§
* Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus Cantoblanco, Madrid
E-28049, Spain; Terry Fox Laboratory, British Columbia Cancer Research Center, Vancouver, British Columbia V5Z 1L3,
Canada; and § Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada
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Abstract |
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To study the effect of continued telomere
shortening on chromosome stability, we have analyzed
the telomere length of two individual chromosomes
(chromosomes 2 and 11) in fibroblasts derived from
wild-type mice and from mice lacking the mouse telomerase RNA (mTER) gene using quantitative fluorescence in situ hybridization. Telomere length at both
chromosomes decreased with increasing generations of
mTER/
mice. At the 6th mouse generation, this telomere shortening resulted in significantly shorter chromosome 2 telomeres than the average telomere length
of all chromosomes. Interestingly, the most frequent fusions found in mTER
/
cells were homologous fusions
involving chromosome 2. Immortal cultures derived
from the primary mTER
/
cells showed a dramatic accumulation of fusions and translocations, revealing that
continued growth in the absence of telomerase is a potent inducer of chromosomal instability. Chromosomes
2 and 11 were frequently involved in these abnormalities suggesting that, in the absence of telomerase, chromosomal instability is determined in part by chromosome-specific telomere length. At various points during
the growth of the immortal mTER
/
cells, telomere
length was stabilized in a chromosome-specific man-ner. This telomere-maintenance in the absence of
telomerase could provide the basis for the ability of
mTER
/
cells to grow indefinitely and form tumors.
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Introduction |
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EUKARYOTIC linear chromosomes are capped by a
special structure known as the telomere. In vertebrates, telomeric DNA consists of tandem repeats
of the sequence TTAGGG (reviewed in Blackburn, 1991).
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
). More than 50 years ago, Barbara McClintock
observed that chromosome ends lacking telomeres have a
tendency to fuse (McClintock, 1941
). 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
).
In most human cells, telomeres shorten with each cell division due to the incomplete replication of linear DNA
molecules and the absence of telomere-elongating mechanisms (reviewed in Greider, 1996). 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
). Cell types that proliferate indefinitely, such as unicellular eukaryotes, germline cells, and
immortal cells, maintain their telomeres at a constant length. 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 a reverse transcriptase that elongates
telomeres by synthesizing TTAGGG repeats onto the 3'
ends of chromosomes. Thus, telomerase can compensate
for the incomplete replication of telomeres (reviewed in
Greider, 1996). 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
; Martín-Rivera et al., 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., 1995
;
Harrington et al., 1997b
; Nakayama et al., 1997
; Gandhi
and Collins, 1998
).
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
). Yeast and mouse strains that lack any of the essential telomerase components do not have detectable telomerase activity and undergo telomere shortening and loss of
viability after a variable number of generations, indicating
that active telomerase is essential to maintain telomere length in vivo (Singer and Gottschling, 1994
; McEachern
and Blackburn, 1996
; Blasco et al., 1997
; Nakamura et al.,
1997
, 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
). Telomerase-independent telomere elongation has been also described in some immortal human cell lines that do not
have detectable telomerase activity (Bryan et al., 1995
,
1997
).
The analysis of telomere length in primary cells from
mouse telomerase RNA (mTER)1 knock out mice that lack
telomerase activity has revealed that telomeres shorten at
a rate of 4.8 ± 2.4 kb per generation, and that this is accompanied by an increase in chromosomal instability (Blasco et al., 1997). The loss of telomere repeats was not
obvious from conventional telomere length measurements
by Southern analysis but was readily apparent using
quantitative fluorescence in situ hybridization (Q-FISH;
Lansdorp et al., 1996
; Zijlmans et al., 1997
; Martens et
al., 1998
). With Q-FISH, the fluorescence intensity at individual telomeres is calculated from digital images using image analysis techniques after quantitative hybridization of denatured telomere target sequences with directly
labeled and highly efficient (CCCTAA)3 peptide nucleic
acid probes. 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.
Despite lacking telomerase activity, mTER/
cells have
divided >500 times in culture (this paper), suggesting that
telomerase activity per se is not essential for immortalization and/or that there might be telomerase-independent
mechanisms maintaining telomeres in these telomerase-deficient cells. Here, we have analyzed telomere length
dynamics at the level of individual chromosomes in primary and immortal cells that lack the mTER gene. Furthermore, we have characterized the type and frequency
of fusions and translocations that accumulate in these cells
as a consequence of proliferation in the absence of telomerase activity. The results of this analysis underscore the
importance of telomeres in the maintenance of genomic stability.
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Materials and Methods |
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Cell Culture
Mouse embryonic fibroblasts (MEFs), were prepared from day 13.5 embryos derived from wild-type (wt) and mTER/
mice from different generations. The details are explained elsewhere (Blasco et al., 1997
). The
first culture directly obtained from embryos was considered as population
doubling 2 (PD 2). Serial cultures were done according to the 3T3 protocol (Todaro and Green, 1963
) in dishes of 10 cm diam, seeding 106 cells every 3 d. The doubling number of each passage was calculated using the
formula PD = log (nf/n0)/log2; where n0 is the initial number of cells (106)
and nf is the final number of cells. The details on the establishment of the
immortal cell lines are explained elsewhere (Blasco et al., 1997
).
