From the Unidad de Investigación, Hospital
Universitario Reina Sofía, 14004 Córdoba, Spain and
¶ Department of Immunology and Oncology, National Centre of
Biotechnology, 28049 Madrid, Spain
Received for publication, July 9, 2002, and in revised form, October 10, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chromosomal stability and cell viability
require a proficient telomeric end-capping function. In particular,
telomere dysfunction because of either critical telomere shortening or
because of mutation of telomere-binding proteins results in increased
apoptosis and/or cell arrest. Here, we show that, in turn, DNA
damage-induced apoptosis results in a dramatic telomere loss. In
particular, using flow cytometry for simultaneous detection of telomere
length and apoptosis, we show that cells undergoing apoptosis upon DNA
damage also exhibit a rapid and dramatic loss of telomeric sequences.
This telomere loss occurs at early stages of apoptosis, because it does
not require caspase-3 activation, and it is induced by loss of the mitochondrial membrane potential ( Somatic cells are exposed to a great variety of DNA damaging
agents, which may result in genomic instability. All organisms are
equipped with mechanisms to recognize and respond to DNA damage, either
by triggering cell cycle arrest or by undergoing cell death by
apoptosis (1). Apoptosis is a form of cell death that has been
described as distinct from necrotic cell death. The biochemical characteristics of apoptosis include (i) loss of plasma membrane phospholipid asymmetry, (ii) activation of a cascade of caspases, and
(iii) loss of the mitochondrial barrier function (2).
Since the seminal work of Müller in the 1930s, we know that the
ends of chromosomes have a special structure that distinguishes them
from DNA breaks and protects them from fusing (3). Telomeres are
composed of tandem repeats of non-coding DNA sequences (TTAGGG in all
vertebrates) and of associated proteins (4, 5). Both a minimal length
of TTAGGG repeats at the telomeres and telomere-binding proteins are
essential to preserve functional telomeres (6). It is likely that
telomeres are protected from cellular activities by their ability to
form a higher order structure known as the T-loop (7). Loss of TTAGGG
repeats during cell division or with increasing age may result in
telomere dysfunction and, therefore, delimit life span. Telomere
shortening is prevented in those cell types that activate the enzyme
telomerase, a reverse transcriptase that synthesizes telomeric repeats
de novo at chromosome ends. Telomerase has two essential
components, a catalytic subunit (hTERT) and a small RNA molecule that
contains the template use to synthesize new telomeric repeats (hTER)
(8). Telomerase activity has been found in germ cells and in most
tumors, but only weak or no activity is detected in normal somatic
cells (9). Ectopic expression of telomerase in somatic mortal cells
results in telomere elongation and indefinite extension of the life
span, indicating that telomerase activity is sufficient for immortal
growth (10). In addition, it has been shown recently (11, 12) that
re-introduction of telomerase is able to specifically elongate
critically short telomeres and to prevent chromosomal instability in
the telomerase-deficient mouse model.
Genes involved in signaling DNA damage are important for apoptosis,
such as p53 (13-15), poly(ADP-ribose) polymerase
(PARP1)1 (16) or the
DNA-dependent protein kinase (DNA-PK) complex (17). In addition,
proteins important for double strand break repair, such as the
components of the DNA·PK and MRE11 complexes, which are involved in
non-homologous end joining of double strand DNA breaks and in
homologous recombination, have been shown to be also located at the
telomeres and to influence telomere function (18-21). A connection
between telomerase activity and resistance to apoptosis has also been
established (22). In particular, inhibition of telomerase and telomere
shortening below a critical length results in apoptosis in various
cell types, whereas induction of telomerase activity is associated with
resistance to apoptosis (23-25). Here, we show that the opposite is
also true and that DNA damage-induced apoptosis results in a dramatic
telomere shortening in those cells undergoing apoptosis. In addition,
we determine that this massive telomere shortening is one of the early
events of DNA damage-induced apoptosis. All together, these results
support a role for telomeres as key sensors of DNA damage.
Cell Culture--
Peripheral blood lymphocytes (PBL) were
obtained from 20 ml of heparinized whole blood donated by 15 healthy
volunteers. PBL were isolated by differential gradient centrifugation
(Ficoll/Hypaque; Amersham Biosciences). Pro-myelocytic leukemia
HL60 cells (ATCC CCL 240) were obtained from the American Type Culture
Collection (Manassas, VA). Cultured cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), and 1% penicillin/streptomycin
(10,000 units penicillin/ml and 10 mg/ml streptomycin). Cells were
grown at 37 °C in a humidified, 5% CO2 atmosphere.
