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Oncogene (2002) 21, 584 ± 591
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Many ways to telomere dysfunction: in vivo studies using mouse models
FermõÂ n A Goytisolo1 and MarõÂ a A Blasco*,1
1
Department of Immunology and Oncology, Centro Nacional de BiotecnologõÂa-CSIC, Campus Cantoblanco, E-28049, Madrid,
Spain
The existence of a capping structure at the extremities of
chromosomes was ®rst deduced in the 1930s by Herman
MuÈller (MuÈller, 1938), who showed that X-irradiation of
Drosophila rarely resulted in terminal deletions or
inversions of chromosomes, suggesting that chromosome
ends have protective structures that distinguish them
from broken chromosomes, which he named telomeres. In
this review, we will focus on mammalian telomeres and,
in particular, on the analysis of di€erent mouse models
for proteins that are important for telomere function,
such as telomerase and various telomere-binding proteins. These murine models are helping us to understand
the consequences of telomere dysfunction for cancer,
aging and DNA repair, as well as, the molecular
mechanisms by which telomeres exert their protective
function.
Oncogene (2002) 21, 584 ± 591. DOI: 10.1038/sj/onc/
1205085
Keywords: telomeres; telomerase; mouse models
Structure and components of the mammalian telomere
Telomere structure
The telomere is a large nucleoprotein complex with a
structure which is di€erent to that of the bulk of the
chromatin (reviewed in Blasco et al., 1999; Blackburn,
2000; de Lange, 2001). Mammalian telomeric DNA is
composed of G-rich tandem repeats of the sequence
TTAGGG, which in humans extend 10 ± 15 Kb at each
chromosome end (de Lange et al., 1990; Harley et al.,
1990). In the mouse, wild-derived strains have telomere
lengths similar to those of humans (Hemann and
Greider, 2000), while established inbred mouse strains
have telomere lengths of approximately 40 Kb (Zijlmans et al., 1997). The average length of TTAGGG
repeats, however, does not seem to matter for a proper
telomere function, unless telomeres shorten below a
minimal functional length, which results in end-to-end
chromosome fusions (Blasco et al., 1997; Lee et al.,
1998).
In addition, the telomere is characterized by having
a 3' G-rich overhang (G-strand overhang), which in
mammals has a length of approximately 200
*Correspondence: MA Blasco; E-mail: [email protected]
nucleotides (McElligott and Wellinger, 1997; Wright
et al., 1997). The G-strand overhang is the substrate
to which telomeric repeats are added by telomerase.
The study of telomerase-de®cient mice, however,
indicated that the formation of the G-strand overhang does not require telomerase activity (Nikaido et
al., 1999; Hemann and Greider, 1999). In fact, the Gstrand overhangs are likely to be a direct consequence of the end replication problem (Ohki et al.,
2001). Importantly, the G-strand overhang can fold
back and invade the duplex telomeric repeats,
displacing one strand and hybridizing to its complementary sequence (Grith et al., 1999). This
higher-order telomere structure has been named the
`T-loop' (Figure 1). The T-loop model provides a
mechanism for the sequestering of the G-strand
overhang which could otherwise activate DNA
damage checkpoints and DNA repair enzymes
(reviewed in Greider, 1999; Collins, 2000). An
additional function of the T-loop could be to prevent
the access of telomerase to the telomere (Grith et
al., 1999; Collins, 2000).
Telomere binding proteins
The ®rst TTAGGG-binding protein identi®ed was
TRF1 (Chong et al., 1995). TRF1 is a negative
regulator of telomere length and its function is
regulated by TIN2 (Kim et al., 1999), and by two
proteins highly homologous to each other, TANK1
(also known as tankyrase) and TANK 2 (Smith et al.,
1998; Kaminker et al., 2001). TIN2 is thought to
enhance TRF1-dependent pairing of telomeric repeats.
