3- and TUNEL-positive cells are indicated with black arrows.

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To further address the possible involvement of MSH2 in

mediating telomere dysfunction due to short telomeres, we

quantified the frequency of cells containing DNA damage, as

determined by the percentage of cells showing γ-H2AX and

53BP1 foci in small intestine sections from late-generation (G2–

G3) single Terc–/– and double Terc–/–/MSH2–/– mice (Experimental

procedures). γ-H2AX and 53BP1 foci have been previously

described to label double-strand breaks as well as critically short

and dysfunctional telomeres (Modesti & Kanaar, 2002; d’Adda

di Fagana et al., 2003). As shown in Fig. 6 (c–f), increasing

generations of Terc–/– mice presented progressively higher

numbers of small intestine cells showing γ-H2AX and 53BP1 foci

as compared to wild-type controls. Importantly, the percentage

of cells with γ-H2AX and 53BP1 foci was not rescued by MSH2

deficiency, suggesting that Terc–/–/MSH2–/– cells bear a similarly

high DNA damage load than single Terc–/– controls. Altogether,

these results indicate that mice doubly deficient in MSH2 and

telomerase do not present a diminished telomere dysfunction

compared to single Terc-deficient mice.

MSH2 deficiency does not lead to increased telomere recombination

Defects in MMR may be triggering telomere recombination

events; therefore, this facilitates the activation of ALT pathways,

similar to what has been previously proposed for yeast strains

lacking telomerase and MSH2, as well as for telomerase-

deficient human colon cancer cell lines mutant for MSH6 (Rizki

& Lundblad, 2001; Bechter et al., 2004). In the above-mentioned

works, the fact that MMR deficiency was able to rescue prolif-

eration defects was interpreted according to the known role of

MMR in inhibiting recombination between imperfect repeats

(Modrich & Lahue, 1996), which in turn could be favoring acti-

vation of ALT pathways for telomere maintenance. In a similar

way, the rescue of proliferative defects and aging pathologies

in Terc–/–/MSH2–/– mice might be due to similar role for murine

MSH2 in repressing telomere recombination. To directly test this

hypothesis, we determined the frequency of telomeric sister

chromatid exchange events (T-SCE) in primary MEF from mice

Fig. 5 MSH2 deficiency does not rescue telomere shortening in Terc–/– mice. (a) Telomere fluorescence distribution in small intestine sections of mice of the indicated genotype as determined by Q-FISH analysis. Mean and median telomere fluorescence and standard deviation values are shown for each genotype. n, number of telomeres used for the analysis from a total of three independent animals per genotype. (b) Telomere length distribution in metaphases of primary MEF (passage 2) as determined by Q-FISH analysis. Mean and median telomere length in kb and standard deviation values are shown for each genotype. n, number of telomeres used for the analysis from a total of one to three independent MEF per genotype. For statistical comparisons between genotypes and generations, see Supporting Table S3. (c) Percentage of signal free ends in metaphases of the indicated genotype. n, number of telomeres used for the analysis from a total of one to two independent MEF per genotype. Bars represent the standard error values among metaphases of the same genotype. Statistical comparisons using the chi-squared test are shown. Gray bars, MSH2+/+ mice; black bars, MSH2–/– mice. (d) Representative images of chromosomes showing telomeres with detectable fluorescence (left) and of chromosomes showing telomeres with undetectable fluorescence or ‘signal-free’ ends (right; see white arrows).

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of the different genotypes using two-color chromosome orien-

tation FISH (CO-FISH) (Bailey et al., 2004; Gonzalo et al., 2006).

