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© 2001 Oxford University Press
Human Molecular Genetics, 2001, Vol. 10, No. 5
519–528
Extra-chromosomal telomeric DNA in cells from Atm–/–
mice and patients with ataxia-telangiectasia
M. Prakash Hande1,+, Adayabalam S. Balajee2, Andrei Tchirkov1, Anthony Wynshaw-Boris3
and Peter M. Lansdorp1,4,§
1Terry
Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada,
for Radiological Research, College of Physicians and Surgeons, Columbia University, 630 West 168th Street,
New York, NY 10032, USA, 3School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
92093-0627, USA and 4Department of Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
2Center
Received 9 November 2000; Revised and Accepted 12 January 2001
Ataxia-telangiectasia (AT) is an autosomally recessive
human genetic disease with pleiotropic defects such
as neurological degeneration, immunodeficiency,
chromosomal instability, cancer susceptibility and
premature aging. Cells derived from AT patients and
ataxia-telangiectasia mutated (ATM)-deficient mice
show slow growth in culture and premature senescence. ATM, which belongs to the PI3 kinase family
along with DNA-PK, plays a major role in signaling
the p53 response to DNA strand breaks. Telomere
maintenance is perturbed in yeast strains lacking
genes homologous to ATM and cells from patients
with AT have short telomeres. We examined the
length of individual telomeres in cells from Atm–/– mice
by fluorescence in situ hybridization. Telomeres
were extensively shortened in multiple tissues of
Atm–/– mice. More than the expected number of
telomere signals was observed in interphase nuclei
of Atm–/– mouse fibroblasts. Signals corresponding
to 5–25 kb of telomeric DNA that were not associated
with chromosomes were also noticed in Atm–/–
metaphase spreads. Extrachromosomal telomeric
DNA was also detected in fibroblasts from AT
patients and may represent fragmented telomeres or
by-products of defective replication of telomeric
DNA. These results suggest a role of ATM in telomere
maintenance and replication, which may contribute
to the poor growth of Atm–/– cells and increased
tumor incidence in both AT patients and Atm–/– mice.
INTRODUCTION
Ataxia-telangiectasia (AT) is a pleiotropic inherited disease
characterized by neurodegeneration, cancer susceptibility,
immunodeficiency, genetic instability, radiation sensitivity
and premature aging. ATM (ataxia telangiectasia mutated), the
gene responsible for AT, is thought to play a crucial role in a
signal transduction network that modulates cell cycle checkpoints, genetic recombination, apoptosis and other cellular
responses to DNA damage. Cells derived from AT patients (1)
and ATM-deficient mice show slow growth in culture and
premature senescence (2–4). Cells from AT individuals
display abnormalities in culture such as cytoskeletal defects
and hypersensitivity to ionizing radiation (5). They also show
chromosome abnormalities in the form of end-to-end associations
involving telomeres (6,7). Telomeres, consisting of (TTAGGG)n
repeats and associated proteins, protect chromosomes from end
fusions, incomplete replication and exonuclease degradation
(8–10). Telomere shortening may play an important role in
tumorigenesis and aging (11). Peripheral blood lymphocytes
from AT patients show accelerated telomere shortening (12)
and dysfunctional telomeres in yeast strains lacking ATM
homolog genes, tel1+ and rad3, caused self-circularization of
chromosomes (13). ATM shows some homology to TEL1 and
MEC1 genes of budding yeast and rad3+ and tel1+ genes in
fission yeast which are known to be involved in telomere maintenance (13,14) and meiotic and mitotic cell cycle checkpoint
control (15). Yeast strains with mutations in genes homologous to
ATM such as TEL1 and MEC1 show altered telomere
chromatin and redistribution of silencing proteins in response
to DNA damaging agents (13,14,16–18). Additionally, the
ATM gene has been implicated in recombination of DNA
sequences and double-strand break repair. Based on the
homology of ATM to TEL1 and MEC1 genes in yeast, it has
been suggested that mutations in ATM could also lead to defective
telomere maintenance (14,16). In this study, we have evaluated
the potential role of the ATM gene in telomere function and
chromosome integrity in wild-type and ATM knockout mice.
