Telomere Length Dynamics and Chromosomal Instability in Cells Derived from Telomerase Null Mice

“Indeed, telomere shortening to a critical length has been proposed to limit the life span of somatic cells in humans (Harley et al., 1990; Counter et al., 1992).”

“It has been recently shown that the introduction of the telomerase catalytic subunit in primary human cells is sufficient to restore enzymatic activity, to elongate and maintain telomeres, and, in some cell types, to sustain immortal growth ( Bodnar et al., 1998; Wang et al., 1998; Kiyono et al., 1998).”

“In such cells the enzyme telomerase seems to be the main mechanism for the maintenance of telomere length (Greider and Blackburn, 1985; Morin, 1989; Yu et al., 1990; Singer and Gottschling, 1994; McEachern and Blackburn, 1996; Nakamura et al., 1997).”

“These mechanisms, which can be either telomerase dependent (reviewed in Shay and Bacchetti, 1997 ) or independent (this paper and Bryan et al., 1995, 1997), can prevent telomeres from further shortening, allowing the immortal growth of cells that have critically short telomeres and a high degree of genetic instability.”

“col (Todaro and Green, 1963) in dishes of 10 cm diam, seeding 10 6 cells ev-”

“, 1998), an RNA molecule that contains the template for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al.”

“The details are explained elsewhere (Blasco et al., 1997).”

“Indeed, telomere shortening to a critical length has been proposed to limit the life span of somatic cells in humans (Harley et al., 1990; Counter et al., 1992).”

“…by a protein catalytic subunit with similarity to other reverse transcriptases (Lingner et al., 1997; Harrington et al., 1997a; Kilian et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997; MartínAddress correspondence to María A. Blasco, Department of Immunology and Oncology, Centro Nacional…”

“It has been shown recently that telomere binding proteins are of crucial importance to prevent end-to-end fusions in human chromosomes ( Van Steensel et al., 1998 ).”

“, 1998), an RNA molecule that contains the template for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al.”

“In such cells the enzyme telomerase seems to be the main mechanism for the maintenance of telomere length (Greider and Blackburn, 1985; Morin, 1989; Yu et al., 1990; Singer and Gottschling, 1994; McEachern and Blackburn, 1996; Nakamura et al., 1997).”

“Greider, C.W., and E.H. Blackburn.”

“Such variation between littermates could be the basis for the fact that only a percentage of KO-G6 mice show defects associated with telomere loss (Lee et al., 1998; Herrera el al., 1999).”

“Telomerase is formed by a protein catalytic subunit with similarity to other reverse transcriptases (Lingner et al., 1997; Harrington et al., 1997a; Kilian et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997; MartínAddress correspondence to María A. Blasco, Department of Immunology and…”

“…been recently shown that the introduction of the telomerase catalytic subunit in primary human cells is sufficient to restore enzymatic activity, to elongate and maintain telomeres, and, in some cell types, to sustain immortal growth (Bodnar et al., 1998; Wang et al., 1998; Kiyono et al., 1998).”

“Telomerase is a reverse transcriptase that elongates telomeres by synthesizing TTAGGG repeats onto the 3 9 ends of chromosomes.”

“Interestingly, it is possible to isolate survivor yeast strains that are able to stabilize their chromosomes by mechanisms that involve telomere elongation by recombination or chromosome circularization (Lundblad and Blackburn, 1993; McEachern and Blackburn, 1996; Nakamura et al., 1997, 1998; Naito et al., 1998).”

“These specialized structures constitute the final 10 kb of all human chromosomes, and the final 12–80 kb of all mouse chromosomes (Lansdorp et al., 1996; Zijlmans et al., 1997).”

“Recent studies in yeast and mice have proven that telomeres are essential to maintain chromosomal stability (Sandell and Zakian, 1993; Blasco et al., 1997; Lee et al., 1998; Naito et al., 1998; Nakamura et al., 1998).”