The strand-specific nature of the CO-FISH yields typically two

telomeric signals of each color (red: lagging; green: leading) per

chromosome in the absence of recombination events. A sister

chromatid exchange within telomeric DNA (T-SCE) leads to the

mixture of red and green fluorescent signals. We counted as

positive T-SCE events only those that were detected with both

the leading and the lagging telomere probes. T-SCE frequencies

in the double mutant Terc–/–/MSH2–/– mice were not significantly

different from those of the single Terc–/– controls (Fig. 7a,b),

suggesting that an increased telomere recombination between

sister telomeres is unlikely to be responsible for the higher pro-

liferation capacity observed in these mice. These results are also

in agreement with the fact that Terc–/–/MSH2–/– mice did not

show either a lower incidence of chromosomes lacking telomere

Fig. 5 Continued

MSH2 and signaling of short telomeres, P. Martinez et al.

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signals or a significant rescue of telomere-associated chromo-

somal aberrations (Figs 5d,e and 6). However, we cannot rule

out subtle differences because in these analyses we examined

only one to two mice per genotype and generation. It should

also be pointed out that CO-FISH allows for the detection of

recombination events only between sister telomeres but not

between sister subtelomeric repeats or between telomeres

located at different chromosomes. The fact that telomere length

and the frequency of H2AX and 53BP1-positive cells is not

significantly rescued by MSH2 deficiency argues, however, that

Fig. 6 Telomere dysfunction and chromosomal instability in MSH2/Terc-deficient cells. (a) Frequency of fusions events and (b) breaks and fragments events in primary MEF metaphases of the indicated genotypes. End-to-end fusion events lacking (– TTAGGG) or not (+ TTAGGG) telomeric signals at the fusion point are indicated. n, number of metaphases used for the analysis from a total of two to four independent MEF per genotype. Bars represent standard error. Statistical comparisons using the chi-squared test are shown. (c) Percentage of γ-H2AX- and (d) 53BP1-positive cells in small intestine sections of mice of the indicated genotype. n, number of mice analyzed per genotype. The total number of cells scored is indicated. Bars represent standard error. Statistical comparisons using the chi-squared test are shown. Gray bars, MSH2+/+ mice; black bars, MSH2−/– mice. For statistical comparisons between generations, see Supporting Table S2. (e) Representative images of γ-H2AX- and (f) 53BP1-positive cells (upper panel) and of the DAPI combined-images (lower panel) in small intestine sections of G3Terc–/–MSH2+/+ and G3Terc–/–MSH2–/–mice.

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MSH2 does not have a major impact on the activation of ALT

pathways involving subtelomeric sequences or telomeres at

different chromosomes.

MSH2 deficiency results in an attenuated p53 and p21 induction in response to short telomeres

A role for MMR proteins in DNA damage signaling, which in

turn could impact on tumorigenesis, has also been proposed.

In particular, MMR proteins have been directly involved in

signaling DNA damage through the p53 pathway (Duckett

et al., 1999; Peters et al., 2003; Luo et al., 2004). To address

whether the rescue of proliferation defects and aging patho-

logies in G1–G3 Terc–/–/MSH2–/– mice could be due to a role for

MSH2 in signaling telomere dysfunction through the p53–p21

cellular senescence axis, we determined p53 and p21 levels by

immunohistochemistry directly on small intestine sections

(Experimental procedures). Successive generations of single

Terc–/– mice showed elevated amounts of p53 and of its down-

stream transcriptional target p21 in the gastrointestinal tract

(Fig. 8a–d). Interestingly, MSH2 deficiency dramatically decreased

p53 and p21 accumulation in the small intestine of G1–G3 Terc–/–/MSH2–/– mice, in agreement with the observed concomitant

increase in proliferation rates (Figs 8a–d and 4c,d). These results

demonstrate an attenuated p53/p21 induction in vivo in

response to telomere shortening in the absence of MSH2.

Finally, we noticed that p21 levels were also diminished by

MSH2 depletion in a Terc+/+ background, suggesting that MSH2

abrogation may also impact on telomere-independent mech-

anisms of p21 induction. Furthermore, the fact that p21 levels

were clearly elevated in G2 and G3 Terc–/–/MSH2–/– mice com-

pared to Terc+/+/MSH2–/– mice indicates that MSH2 deficiency

does not fully abolish p21 accumulation; instead, it attenuates

its induction in the late-generation Terc–/– mice, therefore,

suggesting that MSH2 is at least partially involved in signaling

dysfunctional telomeres.