Mice--
Wild-type and late generation telomerase-deficient
mice, Terc Drugs and Apoptosis Induction--
The inhibitor of caspase-3
protease (acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-DEVD-CHO) was purchased
from BD Biosciences and was used at 100 nM, and
camptothecin (CPT; 10 µM) and etoposide (10 µg/ml)
(Sigma) were used for 6 h at the indicated concentrations. In experiments using UV to induce apoptosis, cells were exposed to 180 mJ/cm2 UV light and further cultured for
8 h in medium.
Telomerase Assay--
S-100 extracts were prepared from
wild-type, DNA·PKcs+/ Transient Transfection of HL60 Cells with p53 Full-length
cDNA--
A HindIII fragment encompassing the human p53
cDNA was cloned into the retroviral vector pLNSX. Transfection of
the plasmid containing the p53 gene or the empty vector was done using
Lipofectin according to the manufacturer (Invitrogen). Human HL60 cells
were plated in six-well culture plates for 24 h at a density of
1 × 105 cells/well. Plasmid DNA (1 µg) and
Lipofectin (6 µg) in culture medium were incubated for 45 min at room
temperature. Plasmid and Lipofectin were then mixed and further
incubated for 45 min before laying the complex over the cells. After
6 h of incubation at 37 °C, the DNA-containing medium was
replaced by fresh medium. Twenty-four h after transfection, cells were
either UV irradiated or not, cultured for another 3, 6, 9, or
14 h, and then lysed in 100 µl of lysis buffer (Promega).
Caspase Activity Determinations--
Caspase-3 activity was
determined by flow cytometry using phycoerythrin-conjugated polyclonal
anti-human active caspase-3 antibodies (Pharmingen). Cells (5 × 105) were washed, fixed, and permeabilized using the FIX & PERM cell permeabilization kit (Caltag Laboratory, Burlingame,
CA). After incubation for 5 min at room temperature, 4 ml of pre-cooled
absolute methanol were added and incubated for 10 min at 4 °C.
Thereafter, cells were washed (5 min, 1500 rpm) in wash buffer
(phosphate-buffered saline + 0.1% NaN3 + 10% autologous
serum). To permeabilize the cells, the supernatant was removed, and 100 µl of permeabilization reagent and 10 µl of
phycoerythrin-conjugated polyclonal, anti-active caspase-3 antibody
(Pharmingen), or isotype control antibody were added. After incubation,
cells were washed and resuspended in 0.5 ml of 1% formaldehyde and
stored at 4 °C until flow cytometric analysis.
Analysis of Mitochondrial Transmembrane Potential--
Loss of
mitochondrial transmembrane potential was monitored using flow
cytometry. Briefly, cells were incubated at 37 °C for 15 min in the
presence of chloromethyl-X-rosamine (0.1 µM, fluorescence at 600 nm; Molecular Probes, Eugene, OR), followed by immediate analysis of fluorochrome incorporation in a FACScan flow cytometer (BD
Biosciences). As a control, cells were incubated throughout culture in
the presence of the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (50 µM; Sigma).
Detection of Reactive Oxygen Species (ROS)--
Hydroethidine at
2 µM (Molecular Probes), a substance that is oxidized by
ROS to become ethidium and to emit red, was used to measure superoxide
anion. Briefly, cells were exposed for 15 min at 37 °C to
hydroethidine. Analyses were performed in a flow cytometer (FACScan).
Immunoblot Analysis of PARP1 Degradation--
Cells (5 × 106) were lysed in 20 mM Hepes, 250 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 0.5 µg/ml benzamidine, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride. Proteins (80 µg/ml) were separated on
an SDS-PAGE polyacrylamide gel and transferred onto a polyvinylidene
difluoride membrane. Western blotting was carried out using a
monoclonal anti-PARP1 antibody (Pharmingen) and anti-mouse antibody
conjugated to horseradish peroxidase. Protein bands were detected by
chemiluminescence. The appearance of an 85-kDa PARP1 cleavage product
was used as a measure of apoptosis.