Our results indicate that the functional inactivation of ATM
leads to telomere shortening, chromosome instability and the
occurrence of extrachromosomal fragments of telomeric DNA
suggestive of an important role for the mammalian ATM gene
in maintaining telomere integrity.
+Present address: Center for Radiological Research, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York,
NY 10032, USA
§To whom correspondence should be addressed at: Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver,
BC V5Z 1L3, Canada. Tel: +1 604 877 6070; Fax: +1 604 877 0712; Email: [email protected]
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Table 1. Telomere fluorescence and chromosomal abnormalities in metaphase chromosomes from Atm–/– mice; results from Q-FISH (19,20) and cytogenetic
analyses from five litters of 129/SvEv mice
ATM genotype
Metaphases
analyzed
Telomere fluorescence (mean ± SD)
p-arm
q-arm
all
Aneuploid
cells
End-to-end
fusions
Deletions
Fragments
+/+ (five mice)
64
37.7 ± 15.7
52.6 ± 22.9
45.0 ± 20.9
1 (1.4)
0
0
3 (4.1)
+/– (five mice)
81
32.0 ± 13.9
39.7 ± 19.2
35.9 ± 17.1
13 (16.0)
8 (9.9)
3 (3.7)
9 (11.1)
–/– (six mice)
95
24.0 ± 11.0
31.7 ± 15.5
27.9 ± 13.9
19 (20.0)
22 (23.2)
14 (14.7)
30 (31.6)
Data are pooled for comparison. Numbers in parentheses indicate events per 100 cells. End-to-end fusions include Robertsonian-like configurations, telomere
associations and dicentric chromosomes; deletions were noticed as chromosomes lacking q-arm telomeres; fragments/breaks include centric fragments, acentric
fragments, chromosome breaks and chromatid breaks.
RESULTS AND DISCUSSION
To study telomeres in mice lacking the ATM gene, the telomere
length was measured using quantitative fluorescence in situ
hybridization (Q-FISH) (19,20) in metaphase chromosomes
from primary cells derived from littermate Atm+/+, Atm+/– and
Atm–/– mice (129/SvEv and 129/SvEv/BlSW genetic background). Atm–/– splenocytes from 129/SvEv mice showed
extensive telomere shortening (Table 1 and Fig. 1A) corresponding to a 35–40% loss of telomeric DNA (from 44 kb in
wild-type to 27 kb in Atm–/– mice). Heterozygous Atm +/– mice
also displayed a significant reduction (20–25%) in telomere
length compared with their Atm+/+ littermates. A similar degree
of telomere shortening was observed in cultured fibroblasts
from lung and skin of Atm–/– compared with wild-type Atm+/+
129/SvEv mice (data not shown). Telomeres in cells from 129/
SvEv/BlSW Atm–/– splenocytes were very heterogeneous in
length and also significantly shorter than in Atm+/+ controls
(Fig. 1B). Chromosome ends without detectable repeats were
frequently observed in Atm–/– but not Atm+/+ cells of mice from
both genetic backgrounds (Fig. 1A, B, E and F). Representative FISH images of metaphase spreads from Atm+/+ and Atm–/–
mouse splenocytes are shown in Figure 1C and D, respectively.
In order to determine the telomere length in non-dividing
cells other than splenocytes, the telomere repeat content of
interphase cells from thymus and bone marrow of the same
mice was measured using FISH of cells in suspension and flow
cytometry (flow-FISH) (21). A significant reduction in
telomere fluorescence in cells from all the homozygous Atm–/– mice
analyzed was observed (Fig. 2), although the telomere length
differences were less dramatic compared with the results
obtained using Q-FISH. Thymocytes from a single male Atm–/–
mouse were an exception and showed slightly elongated
telomeres (Fig. 2). Further analysis revealed that this particular
mouse had a thymic lymphoma, which may explain this observation.