These results were recapitulated in vitro using tissue cultured

MEF and Western blotting (Experimental procedures). Again,

MSH2 deficiency resulted in decreased p21 proteins levels in G2

Terc–/–/MSH2–/– MEF compared to the G2 Terc–/– controls, both

in basal conditions and after ionizing radiation (Fig. 8d,e). In

particular, the accumulation of p21 protein associated to short

telomeres was largely abrogated by the simultaneous absence

of MSH2 and telomerase in G2 Terc–/–/MSH2–/– MEF. As a positive

control for p21 induction in response to DNA damage, we

detected increased p21 levels in response to 10 Gy of gamma

irradiation (Fig. 8e).

These results indicate an attenuated p53 accumulation and

a subsequently decreased p21 induction in response to short

telomeres in the absence of MSH2, similarly to that recently

shown in PMS2, p21 and EXO1 deficiencies arguing that these

factors are in the same pathway to signal cell-cycle arrest in

response to critically short telomeres (Choudhury et al., 2007;

Schaetzlein et al., 2007; Siegl-Cachedenier et al., 2007). Although

a possible rescue of apoptosis associated to critically short

telomeres may be masked by p53-independent apoptosis

Fig. 7 MSH2 deletion does not increase recombination events between sister telomeres. (a) Percentage of telomeric sister chromatid exchange (T-SCE) events in MEF of the indicated genotypes. Gray bars, MSH2+/+ mice; black bars, MSH2–/– mice. n, number of chromosomes used for the analysis from a total of one to two independent MEF per genotype. Bars represent standard error. Statistical comparisons between genotypes using the chi-squared test are shown. (b) Representative CO-FISH images of metaphases hybridized with probes against the leading (green fluorescence) and lagging (red fluorescence) telomere. T-SCE events are indicated with yellow arrows. A T-SCE was considered positive when it was observed with both the leading and lagging strand probes and involved a reciprocal exchange of telomere signal.

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associated to severe chromosomal instability in MSH2/Terc

doubly deficient mice, previous findings using p21/Terc and

PMS2/Terc doubly deficient mice also showed that, at least in

the gastrointestinal tract, cell-cycle arrest is predominant over

apoptosis in eliciting age-related pathologies and decreasing

survival in telomerase-deficient mice (Choudhury et al., 2007;

Schaetzlein et al., 2007; Siegl-Cachedenier et al., 2007). We do

not rule out, however, that a different scenario could be observed

in other tissues.

Discussion

The data presented here strongly support a role for the MSH2

MMR gene in mediating cell-cycle arrest in response to short

and dysfunctional telomeres in the context of the organism,

which in turn impacts on mouse aging. In particular, MSH2

deficiency is able to prevent degenerative pathologies in

telomerase-deficient mice by attenuating a p53/p21-dependent

cell-cycle arrest in response to critically short telomeres. A similar

role has been recently proposed for the PMS2 MMR factor

(Siegl-Cachedenier et al., 2007). Furthermore, these findings go

in line with recent reports showing that abrogation of additional

factors, which are also related to the MMR signaling pathway

(namely, EXO1 and p21), are also able to significantly delay

aging pathologies in telomerase-deficient mice by rescuing

proliferative defects associated to short telomeres (Choudhury

et al., 2007; Schaetzlein et al., 2007). In particular, mice doubly

deficient in Terc and p21 show a rescue of organismal survival

and a rescue of proliferative defects but not of apoptosis, which

is independent of telomere length (Choudhury et al., 2007).