In addition, TIN2 is a negative regulator of telomere
length (Kim et al., 1999). TANK1 and TANK2 have a
catalytic domain homologous to that of poly(ADPribose) polymerase (PARP1), a nuclear enzyme which
is activated by DNA breaks and catalyses the addition
of poly(ADP-ribose) residues to a variety of nuclear
proteins and has a role in the base excision repair
(reviewed in BuÈrkle et al., 2000). PARP1 has also been
proposed to in¯uence telomere function (see later).
TANK1 inactivates TRF1 by poly(ADP-ribosyl)ation
(Smith et al., 1998), and causes telomeric elongation
when overexpressed (Smith and de Lange, 2000).
TANK2, in contrast, causes rapid cell death by
necrosis when overexpressed (Kaminker et al., 2001).
Another TTAGGG-repeat binding protein is
TRF2 (Bilaud et al., 1997; Broccoli et al., 1997).
Mouse models for telomere function
FA Goytisolo and MA Blasco
585
Figure 1 The mammalian telomere is depicted in two alternative conformations. The `closed' conformation is based on the T-loop
model described by Grith et al. (1999). T-loops are likely to be stabilized by telomere-binding proteins TRF1 and TRF2. A
`closed' telomere hides the 3' G-strand overhang from telomerase activity and from other cellular activities, such as degradation and
recombination. Loss of TTAGGG repeats below a critical length or loss of telomere-binding proteins from the telomere could
result in an `open' telomere conformation, which in turn could render the 3' end accessible to telomerase or to other cellular
activities. NHEJ, non-homologous end-joining activities; HR, homologous recombination; MMR, mismatch repair activities
Like TRF1 and TIN2, TRF2 is also a negative
regulator of telomere length (Smogorzewska et al.,
2000). In addition, TRF2 has unique functions at
the telomere such as stabilizing the G-strand overhang, and preventing telomeric fusions (van Steensel
et al., 1998). Furthermore, over-expression of a
TRF2 dominant negative mutant, causes premature
senescence (van Steensel et al., 1998), and activation
of the apoptotic cascade mediated by ATM and p53
(Karlseder et al., 1999). Both TRF1 and TRF2 are
found at telomeric T-loops and their e€ects on
telomere length and end-protection could be
mediated by this property (Grith et al., 1999).
TRF2 has been shown to recruit hRAP1 to human
telomeres. hRAP1 is the homologue of yeast RAP1
protein and its overexpression causes telomere
elongation (Li et al., 2000). In addition, TRF2
recruits the MRE11 complex to human telomeres
(Zhu et al., 2000). The MRE 11 complex is
composed of RAD50, MRE11 and NBS1 and is a
key component of the homologous recombination
(HR) and non-homologous end joining pathways
(NHEJ). Interestingly, in S. cerevisiae, elimination of
the components of the MRE11 complex result in
telomere shortening (Nugent et al., 1998b). Unfortunately, mice de®cient for any of the MRE11
complex proteins are embryonic lethal (Zhu et al.,
2001; Luo et al., 1999; Xiao and Weaver, 1997).
Ku70 and Ku86, together with DNA-PKcs, form an
enzyme called DNA dependent protein kinase (DNAPK), which is involved in DNA double strand break
(DSB) repair by NHEJ and in V(D)J recombination
(reviewed in Smith and Jackson, 1999). Ku proteins
also interact with TTAGGG repeats (Bianchi and de
Lange, 1999; Hsu et al., 1999) and with telomeric
proteins TRF1 and TRF2 (Hsu et al., 2000; Song et
al., 2000). The study of Ku86 and DNA-PKcs de®cient
mice indicated that these proteins also have essential
roles at the mammalian telomere (Bailey et al., 1999;
Samper et al., 2000; Hsu et al., 2000; Goytisolo et al.,
2001). Figure 1 shows di€erent telomeric proteins at
the mammalian telomere.