Since one of the functions of telomeres is to prevent aberrant
chromosome end associations (20,22), the severe telomere
shortening observed in the Atm–/– mice may result in chromosome abnormalities. Indeed, high levels of chromosome
fragmentation (e.g. Fig. 1D–F, asterisks) and end-to-end
fusions were observed in Atm–/– mouse splenocytes compared
with wild-type cells (Table 1). About 15% of metaphase cells
from Atm–/– mice showed deletions characterized by the
complete lack of q-arm telomeres (e.g. Fig. 1E and F, asterisks).
Most likely, the aneuploidy in the Atm–/– cells resulted from
damage to telomeres and/or previous breakage–fusion–bridge
cycles (23). Interestingly, cells from heterozygous mice also
showed higher levels of chromosome instability compared
with the wild-type controls (Table 1).
To localize telomeres and to detect the number and distribution of telomere signals in mitotic cells, fibroblasts derived
from skin or lung tissues of both Atm+/+ and Atm–/– mice were
grown on coverglasses. Cells at a semi-confluent stage were
fixed and hybridized with telomeric peptide nucleic acid
(PNA) probe (see Materials and Methods). Deconvolution
microscopy was used to visualize the telomere signals in the
interphase nuclei of such cells (Fig. 3A–C). The reconstructed
three-dimensional images were used to count the number of
telomere spots in each nucleus. Surprisingly, Atm–/– mouse
fibroblasts showed more than the expected number of telomere
signals in interphase nuclei (Fig. 3B and C). Cells from Atm +/+
mice displayed an average of 74.5 ± 2.0 telomere signals in
their nuclei, close to the expected 80 (40 chromosomes, two
ends each) (Fig. 3A and D). In contrast, Atm–/– mouse fibroblasts showed ∼1.3–2 times more telomere spots, depending on
the threshold fluorescence intensity used to detect telomere
spots (127–181 spots) (Fig. 3D). In view of the short telomeres
in cells from Atm–/– mice, the opposite would have been
expected from limitations in hybridization efficiency or fluorescence detection. Similarly, clustering of telomeres, as
described during meiosis in Atm–/– mice (24), would have yielded
opposite results. In view of the large increase in the number of
telomere signals, aneuploidy alone in Atm–/– cells seems an
unlikely explanation for our observations. Possibly, the surplus
telomere signals represent broken telomere fragments. This
would also explain why the telomere repeat content in cells
from Atm –/– mice measured by flow-FISH (Fig. 2) suggested
less telomere shortening relative to Atm+/+ cells than observed
at chromosome ends by Q-FISH (Fig. 1). Alternatively, a high
proportion of Atm–/– interphase cells could have been in
S-phase or telomeres in Atm –/– cells could be replicated earlier
than those in wild-type cells. To study these possibilities further,
we performed parallel measurements of DNA and TTAGGG
repeats by flow cytometry (Fig. 3E). The results of this
analysis were compatible with a modest increase in the
percentage of Atm–/– fibroblasts in the S- and G2/M-phases of
the cell cycle (Fig. 3E and data not shown). A similar trend was
also seen in bone marrow cells from the Atm–/– mice (data not
shown). The data were furthermore suggestive of early DNA
replication of a subset of telomeres in cells from both normal
and Atm –/– mice.
Human Molecular Genetics, 2001, Vol. 10, No. 5
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Figure 1. Frequency distributions of telomere fluorescence in 129/svEv (A) and 129/svEv/BlSW (B) mice with and without the ATM gene. Data were collected
from Q-FISH studies (19) on metaphase spreads of primary mouse splenocytes of the indicated genotype. The mean and SD for each group of mice is shown. An
asterisk indicates P < 0.001 in a Mann–Whitney rank sum test comparing the telomere length distribution in cells from Atm–/– and Atm+/– mice with that in cells
from Atm+/+ mice. Individual fluorescence values of p and q arms [expressed in telomere fluorescence units (TFU)] were used to generate the histograms. One TFU
corresponds to an estimated 1 kb of telomeric DNA (40). (C–F) FISH analysis of metaphase chromosome spreads from cultured splenocytes of Atm+/+ (C) and
Atm–/– mice (D–F). DNA (DAPI) is shown in blue and TTAGGG repeats are shown in red (representing Cy3-labeled telomere PNA probe). Note the overall
decrease in telomere fluorescence and heterogeneous telomere signals in the Atm –/– mouse chromosomes. Asterisks indicate fusion events (D) and chromosomes
lacking telomeres due to breakage in or outside telomeric DNA (E and F).