Similarly, Terc–/–/EXO1–/– double mutant mice display an improved

organ maintenance and longer lifespan compared to single

Terc–/– mice (Schaetzlein et al., 2007). As mentioned above,

EXO1 is a downstream player of the MMR pathway through its

Fig. 8 MSH2 is involved in signaling telomere dysfunction. Percentage of (a) p53- and (b) p21-positive cells in small intestine sections of mice of the indicated genotypes. The mice analyzed were age-matched ranging from 20 to 50 weeks old. n, number of mice analyzed per genotype. Bars represent standard error. The total number of cells scored for each genotype is shown in brackets. Gray bars, MSH2+/+ mice; black bars, MSH2–/– mice. Statistical comparisons between genotypes using the chi-squared test are shown. For statistical comparisons between generations, see Supporting Table S2. (c) Representative images of small intestine sections of mice of the indicated genotype stained for p53 and p21. Note the higher amount of p21- and p53-positive cells in G3 Terc–/– single mutant mice compared to G3 Terc–/–/MSH2–/– mice. Intestine sections of p53- and p21-deficient mice were used as negative controls. (d) Quantification of p21 protein levels in MEF (passage 2) of the indicated genotypes before (–) and 18 h after (+) ionizing radiation after normalization to actin levels. n, number of independent MEF per genotype used for the analysis. (e) Representative Western blot images of p21 protein levels in primary MEF. Actin was used as a loading control.

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exonuclease activity (Genschel et al., 2000; Jiricny, 2006; Kadyrov

et al., 2006). Furthermore, EXO1 deletion has been recently shown

to impair the upstream induction of DNA damage responses by

reducing single stranded DNA formation and the recruitment

of replication protein A (RPA) and ATR at DNA breaks. In this

regard, a similar rescue of ATR foci was not observed by MSH2

deletion in successive generations of Terc-deficient mice

(Supporting Fig. S5), arguing that rescue of ATR foci in late-

generation Terc-deficient mice by EXO1 abrogation (Schaetzlein

et al., 2007) may be related to a role of this enzyme in additional

DNA repairs pathways where MMR genes are not involved. In

summary, these results suggest a role for the MMR pathway in

mediating the cellular and organismal response to short telo-

meres, thereby impinging on cancer and aging.

Based on these findings, we would like to propose a specu-

lative model by which MMR may be signaling cellular arrest in

response to critical telomere shortening. It is conceivable that

when telomeres reach a critically short length, annealing of the

single DNA G-strand overhang to its complementary 3′–5′strand during T-loop formation (de Lange, 2005) may be forced

to take place at the vicinity of subtelomeric regions where

degenerated telomeric repeats are found. This would lead to

generation of heteroduplex DNA containing mismatches (i.e.

TTAGGG repeats would anneal to degenerated subtelomeric

repeats), which could then be signaled through components of

the MMR pathway, such as MSH2 and PMS2, as well as through

downstream MMR effectors, such as p21 (Choudhury et al.,2007; Siegl-Cachedenier et al., 2007; this paper). Activation of

the MMR pathway will then induce a DNA damage response

that imposes a p53/p21-dependent cell cycle arrest in the

gastrointestinal tract epithelium in the case of telomerase-

deficient mice. In telomerase-null mice simultaneously deficient

for MMR components (PMS2, MSH2) or for its downstream

effectors (p53, p21), this DNA damage response is attenuated

resulting in normal cell proliferation rates and maintenance of

tissue fitness. At the same time, rescue of cell-cycle arrest may

lead to increased cancer incidence depending on the oncogenic

activity of the MMR mutation. In this regard, MSH2 deletion has

been previously shown to be more potent in inducing tumori-

genesis than PMS2, EXO1 or p21 deletion (Baker et al., 1995;