Fluorescence In Situ Hybridization with PNA-telomere Probe
Plates containing primary MEFs or established cell lines were treated with
colcemid (0.1 µg/ml) for 4-5 h and subsequently trypsinized and spun for
8 min at 120 g. After hypotonic swelling in sodium citrate (0.03 M) for 25 min at 37°C, cells were fixed in methanol/acetic acid (3:1). After 2-3 additional changes of fixative, cell suspensions were dropped on wet, clean
slides and dried overnight. FISH with Cy-3 labeled (CCCTAA)3 peptide-nucleic acid, and subsequent quantitative analysis of digital images were performed as described (Zijlmans et al., 1997). The slide coordinates of the metaphase images captured were noted to relocate the same metaphase after FISH with minor satellite DNA (see below) and mouse
chromosome probes (Oncor).
Quantitative Image Analysis
Digital images were recorded with a MicroImager MI1400-12 camera
(Xillix) on an Axioplan fluorescence microscope (Zeiss). Microscope control and image acquisition was performed with a dedicated software (SSM;
Xillix). Separate DAPI and Cy-3 images were subjected to telomere fluorescence analysis by using ad dedicated computer program, TFLTELO
(Martens et al., 1998). Chromosomes and telomeres were identified
through segmentation of the DAPI image and the Cy-3 image, respectively. Both images were combined and corrected for pixel shifts. The integrated fluorescence intensity for each telomere was calculated after correction for image acquisition exposure time. Finally, the integrated
fluorescence intensity of individual telomeres is expressed in a table for
each chromosome, which can be subjected to editing. Each metaphase of
40 chromosomes (in the mouse) yields 160 telomere spots and a typical
analysis of 15-20 metaphases produces several thousand telomere fluorescence values. Because of the large number of data points in Q-FISH analysis, the standard error of mean telomere fluorescence estimates is typically small (for example, less than a few percent of the average) despite
considerable variation in individual telomere fluorescence values.
Details of the calibration procedures used to reliably measure telomere
fluorescence intensity are explained elsewhere (Martens et al., 1998). In
brief, two levels of calibration values were used. First, to correct for daily
variations in lamp intensity and alignment, images of fluorescent beads
(orange beads, size 0.2 µm; Molecular Probes) were acquired and similarly analyzed with the analysis software. Second, relative telomere fluorescence units (TFU) were extrapolated from the plasmid calibration. For this, we hybridized and analyzed plasmids with a defined (TTAGGG)n length of 0.15, 0.4, 0.8, and 1.6 kb. There was a linear correlation (r2 = 0.99) for plasmid fluorescence intensity and (TTAGGG)n length with a
slope of 48.7 (Martens et al., 1998
). Therefore, the calibration corrected
telomere fluorescence intensity (ccTFI) of each telomere was calculated
according to the formula: ccTFI = (Bea1/Bea2) × (TFI/48.7), where Bea1
equals the fluorescence intensity of beads when plasmids were analyzed,
Bea2 equals the fluorescence intensity of beads when sample x was analyzed, and TFI equals unmodified fluorescence intensity of a telomere in
sample x.
A restriction of this calibration method is that the actual telomeres are outside the range of (TTAGGG)n length of the plasmids. The assumption is made that the linear correlation obtained between fluorescence intensity and telomere insert size in plasmid is maintained in the higher range in chromosomal DNA.
FISH with Minor Satellite DNA
The murine minor satellite DNA probe was a gift from Drs. J.B. Rattner
and Shu-Lin Liu (Calgary University, Calgary, Canada). The minor satellite probe labels all centromeres near the kinetochore except mouse chromosome Y (Wong and Rattner, 1988). The probe was labeled with Spectrum-green dUTP by nick translation and then precipitated with ethanol,
using salmon sperm DNA as carrier. The probe was dissolved in a hybridization buffer containing 30% deionized formamide, 2× SSC, 10% dextran sulphate and 50 mM phosphate buffer (pH 7.0) to a concentration of
20 ng/µl. The slides used for telomere FISH were washed and then hybridized with the minor satellite DNA probe. The hybridization was performed as described (Hande et al., 1996
). Slides were incubated with pepsin
(0.005%) in 10 mM HCl for 10 min at 37°C, washed with PBS containing
50 mM MgCl2, and then treated with 1% formaldehyde in PBS/MgCl2 for 10 min at room temperature. After one more wash in PBS, slides were
dehydrated in a 70, 90, and 100% ethanol series. The labeled probe was
diluted with hybridization buffer to a final concentration of 4 ng/µl, and 20 µl were added on each slide. The probe and the target DNA were denatured simultaneously at 80°C, for 3.5-4.0 min. Hybridization was carried
out overnight at 37°C in a moist chamber. After hybridization, the slides
were washed three times with 30% formamide, 2× SSC buffer (pH 7.0)
for 5 min at 37°C, followed by two washes in 2× SSC at room temperature. The slides were dehydrated and embedded with Vectashield mounting medium (Vector Labs) containing 1 µg/ml propidium iodide. About
15-25 metaphases per group that had already been used for telomere
measurements were relocated and observed under the microscope for
characterizing the end-to-end fusions.