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Figure 2. Results from telomere fluorescence analysis by flow-FISH (21) of primary cells from spleen, bone marrow and thymus. Fluorescence values are
expressed as the mean number of molecules of equivalent soluble fluorochromes (MESF). Vertical bars indicate the standard deviation of the mean. The statistical
significance using Student’s t-test was: a, P = 0.023; b, P = 0.025; c, P = 0.007; *, male Atm–/– mouse that showed a thymic lymphoma.
To further study the excess number of telomere spots seen in
interphase cells, we performed telomere FISH on metaphase
chromosomes from fibroblasts that were fixed in situ to
preserve cytoplasm (Fig. 4). In the cells from Atm–/– mice, we
observed that >80% of the mitotic metaphase cells displayed
excess telomere signals that were clearly dissociated from the
bulk of chromosomal DNA (Fig. 4C and D). Such extrachromosomal spots were rarely observed in Atm +/+ cells (Fig.
4A and B). The presence of extra telomere signals that were
not associated with chromosomes but located within the cell
was also noticed in 60–75% of the metaphases derived from
bone marrow cells of Atm–/– mice. Extra chromosomal
telomere signals observed in the metaphase cells of Atm–/– mice
correlated with the increased telomere spots observed in the
interphase nuclei in these cells. The extra spots could represent
broken telomeres or defective replication intermediates such as
single-strand G-rich repeats. However, no signals were
observed in Q-FISH experiments if heat denaturation was
omitted from the protocol, suggesting a double-strand nature of
the extra-chromosomal telomere signals.
Primary fibroblasts from three normal individuals and four
different AT patients were also analyzed to study the presence
of extra-chromosomal telomeric DNA in human cells. The
fibroblasts were grown on chamber slides and hybridized with
Cy3-labeled PNA-telomeric probe. Although the presence of
extra-chromosomal telomeric DNA in cells from human AT
patients was not as pronounced as in cells from Atm–/– mice,
∼40–50% of the cells analyzed showed a significantly
increased number of telomere fluorescence spots not associated
with the chromosome ends relative to normal fibroblasts
(Table 2). An example of a metaphase spread from a
homozygous AT patient is shown in Figure 5. The presence of
extra-chromosomal telomeres in cells from both AT patients
and Atm–/– mice supports an important role of the ATM gene in
maintaining telomere integrity. This notion is supported by
control experiments with PNA probes specific for alphoid
sequences showing bright staining at centromeres but no extrachromosomal signals in AT-deficient cells from either human
or murine origin (data not shown).
To find out whether the extra chromosomal telomeres
observed in Atm –/– cells are present in the nucleus or in the
cytoplasm, we isolated cytoplasmic and nuclear DNA by high
salt extraction. In order to verify that the cytoplasmic DNA
was free of nuclear DNA contamination and vice versa, dot
blot hybridization was performed on equal amounts of
cytoplasmic and nuclear DNA blotted onto Hybond-N
membrane and hybridized digoxigenin-labeled mitochondrial
and ribosomal DNA probes. As expected, the hybridization
signal for mitochondrial DNA was confined to the cytoplasmic
fraction and that of ribosomal DNA to the nuclear fraction,
indicating that there was little contamination of cytoplasmic
DNA with nuclear DNA (Fig. 6A). The results of similar
hybridizations with 32P-end-labeled telomere probe were in
agreement with the FISH data and showed the presence of
increased amounts of telomeric DNA in the cytoplasma of
primary fibroblasts and thymocytes from Atm–/–compared with
Atm+/+ mice (Fig. 6B). Similar results were obtained when
undigested nuclear and cytoplasmic DNA isolated from Atm–/– and
Atm+/+ cells was run on agarose gel, transferred to nylon
membrane and hybridized with a (TTAGGG)3 probe (Fig. 6C).