de Wind et al., 1995; Prolla et al., 1998; Wei et al., 2003), in

agreement with the fact that MSH2 deficiency but not p21,

EXO1 or PMS2 deficiency abolishes the tumor suppressor

activity of short telomeres (Choudhury et al., 2007; Schaetzlein

et al., 2007; Siegl-Cachedenier, 2007; this paper). The tumor

prone phenotype associated to MSH2 deficiency explains that

the survival of MSH2–/–/Terc–/– mice is not prolonged compared

to single Terc–/– mouse counterpart, even though degenerative

pathologies in these mice are greatly prevented. These results

suggest that telomerase inhibitors would not be effective in the

treatment of tumors bearing MSH2 mutations. This situation is

similar to that previously described for Terc–/–/p53–/– mice (Chin

et al., 1999). In both cases, MSH2 and p53 deficiency leads to

rapid and robust tumor development (all mice die with tumors

within the first 50 weeks of life), which is not impaired by

telomerase deficiency and the presence of critically short

telomeres. Indeed, telomere dysfunction accelerates p53-induced

carcinogenesis by promoting chromosomal instability (Artandi

et al., 2000). Similarly, mutations in MSH2 are by far the most

aggressive, leading to tumorigenesis, compared to other MMR

components, in agreement with the fact that MSH2–/– animals

lack all mismatch recognition functions and have a higher

chromosomal instability (de Wind et al., 1995). Of notice,

together with p53 and MSH2 deletions, TRF2 overexpression is

the third genetic scenario, which abolishes the tumor suppressor

activity of short telomeres (Blanco et al., 2007).

Finally, although to the best of our knowledge no direct physical

interaction between MMR components and telomeric DNA has

been reported to date, the telomere-interacting protein WRN

has been described to interact with MMR components. In particular,

when MutSα and MutSβ encounter DNA heteroduplexes,

they recruit the WRN DNA helicase to disrupt the joined DNA

molecule (Saydam et al., 2007). Interestingly, WRN interacts

with TRF2 and can be detected at telomeres (reviewed in

Opresko et al., 2002; Crabbe et al., 2004; Opresko et al., 2004;

de Lange, 2005). In addition, human MutSα has been shown

to bind efficiently to G-quadruplex DNA (Larson et al., 2005),

a DNA structure that is also proposed to form at telomeric

repeats.

In conclusion, the results shown here strongly support an

important role of the MMR pathway in the upstream signaling

events in response to dysfunctional short telomeres and indicate

that at least at the gastrointestinal tract, cell-cycle arrest is

predominant over apoptosis in eliciting age-related pathologies

and decreasing survival in telomerase-deficient mice.

Experimental procedures

Generation of mice and primary mouse cells

To generate MSH2–/–/Terc–/– mice, MSH2–/– mice (Reitmair et al.,1995) were crossed with Terc–/– animals (Blasco et al., 1997) and

the resulting double heterozygous breeding pairs were used to

generate G1 of single Terc–/– and of double Terc–/–/MSH2–/–.

Mouse colonies of successive generations, G2 and G3, were

generated by intercrossing G1 and G2 MSH2+/–/Terc–/– pairs,

respectively. Genotyping was performed as described elsewhere

(Reitmair et al., 1995; Blasco et al., 1997). Mice were maintained

at the Spanish National Cancer Centre under specific-pathogen-

free conditions in accordance with the recommendations of the

Federation of European Laboratory Animal Science Associations.

Primary MEFs were prepared from day 13.5 embryos, as

previously described (Blasco et al., 1997).

Telomere length analysis by Q-FISH

Exponentially growing primary MEFs were incubated with

0.1 μg mL–1 colcemide (Gibco, Paisley, UK) for 4 h at 37 °C and

then fixed in methanol : acetic acid (3 : 1). Q-FISH was performed

as described (Herrera et al., 1999; Samper et al., 2000). To correct

MSH2 and signaling of short telomeres, P. Martinez et al.

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for lamp intensity and alignment, images from fluorospheres

(fluorescent beads; Molecular Probes, Carlsbad, CA, USA) were

analyzed using the TFL-Telo software (gift from Dr Peter

Lansdorp). Telomere fluorescence values were extrapolated

from the telomere fluorescence of lymphoma cell lines LY-R (R

cells) and LY-S (S cells) with known telomere lengths of 80 and

10 kb, respectively. There was a linear correlation (r2 = 0.999)

between the fluorescence intensity of the R and S telomeres.