Telomere Length Measurement by Terminal Restriction Fragment
(TRF) Analysis--
DNA was isolated by proteinase K digestion and
phenol/chloroform extraction. DNA samples were digested with the
restriction enzymes HinfI and RsaI (Roche
Molecular Biochemicals). Aliquots of undigested and digested DNA were
resolved by 0.5% agarose gel electrophoresis (70 V, 2 h) and
examined by ethidium bromide staining for the absence of nonspecific
degradation and complete digestion, respectively. A total of 1.5 mg of
each digested sample was resolved by 0.7% agarose gel electrophoresis
(40 V, 40 h). DNA was Southern blotted onto a nitrocellulose
membrane (Hybond-C Extra; Amersham Biosciences) and probed as described
previously (20, 25) with minor modifications. The membranes were
hybridized at 42 °C overnight with a end-labeled
32P-(TTAGGG)5 oligonucleotide telomere probe in
a buffer containing 25% formamide, Denhardt's solution, saline/sodium
phosphate/EDTA, 0.1% SDS, and 100 mg/ml denatured salmon sperm
DNA. After a 15-min stringency wash at 42 °C in 0.23 SSC, 0.1% SDS
the autoradiography signal was digitalized in a phosphorimage scanner
(Fuji) using ImageGauge software. All lanes were subdivided into
intervals of ~1 to 2 mm. The mean size of the TRF was estimated using
the formula (ODi × Li)/(ODi), where ODi is the density reading
from interval I, and Li is the size in kbp of the interval relative to
the markers. Mean TRF length was determined over the range of 2.3 to
23.1 kbp markers (broad range) and also on the basis of the intensity
of the signal (narrow range), where the intervals averaged were those intervals that were higher than 1% of the total signal in that lane.
The median and mode values were also derived on the basis of the narrow
range determination.
Flow Cytometric Detection of Telomere Fluorescence in Situ
Hybridization (FISH) (Flow-FISH)--
For Flow-FISH, 1 × 106 cells were resuspended in hybridization buffer
containing 70% deionized formamide, 10 mM Tris, pH 7.0, 10% fetal calf serum, and 0.3 µg/ml of the telomere-specific
fluorescein isothiocyanate-conjugated probe (fluorescein
isothiocyanate-O-CCCATAACTAAACAC-NH2). DNA from samples was
heat-denatured for 10 min at 80 °C in a Thermomixer 5436 (Eppendorf,
Netheler, Germany) followed by hybridization for 2 h at room
temperature. Cells were washed in washing buffer containing 70%
deionized formamide, 10 mM Tris, pH 7.0, 10% fetal calf
serum, 0.1% Tween 20. After incubation for 1 h at room
temperature, cells were washed and resuspended in phosphate-buffered
saline, 10% fetal calf serum, RNase A at 10 µg/ml (Roche Molecular
Biochemicals), and propidium iodide, incubated for 1 h at room
temperature, washed, and analyzed in a FACScan flow cytometer. The
telomere fluorescence signal was defined as the mean fluorescence
signal in G0/G1 cells after subtraction of the
background fluorescence signal (FISH procedure without probe). The flow
cytometer was calibrated every day using fluorescein
isothiocyanate-labeled fluorescence Sphero microparticles (Pharmingen).
The resulting calibration curve was used for correction of experimental
fluorescence values in each experiment. Green fluorescence was measured
on a linear scale, and results were expressed in molecular equivalents
of soluble fluorochrome units (kMESF) (26). The relative telomere
length value was calculated by comparing the kMESF values to those
obtaining using different cell lines as telomere length controls.
TUNEL Assay--
Apoptosis was measured using a kit based on the
TUNEL (Roche Molecular Biochemicals). In accordance with the
manufacturer's instructions, 106 cells fixed with 4%
paraformaldehyde for 30 min at room temperature, washed, and
permeabilized for 2 min in ice with 0.1% Triton X-100. After washing,
cells were decanted and resuspended in 50 µl of TUNEL reaction
mixture (5 µl TUNEL enzyme containing terminal deoxynucleotidyltransferase, mixed with 45 µl of TUNEL label
containing phycoerythrin-dUTP and dNTP nucleotides) or in 50 µl of
TUNEL Label as negative control. After 60 min at 37 °C in a humid
atmosphere, cells were washed three times in wash buffer
(phosphate-buffered saline + 0.1% NaN3 + 10% autologous
serum) and analyzed by flow cytometry.
Statistical Analysis--
Differences between means were
analyzed by analysis of variance followed by the Duncan test. Results
are presented as the mean ± S.D. of experiments performed in triplicate.
Cells Undergoing Apoptosis Show Dramatically Shortened
Telomeres--
To determine whether DNA damage directly affects
telomere length, we simultaneously measured telomere length and
apoptosis in normal human PBL, as well as in a human tumor cell line,
HL60 (see "Experimental Procedures"), or after treatment with
various DNA damaging agents including CPT, etoposide (Topo 1), and UV radiation (Rad UV). For telomere length determinations, we performed quantitative FISH hybridization using a telomere-specific
peptide nucleic acid probe, and measured telomere fluorescence
by flow cytometry (Flow-FISH; see "Experimental Procedures"). The
intensity of telomeric fluorescence is proportional to telomere length
(see "Experimental Procedures"). Fig.