“, 1998), an RNA molecule that contains the template for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al.”

“…for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998).”

“…been recently shown that the introduction of the telomerase catalytic subunit in primary human cells is sufficient to restore enzymatic activity, to elongate and maintain telomeres, and, in some cell types, to sustain immortal growth (Bodnar et al., 1998; Wang et al., 1998; Kiyono et al., 1998).”

“In such cells the enzyme telomerase seems to be the main mechanism for the maintenance of telomere length (Greider and Blackburn, 1985; Morin, 1989; Yu et al., 1990; Singer and Gottschling, 1994; McEachern and Blackburn, 1996; Nakamura et al., 1997).”

“Telomerase is formed by a protein catalytic subunit with similarity to other reverse transcriptases (Lingner et al., 1997; Harrington et al., 1997a; Kilian et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997; MartínAddress correspondence to María A. Blasco, Department of Immunology and…”

“The loss of control of the chromosomal number or aneuploidy is an alteration commonly observed in cultured cells (Zindy et al., 1997).”

“FISH with Cy-3 labeled (CCCTAA) 3 peptidenucleic acid, and subsequent quantitative analysis of digital images were performed as described (Zijlmans et al., 1997).”

“, Rad 52 (McEachern and Blackburn, 1996) or in DNA repair, such as DNA-PK, ATM, Rad 3, tel11, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres (Greenwell et al., 1995; Boulton and Jackson, 1996; Metcalfe et al., 1996; Nugent et al., 1998; Naito et al., 1998).”

“…for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998).”

“The Journal of Cell Biology, Volume 144, 1999 594 ficient to prevent telomere erosion as it has been proposed previously for other cell types (Counter et al., 1994; Chiu et al., 1996) or, alternatively, that telomere length in these cultured cells is not tightly regulated around a fixed length.”

“…and Blackburn, 1996) or in DNA repair, such as DNA-PK, ATM, Rad 3, tel11, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres (Greenwell et al., 1995; Boulton and Jackson, 1996; Metcalfe et al., 1996; Nugent et al., 1998; Naito et al., 1998).”

“…for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998).”

“…and Blackburn, 1996) or in DNA repair, such as DNA-PK, ATM, Rad 3, tel11, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres (Greenwell et al., 1995; Boulton and Jackson, 1996; Metcalfe et al., 1996; Nugent et al., 1998; Naito et al., 1998).”

“, 1998), an RNA molecule that contains the template for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al.”

“The Journal of Cell Biology, Volume 144, 1999 594 ficient to prevent telomere erosion as it has been proposed previously for other cell types (Counter et al., 1994; Chiu et al., 1996) or, alternatively, that telomere length in these cultured cells is not tightly regulated around a fixed length.”

“…and Blackburn, 1996) or in DNA repair, such as DNA-PK, ATM, Rad 3, tel11, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres (Greenwell et al., 1995; Boulton and Jackson, 1996; Metcalfe et al., 1996; Nugent et al., 1998; Naito et al., 1998).”

“, 1998), an RNA molecule that contains the template for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al.”

“Similarly, other studies have suggested a role of telomeres and telomere binding proteins in chromosome segregation and meiosis (Bass et al., 1997; Chikashige et al., 1997; Chua et al., 1997; Conrad et al., 1997, Cooper et al., 1998; Naito et al., 1998).”

“Recent studies in yeast and mice have proven that telomeres are essential to maintain chromosomal stability (Sandell and Zakian, 1993; Blasco et al., 1997; Lee et al., 1998; Naito et al., 1998; Nakamura et al., 1998).”

“Similarly, other studies have suggested a role of telomeres and telomere binding proteins in chromosome segregation and meiosis (Bass et al., 1997; Chikashige et al., 1997; Chua et al., 1997; Conrad et al., 1997, Cooper et al., 1998; Naito et al., 1998).”