We recorded the images using a Cohu CCD camera (San Diego,

CA, USA) on a fluorescence microscope (DMRb, Leica, Wetzlar,

Germany). A mercury vapour lamp (CS 100 W-2; Philips,

Amsterdam, the Netherlands) was used as a source. We captured

the images using the Leica Q-FISH software in a linear acquisi-

tion mode to prevent oversaturation of fluorescence intensity.

We used the TFL-Telo software to quantify the fluorescence

intensity of telomeres from at least 10 metaphases for each data

point (Zijlmans et al., 1997).

For Q-FISH analysis on small intestine sections, de-paraffinated

sections were hybridized with a PNA-telomeric probe and

telomere fluorescence was determined as previously described

(Herrera et al., 1999; Gonzalez-Suarez et al., 2000). More

than 200 nuclei from each genotype were captured at

×100 magnification using a Leica CTR MIC microscope and a

Cohu High Performance CCD camera. Telomere fluorescence

was integrated using spot Integrated Optical Density analysis

in the TFL-TELO program (kindly provided by Dr Landsdorp,

Vancouver, BC, Canada) (Zijlmans et al., 1997).

TRF-based telomere length analysis of MEF

Primary MEF of the indicated genotypes were included in

agarose plugs following instructions provided by the manufacturer

(Bio-Rad, Munich, Germany) and TRF analysis was performed

as previously described (Blasco et al., 1997).

Cytogenetic analysis using telomere Q-FISH on metaphases

For analysis of chromosomal aberrations, at least 50 metaphases

per genotype were analyzed by superimposing the telomere

image on the 4′,6-diamidino-2-phenylindole (DAPI) image using

the TFL-telo software.

Telomere recombination measurements using CO-FISH

Exponentially growing primary MEFs were subcultured in the

presence of 5′-bromo-2′-deoxyuridine (Sigma, St Louis, MO,

USA) at a final concentration of 1 × 10–5 M, and then allowed

to replicate their DNA once at 37 °C for 24 h. Colcemide was

added at a concentration of 1 μg mL–1 during the last 4 h. Cells

were then recovered and metaphases prepared as described

(Samper et al., 2000). CO-FISH was performed as described

(Bailey et al., 2004; Gonzalo et al., 2006) using first a (TTAGGG)7probe labeled with Cy3 and then a second (CCCTAA)7 probe

labeled with Rhodamine Green (Applied Biosystems, Bedford,

MA, USA). Metaphase spreads were captured on a Leitz Leica

DMRB fluorescence microscope.

Histopathology and immunohistochemistry

Small intestine sections were fixed in 10% buffered formalin,

embedded in paraffin wax and sectioned at 2–3 μm. Immuno-

histochemistry was performed on de-paraffinated intestine

sections processed with 10 mM sodium citrate (pH 6.5) and

cooked under pressure for 2 min. Slides were washed in water,

then in buffer TBS Tween 20 0.5%, blocked with peroxidase,

washed with TBS Tween 20 0.5% again and blocked with fetal

bovine serum followed by another wash. Then the slides were

incubated with the primary antibodies: rabbit monoclonal to

Ki-67 antibody (prediluted, SP6, Master Diagnostica, Madrid,

Spain), rabbit polyclonal to active caspase-3 at 1 : 200 (R&D

Systems, Minneapolis, MN, USA), rabbit polyclonal to p53 at

1 : 200 (CM5, Novocastra, Newcastle upon Tyne, UK) or goat

polyclonal to p21 at 1 : 250 (C-19-G, Santa Cruz Biotechnology,

Santa Cruz, CA, USA). Slides were then incubated with second-

ary antibodies conjugated with peroxidase from Dako (Glostrup,

Denamark), goat anti-rabbit (1 : 50) in the case of Ki-67, active

caspase-3 and p53 and rabbit anti-goat in the case of p21.

For signal development DAB (Dako) was used as a substrate.