1A shows a representative
example of simultaneous analysis by flow cytometry of telomere
fluorescence and apoptosis in HL60 cells that were either untreated or
treated with CPT (Fig. 1A). In the absence of CPT treatment,
telomeres showed the characteristic distribution of fluorescence and no apoptosis was detected in these cells (R1 peak in Fig. 1A,
upper panel). Following CPT treatment, telomeric Flow-FISH
showed a two-peak bimodal distribution of cells, depending on their
telomere length/fluorescence, one peak showing normal-length telomeres (R1) and another with dramatically shortened telomeres (R2), as indicated by their lower telomeric fluorescence intensity. On cell
sorting of the two peaks and determination of apoptosis, the R2 peak
was shown to contain apoptotic cells, whereas the R1 peak contained
normal viable cells, indicating that those cells that are undergoing
apoptosis also show a dramatic telomere shortening (Fig 1B,
bottom panel). Indeed, all forms of DNA damage tested resulted in increased apoptosis, concomitant with a significant telomere shortening in both the normal PBL and the HL60 cell
line (Table I). Table I also shows
that those cells with higher rates of apoptosis also had the shortest
telomeres (see apoptosis/telomere ratios). In some cases, Flow-FISH
results were confirmed by Southern blot analysis of TRF, which contain
the TTAGGG telomeric repeats, as well as various lengths of
subtelomeric sequences. Fig. 1B shows that treatment with
two other DNA damaging agents, UV and CPT, resulted in the appearance
of shorter TRFs, indicating telomere shortening in the treated
cells.
A time course study performed in HL60 cells treated with CPT showed
that shortening in telomere length is already evident after 2 h in
culture and that it peaks after 6 h (Fig.
2). Associated with the decrease in
telomere length, an increase was also found in the percentage of
apoptotic cells (16 ± 3% through 71 ± 10%) following
12 h of cell culture (Fig. 2). In all experiments more than 90%
of the apoptotic cells were found in the low telomere subset; this is
in contrast to that observed in the cell subset with normal
telomere length in which the apoptosis was less than 5% (data not
shown).
The Telomere Shortening Associated with DNA Damage-induced
Apoptosis Is Independent of Caspase-3 Activation--
One of the
molecular events associated with DNA damage-induced apoptosis is the
cleavage of caspase-3 pro-enzyme into a caspase-3 active form. To
determine whether the cells showing short telomeres upon DNA damage
also express the active form of caspase-3, flow cytometry was used to
simultaneously detect telomere fluorescence and caspase-3 activity (see
"Experimental Procedures"). Five different experiments were carried
out using CPT as the DNA damage agent, and a representative set
of results is shown in Fig.
3A. The fraction of
CPT-treated cells showing the shortest telomeres by Flow-FISH (R2 peak
in Fig. 3A) also exhibited the higher levels of caspase-3 activity (Fig. 3A).
To determine whether the telomere shortening associated with DNA
damage-induced apoptosis was dependent on caspase-3 activation, we
treated the cells with Ac-DEVD-CHO, a caspase-3 inhibitor. As expected,
inhibition of caspase-3 activation by Ac-DEVD-CHO prevented both
caspase-3 activity and apoptosis in CTP-treated cells (Fig.
3B). However, Ac-DEVD-CHO did not prevent telomere shortening in CTP-treated cells (Fig. 3B).
Mitochondria Are Key Regulators of Apoptosis-induced Telomere
Degradation--
The relationship between apoptosis-induced telomere
shortening and mitochondrial function was also addressed using flow
cytometry. In particular, we focused on two key mitochondrial events
that occur during apoptosis: (i) the generation of ROS, and (ii) the loss of mitochondrial membrane potential ( Relationship between Apoptosis-induced Telomere Shortening and
PARP1 Degradation--
We found here that the subset of CPT-treated
cells that exhibited shorter telomere length (peak R2) also showed
PARP1 cleavage (Fig. 5, lane
2). In contrast, PARP1 cleavage was not observed in the subset of
cells with normal telomere length (Fig. 5, lane 1).
Telomere Shortening Associated with DNA Damage-induced Apoptosis Is
Independent of p53 Status--
The human tumor cell line HL60 used for
this study lacks the p53 gene because of a deletion (27). To determine
the effect of p53 in apoptosis-induced telomere shortening, we
transiently transfected wild-type p53 into HL60 cells, and these cells
were named HL60SN3. As expected, greater susceptibility to apoptosis was observed in p53-transfected HL60 cells (HL60SN3) upon DNA damage
than in HL60 control cells, apoptosis being observed in 87.6 and 69.1%
of cells in each case (Fig. 6). Average
telomere length was also shorter in the p53-transfected HL60 (HL60SN3) cells than in the HL60 p53-null cells, whether they were untreated or
had been treated with CPT (see Table II
and Fig. 6), which agrees with the higher rates of apoptosis in the
p53-positive cells (Fig. 6). However, the ratio of apoptosis to
telomere length (Table II) was similar in both cases, suggesting that
p53 status does not influence the telomere shortening associated with
DNA damage-induced apoptosis, at least in HL60 cells (Table
II).