The study of human premature aging syndromes has
contributed to the identi®cation of proteins that a€ect
telomeric function in mammals, such as ATM, WRN
and BLM (reviewed in Guarente, 1997). In Ataxia
telangiectasia (AT), ATM (AT mutated) is dysfunctional. AT includes ataxia, defects in the immune
system, endocrine disorders, infertility, premature aging
of the skin and hair, and a high incidence of cancer
(Shiloh, 1995; Smilenov et al., 1997). ATM is activated
by DNA damage, and mediates phosphorylation of
p53 and cell cycle arrest via p21 (Barlow et al., 1997;
Banin et al., 1998). In addition, AT cells show
chromosomal abnormalities and an accelerated rate
of telomere shortening (Pandita et al., 1995; Metcalfe
et al., 1996), suggesting that ATM has a major role at
the mammalian telomere. In this regard, ATM de®cient
mice also show telomeric phenotypes (see later).
Another group of human premature aging syndromes that a€ect telomere function include Bloom's
and Werner syndromes, the respective genes mutated
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Mouse models for telomere function
FA Goytisolo and MA Blasco
586
are BLM and WRN, that encode two members of the
RecQ family of DNA helicases (reviewed Wu and
Hickson, 2001). BLM co-localizes with telomeric
repeats (Yankiwski et al., 2000), and re-introduction
of telomerase prevents accelerated senescence of
Werner ®broblasts (Wyllie et al., 2000), suggesting that
the phenotype of these diseases may be partially
explained by telomere dysfunction.
A novel protein, Pot 1, has been found to interact
with the G-strand overhang both in yeast and in
mammals (Baumann and Cech, 2001).
The study of knock-out models for all telomeric
proteins is crucial to understand how telomeres work,
as well as to study their role in the processes of cancer
and aging. Recently, signi®cant understanding of
telomere function has been achieved studying mice
with critically short telomeres (Terc7/7 mice) or mice
de®cient for proteins that been proposed to have a role
at the telomere, such as Ku86, DNA-PKcs, PARP1,
and ATM.
Telomerase: the cellular enzyme that elongates telomeres
Severe telomere dysfunction in Ku86-deficient mice
Telomerase synthesizes telomeres de novo, hence
preventing telomere shortening in those cells where it
is expressed at suciently high levels. Telomerase
consists of two essential components, a reverse
transcriptase known as Tert (Telomerase Reverse
Transcriptase) and an RNA molecule or Terc
(Telomerase RNA component), which contains the
template for the synthesis of new telomeric repeats
(reviewed in Nugent and Lundblad, 1998a; Collins,
2000). Telomerase activity is upregulated in the vast
majority of human tumors compared to normal
somatic tissues (reviewed in Shay and Bacchetti,
1997), and its inhibition in human tumor cell lines
leads to telomere shortening and loss of cell viability
(reviewed in Zumstein and Lundblad, 1999), suggesting
that telomerase inhibition could be an e€ective way to
abolish tumor growth by provoking telomere shortening to a critical length. The exact mechanisms that
regulate the access of telomerase to the telomere are
still unknown, however, T-loops and various telomere
binding proteins have been proposed to regulate this
process (see above).
In yeast, Ku de®ciency results in loss of telomeric
repeats, loss of telomere clustering, loss of telomeric
silencing and deregulation of the G-strand overhang
(Boulton and Jackson, 1996, 1998; Laroche et al., 1998;
Gravel et al., 1998; Nugent et al., 1998b). The analysis
of Ku86 de®cient mice, however, depicts a very
di€erent scenario. Although Ku86 de®ciency in the
mouse results in telomeric fusions (Bailey et al., 1999;
Hsu et al., 2000; Samper et al., 2000), they are
characterized by showing long TTAGGG segments at
the fusion point (Samper et al., 2000). This suggests
that they are not the result of telomere shortening
below a minimum length, but rather that, in the
absence of Ku86, telomeres are no longer protected
from fusing. In addition, Ku86 de®ciency in the mouse
results in signi®cant telomere lengthening without
a€ecting the length of the G-strand overhang (Samper
et al., 2000). This telomere elongation phenotype
suggests that Ku86 impairs the access of elongating
activities to the telomere, similar to that proposed for
TRF1, TIN2, and TRF2 proteins (Smogorzewska et
al., 2000; Kim et al., 1999; Samper et al., 2000). Hence,
Ku86 could be contributing to maintain the telomere in
some sort of `closed' conformation, a candidate
structure would be the T-loop (Figure 1), although
there is no direct experimental evidence for this.