The mechanism responsible for the presence of extrachromosomal telomeric DNA in cells with defective ATM
observed in this study is not known. One possibility is that the
loss of ATM leads to defective replication of telomeric DNA,
which results in the breakage of telomeric DNA. Previous
studies in telomerase-negative human cell lines have also
described the presence of extra-chromosomal telomeres
(25,26). However, Atm–/– mice were shown to have normal
levels of telomerase as reported in a recent study (24). Therefore,
the telomere dysfunction/regulation noticed in our study could
be due to a telomerase-independent process. One intriguing
possibility is that telomeric DNA is particularly vulnerable to
breakage. It has been reported that single-strand breaks preferentially accumulate at telomeres (27–29) and that DNA repair
at telomeres is defective (30). In the absence of ATM, broken
single- or double-strand breaks in telomeres may not be
detected and/or repaired (31), possibly resulting in the observed
fragments and accelerated telomere shortening (32).
Human Molecular Genetics, 2001, Vol. 10, No. 5
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Figure 3. (A) Telomere signal distribution in interphase nuclei of Atm+/+ fibroblasts (see Materials and Methods). Normally ∼70–95 telomere spots are detected in
the wild-type cells (D). (B and C) Telomere spots in interphase nuclei of Atm–/– mouse fibroblasts. Note the increased number of telomere signals (B) and occasional telomere clustering (C) in Atm –/– mouse cells. (D) Distribution of telomere signals in interphase nuclei of Atm+/+, Atm+/– and Atm–/– fibroblasts. Telomere
FISH was performed on fibroblasts grown on coverslips. Image analysis of images acquired under identical conditions was performed using two different settings
(see Materials and Methods). *, P = 0.01; **, P < 0.003. (E) Measurements of DNA and TTAGGG repeat content in single Atm+/+, Atm+/– and Atm–/– mouse fibroblasts using FISH combined with flow-cytometry (see Materials and Methods). Shown are dot plot histograms of PI versus PNA–FITC (telomere fluorescence) as
well as fluorescence histograms of total DNA (PI, top) and telomeric DNA (FITC, side).
Studies in yeast have suggested that the ATM homologs
TEL1 and MEC1 are required to prevent the degradation of
telomeric DNA from exonucleases and allow telomerase
access to telomeric DNA (33). In mammalian cells, degradation
of the telomeres by exonucleases could explain the telomere
shortening observed in cells from patients with AT (12) and in
the Atm–/– mice reported here. A recent study has shown that
presence of double-strand breaks inhibits DNA replication in
an ATM-dependent checkpoint pathway (34).
Our observations suggest that telomeric DNA fragments
accumulate in Atm–/– cells, possibly due to defective replication,
recombination or repair of telomeric DNA. ATM is clearly
involved in the p53-mediated DNA damage response
following telomere fusions and chromosome breakage in
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Figure 4. Telomere FISH using FITC-labeled (CCCTAA) 3 probe on murine metaphase fibroblasts grown on microscope slides. Mitotic cells were arrested at metaphase by colcemid treatment for 3–4 h. Hypotonic swelling was performed on the slides by incubation for 5 min with 0.03 M sodium citrate at 4°C. Cells were
fixed in situ by methanol:acetic acid treatment (6–7 drops of formaldehyde were added to 10 ml of fixative to preserve the cytoplasm) for 10 min. Slides were
processed for telomere FISH immediately or stored in cold ethanol at –20°C until use. Intact metaphase chromosomes are easily observed in Atm+/+ (A and B) and
Atm–/– (C and D) fibroblasts using PI as a counterstain. Images were acquired using a Zeiss Axioplan-2 fluorescence microscope equipped with separate PI and
FITC filters and a cooled charge device (CCD) camera (Sensicam). Separately captured PI (A and C) and FITC (B and D) images were superimposed using Northern Eclipse software (Empix Imaging). Images were processed using Adobe Photoshop software and pseudocolors to simultaneously visualize both chromosomes
and telomere signals. The magnification from both cell types was similar. Note the abundant signals obtained with the telomere probe that are not associated with
chromosomes within the cell boundary. Asterisks point to chromatid breaks. Double asterisks could represent telomeres at chromatid breakage sites.