Changes in Telomerase Activity Associated with DNA Damage-induced
Apoptosis and Telomere Shortening--
To study whether telomere
shortening triggered by apoptosis could be due, at least in part, to
telomerase inhibition in these cells as a consequence of apoptosis, we
studied telomerase activity using the telomeric repeat amplification
protocol assay in HL60 cells before and after treatment with CPT (see
"Experimental Procedures"). Following CPT treatment, a moderate
decrease in telomerase activity could be detected in HL60 cells (Fig.
7).
Cells Derived from Terc Cells with short telomeres are more sensitive to DNA damage
(28-30). In this study, we have demonstrated that cells undergoing apoptosis show dramatically shortened telomeres.
Our results indicate that caspase-3 activation is not necessary for the
telomere shortening that is associated with DNA damage-induced apoptosis. This also indicates that telomere shortening is one of the
early events that occur in apoptosis, prior to the activation of
caspases. In this sense a recent report demonstrated that apoptosis induced by hydroxyl radical is not associated with caspase activation (31).
Two key mitochondrial events that occur during apoptosis are (i) the
generation of ROS, and (ii) the loss of mitochondrial membrane
potential ( PARP1 is activated during the DNA damage response, and it is involved
in the base-excision repair (16), being cleaved by caspases into two
fragments of 115 and 85 kDa, respectively, during apoptosis. PARP1 has
been also suggested as a potential regulator of telomere length and
function, although a recent analysis of telomere length and function in
a PARP1-deficient mouse suggested that PARP1 activity is not directly
involved in telomere length maintenance or in chromosome end-capping
(12). Our data support the idea of a relationship between
apoptosis-induced telomere shortening and PARP1 degradation. These
results further support an association between short telomeres and apoptosis.
p53 is a key of damaged DNA and is capable of eliciting either
apoptosis or cell cycle arrest, depending on the type of cell involved
(14-15). p53 is also a sensor of short telomeres, because the
phenotypes associated with telomeric dysfunction in the
telomerase-deficient mouse model, Terc Our results also suggest that lower telomerase activity is associated
with the process of apoptosis. However, this mild decrease in
telomerase activity is unlikely to account for the massive telomere
loss that can be observed in apoptotic cells, and further mechanisms
will have to be invoked.
Cells derived from G5 Terc In summary, we show here that cells that suffer apoptosis after DNA
damage exhibit a dramatic loss of telomere length compared with
non-apoptotic cells. Furthermore, such telomere shortening does not
require caspase-3 activation and can be directly induced by
mitochondrial membrane depolarization. All in all, these observations suggest that telomere shortening is one of the early events in DNA
damage-induced apoptosis. We also show here that p53 does not influence
the telomere shortening associated with apoptosis. This is in agreement
with previous observations that telomeric dysfunction triggers
apoptosis in human tumor cell lines independently of their p53 status
(23). Finally, we show that PBL derived from late generation
telomerase-deficient mice, which have critically shortened telomeres,
are more sensitive to DNA damage-induced apoptosis than the wild-type
controls. These results thus indicate that (i) dramatic telomere
shortening occurs as an early event of DNA damage-induced apoptosis,
and that, in turn, (ii) this telomeric dysfunction may be one of the
key events that signal the cellular responses associated with the
apoptotic process in a way that is independent of p53 status.
m) and
production of reactive oxygen species. These observations
suggest a direct effect of mitochondrial dysfunction on telomeres.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, were described elsewhere (11).
, and DNA·PKcs
/
primary mouse embryo fibroblast cultures, and a modified version of the telomeric repeat amplification protocol assay was used to
measure telomerase activity as described (11). An internal control for
PCR efficiency was included (TRAPeze kit Oncor).
-Galactosidase activity was determined by an
O-nitrophenol-beta-D-galactopyranoside substrate assay (Promega
-galactosidase enzyme assay) and is expressed relative to the amount
of protein (Bio-Rad protein assay) recovered from each well.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Telomere length determinations by
flow cytometry (Flow-FISH) or by terminal restriction fragment analysis
(TRF). A, representative histograms of telomere fluorescence
in untreated ( CPT) or camptothecin-treated
(+CPT) cells. After CPT treatment two different subsets of
cells were observed according to their telomere length/fluorescence,
the R1 peak showing normal length telomeres and the R2-peak showing
short telomeres. TUNEL assay indicates that those cells with short
telomeres (R2) are undergoing apoptosis compared with the subpopulation
of cells with normal telomeres (R1). B, representative
examples of TRF analysis of untreated and UV-, and CPT-treated cells.