It is relevant to note that Ku86 de®cient mice show
similar phenotypes to those of late generation telomerase de®cient mice (see later) such as, end-to-end
fusions, infertility, small size and decreased viability
with age (Nussenzweig et al., 1996; Vogel et al., 1999).
Furthermore, Ku86 de®ciency can suppress tumor
growth in a wild-type p53 background (Di®lippantonio
et al., 2000), similar to that reported for late generation
telomerase de®cient mice (Greenberg et al., 1999;
GonzaÂlez-SuaÂrez et al., 2000; Artandi et al., 2000;
Rudolph et al., 2001). Hence, the role of Ku86 protein
in aging, cellular proliferation, and transformation
could be mediated by its essential function at the
telomere (Table 1).
Telomerase-independent elongating activities at the
mammalian telomere
Human cell lines and tumors that lack telomerase
activity, however, are able to maintain or elongate
their telomeres by alternative mechanisms, which have
been termed ALT (Bryan et al., 1995; 1997). In
mammalian ALT cells, DNA sequences are copied
from telomere to telomere suggesting that ALT
involves HR (Dunham et al., 2000). In S. cerevisiae,
there are two HR pathways involved in ALT, which
depend on either RAD50 or RAD51 (Lundblad and
Blackburn, 1993; Le et al., 1999; Chen et al., 2001). In
addition, RecQ helicases (WRN and BLM in
mammals) are required for ALT in yeast (Cohen
and Sinclair, 2001; Huang et al., 2001; Johnson et al.,
2001). ALT can also be enhanced by eliminating the
mismatch repair (MMR) pathway in yeast, as the
MMR machinery inhibits HR (Rizki and Lundblad,
2001). It is foreseeable that in the next few years a
considerable e€ort will be devoted to determine the
roles of HR, MMR, WRN and BLM proteins in
mammalian ALT by studying the di€erent mouse
models for these proteins.
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Mouse models for telomere function
Differential roles of Ku86 and DNA-PKcs at the
mammalian telomere
The role of DNA-PKcs at the telomere has been
addressed using two mouse models: (i) the Scid mouse,
which carries an inactivating mutation in the catalytic
Mouse models for telomere function
FA Goytisolo and MA Blasco
Table 1
Mouse model*
Phenotypes of mice de®cient for telomerase activity or other proteins with a function at the telomere
Growth defects and
Aging phenotypes
Tumorigenesis
Fertility
Sensitivity to
DNA damage
Molecular defect
in DNA repair
Telomeric
dysfunction
Late generation
Terc 7/7
(Blasco et al., 1997)
enhanced
reduced except
in combination with
p53 deficiency
decreased
decreased
moderately
sensitive
slow rate of repair
(undetermined
repair pathway)
telomere shortening
end-to-end fusions
Ku86 7/7
(Zhu et al., 1996)
enhanced
reduced except
in combination with
p53 deficiency
decreased
very
sensitive
deficient in NHEJ
elongated telomeres
end-to-end fusions
DNA PKcs 7/7
(Taccioli et al., 1998)
normal
normal
normal
very
sensitive
deficient in NHEJ
normal length
mild end-to-end
fusion phenotype
PARP D2
(Wang et al., 1995)
normal
normal and increased
in combination with
p53 deficiency
normal
sensitive
deficient in
base-excision repair
telomere shortening
end-to-end fusions
PARP D4
(MeÂnissier de Murcia
et al., 1997)
normal
normal and decreased
in combination with
p53 deficiency
normal
sensitive
deficient in
base-excision repair
normal length
mild end-to-end
fusion phenotype
ATM 7/7
(Barlow et al., 1996)
enhanced
increased and further
increased in combination
with p53 deficiency
decreased
sensitive
undetermined
repair pathway
telomere shortening
extrachromosomal
telomeres
587
* Original description of the mice. The references for the di€erent phenotypes are cited in the text
Table 2
Mouse model
Wild-type
Late generation Tert 7/7
Ku86 7/7
DNA PKcs 7/7
PARP1 D2
PARP1 D4
Telomeric phenotypes in mouse models for telomeric proteins
Number of
end-to-end fusions
per metaphase
Number of
breaks and fragments
per metaphase
0.011
0.2 ± 0.3
0.187
0.065
0.25
0.026
n.d.