mammalian cells (35). ATM is also a part of p53- and p21mediated cell cycle checkpoint pathways in response to doublestrand DNA breaks (for a review see ref. 1). Recent data has
also implicated ATM in DNA damage repair, since ATM
directly phosphorylates BRCA1 after ionizing radiation exposure
(36). Any of these functions of ATM could participate in the
maintenance of telomeres and telomere length in cultured cells.
Irrespective of the precise mechanisms involved, the excess
telomere signals observed in Atm–/– cells reported here support
a major role of ATM in the replication and maintenance of the
telomeric DNA in mammalian cells.
MATERIALS AND METHODS
Mice and cells
Atm+/+, Atm+/– and Atm–/– mice of 129/SvEv and 129SvEv/
BlSW (NIH Black Swiss) strains were either purchased from
Jackson Laboratories or obtained from the National Institutes
of Health (A.W.-B.’s laboratory). A total of 16 mice from five
litters (five Atm+/+, five Atm+/– and six Atm–/– mice all derived
from Atm+/– crosses) were used in the study. Animals were
sacrificed at 8–12 weeks of age and the tissue samples were
Human Molecular Genetics, 2001, Vol. 10, No. 5
527
Table 2. Extra-chromosomal telomere fluorescence in human fibroblasts with a heterozygous (AG03059) or homozygous (AG04405, AG04405, GM05823 and
GM02052) defect in the ATM gene
Normal fibroblasts (BJ)
Number of metaphases analyzed
Mean
SD
Standard error
Median
Maximum
Minimum
15
1.7
1.9
0.5
1
6
0
P-value
Normal fibroblasts (WI38)
18
1.6
1.7
0.4
1
5
0
Normal fibroblasts (MRC5)
20
1.4
1.6
0.4
1
5
0
A-T +/– fibroblasts (AG03059)
18
2.9
3.1
0.7
2
10
0
Not significant
A-T –/– fibroblasts (AG04405)
15
3.7
3.5
0.9
3
14
0
0.05
A-T –/– fibroblasts (AG03058)
15
3.9
2.9
0.7
4
9
0
0.02
A-T –/– fibroblasts (GM05823)
19
4.3
3.2
0.7
4
12
0
0.001
A-T –/– fibroblasts (GM02052)
18
3.9
2.7
0.6
3
8
0
0.001
A Mann–Whitney rank sum test was used to calculate whether the number of extra-chromosomal spots [defined as distinct fluorescence signals with a size and
fluorescence intensity >0.5 TFU, comparable to the telomere signals on chromosomes in that metaphase spread (see arrows in Fig. 5)] in the AT cells was significantly increased relative to the normal (BJ, WI38 and MRC5) fibroblasts.
tissues, tissues were cut into small pieces and treated with
0.25% trypsin-citrate saline. After 30 min, trypsin was
removed and fresh Dulbecco’s modified Eagle’s medium with
10% fetal bovine serum (FBS) was added to the culture dishes.
Once sufficient numbers of fibroblasts were released from the
tissues and attached to the culture dishes, tissue pieces were
removed. The fibroblasts were subcultured and used in the
analysis.
Human (WI38, MRC5) and AT (GM05823 and GM02052)
primary fibroblasts were obtained from Coriell Cell Repository.
All the cells were routinely maintained in 2× Eagle’s minimal
essential medium supplemented with 15% FBS (Gibco BRL),
vitamins, essential amino acids, non-essential amino acids and
antibiotics. The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.