Notice that upon treatment with DNA damaging agents, lower molecular
weight TRFs appear (short TRFs) in the treated cells, in
agreement with telomere shortening in these cells.
DNA damage is associated with telomere shortening
View larger version (14K):
[in a new window]
Fig. 2.
Time course study performed in HL60 cells
treated with CPT. After CPT treatment, telomere length and
apoptosis were studied in the same experiment. As this figure shows,
the decrease in telomere length was associated with the increase in
apoptosis.
View larger version (22K):
[in a new window]
Fig. 3.
Caspase3 activity determinations using flow
cytometry in untreated ( CPT) or CPT-treated cells
(+CPT). A, upon CPT treatment,
caspase-3 activity is greater in the subpopulation of cells with short
telomeres (R1 peak) than in those showing normal length
telomeres (R2 peak). B, effect of caspase-3
inhibitor Ac-DEVD-CHO on caspase activity, apoptosis, and telomere
length. Ac-DEVD-CHO treatment inhibits both caspase-3 activation and
apoptosis in CPT-treated cells but does not inhibit telomere
shortening. These observations indicate that telomere shortening occurs
prior to caspase-3 activation.
m). Fig.
4A shows that the subset of
CPT-treated cells showing short telomeres (peak R2) also exhibited a
decrease in
m, again indicating that cells undergoing
apoptosis have shorter telomeres (Fig. 4A, upper
panel). Treatment with CPT also increased ROS production,
coincidental with increased apoptosis and concomitant with telomere
shortening (Fig. 4B). Furthermore, complete uncoupling of
the mitochondrial membrane potential by addition of protonophore mC1CCP
further increased ROS production, accelerated apoptosis, and
dramatically increased the percentage of cells with very short
telomeres (Fig. 4). Indeed, all CPT-treated cells were in the R2 peak
of short telomeres when mC1CCP was used (Fig. 4A).
View larger version (19K):
[in a new window]
Fig. 4.
Involvement of the mitochondria in the
telomere shortening associated to DNA damage induced apoptosis.
A, upon CPT treatment, the subset of cells showing shorter
telomeres also show a decreased m. Upon CPT + mC1CCP
treatment, the majority of cells underwent telomere shortening
(R2 peak only) compared with the cells treated only with
CPT. Furthermore, the CPT + mC1CCP-treated cells showed uncoupling
of the mitochondria membrane potential (bottom panel).
B, effect of uncoupling of the mitochondrial potential on
ROS generation, apoptosis, and telomere length. Treatment of cells with
CPT increased ROS production and apoptosis and resulted in telomere
shortening. These effects were further increased when cells were
treated with CPT + mC1CCP, indicating that loss of the mitochondrial
membrane potential directly affects telomeric function.
View larger version (42K):
[in a new window]
Fig. 5.
Degradation of PARP1 in cells undergoing
apoptosis after CPT treatment. Lane 1 shows normal PARP1
status in untreated cells showing normal length telomeres. Upon CPT
treatment, those cells showing short telomeres (R2 peak)
also showed PARP1 degradation into the 115- and 85-kDa fragments
(lane 2).
View larger version (19K):
[in a new window]
Fig. 6.
Telomere shortening associated with DNA
damage induced apoptosis in cells that either lack (HL60) or have
reconstituted wild-type p53 activity (HL60SN3).
CPT treatment of P53-proficient cells (HL60SN3; bottom
panel), resulted in a higher percentage of apoptotic cells as
determined by TUNEL than in the case of the p53-deficient cells
(HL60; upper panel). The apoptotic cell fraction
showed a similar telomere shortening (R2 peak) in HL60 and
HL60SN3 cells. See Table II for quantifications.
Simultaneous study of apoptosis and telomere length in wild-type and
null p53 HL60 cells
View larger version (54K):
[in a new window]
Fig. 7.
Telomerase activity measured using the
telomeric repeat amplification protocol assay in untreated
( CPT) and CPT-treated (+CPT)
cells. Extracts were pretreated (+) or not (
) with RNase, to
check for specificity of the telomeric repeat amplification protocol
reaction. The arrow indicates the internal control
(IC) for PCR efficiency.