FISH with Mouse Chromosome Paint Probes
Chromosome painting probes for chromosomes 2 and 11 were used to follow telomere length in these chromosomes. The same slides that were analyzed after FISH with telomere probe and minor satellite DNA were processed for the third FISH with chromosome painting probes. The chromosome painting probes were purchased from Oncor. FISH was performed according to the manufacturer's instructions. About 15-25 metaphases were relocated using the known coordinates in the microscope and analyzed.
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Results |
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Telomere Dynamics of Individual Chromosomes in
mTER/
Telomerase-deficient Embryos from
Different Generations
Primary cells (passage 1 mouse embryonic fibroblasts,
MEFs) derived from wt embryos and from mTER/
embryos from the 1st (G1) to the 6th (G6) generation were
obtained following the scheme previously described (Blasco
et al., 1997
). The telomere length of individual chromosomes (chromosomes 2 and 11) was measured using
Q-FISH and chromosome painting. Chromosome 2 was
chosen because it consistently has relatively short telomeres in several mouse strains (Hande, M.P., and P. Lansdorp, unpublished results; this paper). Chromosome 11 is
the mouse homologue of human chromosome 17, which
was found to have relatively short telomeres in all individuals analyzed to date (Martens et al., 1998
).
Fig. 1 shows the mean and standard error of telomere
fluorescence intensity of all telomeres together (average
of q- and p-arms), and also of q- and p-arms separately
from primary MEFs of both wt and mTER/
embryos of
the 2nd (KO2-G2), 4th (KO7-G4), and 6th generation (littermate embryos KO9-G6 and KO11-G6; littermate embryos KO1-G6 to KO4-G6 and embryo KO5-G6). Despite
considerable variation between individual telomere fluorescence values (see for example Fig. 2 D), the large number of data points (>1,000) resulted in insignificant standard error values in the telomere values of all chromosomes. The standard error was also small for individual telomeres on chromosomes 2 and 11, with smaller
number of data points (50-100; see Figs. 1 A and 2, B and
C). The standard error rather than the standard deviation
is shown for clarity and presentation purposes only. The
average telomere fluorescence of all chromosomes decreased linearly during successive generations of mTER
/
mice. The average telomere shortening was 3.9 kb per
generation (calculated as described in Fig. 1 B). This
shortening affected both q-telomeres (telomeres of the
q-arms) that showed a shortening of 4.17 kb per generation, and p-telomeres (telomeres of the p-arms) that
showed a shortening of 3.7 kb per generation. As a result, the difference in telomere length between p- and q-arm telomeres was maintained throughout the six mouse generations (Fig. 1 B). The loss of telomere repeats in mTER
/
mice resulted in an average length of 14.5 and 22.4 kb for
p- and q-telomeres, respectively, in cells derived from the
6th generation. When we measured the mean telomere
fluorescence of chromosome 2, the estimated rate of telomere shortening per generation was 3.4 kb for both 2q-
and 2p-telomeres (Fig. 1, A and B). This telomere shortening resulted in 6th generation 2p- and 2q-telomeres of an average length of 7.6 kb and 16.2 kb, respectively (embryo KO9-G6 had an estimated 2p-telomere length of only
0.15 ± 0.1 kb), shorter than the average of all chromosomes. In the case of chromosome 11, the average telomere fluorescence of 11q and 11p-telomeres decreased at
an average rate of 5.2 and 5.6 kb per generation, respectively, up to the 4th generation (Fig. 1, A and B). Interestingly, from the 4th (embryo KO-G4) to the 6th generation
(the average of seven different embryos) we did not detect
the expected telomere shortening in any of chromosome
11 telomeres (Fig. 1 B). In contrast, there was a 6-kb increase in the telomere length at the 6th generation (Fig. 1,
A and B). Altogether, these results suggest that in the absence of telomerase activity, telomere shortening occurs at
a similar rate in all chromosome ends. However, it appears
that mechanisms that prevent telomere shortening in the
absence of telomerase act differentially on different telomeres. In our study, chromosome 11 telomeres did not
show the predicted shortening with increasing generations in seven different embryos, while chromosome 2 telomeres continued to shorten throughout the six generations
of mTER
/
mice (see Discussion). In this study, we cannot rule out that telomerase independent mechanisms of
telomere maintenance are also operating in early generation mTER
/
or wt mice.
|
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Interestingly, in the different mTER/
cells derived
from 6th generation embryos, we observed a marked heterogeneity in the mean telomere fluorescence. This heterogeneity affected both chromosome 2 and 11 telomeres and
was also observed in cells derived from littermate embryos. This variation in telomere length could be the basis
for the variable penetrance of the phenotypes described in
6th generation mTER
/
mice (Lee et al., 1998
; Herrera et
al., 1999
).
Telomere Dynamics of Individual Chromosomes in
Spontaneously Immortalized mTER/
Cell Lines
Serial passage of mouse embryonic fibroblasts allows the
selection of oligoclonal populations with the capacity to
stably proliferate in culture. We had previously described
that serial passage of mTER/
MEFs according to a 3T3
protocol resulted in the selection of immortal cell lines in a
manner similar to that of mTER+/+ MEFs, indicating that
telomerase activity is not essential for the immortalization
of mouse cells (Blasco et al., 1997
). Although some differences were observed in the growth rate between wt and mTER
/
cell lines, all cultures have shown a continued
growth that exceeded 500 PDs for G1 MEFs and 250 PDs
for G6 MEFs (not shown). To understand the basis for the
continuous growth of these telomerase negative cells we
have analyzed their telomere dynamics. Fig. 2 A shows the
mean telomere fluorescence of q- (black bars) and p-telomeres (gray bars), separately, during increasing PDs of wt
and mTER
/
cells. We calculated that telomeres of wt
cells, Wt14, underwent a modest shortening at an estimated rate of 24.8 bp per PD (Fig. 2 A). The occurrence of
telomere shortening in wt MEFs could indicate that the
level of telomerase activity present in these cells is not sufficient 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.