FISH on interphase nuclei and telomere spot analysis
Figure 5. Example of extrachromosomal telomere signals (arrows) in a metaphase spread obtained from fibroblasts of a patient with AT (AG 03058) visualized using standard Q-FISH procedures (40). Although spots that were
apparently not attached to metaphase chromosomes were occasionally also
observed in normal fibroblasts (Table 2), the frequency of such extrachromosomal events in standard metaphase preparation was significantly higher in
cells from patients with AT.
collected aseptically. Isolation and culturing of splenocytes
were described previously (37). For metaphase preparations,
splenocytes were grown for 40–42 h in the presence of
mitogens and treated with colcemid for 2 h before harvesting
(37). Bone marrow cells and thymocytes were isolated by
standard techniques. To release fibroblasts from lung and skin
Fibroblasts derived from skin/lung were grown on coverglasses until the cells were semiconfluent. Cells were then
fixed with 4% paraformaldehyde and dehydrated using cold
ethanol series. Coverglasses were used either immediately
before telomere FISH or after storage at –20°C. The method
used for FISH was as described (20) except that 90% glycerol/
DABCO was used as mounting medium for DAPI. The cells
were analyzed using a Delta Vision deconvolution optical
sectioning microscope. A stack of images (0.2 µm intervals)
was acquired using Cy-3 for telomeres and DAPI for DNA for
both the Atm+/+ and Atm–/– fibroblasts under similar exposure
conditions. Approximately 20 images were acquired for each
genotype studied. The three-dimensional reconstruction of the
acquired images was performed using Delta Vision deconvolution
software. Telomere spots were counted using two different
threshold settings to segment telomere spots from the background.
DNA analysis combined with flow-FISH
For simultaneous analysis of DNA as well as telomere fluorescence, DNA staining and flow-FISH methods were combined.
To obtain clear DNA histograms, cells were fixed in absolute
ethanol (–20°C) for 12–16 h before processing for flow-FISH.
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Human Molecular Genetics, 2001, Vol. 10, No. 5
Figure 6. Dot blot analysis of Atm +/+ and Atm –/– murine cells. (A) Cytoplasmic and nuclear DNA was isolated from Atm+/+ and Atm–/– fibroblasts and hybridized
with digoxigenin-labeled mitochondrial and ribosomal DNA probes. The hybridization signals indicate a good separation of cytoplasmic and nuclear DNA. (B)
Dot blot analysis of telomeric DNA in cytoplasmic and nuclear DNA isolated from Atm+/+ and Atm–/– fibroblast and thymocytes. The filters were analyzed after
hybridization with 32P-labeled (TTAGGG) 3 telomere probe. Note that telomeric DNA is detected in the cytoplasma of Atm–/– but not Atm+/+ cells. (C) Southern blot
analysis of cytoplasmic and nuclear DNA isolated from Atm+/+ and Atm–/– cells. Samples were run on a 1% agarose gel and subjected to Southern blot analysis with
32P-labeled (TTAGGG) probe (see Materials and Methods). The blots of cytoplasmic DNA were overexposed to show visible signals. Gels from nuclear and cyto3
plasmic DNA were subjected to autoradiography films for different exposure time. The differences in the amount of DNA that was loaded on the gels and the
differences in exposure time make these blots/gels unsuitable for quantitative analysis.
Human Molecular Genetics, 2001, Vol. 10, No. 5
Then the cells were pelleted and resuspended in 1 ml phosphate-buffered saline. The subsequent steps were as described
for flow-FISH with fluorescein isothiocyanate (FITC)-labeled
telomeric PNA probe (21) and finally DNA staining was done
using propidium iodide (PI) at a concentration of 0.2 µg/ml.
Isolation of cytoplasmic and nuclear DNA
DNA was isolated from fibroblast and thymocytes from Atm–/–
and Atm+/+ mice. The cells were suspended in ice-cold buffer
(0.15 M NaCl, 10 mM Tris–HCl pH 7.4, 2 mM MgCl2 and
1 mM dithiothreitol) and homogenized by repeated pipetting.