/
Mice with Short Telomeres
Exhibit Increased Apoptosis after CPT Treatment--
Lymphocytes
derived from wild-type, second-generation (G2) and
fifth-generation (G5) Terc
/
mice that lack
telomerase activity were used to further investigate the relationship
between susceptibility to apoptosis and telomerase length (11).
Although G2 Terc
/
mice have telomeres that
are similar in length to those of wild-types, G5
Terc
/
mouse telomeres are an average of 40% shorter
than those of the wild-type controls (11). Furthermore, G5
Terc
/
mice display a proportion of chromosomes that
lack detectable TTAGGG repeats at the telomeres, resulting in greater
chromosomal instability (11). After CPT treatment (see "Experimental
Procedures"), apoptosis occurs in 46 ± 9% of G5
Terc
/
lymphocytes, compared with only 6 ± 2% of
wild-type lymphocytes (Fig. 8).
Similarly, phytohemagglutinin A stimulation also resulted in a
rise in lymphocyte apoptosis in G5 Terc
/
mice, in contrast to what we observed in lymphocytes from wild-type mice (Fig. 8).
View larger version (20K):
[in a new window]
Fig. 8.
Determination of percentage of apoptotic
cells in untreated, treated with CPT, or treated with
phytohemagglutinin A mouse primary lymphocytes derived from wild-type,
second-generation (G2) and fifth-generation
(G5) Terc /
mice, which lack telomerase
activity and show progressive telomere shortening with increasing mice
generations. Cells from G5 Terc
/
mice
displayed increased apoptosis after CPT or phytohemagglutinin A
treatment compared with similarly treated cells from wild-type or
second-generation mice.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m). We have demonstrated that cells with short telomeres showed a drop in
m. We have also
observed increased ROS production, coinciding with increased apoptosis
and concomitant with telomere shortening. These results support a key
role for the mitochondria in telomere length metabolism.
/
, are delayed
when in a p53-deficient background (11, 32). Our results show that
telomere shortening associated with DNA damage-induced apoptosis is
independent of p53 status.
/
mice with short
telomeres exhibit increased apoptosis after CPT treatment. These
results suggest that telomere shortening renders lymphocytes more
susceptible to apoptosis after treatment with DNA damaging agents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants 72/01 and 68/01 from the Junta de Andalucía, Fundación Nefrológica and Grants FIS 00/0788 (to J. C.) and 00/0701 (to R. R.). The Blasco laboratory was funded by SWISS BRIDGE AWARD 2000, by the Ministry of Science and Technology, Spain (PM97-0133), by European Union (EU) Grants EURATOM/991/0201, FIGH-CT-1999-00002, and FIS5-1999-00055, and by the Department of Immunology and Oncology (DIO). The DIO was funded by the Spanish Council for Scientific Research and by Pharmacia Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Unidad de Investigación, Hospital Universitario Reina Sofía, Avda. Menéndez Pidal S/N, Cordoba 14004, Spain. Tel.: 34-957-010452; Fax: 34-957-010452; E-mail: rramirez@hrs.sas.junta-andalucia.es.
Pre-doctoral fellow of the EU.
** Supported by the DIO.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M206818200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PARP1, poly(ADP-ribose) polymerase; PBL, peripheral blood lymphocytes; FISH, fluorescence in situ hybridization; CPT, camptothecin; ROS, reactive oxygen species; TRF, terminal restriction fragment; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zhou, B. B., and Elledge, S. J. (2000) Nature 408, 433-439[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Roy, S.,
and Nicholson, D. W.
(2000)
J. Exp. Med.
192,
647-658 |
3. | Müller, H. J. (1938) Collect. Net. Woods Hole. 13, 181-198 |
4. | McEachern, M. J., Krauskopf, A., and Blackburn, E. H. (2000) Annu. Rev. Genet. 34, 331-358[CrossRef][Medline] [Order article via Infotrieve] |
5. |
de Lange, T.
(2001)
Science
292,
1171-1175 |
6. |
Blackburn, E. H.
(1990)
J. Biol. Chem.
265,
5919-5921 |
7. | Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., and de Lange, T. (1999) Cell 97, 503-514[Medline] [Order article via Infotrieve] |
8. | Collins, K. (2000) Curr. Opin. Cell Biol. 12, 378-383[CrossRef][Medline] [Order article via Infotrieve] |
9. | Shay, J. W., and Bacchetti, S. (1997) Eur. J. Cancer. 33, 787-791[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Bodnar, A. G.,
Ouellette, M.,
Frolkis, M.,
Holt, S. E.,
Chiu, C. P.,
Morin, G. B.,
Harley, C. B.,
Shay, J. W.,
Lichtsteiner, S.,
and Wright, W. E.