“…for the synthesis of telomeric repeats (Greider and Blackburn, 1989; Singer and Gottschling, 1994; Blasco et al., 1995; Feng et al., 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998).”

“, Rad 52 (McEachern and Blackburn, 1996) or in DNA repair, such as DNA-PK, ATM, Rad 3, tel11, and TEL 1, have been proposed as candidates because their mutation results in accelerated loss of telomeres (Greenwell et al., 1995; Boulton and Jackson, 1996; Metcalfe et al., 1996; Nugent et al., 1998; Naito et al., 1998).”

“Interestingly, it is possible to isolate survivor yeast strains that are able to stabilize their chromosomes by mechanisms that involve telomere elongation by recombination or chromosome circularization (Lundblad and Blackburn, 1993; McEachern and Blackburn, 1996; Nakamura et al., 1997, 1998; Naito et al., 1998).”

“In addition, the study of Tetrahymena mutants with an altered telomerase RNA template suggests an important role of telomeres in chromatid separation during anaphase (Kirk et al., 1997).”

“Similarly, other studies have suggested a role of telomeres and telomere binding proteins in chromosome segregation and meiosis (Bass et al., 1997; Chikashige et al., 1997; Chua et al., 1997; Conrad et al., 1997, Cooper et al., 1998; Naito et al., 1998).”

“Moreover, there are a variety of human established cell lines that do not show detectable telomerase activity ( Bryan et al., 1995, 1997 ).”

“Similarly, other studies have suggested a role of telomeres and telomere binding proteins in chromosome segregation and meiosis (Bass et al., 1997; Chikashige et al., 1997; Chua et al., 1997; Conrad et al., 1997, Cooper et al., 1998; Naito et al., 1998).”

“300 divisions, suggesting that the growth of ES cells is telomerase dependent (Niida et al., 1998).”

“This variation in telomere length could be the basis for the variable penetrance of the phenotypes described in 6th generation mTER 2 / 2 mice (Lee et al., 1998; Herrera et al., 1999).”

“The hybridization was performed as described (Hande et al., 1996).”

“, 1995; McEachern and Blackburn, 1995), and other associated proteins (Collins et al., 1995; Harrington et al., 1997b; Nakayama et al., 1997; Gandhi and Collins, 1998).”

“Q-FISH has become the method of choice for the analysis of telomere length in the mouse (Lansdorp, 1997) and for further details the reader is referred to the references above.”

“Here we find that ditions (Hande et al., 1999). the ability of the mTR2/2 mice to respond to various physiological stresses is compromised and declines Progressive Increase in the Rate of Spontaneous with increasing age of the mouse.”

“) mTRϪ/Ϫ mice (Table 1) (Blasco et al., 1997; Hemann et al., 2001b; Lee et al., 1998).”

“The technique is well established to study telomere biology in many settings (8, 81).”

“In addition, our finding that mTR −/− MEFs do not senesce in 3% oxygen, in contrast to telomerase-negative human cells, is most probably caused by the ability of mouse chromosomes to stabilize telomeres in the absence of telomerase until alternative telomere maintenance pathways are induce”

“The severity of these degenerative phenotypes parallels the degree of telomere dysfunction in successive Terc −/− generations, as measured by the number of chromosomal fusions and anaphase bridges and, in a more recent study, the number of DNA damage foci at telomere”

“ALT mechanisms are also activated in Terc -deficient mice in cultured mouse embryonic fibroblasts and embryonic stem cell”

“In mammals, the telomerase-deficient mouse model has been instrumental in demonstrating telomere maintenance in the absence of telomerase, in both cultured mouse embryonic fibroblasts (MEFs”

“To date, clear evidence for ALT activity has only been found in abnormal situations, including human tumors, immortalized human cell lines (Table 1), and in telomerase-null mouse cell lines (Bryan et al., 1995, 1997a; Hande et al., 1999; Niida et al., 2000).”