In contrast to wt cells, mTER/
cell lines derived from
1st (KO16-G1 and KO19-G1), 2nd (KO2-G2), and 4th
generation (KO7-G4) embryos, showed a marked decrease in the telomere fluorescence of both p- and q-telomeres (Fig. 2 A). The estimated average telomere loss in
the different cell lines ranged between 65 and 108 bp per
PD, similar to the shortening rate described for human
cells that do not express telomerase. This indicates that in
these mTER
/
cells that had escaped senescence and are
immortal, the mean telomere length continues to shorten
with increasing passage number. Interestingly, the rate of
telomere shortening at both p- and q-telomeres decreased
at later passages (PDs 215 and 322) of the KO16-G1 cell
line, suggesting the activation of telomere maintenance mechanisms when telomeres shorten to a critical length
(Fig. 2 A). In the case of two different mTER
/
cell lines,
KO9-G6 and KO11-G6, derived from 6th generation embryos, the telomere fluorescence at both p- and q-telomeres was maintained or increased during the different
PDs analyzed (Fig. 2 A). In KO9-G6 cells, p- and q-telomeres were maintained at an average length of 10.8 and
24.8 kb, respectively, and in KO11-G6 cells at an average
length of 16.3 and 25.7 kb. These observations point to
telomerase-independent mechanisms for the maintenance
of telomeres in the immortal cells derived from the 6th
generation mTER
/
MEFs.
Representative FISH images of metaphase spreads from
wt and mTER/
cell lines at early and late passages are
shown in Fig. 3. As previously described for immortal
MEF cultures (Zindy et al., 1997
), most of the cell lines
studied here were aneuploid at late passages (Fig. 3, A, C,
and D). Fig. 3 A shows two metaphases of Wt14 cells before, PD 2, and after immortalization, PD 243. At PD 243, all chromosome ends had TTAGGG repeats and the cells did not show an increase of end-to-end fusions except for a
very long chromosome that was clonal (indicated by an arrowhead in Fig. 3 A). Metaphases of KO16-G1 and KO7-G4 mTER
/
cell lines (Fig. 3, B and C, respectively) show
a decrease in telomere fluorescence when early and late
PDs are compared. In contrast, KO9-G6 cells showed a
similar telomere fluorescence signal at both early, PD 2, and late, PD 88, PDs, in agreement with the observation
that the mean telomere length is maintained in these cells
(Fig. 3 D, see above). Finally, all mTER
/
cell lines contain many chromosomes lacking detectable telomere signal at late PDs, as well as a significant increase of end-to-end fusions (Fig. 3 arrows; see below).
|
We have also studied the telomere fluorescence of chromosomes 2 and 11 as a function of the accumulated number of cell doublings (Fig. 2, B and C). When telomere fluorescence of both chromosomes 2 and 11 was measured in
the wt cell line Wt14, we observed a slight decrease with
increasing PDs, (Fig. 2, B and C). The calculated average
rate of telomere shortening for chromosomes 2 and 11 in the Wt14 cells was 10 and 11 bp per PD, respectively. Interestingly, when we studied telomere dynamics of chromosome 2 and 11 in the different generation of mTER/
cell lines, we could not detect the predicted pattern of
telomere shortening with increasing PDs (Fig. 2, B and C).
The length of p- and q-telomeres at chromosomes 2 and 11 with increasing PDs suggests the activation of telomere
maintenance mechanisms at different points during the
growth of the cell lines. In this regard, it is interesting to
note that in KO16-G1 cells, 11q-telomeres did not shorten
from PD 19 to PD 81 or from PD 215 to PD 322. However,
11p-telomeres continued to shorten to an average length of only 5.6 kb at PD 215 and then were stabilized. The involvement of 11p telomere in the chromosomal instability
of this cell line will be discussed later in this paper. The
maintenance of telomere length was particularly clear in
cumulative PDs of the two cell lines derived from the 6th
generation mTER
/
embryos (Fig. 2, B and C). Interestingly, in these cells telomeres from different chromosomes
were maintained at different length and, in general, chromosome 2 telomeres were stabilized at shorter lengths
than chromosome 11 telomeres (Fig. 2, B and C).
Fig. 2 D shows the distribution of fluorescence intensity
values for 2q, 2p, 11q, and 11p telomeres with increasing
PDs in wt (Wt14) and in mTER/
cell lines from the first
(KO16-G1) and from the 6th (KO9-G6 and KO11-G6)
generation. The telomere fluorescence in wt cells at 2p, 2q,
11p, and 11q telomeres with increasing PDs remained similar, in agreement with the fact that, overall, telomeres
were maintained in this cell line. In contrast, the number
of telomeres with low fluorescence values (0-10 TFU) increased with passage number in the mTER
/
cell lines.