(1998)
Science
279,
349-352 |
11. | Blasco, M. A., Lee, H. W., Hande, M. P., Samper, E., Lansdorp, P. M., DePinho, R. A., and Greider, C. W. (1997) Cell 91, 25-34[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Samper, E.,
Flores, J. M.,
and Blasco, M. A.
(2001)
EMBO Rep.
2,
800-807 |
13. | Wang, B. (2001) J. Radiat. Res. 42, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
14. | Andoh, T. (2000) Cell. Biochem. Biophys. 33, 181-188[CrossRef][Medline] [Order article via Infotrieve] |
15. | Taylor, W. R., and Stark, G. R. (2001) Oncogene 20, 1803-1815[CrossRef][Medline] [Order article via Infotrieve] |
16. | Bürkle, A., Schreiber, V., Dantzer, F., Oliver, F. J., Niedergang, C., de Murcia, G., and Ménissier de Murcia, J. (2000) in DNA Damage and Stress Signaling to Cell Death (de Murcia, G. , and Shall, S., eds) , pp. 80-124, Oxford University Press, Oxford, United Kingdom |
17. |
Smith, G. C.,
and Jackson, S. P.
(1999)
Genes Dev.
13,
916-934 |
18. |
Samper, E.,
Goytisolo, F.,
Slijepcevic, P.,
van Jul, P.,
and Blasco, M. A.
(2000)
EMBO Rep.
1,
244-252 |
19. |
Hsu, H. L.,
Gilley, D.,
Galande, S. A.,
Hande, M. P.,
Allen, B.,
Kim, S. H., Li, G. C.,
Campisi, J.,
Kowhi-Shigematsu, T.,
and Chen, D. J.
(2000)
Genes Dev.
14,
2807-2812 |
20. |
Goytisolo, F. A.,
Samper, E.,
Edmonson, S.,
Taccioli, G. E.,
and Blasco, M. A.
(2001)
Mol. Cell. Biol.
21,
3642-3651 |
21. | Zhu, X. D., Kuster, B., Mann, M., Petrini, J. H., and de Lange, T. (2000) Nat. Genet. 25, 347-352[CrossRef][Medline] [Order article via Infotrieve] |
22. | Mattson, M. P., Fu, W., and Zhang, P. (2001) Mech. Ageing. Dev. 122, 659-671[CrossRef][Medline] [Order article via Infotrieve] |
23. | Hahn, W. C., Stewart, S. A., Brooks, M. W., York, S. G., Eaton, E., Kurachi, A., Beijersbergen, R. L., Knoll, J. H., Meyerson, M., and Weinberg, R. A. (1999) Nat. Med. 5, 1164-1170[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Herbert, B.,
Pitts, A. E.,
Baker, S. I.,
Hamilton, S. E.,
Wright, W. E.,
Shay, J. W.,
and Corey, D. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14276-14281 |
25. | Holt, S. E., Glinsky, V. V., Ivanova, A. B., and Glinsky, G. V. (1999) Mol. Carcinog. 25, 241-248[CrossRef][Medline] [Order article via Infotrieve] |
26. | Henderson, L. O., Marti, G. E., Gaigalas, A., Hannon, W. H., and Vogt, R. F., Jr. (1998) Cytometry 33, 97-105[CrossRef][Medline] [Order article via Infotrieve] |
27. | Shimizu, T., and Pommier, Y. (1997) Leukemia 11, 1238-1244[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Goytisolo, F. A.,
Samper, E.,
Martin-Caballero, J.,
Finnon, P.,
Herrera, E.,
Flores, J. M.,
Bouffler, S. D.,
and Blasco, M. A.
(2000)
J. Exp. Med.
192,
1625-1636 |
29. | Wong, K. K., Chang, S., Weiler, S. R., Ganesan, S., Chaudhuri, J., Zhu, C., Artandi, S. E., Rudolph, K. L., Gottlieb, G. J., Chin, L., Alt, F. W., and DePinho, R. A. (2000) Nat. Genet. 26, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Lee, K. H.,
Rudolph, K. L., Ju, Y. J.,
Greenberg, R. A.,
Cannizzaro, L.,
Chin, L.,
Weiler, S. R.,
and DePinho, R. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3381-3386 |
31. | Ren, J. G., Xia, H. L., Just, T., and Dai, Y. R. (2001) FEBS Lett. 488, 123-132[CrossRef][Medline] [Order article via Infotrieve] |
32. | Chin, L., Artandi, S. E., Shen, Q., Tam, A., Lee, S. L., Gottlieb, G. J., Greider, C. W., and DePinho, R. A. (1999) Cell 97, 527-538[Medline] [Order article via Infotrieve] |