“FISH was performed on metaphase spreads using telomere and centromere specific peptide nucleic acid (PNA) probes (Applied Biosystems) labelled with Cy-3 and FITC respectively as explained earlier [38,39].”

“The murine ALT lines that arise readily in the absence of telomerase maintain atypically short rather than long telomeres (Hande et al., 1999), suggesting some species-speci®city in ALT pathway requirements.”

“Disease states associated with telomerase deficiency appear earlier in mice with short telomeres”

“Mammalian telomeres consist of repetitive G-rich sequences and associated proteins, which cap the ends of linear chromosomes and maintain chromosomal stabilit”

“This contributes to genomic instability (Hande et al. 1999).”

“These findings agree with previous reports showing activation of ALT pathways in late-generation Terc −/− cell”

“, resulting in cycles of chromatin bridge breakage and fusio”

“The technique is well established to study telomere biology in many settings [52, 53] and may be a good choice for measuring telomere length in rare cells [54]; the main characteristics of the assay are summarized in Table 1.”

“A likely explanation for this anomalous finding is that shortened telomeres have a dual effect: they initiate checkpoint signals that provoke a cell-cycle arrest or trigger apoptosis, but they can also cause chromosomal instabilit”

“In addition to DNA breaks, anaphase bridges can be caused by abnormal shortening of the telomeres (de Lange 2002; Hande et al. 1999) or by persistent chromatid cohesion (Coelho et al.”

“Chromosome 2 was chosen for the study because, in the mouse, this chromosome has shorter telomeres than average (Hande et al., 1999).”

“Indeed, impairment of telomere function through telomere attrition or loss of telomere binding proteins leads to the triggering of DNA damage responses and to telomeres engaging in inappropritate recombination events with one another and with other loci.(183,184) In mammals, telomeres are covered by binding proteins (…”

“Telomeres are specialized structures consisting, in human cells, of TTAGGG repeat sequences (4, 33) which, together with specific binding proteins (5), form caps at the ends of chromosomes that are essential for chromosome stability (11, 17).”

“Telomeres were detected with a Cy3-conjugated Telomere PNA probe (Cy3-OO-(CCCTAA)3; Panagene, Korea) by FISH as described in Hande et al (1999), with the only modification of a DNA denaturation step in 0.5M NaOH 1M NaCl for 8min.”

“In this regard, recombination-based ALT pathways are activated in telomerase-deficient mice (Blasco et al, 1997; Hande et al, 1999; Rudolph et al, 1999; Herrera et al, 2000; Niida et al, 2000; Chang et al, 2003).”

“A cardinal indicator of telomere dysfunction is endto-end chromosomal fusion, which becomes more frequent in later-generation Terc / mice (15).”

“1B; Blasco et al. 1997; Hande et al. 1999; Hemann et al. 2001).”

“Quantitative fluorescence in situ hybridisation (qFISH) was performed using telomere sequence-specific peptide nucleic acid (PNA) probe labelled with Cy3 as described [17,18].”

“Indeed, oncogenic transformation is increased manifold in telomerase knockout mice (Blasco et al., 1997; Hande et al., 1999).”

“Activation of ALT in tumor cells is a much rarer event than activation of telomerase (20), and may normally occur under selective pressure when expression of the enzyme is prevented (38,39).”

“They exhibited defective spermatogenesis and haematopoiesis, coupled with telomere-length reductions and abnormal cytogenetic profiles, indicating an essential role for telomerase and telomeres in the maintenance of genomic integrity and in the long-term viability of high-renewal tissues [29, 64].”

“Fluorescence beads (Molecular Probes, Eugene, OR, USA) were used as quantification standards, as described previously (Hande et al., 1999).”

“cgi?l=21748"> Terc , are also able to undergo immortalizatio”

“The existence of telomere-rescue mechanisms in late generation mTR–/– mice is supported by previous reports showing that late generation mTR–/– cells in culture can maintain telomeres without telomerase (Hande et al., 1999).”