Interestingly, in the mTER
/
cell lines the heterogeneity
in fluorescence intensity values increased with increasing
PDs for some telomeres (i.e., 11q telomeres in KO9-G6
cell line), again suggesting the existence of alternative telomere maintenance mechanisms in these cells.
Analysis of End-to-End Fusions
To analyze the nature of the chromosomal fusions promoted by the absence of telomerase, we performed FISH
on wt and mTER/
metaphases using telomeric and centromeric probes, as well as chromosomes 2 and 11 painting
probes (Materials and Methods). Fig. 4 shows the diagrams of the different fusions characterized in this study
together with representative images. End-to-end fusions
were classified into different types according to their structure as shown in Fig. 4. Types I, II, and III involve p-to-p arms fusions. Type I fusions contain telomeric repeats at
the fusion point (Fig. 4 a) and two copies of minor satellite
centromere repeat sequences (b). Type II fusions do not
contain detectable telomeric sequences at the fusion point
(Fig. 4 a) and yield two centromere signals (b). Type III
fusions lack telomeric signals at the fusion point (Fig. 4 a)
and only have one centromere signal (b). Type IV and V
fusions involve q-to-q arm fusion, and have or lack detectable telomeric signals at the fusion point, respectively. Finally, type VI involves p-to-q arm fusion. In some cases,
we performed chromosome painting to determine whether
the fusions were homologous (for example, chromosome
2-to-chromosome 2 in panel c of type II fusions) or nonhomologous (for example, chromosome 11 to an undetermined chromosome in panel c of type VI fusions).
|
Chromosomal Instability in Primary mTER/
Cells
No fusions were detected in metaphases analyzed from
early passage primary wt cells (Table I). In the case of
mTER/
primary cells, the frequency of fusions increased
significantly from 0.07 fusions per metaphase in mTER
/
cells from the 1st generation (KO19-G1) to an average of
1.04 fusions (range from 0.5 to 1.72) per metaphase in
seven independently derived mTER
/
cells from 6th generation embryos (KO9-G6, KO11-G6, and KO1-G6 to
KO5-G6; Table I). Interestingly, cytogenetic analysis of
the cells derived from seven independent 6th generation
embryos revealed that an average of 41% of the fusions
was type II fusions. Chromosome painting showed that
70% of these type II fusions were homologous fusions involving 2p (2p-to-2p fusions; see Fig. 4 for example) and
only 2% involved chromosome 11. The dramatic increase
in chromosome 2 but not chromosome 11p-arm fusions in
6th generation mTER
/
cells, is probably the consequence of 2p-telomeres shortening from 26.0 ± 2.8 kb in
the wt cells, to an average of 7 kb, shorter than the average
of all telomeres in cells from the 6th generation. In this regard, in 6th generation cells derived from embryo KO9-G6, 2p-telomeres were only an estimated 0.15 kb long and
100% of type II fusions were 2p-to-2p fusions. The homologous nature of these fusions indicates that they are likely
to be the result of a failure to separate sister chromatids
during mitosis (see model in Fig. 5). Interestingly, fusions
involving chromosome 2 were stably maintained in two
different G6 cell lines studied, KO9-G6 and KO11-G6, at
least for >80 PDs (Table II, see Discussion). Other fusions
found in primary KO9-G6 cells included type I fusions (35%), and less frequently, type III (8%) and type V
(18.4%) fusions (see Fig. 4 for examples and Table I for
data). Taken together, chromosome 2 seems to be more
frequently involved in chromosome fusions than other
chromosomes in the mTER
/
MEFs, although we can not
rule out that other chromosomes might occasionally be involved in fusions in mTER
/
cells (Lee et al., 1998
).
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|
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Chromosomal Instability in mTER/
Cell Lines
To study the consequences of continuous proliferation in
the absence of telomerase activity on chromosomal stability, we also analyzed chromosomal aberrations in wt and
mTER/
cell lines as a function of the accumulated number of PDs (Table II). The wt cell line, Wt14, did not show
any end-to-end chromosome fusions in the first 20 PDs,
except for a very long chromosome that was clonal. We
determined that this long chromosome was the result of a
terminal translocation between chromosome 11 and another chromosome (see arrowhead in Fig. 3 A), and was
stably transmitted throughout all the PDs analyzed. In
later passages of the Wt14 cell line (PD 350), a low percentage of p-arm fusions, 0.2 per metaphase, was also
detected. These fusions could be a consequence of the
moderate telomere shortening detected in these cells (see above).