Sections were lightly counterstained with haematoxylin and

analyzed by light microscopy. Apoptosis was also determined

by in situ hybridization TUNEL kit according to the manufacturer’s

instruction (ApopTag, Chemicon, Temecula, CA, USA).

Phosphorylated H2AX foci were detected using a mouse

monoclonal anti-phospho-histone H2AX antibody (1 : 500;

Upstate Biotechnology, Lake Placid, NY, USA). 53BP1 foci were

detected using a rabbit polyclonal anti-53BP1 (1 : 100; Novus

Biologicals, Littleton, CO, USA). ATR foci were detected using a

mouse monoclonal ant ATR (1 : 100; Cell Signalling Technology,

Danvers, MA, USA). After incubation with Cy3-goat anti-mouse

or 488 Alexa-goat anti-rabbit antibody (1 : 400; Jackson Immuno-

Research Laboratories, West Grove, PA, USA), for 30 min at

room temperature, slides were mounted in Vectashield with

DAPI. Images were obtained using a fluorescence microscope

(Leica DMRB).

The intestinal histopathology was analyzed in sections of

duodenum, cecum and colon. Five fields at ×20 were evaluated

by a pathologist in a blind manner. The length of the intestinal

wall, number and distribution of the glands, inflammation,

ulcerations, and compensatory glandular hyperplasia were

evaluated. Intestinal lesions were classified as mild, moderate

or severe lesions according to the pathological findings. Mild

intestinal lesions were characterized by focal atrophy, loss of

mucosal glands and few inflammatory cells in the lamina propia.

Moderate intestinal lesions showed diffuse atrophy of the

mucosal glands with focal hyperplasia and cysts in some crypts.

Some ulcers and a moderate cellular infiltrate of lymphocytes,

plasma cells and macrophages are present in lamina propia and

submucosa. Severe intestinal lesions are characterized by severe

atrophy and focal regenerative hyperplasia of glands. The intestine

MSH2 and signaling of short telomeres, P. Martinez et al.

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15

is ulcerated and has marked infiltration of mixed inflammatory

cells in the lamina propia, submucosa and muscular layer with

a narrow intestinal lumen. Chronic peritonitis could be present.

Western blots

Whole-cell extracts were prepared from non-irradiated and

irradiated primary MEF as described (Blanco et al., 2007).

Protein concentration was determined using the Bio-Rad

DC Protein Assay (Bio-Rad). Fifty micrograms of each extract

were separated in 4–20% gradient sodium dodecyl sulfate–

polyacrylamide gels by electrophoresis. After transfer, the

membranes were incubated with an anti-p21 C-19 poly-

clonal antibody (1 : 500) (Santa Cruz Biotechnology) and

anti-β-actin monoclonal (1 : 10.000) (Sigma). Antibody

binding was detected after incubation with a secondary

antibody coupled to horseradish peroxidase using enhanced

chemiluminescence.

γγγγ-Irradiation of primary MEF

A total of 3 × 105 MEFs were seeded in 60-mm plates and

incubated overnight at 37 °C. Then, MEFs were treated with

10 Gy by using a 137Cs source (MARK 1–30 Irradiator; Shepherd

& Associates, San Fernando, CA, USA) at a rate of 2.11 Gy min–1.

After removing the medium, cells were washed and fresh

medium was added. To analyze the effect of γ-irradiation on

protein expression, cells were incubated for 18 h at 37 °C and

then recovered and frozen for further analysis by Western blot.

Statistical analysis

A log rank test was used to calculate statistical differences in

median survival of the different mouse cohorts. A Student’s

t-test was used to calculate the statistical significance of the

observed differences in mean lifespan, The Wilcoxon–Mann–

Whitney rank sum test was used for statistical comparisons of

the mean telomere length in MEFs and small intestine sections.

A chi-squared test was used to calculate statistical significance

in pathologies, signal-free ends, chromosomal aberrations,

T-SCE, γH2AX, 53BP1, apoptosis, proliferation, as well as p21

and p53 expression.