“However, telomeres shorten progressively and this shortening is accompanied by an increase in chromosomal abnormalities (Blasco et al., 1997; Hande et al., 1999b).”

“Metaphase spreads at selected PDL from wild-type (WT) and mTER ES cells were hybridized as described earlier (17, 18).”

“This result contrasts with what is seen in wild-type strains and with the slower 2–7-kb loss of sequences per generation that is seen in the murine telomerase RNA-knockout strain”

“This was due mainly to the absence of telomerase activity and was associated with an increase in aneuploidy and end-to-end chromosomal fusions (Blasco et al., 1997; Hande et al., 1999).”

“In contrast, chromosomes from telomerase null cells with shortened telomeres form increased numbers of p-p arm fusions (58).”

“Telomeraseknockout mouse models, in which animals possess critically short telomeres, exhibit increased cancer rates (9-11).”

“2) suggested less telomere shortening relative to Atm+/+ cells than observed at chromosome ends by Q-FISH (Fig.”

“Most of these unfavorable effects presumably resulted from the fusion of the chromosomes, various translocations and loss of certain chromosomal sites due to progressive loss of the telomeric DNA (Lee et al., 1998; Hande et al., 1999; Herrera et al., 1999).”

“that are null for the telomerase RNA gene and have no detectable telomerase activity (14).”

“Accordingly, we used the absence of a telomere fluorescence signal on a chromosome end as an indicator of telomere dysfunction (Blasco et al., 1997; Hande et al., 1999; Hemann et al., 2001).”

“Telomere shortening in cells from telomerase-deficient mice results in loss of telomere function and consequently genetic instability with chromosomal end-toend fusions (47).”

“The relationship between telomere length and chromosome fusions in mammalian cells became evident in telomerase-deficient mice with short telomeres where a high frequency of Robertsonian fusions was observed (8,27).”

“By using the PNA-telomere probe [Hande et al., 1999] no interstitial telomeric signals were detected, although the probe hybridized to the Yq telomeric sequences (Fig.”

“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…”

“The average telomere length decreases nonlinearly in the presence of symmetric stem cell self-renewal The average telomere length decreases in most human tissues with age (Harley et al., 1990).”

“…data in the literature support a correlation between exaggerated shortening and telomere dysfunction (Counter et al., 1992; Blasco et al., 1997; Hande et al., 1999; Gisselsson et al., 2001; Hackett et al., 2001), this relationship has never been directly shown for specific chromosome…”

“Hande et al. (1999) shed some light on this conundrum by showing that, in immortal cells from telomerase-null mice, telomere length became stabilized in a chromosome-specific manner, indicating that a telomerase-independent telomere maintenance mechanism can be activated in mouse cells.”

“Telomeres are known to play a key role in chromosome segregation (Sandell and Zakian 1993; Blasco et al. 1997; Kirk et al. 1997; Hande et al. 1999).”

“Also, chromosomal instability increases in fibroblasts derived from older telomerase (mouse transferase RNA, mTER)-null mice that have compromised telomerase activity [83].”

“F or p er so na l u se o nl y. Acentric chromosome: a chromosome lacking a centromere Dicentric chromosome: a chromosome with two centromeres produce acentric or dicentric chromosomes that can attach improperly to the mitotic spindle and missegregate (Burrell et al. 2013, Hande et al. 1999).”

“Degradation of telomeres has been shown to lead to whole chromosomal instability, via telomere end fusion, and the generation of breakage–fusion–bridge cycles within chromosomes, which leads to gene amplification and gene dosage imbalance, an important risk factor for cancer (53–55) .”

“Increased chromosome fusion and chromosome instability has been observed in cells from telomerasedeficient mice ( 27 ) and in mammalian cells with a variety of mutations that affect telomere function, including DNA-PKcs (4, 24), Ku (4, 31), and TRF2 (79).”

“Vol. 14, 556–570, February 2003”