Many ways to telomere dysfunction: in vivo studies using ... - Nature

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Oncogene (2002) 21, 584 ± 591 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

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 dierent 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 dierent 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 (Grith 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 (Grith 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

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Figure 1 The mammalian telomere is depicted in two alternative conformations. The `closed' conformation is based on the T-loop model described by Grith 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 eects on telomere length and end-protection could be mediated by this property (Grith 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 dierent telomeric proteins at the mammalian telomere. The study of human premature aging syndromes has contributed to the identi®cation of proteins that aect 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 aect telomere function include Bloom's and Werner syndromes, the respective genes mutated Oncogene


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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 suciently 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 eective 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 dierent 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 aecting 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 eort will be devoted to determine the roles of HR, MMR, WRN and BLM proteins in mammalian ALT by studying the dierent mouse models for these proteins. Oncogene

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


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

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* Original description of the mice. The references for the dierent 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 aect 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 aect telomere length, and furthermore, does not result in a dramatic increase in end-to-end fusions. Oncogene


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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 dierent 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 dierent 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


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 eective way to prevent tumor growth. Futhermore, telomerase inhibition even when telomeres are suciently 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 dierent levels. We are still very far from understanding the precise molecular and functional interactions between the dierent 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 dierent 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

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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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