A dramatic increase in end-to-end fusions was observed
with increasing PDs in all the mTER/
cell lines studied
(see also Fig. 3, B-D for examples of metaphases). This
chromosomal instability furthermore increased with the
generation number (Table II). The high frequency of p-arm fusions (89%) versus q-arm fusions (11%) in the cell
lines could be due to the fact that mouse p-telomeres are
shorter than q-telomeres. Whereas type I and type II fusions were the most common fusions in primary cells (Table I), type III fusions (with only one pair of centromere signals) were the most abundant in the cell lines (65%; see
also Table II). Interestingly, 80% of the type II fusions detected in the two different G6 cell lines, KO9-G6 and
KO11-G6 involved chromosome 2, in agreement with the
observation that chromosome 2 telomeres were shorter
than the average of all telomeres in the 6th generation
MEFs and/or that there is an increased stability of fusions
involving chromosome 2 relative to fusions involving other
chromosomes. By chromosome painting, we determined
that type III fusions present in mTER
/
cell lines, usually
involve nonhomologous chromosomes. Interestingly, 20%
of these fusions involved chromosome 11p fused to other
chromosomes. In the KO16-G1 PD 81 cell line, >75% of
all type III fusions involved chromosome 11, in agreement
with the fact that 11p-telomeres were specially short in this
particular cell line (for example, Figs. 2 A and 4). Type VI
fusions, visualized as chromosome rings, were also present
in mTER
/
cell lines (for examples see Fig. 4, B1 and B2).
Other chromosomal rearrangements appear at late passage in mTER
/
cells. For example, in the case of KO16-G1 (PD 215) cells, these rearrangements included reciprocal and terminal translocations involving chromosomes 2 or 11. The frequency of such chromosome exchanges was
0.06 and 0.1 per metaphase, respectively (not shown).
Fusion between nonhomologous chromosomes could
originate by the simultaneous existence of two different
chromosomes with critically short telomeres. To estimate
the minimal telomere length that triggers chromosome fusions, we have calculated the mean telomere length of intrachromosomal telomere repeats in all fusions involving the p-arm of one chromosome and q-arm of a different
chromosome (Type VI fusions; see Fig. 4) and in terminal
translocations detected at PD 215 and at PD 322 of the
KO16-G1 cell line (Type I fusions were excluded from the
analysis). The average length of intrachromosomal telomere repeats (not considering the type I fusions) was 2.3 kb (ranging between 0.1 and 5.7 kb), indicating that this
length is not sufficient to prevent chromosome fusions in
mouse cells. Altogether, these results suggest that end-to-end fusions and other chromosomal aberrations detected
in the mTER/
cell lines are the result of telomere shortening to a critical length, and that chromosomes 2 and 11 are commonly involved in these fusions.
![]() |
Discussion |
---|
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---|
To study the role of telomeres and telomerase, we have
analyzed telomere length and chromosomal stability in
mouse cells deficient for telomerase activity. Previously,
we have shown that the average telomere length of mice
genetically deficient in the RNA component of telomerase
(mTER) decreases with each generation number (Blasco et al., 1997). However, somatic cells derived from these
mice have been able to grow in culture beyond 500 PDs
and were shown to be transformed and form tumors in
nude mice (Blasco et al., 1997
; this paper). These findings
have raised the question of how telomeres are maintained
in late generation mTER
/
cells in the absence of telomerase. Indeed, deletion of the mTER gene in embryonic
stem (ES) cells was recently reported to result in a severe
growth defect after >300 divisions, suggesting that the
growth of ES cells is telomerase dependent (Niida et al., 1998
). In our study we found no growth inhibition in primary somatic cells from mTER
/
mice, suggesting that
the telomerase requirement for continued growth may
vary between cell types. To address questions about the
mechanism of telomere maintenance in primary somatic
cells from mTER
/
mice, the length of individual telomeres
was measured using quantitative FISH and chromosome
painting. Chromosome painting with whole chromosome probes unequivocally identifies mouse chromosomes and
has allowed us to follow telomere length of particular
chromosomes and their involvement in chromosome fusions.
Telomere Dynamics in Primary Cells
We concentrated our attention on chromosomes 2 and 11. Initial banding analysis of mTER/
6th generation MEFs
used in an earlier study (Blasco et al., 1997
) suggested that
chromosome 2 was frequently involved in fusions (unpublished results). Therefore, we sought to analyze the telomere length dynamics of this chromosome in mTER
/
cells. Chromosome 11 is the mouse homologue of human
chromosome 17. Recently, it was found that chromosome
17p has relatively short telomeres in normal human cells
(Martens et al., 1998
). The average telomere length of all
chromosomes decreased uniformly at a rate of 3.9 kb per
mouse generation in a number of independent groups of
telomeres such as p-telomeres and q-telomeres, as well as
individual 2p- and 2q-telomeres. This uniform and constant rate of shortening indicates that probably all telomeres are subjected to the same erosive processes in vivo.
In the case of chromosome 2p, telomeres shortened to an
average length of 7 kb in the 6th generation; however,
some 6th generation cells (KO9-G6) had an average 2p
telomere length of only 150 bp. Heterogeneity in telomere length was also observed for 2q, 11p, and 11q telomeres in
different KO-G6 mice. 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). Interestingly, the
average telomere length of chromosome 11 increased ~6
kb from the 4th to the 6th generation. This telomere lengthening in the absence of telomerase activity is suggestive of alternative telomere maintenance mechanisms.
Such mechanisms may act in a telomere chromosome-specific manner because telomere 11p and 11q but not chromosome 2 telomeres showed this type of elongation upon
analysis of the average telomere length in seven different
6th generation mTER
/
embryos.