Nonidet P-40 was added to the homogenate. After incubation
for 30 min on ice, the homogenate was spun at 15 000 g for
5 min. The supernatant was used as a cytoplasmic DNA
extract. The nuclear pellet was resuspended in NET buffer
(150 mM NaCl, 10 mM EDTA and 50 mM Tris–HCl pH 7.5)
containing 100 µg/ml proteinase K and 0.5% SDS. The DNA
was isolated using high salt as described (38).
Dot blot hybridization and Southern blot hybridization of
cytoplasmic and nuclear DNA
The efficiency of fractionation of cytoplasmic and nuclear
DNA was determined by dot blot hybridization using digoxigenin-labeled mitochondrial and ribosomal DNA probes.
Human mitochondrial DNA probe spanning the region from
5905–7433 bp was generated by PCR. The ribosomal DNA
pABB probe (kindly provided by J.E. Sylvester, University of
Pennsylvania, PA) is a 1.4 kb human 28S rDNA which is
homologous to mouse sequence. Mitochondrial and ribosomal
DNA probes (500 ng of each) were labeled with digoxigenin
using the high prime labeling kit of Roche Diagnostics. Cytoplasmic and nuclear DNA samples (∼250 ng) derived from
Atm+/+ and Atm–/– mice were dot blotted onto Hybond-N
membrane (Amersham Pharmacia Biotech). The conditions for
pre-hybridization, hybridization, washing and immunological
detection of the hybridization signal were essentially according
to the instructions of the manufacturer. The hybridization
signal was detected by enhanced chemiluminescence followed
by exposure to X-ray film.
To study the presence of telomeric DNA in cytoplasmic and
nuclear DNA, ∼1–2 µg of fractionated DNA isolated from
Atm–/– and Atm+/+ mice was dot blotted on to Hybond-N
membranes. The oligonucleotides (TTAGGG)3 and
(CCCTAA)3 were obtained from Gibco BRL. DNA probes
(250 ng) were 5′ end-labeled by the exchange reaction using
T4 polynucleotide kinase (Roche) following the manufacturer’s instructions. The unincorporated nucleotides were
removed by Sephadex G-50 column chromatography. The
filters were hybridized overnight at 37°C in the hybridization
solution (Gibco BRL) containing 0.5% SDS and 10 mM
EDTA. After hybridization, the filters were washed for 30 min,
once in 2× SSC/0.1% SDS and twice in 0.5× SSC/0.1% SDS.
The filters were then exposed to autoradiography films at –80°C
for a few hours.
For Southern hybridization, ∼1–2 µg of cytoplasmic and
nuclear DNA isolated from Atm–/– and Atm+/+ mice were digested
with HinfI and RsaI (Gibco BRL) according to the instructions
of the manufacturer. The DNA was fractionated on a 1.2%
agarose gel and transferred to Hybond-N membrane using capillary blotting in a standard protocol (39). The oligonucleotides
529
(TTAGGG)3 and (CCCTAA)3 were obtained from Gibco BRL.
DNA probes (250 ng) were 5′ end-labeled using T4 polynucleotide kinase (Roche) following the instructions from the manufacturer. The unincorporated nucleotides were removed by
Sephadex G-50 column chromatography. The filters were
hybridized overnight at 37°C in hybridization solution (Gibco
BRL) containing 0.5% SDS and 10 mM EDTA. After hybridization, the filters were washed for 30 min, once in 2× SSC/0.1%
SDS and twice in 0.5× SSC/0.1% SDS. The filters were then
exposed to autoradiography films at –80°C for a few hours.
ACKNOWLEDGEMENTS
Denise Larson, Cam Smith, Viktoriya Dobrovinska and Elizabeth Chavez are thanked for assistance. We thank Drs Howard
Lieberman and Tej Pandita for reagents. Drs David J Brenner
and Charles R. Geard are acknowledged for their support. This
study was supported by grants from the Canadian Institute of
Health Research (MOP 38075), the NIH (R01A129524) and
by a grant from the National Cancer Institute of Canada with
funds from The Terry Fox Run.
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