0.425
0.036
0.100
0.021
0.168
site of the protein (Kirchgessner et al., 1995), and (ii)
DNA-PKcs7/7 mice which are null for DNA-PKcs
protein (Taccioli et al., 1998). Similar to Ku86
de®ciency, DNA-PKcs absence results in end-to-end
fusions with TTAGGG repeats at the fusion point,
indicating telomere dysfunction in the absence of
telomere shortening (Goytisolo et al., 2001). However,
the frequency of end-to-end fusions detected in DNAPKcs7/7 cells is signi®cantly lower than in Ku867/7
cells, suggesting that Ku86 is more important for endcapping than DNA-PKcs (Goytisolo et al., 2001)
(Table 2). Furthermore, in contrast to Ku86 de®ciency,
DNA-PKcs de®ciency did not a€ect telomere length
(Goytisolo et al., 2001). In the case of Scid mice,
however, telomeres are elongated (Hande et al., 1999b;
Goytisolo et al., 2001), indicating that Scid and DNAPKcs mice are not equivalent in terms of telomere
function and that the Scid mice may carry other
alterations in addition to the DNA-PKcs mutation
(Goytisolo et al., 2001). Overall, the telomeric
Telomere length
normal
40% shorter
20% elongated
normal
30% shorter
(elongated in
combination with
p53 deficiency)
normal
Length
G-strand overhang
normal
normal
normal
normal
normal
normal
phenotype of DNA-PKcs7/7 cells is milder than that
of Ku867/7 cells, and this ®nding is consistent with the
greater severity of phenotypes in the Ku867/7 mouse
than in the DNA-PKcs de®cient mice (Table 1).
PARP1 deficient mice: controversial role of PARP1 at
mammalian telomeres
It has been reported in the past that PARP1 de®ciency
in the mouse (PARPD2 described in Wang et al., 1995)
causes a drastic telomere shortening and numerous
end-to-end fusions (d'Adda di Fagagna et al., 1999). A
more recent study has re-addressed this work using
both Q-FISH and SKY analysis on a similar PARP1
de®cient mouse strain (PARPD4 described in MeÂnissier
de Murcia et al., 1997; Samper et al., 2001a). In
contrast to the previous study, Samper et al. (2001a)
showed that elimination of PARP1 does not signi®cantly a€ect telomere length, and furthermore, does
not result in a dramatic increase in end-to-end fusions.
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Mouse models for telomere function
FA Goytisolo and MA Blasco
588
Table 2 compares the results obtained in the two
studies (d'Adda di Fagagna et al., 1999; Samper et al.,
2001a). The absence of a dramatic telomere phenotype
associated to PARP1 de®ciency, however, is more
consistent with the fact that neither of the two PARP7/7
strains studied show proliferative defects or age-related
phenotypes (MeÂnissier de Murcia et al., 1997; Wang et
al., 1995) (Table 1).
ATM deficient mice
A murine model for AT has been created by disrupting
the ATM gene. These mice present various disease
states associated with aging, including malignancy
(Barlow et al., 1996). A detailed analysis of telomere
function in ATM7/7 mice showed telomere shortening
and the appearance of extrachromosomal TTAGGG
repeats (Hande et al., 2001). Elimination of ATM
homologues in S. pombe also causes a drastic increase
in chromosomal instability and loss of telomeric
repeats (Naito et al., 1998).
