Massive Telomere Loss Is an Early Event of DNA Damage-induced Apoptosis*

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 partic-ular, 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 ( (cid:1)(cid:2) m ) and production of reactive oxygen species. These observations suggest a direct effect of mitochondrial dysfunction on telomeres.

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 Mu ¨ller 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)(14)(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 DNA⅐PK 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)(24)(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.
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 M) and etoposide (10 g/ml) (Sigma) were used for 6 h at the indicated concentrations.In experiments using UV to induce apoptosis, cells were exposed to 180 mJ/cm 2 UV light and further cultured for 8 h in medium.
Telomerase Assay-S-100 extracts were prepared from wild-type, DNA⅐PKcs ϩ/Ϫ , and DNA⅐PKcs Ϫ/Ϫ 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).
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 ϫ 10 5 cells/well.Plasmid DNA (1 g) and Lipofectin (6 g) 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 l of lysis buffer (Promega).␤-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.
Caspase Activity Determinations-Caspase-3 activity was determined by flow cytometry using phycoerythrin-conjugated polyclonal anti-human active caspase-3 antibodies (Pharmingen).Cells (5 ϫ 10 5 ) 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 ϩ 0.1% NaN 3 ϩ 10% autologous serum).To permeabilize the cells, the supernatant was removed, and 100 l of permeabilization reagent and 10 l 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 M, 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 M; Sigma).
Detection of Reactive Oxygen Species (ROS)-Hydroethidine at 2 M (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 ϫ 10 6 ) were lysed in 20 mM Hepes, 250 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 2 g/ml leupeptin, 2 g/ml aprotinin, 0.5 g/ml benzamidine, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.Proteins (80 g/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 32 P-(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 ϳ1 to 2 mm.The mean size of the TRF was estimated using the formula (ODi ϫ 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 ϫ 10 6 cells were resuspended in hybridization buffer containing 70% deionized formamide, 10 mM Tris, pH 7.0, 10% fetal calf serum, and 0.3 g/ml of the telomerespecific fluorescein isothiocyanate-conjugated probe (fluorescein isothiocyanate-O-CCCATAACTAAACAC-NH 2 ).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 g/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 G 0 /G 1 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, 10 6 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 l of TUNEL reaction mixture (5 l TUNEL enzyme containing terminal deoxynucleotidyltransferase, mixed with 45 l of TUNEL label containing phycoerythrin-dUTP and dNTP nucleotides) or in 50 l 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 ϩ 0.1% NaN 3 ϩ 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 Ϯ 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 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 Ϯ 3% through 71 Ϯ 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 CTPtreated cells (Fig. 3B).
Mitochondria Are Key Regulators of Apoptosis-induced Telomere Degradation-The relationship between apoptosis-in- 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 (⌬ m ).Fig. 4A shows that the subset of CPTtreated 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).
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 wildtype 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

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.associated with DNA damage-induced apoptosis, at 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 Ϫ/Ϫ Mice with Short Telomeres Exhibit Increased Apoptosis after CPT Treatment-Lymphocytes derived from wild-type, second-generation (G 2 ) and fifth-gen-eration (G 5 ) Terc Ϫ/Ϫ mice that lack telomerase activity were used to further investigate the relationship between susceptibility to apoptosis and telomerase length (11).Although G 2 Terc Ϫ/Ϫ mice have telomeres that are similar in length to those of wild-types, G 5 Terc Ϫ/Ϫ mouse telomeres are an average of 40% shorter than those of the wild-type controls (11).Furthermore, G 5 Terc Ϫ/Ϫ 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 Ϯ 9% of G 5 Terc Ϫ/Ϫ lymphocytes, compared with only 6 Ϯ 2% of wild-type lymphocytes (Fig. 8).Similarly, phytohemagglutinin A stimulation also resulted in a rise in lymphocyte apoptosis in G 5 Terc Ϫ/Ϫ mice, in contrast to what we observed in lymphocytes from wild-type mice (Fig. 8).

DISCUSSION
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 neces- 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 (⌬ 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.
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 Ϫ/Ϫ , 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.
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 G 5 Terc Ϫ/Ϫ 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.
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 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.

FIG. 1 .
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 (ϪCPT) or camptothecin-treated (ϩCPT) 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.

FIG. 2 .
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.

FIG. 4 .FIG. 5 .FIG. 6 .
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 ϩ mC1CCP treatment, the majority of cells underwent telomere shortening (R2 peak only) compared with the cells treated only with CPT.Furthermore, the CPT ϩ 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 ϩ mC1CCP, indicating that loss of the mitochondrial membrane potential directly affects telomeric function.

FIG. 7 .FIG. 8 .
FIG. 7. Telomerase activity measured using the telomeric repeat amplification protocol assay in untreated (؊CPT) and CPT-treated (؉CPT) cells.Extracts were pretreated (ϩ) or not (Ϫ) with RNase, to check for specificity of the telomeric repeat amplification protocol reaction.The arrow indicates the internal control (IC) for PCR efficiency.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, secondgeneration (G 2 ) and fifth-generation (G 5 ) Terc ؊/؊ mice, which lack telomerase activity and show progressive telomere shortening with increasing mice generations.Cells from G 5 Terc Ϫ/Ϫ mice displayed increased apoptosis after CPT or phytohemagglutinin A treatment compared with similarly treated cells from wild-type or second-generation mice.

TABLE I
DNA damage is associated with telomere shortening Results are mean Ϯ S.D. n ϭ 10.
a Double staining in cells sorted from telomere length.

TABLE II
Simultaneous study of apoptosis and telomere length in wild-type and null p53 HL60 cells Results are mean Ϯ S.D. n ϭ 10.
a Ratio of % apoptotic cells/telomere length.