Acknowledgments

We thank R. Serrano and E. Santos for mouse care and

genotyping. P.M. is a ‘Ramon y Cajal’ senior scientist. I.S.-C. is

a Predoctoral Fellow from the Spanish Ministry of Education and

Culture (MEC). M. A. Blasco’s laboratory is funded by the MEC

(SAF2005-00277, GEN2001-4856-C13-08), by the Regional

Government of Madrid (GR/SAL/0597/e2004), European Union

(Telosens-CT-2002-00217, Intact LSHC-CT-2003-506803,

Zincage FOOD-CT-2003-506850, RISC-RAD FI6R-CT-e2003-

508842, Mol Cancer Med Lshc-CT-2004-502943) and the

Spanish Association Against Cancer.

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Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Fig. S1 Kaplan–Meier survival curves of the different mouse

cohorts. (A) MSH2+/+/Terc+/+, G1MSH2+/+/Terc–/–, G2MSH2+/+/Terc–/– and G3MSH2+/+/Terc–/–; (B) MSH2–/–/Terc+/+, G1MSH2–/–/Terc–/–, G2MSH2–/–/Terc–/–- and G3MSH2–/–/Terc–/–. n, number of

mice of each genotype included in the analysis. Statistical

comparisons among genotypes using the log rank test are

shown. For statistical comparisons between generations, see

Supporting Table S1.

Fig. S2 Histopathological analysis of mice of the different

genotypes at their time of death. Percentage of mice of the

indicated genotype showing degenerative pathologies in the

indicated organs at time of death. Intestinal lesions include atrophy,

hyperplasia, cysts, hemorrhage, edema and lymphoid infiltrations

MSH2 and signaling of short telomeres, P. Martinez et al.

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

17

(see also Fig. 2). Kidney lesions include atrophy, tubular

degeneration, amyloidosis, glomerulonephritis, hydronephrosis,

intersticial nephritis, cysts and lymphoid infiltrations. Spleen

lesions include atrophy, amyloidosis, lymphoid and myeloid

hyperplasia. Liver lesions include vacuolar degeneration, necrosis,

cyst and lymphoid infiltration. Testis, seminal vesicle, ovary and

uterus compose the reproductive tract group. The reproductive

tract lesions include atrophy, cyst, hyperplasia of Leydig cells,

inflammation of seminal vesicles and cystic endometrial hyper-

plasia. Heart lesions include congestion, ventricular dilatation

and myocardiopathy. Lung lesions include congestion,

hemorrhage, alveolar hyperplasia and lymphoid infiltration.

Statistical comparisons between genotypes using the chi-squared

test are shown.

Fig. S3 Representative combined images of telomere flu-

orescence and DAPI staining on small intestine sections of mice

of the indicated genotype as determined by Q-FISH.

Fig. S4 Telomere length as determined by TRF in MEF of the

indicated genotypes. No significant differences in TRF fragment

size were detected between single Terc–/– and double Terc–/–

MSH2–/– mice of the indicated mouse generations.

Fig. S5 (a) Percentage of ATR-positive cells in small intestine

sections of mice of the indicated genotypes. n, number of mice

analyzed per genotype. The total number of cells scored is

indicated. Bars represent standard error. Statistical comparisons

using the chi-squared test are shown. Gray bars, MSH2+/+ mice;

black bars, MSH2–/– mice. For statistical comparisons between

generations, see Supporting Table S2. (b) Representative images of

ATR-positive cells (upper panel) and of the DAPI combined-

images (lower panel) in small intestine sections of G3Terc–/–

MSH2+/+ and G3Terc–/– MSH2–/– mice.

Table S1 Statistical significance in median survival and mean

lifespan analysis by log rank and Student’s t-test, respectively

Table S2 Comparisons between generations. Statistical

significance in immunohistochemistry and in situ hybridization

determinations by chi-squared test

Table S3 Statistical significance in Q-FISH analysis in small

intestine sections

Table S4 Statistical significance in Q-FISH analysis in MEF

metaphases

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directed to the corresponding author for the article.