The telomerase deficient mouse models: a minimal length
of TTAGGG repeats is required for telomere function
Several telomerase de®cient mice have been generated
in which expression of either Terc (Blasco et al., 1997;
Niida et al., 1998), or Tert (Liu et al., 2000; Nikaido et
al., 1999), has been eliminated. Most of the studies,
however, have been carried out on the model ®rst
described (Blasco et al., 1997), which was obtained by
the elimination of the gene encoding for murine Terc
(Blasco et al., 1995).
Terc7/7 mice have been studied in two di€erent genetic
backgrounds: in the original mixed C57BL6/129Sv background with an average telomere length of 40 Kb (Blasco
et al., 1997), and in a C57BL6 background with an
average telomere length of only 25 Kb (Herrera et al.,
1999b). In both cases, successive generations of Terc7/7
mice show progressive telomere shortening at a rate of 3 ±
5 Kb per generation, and increased end-to-end fusions
(Blasco et al., 1997; Lee et al., 1998; Herrera et al., 1999b).
In agreement with the initial shorter telomere length, only
four generations of C57BL6 Terc7/7 mice were obtained
compared to six generations in the C57BL6/129Sv
background (Herrera et al., 1999b; Lee et al., 1998). In
addition, the severity of phenotypes was greater in the
C57BL6 Terc7/7 mice than in the C57BL6/Sv129 mice
(Herrera et al., 1999b; Rudolph et al., 1999). These
observations indicate that (i) the number of generations
that can be derived in the absence of telomerase is directly
proportional to the initial telomere length and that (ii)
telomeres have to shorten below a threshold length to
become dysfunctional. In both backgrounds the phenotypes associated to telomere dysfunction included: (i)
partial embryonic mortality due to a defective closure of
the neural tube (Herrera et al., 1999a); (ii) small size and
severe intestinal atrophy (Herrera et al., 1999b; Rudolph
et al., 1999); (iii) spleen atrophy and reduced proliferation
of B and T lymphocytes upon mitogenic stimulation
(Herrera et al., 1999b; Lee et al., 1998); (iv) as well as,
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impaired germinal center function upon immunization
(Herrera et al., 2000). Overall, these results support a
essential role of telomerase in highly proliferate organs
(Lee et al., 1998). A recent study has addressed the
capability of telomerase to `repair' critically short
telomeres by re-introducing telomerase activity onto the
late generation telomerase-de®cient mice (Samper et al.,
2001b). This study demonstrates that telomerase is able to
recognize the shortest telomeres and to elongate them to
di€erent lengths, thus preventing the occurrence of endto-end fusions and the appearance of phenotypes in these
mice (Samper et al., 2001b).
Terc7/7 mice: a murine model for telomerase inhibition in
human cancer
Initial studies showed that late generation Terc7/7
MEFs (mouse embryonic ®broblasts) could immortalize and be transformed in culture, suggesting the
activation of ALT in these cells (Blasco et al., 1997).
Indeed, these cells were shown to stabilize or elongate
their telomeres and to accumulate numerous fusions
and translocations (Hande et al., 1999a), indicating
that continued cell culture in the absence of telomerase
is a potent inducer of chromosomal instability, a key
event in cellular transformation (Hande et al., 1999a).
In contrast to cells in culture, however, late
generation Terc7/7 mice show severe proliferative
defects and no survivor mouse lines can be selected.
Nevertheless, evidence for ALT in the context of the
mouse was obtained from studying the germinal
centers of immunized late generation Terc7/7 mice,
where it was possible to select splenocyte populations
which showed very long telomeres (Herrera et al.,
2000). However, the fact that these mice show severe
immune system defects indicates that although ALT
can be selected in certain Terc7/7 cell types it cannot
rescue the phenotypes in the context of the organism.