Telomere Dynamics in Immortal Cells
Additional evidence for the operation of telomerase-independent telomere maintenance mechanisms was obtained
from the study of serially passaged cells. Serial passage of
mouse embryonic fibroblasts allows the selection of oligoclonal populations with the capacity to stably proliferate in
culture. This experimental system allows the analysis of
telomere dynamics in a context less restrictive than the
entire organism. The telomere length in wt embryonic fibroblasts was stabilized after 100 PDs after an initial modest decline. In contrast, the average telomere length of
mTER/
cultures derived from embryos of the 1st or 4th
generation continued to decrease. As the cells spontaneously escaped senescence (around PD 10), this shortening
occurred at a similar rate to the one estimated in primary
cells during successive generations of mTER
/
mice
(Blasco et al., 1997
). When individual telomeres were followed during successive passages, a variable telomere
shortening was observed, and not all telomeres were affected to the same extent. These observations support the
existence of telomerase-independent and chromosome-specific telomere maintenance mechanisms in these cells.
Previous studies in yeasts have suggested the existence
of telomerase-independent mechanisms that are sufficient
to ensure yeast viability (McEachern and Blackburn, 1996;
Nakamura 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
). Telomere
maintenance in these cells has been proposed to occur
through alternative mechanisms (ALT; Bryan et al., 1995
,
1997
). The fact that human ALT cells were not engineered to be genetically deficient for telomerase genes left open
the possibility that discrete bursts of telomerase activity
could be compensating for telomere shortening in these
cells. However, our studies in mTER
/
mouse cells unequivocally prove the existence of mechanisms in mammalian cells that are capable of maintaining telomeres in the absence of telomerase. The genes responsible for telomerase-independent telomere maintenance are currently
not known. Several genes involved in DNA recombination, i.e., Rad 52 (McEachern and Blackburn, 1996
) or in
DNA repair, such as DNA-PK, ATM, Rad 3, tel1+, 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
). Telomerase-deficient mTER
/
cells are a good system to identify genes
involved in telomere maintenance that could be essential
to sustain tumor growth in the absence of telomerase. The
targeting of both telomerase and gene products involved
in alternative telomere-maintenance mechanisms could be
used to prevent cell proliferation beyond a threshold telomere length. Such telomere-directed strategies may
offer a promising approach to cancer therapy.
Telomere Shortening and Chromosomal Instability
In 1941, Barbara McClintock made the observation that
chromosomes that lack telomeres have a tendency to fuse
and concluded that one of the functions of the telomeric
complex is to prevent end-to-end fusions of chromosomes.
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). 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
).
We have performed a detailed cytogenetic analysis to
examine the relationship between chromosomal fusions
and telomere shortening in mammalian cells. We could
not observe chromosomal fusions in primary cells derived
from wt embryos, thus estimating that the frequency of fusions is lower than 0.04 per metaphase. In contrast, primary cells derived from mTER/
embryos showed a dramatic increase in chromosomal fusions. The frequency of
chromosomal fusions increased with the generation number from <0.04 fusions per metaphase in wt mice to 1.72 fusion per metaphase in some mTER
/
cells from the 6th
generation (~40 times the estimated maximum frequency
in wt mice). We performed a similar study in serially cultured cells. The loss of control of the chromosomal number or aneuploidy is an alteration commonly observed in
cultured cells (Zindy et al., 1997
). Indeed, most of the established cell populations that we have generated in this
study were aneuploid, independently of the presence or
absence of telomerase activity. Despite being aneuploid,
established wt cells presented a very low level of chromosomal fusions (0.20 fusions per metaphase in cultures that have undergone 350 PDs). In comparison, mTER
/
cells
showed a very high proportion of chromosomal fusions
(for example, 8-9 fusions per metaphase in mTER
/
cultures of the 1st generation that have undergone 325 PDs), >40-fold the number of fusions found in wt cell lines.
These observations indicate that telomere shortening promotes the accumulation of chromosomal fusions both in
vivo and in vitro. Recently, it was shown that the junctions
of human dicentric chromosomes are typically characterized by the absence or a low number of telomere repeats (Wan et al., 1999
), providing further support for a critical
role of telomere shortening in chromosomal instability.
To investigate the mechanisms by which telomeres prevent end-to-end fusions, we performed a detailed FISH
analysis of the chromosomal fusions observed in mTER/
cells using whole chromosome and centromeric minor satellite probes. The results obtained from this study are different from the initial characterization of end-to-end fusions carried out in mTER
/
mice (Lee et al., 1998
). In
these studies, chromosome 3 was found to be frequently
involved in fusions of chromosomes derived from splenocytes of one mTER
/
G6 mouse. Possible explanations
for this discrepancy range from cell type specificity to
technical artefacts. In our study with primary mouse embryonic fibroblasts, most of the end-to-end chromosome fusions detected in mTER
/
primary cells from the 6th
generation, i.e., KO9-G6, unequivocally involved 2p-telomeres as determined by chromosome painting and in
agreement with the fact that 2p-telomeres were shorter
than average in these cells. Moreover, the emergence of
2p-to-2p fusions with increasing passage number in the
KO11-G6 cell line also supports the involvement of chromosome 2 in the initial chromosomal instability detected
in mTER
/
cells. It is tempting to speculate that the frequent occurrence of this particular abnormality is related
to the relatively short length of telomeres on this particular chromosome arm in several mouse strains (Hande,
M.P., M.A. Blasco, and P. Lansdorp, unpublished observations) as it seems unlikely that fusions involving 2p may have occurred as isolated clonal events in seven independently derived embryos. While the majority of the primary
fusions seem to involve chromosome 2, we cannot rule out
the involvement of other chromosomes, such as chromosome 3 or 14 (Lee et al., 1998
).