The chromosomal instability caused by telomere
shortening, has been suggested to trigger a very modest
increase of spontaneous lymphomas and carcinomas in
these mice, also invoking ALT activation (Rudolph et al.,
1999). However, more recent studies indicate that short
telomeres suppress tumor progression. In particular, late
generation Terc7/7 mice show signi®cantly less skin
tumors than wild-type controls upon chemical carcinogenesis of the skin (Gonzalez-Suarez et al., 2000). This
tumor suppressor phenotype coincides with p53 upregulation in Terc7/7 papillomas, that may be sensing short
telomeres as damaged DNA and contributing to cessation
of growth (Gonzalez-Suarez et al., 2000). Genetic
evidence that p53 may act as a sensor for short telomeres
came from the study of Terc7/7/p537/7 mice (Chin et al.,
1999). Elimination of p53 in late generation Terc7/7 mice
accentuates the appearance of chromosomal instability
and can increase the tumor incidence in the case of
Terc7/7/p53+/7 mice (Artandi et al., 2000). This is in
contrast with the previous observation that telomerase
inhibition in human tumor cell lines results in telomere
shortening and massive cell death irrespectively of the
p53 status (Hahn et al., 1999; Zhang et al., 1999). This
Mouse models for telomere function
FA Goytisolo and MA Blasco
apparent contradiction between human and murine
cells could be attributed to the fact that end-to-end
fusions resulting from critical telomere shortening are
quite stable in murine primary cells (Hande et al.,
1999a), possibly as a consequence of having acrocentric
chromosomes. In addition, telomerase de®ciency per se,
or in combination with de®ciencies in tumor suppressor
genes other than p53, signi®cantly reduce carcinogenesis as indicated by the study of intestinal carcinomas
in Terc7/7 mice that also carry the Apcmin mutation
(Su et al., 1992; Rudolph et al., 2001) or in mice
de®cient for Terc and for the INK4A locus (a locus
which includes two potent tumor suppressors p16 and
p19ARF) (Serrano et al., 1996; Greenberg et al., 1999).
All together, these results indicate that telomerase
inhibition and telomere shortening is an e€ective way
to prevent tumor growth. Futhermore, telomerase
inhibition even when telomeres are suciently long
(i.e. early generation Terc7/7 mice), has been shown to
have a negative impact on skin tumorigenesis (GonzaÂlez-SuaÂrez et al., 2000). Conversely, constitutive high
levels of telomerase in the skin of Tert-transgenic mice
increases the incidence of tumors upon chemical
carcinogenesis (GonzaÂlez-SuaÂrez et al., 2001), suggesting that high telomerase activity, hence telomere
maintenance, may be signaling proliferation or survival
by still-to-be de®ned mechanisms.
The effect of DNA damaging agents on the Terc7/7
mouse
Two studies addressed the role of telomeres on the
organismal response to DNA damaging agents (Goytisolo et al., 2000; Wong et al., 2000). In both studies,
late generation Terc7/7 mice when irradiated with
gamma-irradiation showed an enhanced mortality
(Goytisolo et al., 2000; Wong et al., 2000). The cause
of death was radiation toxicity in the gastrointestinal
tract, lymphoid organs and kidney mortality (Goytisolo et al., 2000). The moribund late generation Terc7/7
mice show higher chromosomal damage and greater
apoptosis than wild-type controls (Goytisolo et al.,
2000; Wong et al., 2000). Finally, late generation
Terc7/7 mice show normal frequencies of sister
chromatid exchange and normal V(D)J recombination
suggesting that the DNA double strand break repair
pathways are essentially intact (Goytisolo et al., 2000).
It has been suggested that p53 is required for the
enhanced response of late generation Terc7/7 cell to
DNA damaging reagents (Lee et al., 2001). These
results may have important implications for the
radiotherapy of cancer, as tumors treated with
telomerase inhibitors could lead to a telomere loss,
which is likely to increase the sensitivity of these
tumors to radiotherapy (Goytisolo et al., 2000).
How does it all ®t at the end?
The study of cell culture models for TRF1, TRF2,
TANK1, TANK2, TIN2 and hRAP1 function, together
with the characterization of Terc, Tert, Ku86, DNAPKcs, PARP1, ATM de®cient mice, indicates that
telomere function is regulated at many di€erent levels.
We are still very far from understanding the precise
molecular and functional interactions between the
di€erent activities at the telomere, however, the current
view is that both a minimal length of telomeric repeats and
the telomere binding proteins are necessary for proper
telomere function (Figure 1). On one hand, telomere
shortening may result in TTAGGG-exhausted telomeres
and loss of telomeric proteins, thus disrupting the
telomere structure and exposing the end to telomerase
or to other cellular activities, such as exonucleases or
DNA repair activities (Figure 1). It has been demonstrated recently that telomerase is able to recognize
critically short telomeres in the context of a mouse and
elongate them to di€erent lengths, thus preventing endto-end chromosomal fusions (Samper et al., 2001b). If
telomerase activity is not present in the cell, however,
short telomeres result in end-to-end fusions and loss of
cell viability, as demonstrated using the telomerasede®cient mouse model (Blasco et al., 1997; Lee et al.,
1998). On the other hand, telomere binding proteins can
in¯uence telomere length, as it has been shown for TRF1,
TRF2, TIN2, TANK1, hRAP1 and Ku86 (van Steensel
and de Lange, 1997; Smogorzewska et al., 2000; Samper et
al., 2000; Kim et al., 1999; Smith et al., 1998; Li et al.,
2000), probably by regulating the telomeric structure (i.e.,
formation of T-loops), and thus also determining the
accessibility of telomerase or other elongating activities
(i.e., HR) to the end (Figure 1). In this regard, the
telomerase enzyme is able to recognize speci®cally those
telomeres that are short and that need to be elongated
(Samper et al., 2001b). However, whether telomeric
proteins regulate the access of telomerase itself or of
other elongating activities to the telomere such as HR, has
yet to be demonstrated using double knockout mice for
these proteins and telomerase activity.
The study of murine models has allowed establishing
the consequences of telomere dysfunction for aging and
cancer. In this regard, mice with severe telomeric
phenotypes (i.e., end-to-end fusions) such as the Ku86
de®cient mice (Nussenzweig et al., 1996; Vogel et al.,
1999), and the telomerase null mouse (Herrera et al.,
1999a,b; 2000; Lee et al., 1998; Rudolph et al., 1999),
show severe defects in highly proliferative tissues,
diseases associated to premature aging, as well as a
tumor resistant phenotype which can only be rescued
in the absence of p53 protein. This indicates that p53 is
one of the main mediators of telomere dysfunction and
that either telomere shortening or mutation of a
telomere protein result in similarly severe telomeric
phenotypes (Tables 1 and 2). In contrast, mice that
have very mild or absent telomeric phenotypes, such as
the DNA-PKcs and PARP1 de®cient mice do not show
pathologies associated to premature aging and have a
normal tumor incidence (Tables 1 and 2)(Goytisolo et
al., 2001; Samper et al., 2001a).
The ATM de®cient mice could be an example of a
mouse model that is impaired in both telomere
function and in signaling DNA damage. This would
589
Oncogene
Mouse models for telomere function
FA Goytisolo and MA Blasco
590
explain that even though these mice have a severe
telomeric phenotype (i.e. telomere shortening and
chromosomal aberrations), which correlates with premature aging pathologies, they also have a higher
incidence of spontaneous tumors. This is similar to that
described for Ku867/7/p537/7 and Terc7/7/p537/7
mice, and suggests that both ATM and p53 may be
signaling dysfunctional telomeres as damaged DNA,
hence triggering cell cycle arrest or apoptosis.
Acknowledgments
We thank Manuel Serrano for helpful comments. Research
at the laboratory of MA Blasco is funded by Swiss Bridge
Award 2000, by grants PM97-0133 from the Ministry of
Science and Technology (MCT), Spain, and by grants
FIGH-CT1999-00009, FIGH-CT-1999-00002 and QLG11999-01341, from the EU, and by the Department of
Immunology and Oncology (DIO). The DIO was founded
and is supported by the Spanish Research Council (CSIC)
and by the Pharmacia Corporation.
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