As illustrated in the model shown in Fig. 5, telomere loss
or telomere shortening to a critical length could trigger either end-to-end fusions between sister chromatids or a
failure to separate them during mitosis. As a consequence
of either situation, the sister chromatids could be drawn to
the same spindle pole at anaphase resulting in chromosome nondisjunction or missegregation (see Fig. 5 for
model). Alternatively, the fusion product could break at
fragile sites and trigger breakage-fusion-bridge cycles. In the first case, one of the daughter cells (daughter cell 1 in the model) will end up with a Robertsonian fusion-like
chromosome (fusion types I or II) or a dicentric chromosome (fusion types IV or V), and the other cell (daughter
cell 2) will lose the chromosome. Further rounds of cell division might result in the maintenance of the fusion provided that the sister chromatids segregate normally with
two centromeres, or that one of the centromeres is inactivated (type III fusions). In this regard, in 6th generation
mTER/
cells 16% of the p-fusions are type II fusions
and were stably transmitted for >88 PDs. Interestingly,
q-arm fusions were very infrequent in mouse cells. Although the fact that q-arm telomeres are generally longer
than p-arm telomeres may in part responsible for this observation, q-arm fusions may also be more likely to form
an anaphase bridge and may thus be less stable than p-arm fusion products. Altogether, these results indicate that
telomeres protect chromosomes from end-to-end fusion
events in vivo by maintaining telomere length above a critical threshold. This observation supports the important
role of telomeres in chromosome segregation as previously proposed (Kirk et al., 1997
; Bass et al., 1997
; Chikashige et al., 1997
; Chua et al., 1997; Conrad et al., 1997
).
When chromosomal instability was studied in the spontaneously immortalized mTER/
cell lines, we found a
dramatic increase in nonhomologous type III end-to-end
fusions, in p-to-q arms fusions and in various types of
translocations. Again, 2p and 11p telomeres were involved in many of these fusions in the different cell lines studied
(derived from different embryos), suggesting that the involvement of these chromosomes in fusions is a general
phenomena for mouse fibroblasts. The existence of these
chromosomal abnormalities in mTER
/
cell lines can be
explained by breakage-fusion-bridge cycles triggered by
different chromosomes with critically short telomeres (reviewed in de Lange, 1995
).
In general, the types of chromosomal aberrations detected in mTER/
cell lines are similar to the ones described in tumor cells, supporting the notion that telomere
loss is one of the main inducers of chromosomal instability
in tumors (reviewed in de Lange, 1995
). In agreement with
this, tumors generally have short telomeres (reviewed in
de Lange, 1995
) probably because telomere loss is not
compensated at the earlier steps of tumorigenesis. Telomere shortening would lead to chromosomal instability associated with end-to-end fusions and translocations of the
types described in this work, favoring the allelic loss of tumor suppressor genes, such as p53 in the case of mouse
chromosome 11 or human chromosome 17 (Martens et al.,
1998
). Our data agrees with the idea that when telomeres
are critically short and there is a high degree of chromosomal instability telomere-maintenance mechanisms are activated and selected (see Fig. 6 for model). 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. In this regard, mTER
/
mice from
late generations show an increased cancer incidence than
the wt counterparts (Rudolph et al., 1999
).
|
![]() |
Footnotes |
---|
Received for publication 29 June 1998 and in revised form 15 January 1999.
Address correspondence to María A. Blasco, Department of Immunology
and Oncology, Centro Nacional de Biotecnología/CSIC, Campus Cantoblanco, Madrid E-28049, Spain. Tel.: 34 91 585 4846. Fax: 34 91 372 0493. E-mail: mblasco{at}cnb.uam.es
We are indebted to Juan Martín-Caballero for deriving the different
mTER/
mouse generations and to Jessica Freire for the genotyping
work. We specially thank Manuel Serrano, Fermín Goytisolo, Carlos Martinez, and Carol Greider for their support.
We are grateful to Dr. Carol Greider in whose laboratory M.A. Blasco
derived the mTER/
cell lines with the support of National Institutes of
Health (NIH) grant POI-CA 13106 to Carol Greider. E. Samper is recipient of a predoctoral fellowship from The Regional Government of
Madrid. Research in the laboratory of P.M. Lansdorp is supported by
NIH grants ROIAI29524 and GM56162, and by a grant from the National Cancer Institute of Canada with funds from The Terry Fox Run. Research
at the laboratory of M.A. Blasco is funded by grants PM95-0014 and
PM97-0133 from the Ministry of Education and Culture, Spain, by grant
08.1/0030/98 from The Regional Government of Madrid and by the Department of Immunology and Oncology. The Department of Immunology
and Oncology was founded and is supported by the Spanish Research
Council (CSIC) and by Pharmacia and Upjohn.
![]() |
Abbreviations used in this paper |
---|
MEF, mouse embryonic fibroblast; mTER, mouse telomerase RNA gene; PD, population doubling; Q-FISH, quantitative fluorescence in situ hybridization; TFU, telomere fluorescence units; wt, wild-type.
![]() |
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