Cisplatin is a chemotherapeutic agent for the treatment of neuroblastoma, a common solid extracranial malignancy of childhood and infancy. Cisplatin binds to DNA to cause a biological effect by forming cisplatin-DNA adducts and is a potent inducer of apoptosis.1, 2 The major DNA adduct formed is the bifunctional intrastrand cross-link between adjacent guanines. It is unclear how the cisplatin-DNA adducts induce cytotoxicty, though this is widely attributed to the formation of various types of cross-links, especially intrastrand cross-links,3 which are by far the most frequent lesions,4 although interstrand cross-links might be more cytotoxic.5
Telomeres, the DNA-protein structures that form the ends of chromosomes, play an important role for the stability of the genome. Their shortening with each round of DNA replication is caused by different mechanisms, one of these being their sensitivity to DNA damage.6 Cisplatin has been shown to bind preferentially to runs of 2 or more consecutive guanines.7 Human telomeric DNA contains triple runs of guanines at high density, therefore cisplatin may preferentially target telomeric DNA. Telomeres end in single stranded overhangs of the G-rich strand, which appear to be essential for telomeric higher order structure8 and for the generation of DNA damage signals from telomeres.9, 10, 11 Telomerase, a reverse transcriptase that uses an RNA template to add telomeric repeats onto the ends of chromosomes,12, 13 is expressed in 94% of neuroblastomas14 and has been shown to be a powerful independent prognostic factor.15 Thus a specific role of the telomere/telomerase complex in mediating cisplatin-induced neuroblastoma cell death may be expected and has been more or less directly indicated by some data,16, 17, 18, 19, 20, 21, 22 but refuted by others.23, 24, 25 Given these contradictory results, we decided to examine whether there is a preferential role for the telomere/telomerase complex in cisplatin-induced tumour, and especially neuroblastoma cell apoptosis. No indications for a causal involvement were found.
Materials and methods
Cell culture and drug solutions
The neuroblastoma cell line SHSY5Y26 was from the ATCC, USA, and acute lymphoblastic T cells 1301 were from Dako, Denmark. HeLa 229 cells were obtained from A. Buerkle, Konstanz. Cells were grown in RPMI medium plus 10% foetal calf serum at 37°C under air plus 5% CO2. Cisplatin was freshly dissolved in DMSO prior to each experiment and immediately diluted into medium. Concentrations of DMSO were kept below 1%. All reagents were acquired from Sigma-Aldrich (Poole, UK).
Nonadherent and adherent cells were stained with 10 μg/ml propidium iodide for 15 min at 4°C to determine the number of cells with intact plasma membrane. Apoptosis was assessed in a PAS-PPCS flow cytometer (Partec GmbH, Munster, Germany) as the fraction of cells with low forward and high sideward light scatter.27
H2A.X immunofluorescence staining
Cells grown on coverslips were fixed with 2% paraformaldehyde in PBS for 10 min and permeabilised in PBG (0.2% cold water fish skin gelatine, 0.5% BSA in PBS) with 0.5% Triton X-100 for 45 min at room temperature. Cells were then incubated with anti-phosphor histone H2A.X (Upstate, Cat no. 05-636) in PBG with 0.5% Triton X-100, washed and incubated with Alexa fluor 594 goat anti-mouse IgG (Molecular probes, Cat no. A11005) in PBG with 0.5% Triton X-100.
Cells grown on coverslips were immunostained as described above. Cells were subjected to 3 cycles of washing in PBS and fixation in 1 ml of methanol:acetic acid 3:1, air dried, baked at 60°C for 1 hr, rehydrated in 2× SSC at 37°C for 2 min and dehydrated in a series of 70, 80 and 95% ethanol for 2 min each. Cells were treated with 20 μl of Cy-3 labelled telomere-specific (C3TA2)3 peptide nucleic acid (PNA) probe (4 ng/μl) in 70% formamide/2× SSC. Both probe and cellular DNA were co-denatured at 75°C for 10 min in a moist chamber containing 1× SSC, hybridised for 2 hr in 1× SSC at 37°C and washed with 2× SSC/0.5% tween for 10 min. Cells were again incubated with the secondary antibody for 45 min and washed with PBS. Coverslips were mounted in Vectashield mounting solution containing DAPI.
Fluorescence detected alkaline DNA unwinding assay
DNA strand breaks were measured using the semiautomated fluorescence detected alkaline DNA unwinding (FADU) assay.28 The assay detects single and double strand breaks by their stimulating effect on the rate of DNA denaturation in alkaline solution. Briefly cells were treated with cisplatin for 2 hr, washed in PBS and lysed in 0.25 M meso-inositol, 1 M MgCl2 and 10 mM Na2P04/NaH2PO4, pH 7.2. The lysed cells were transferred onto a 96-well plate (replicates of 8–10) where at 4°C a denaturation buffer (9 M urea, 10 mM NaOH, 25 mM CDTA (trans-1,2-diaminocyclohexane-N,N,N′N′-tetraacetic acid), 0.1% SDS) was added, followed by an alkaline solution (200 mM NaOH, 40% denaturation buffer) at 37°C for 90 min. Remaining double stranded DNA was stained with the intercalating dye SYBR green (diluted 1:25,000 in 13 mM NaOH) and fluorescence was measured in a fluorescence reader Spectrafluor Plus (Tecan, Crailsheim, Germany) at λex 492 nm and λem 520 nm. The fluorescence intensity is inversely correlated to the number of DNA strand breaks present at the time of lysis. DNA damage percentage was calculated as 100(P0 – Px)/P0, with P0 being fluorescence intensity of an untreated sample and Px, fluorescence intensity of the treated cell sample.
Telomere and overhang lengths
Treated cells were embedded in 0.65% low melting agarose plugs at a density of 107 cells/ml before treatment with proteinase K.29 DNA was completely digested by Hinf1 (60 U/plug; Roche) at 37°C. Plugs were analysed in a 1% agarose gel by pulsed field gel electrophoresis (Biorad) at 3 V/cm for SHSY5Y cells and 5.5 V/cm for 1301 cells for 17 hr with a switching time of 2–10 in 0.5× TBE. To measure telomere restriction fragment lengths in SHSY5Y the gel was dried at room temperature, denatured (1.5 M NaCl, 0.5 M NaOH) for 30 min, neutralised (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4) for 30 min and in-gel hybridised with 32P-γ-ATP (TTAGGG)4 at 43°C for 16 hr. For the 1301 cells, gels were Southern blotted to Hybond-N+ membranes and hybridised with 32P-γ-ATP (TTAGGG)4 at 43°C for 16 hr. All gels/blots were washed 4 times in 0.2× SSC/0.2× SSC and 0.1% SDS at 43°C for 30 min each and then exposed to a phosphoimager screen overnight. Signals were visualised using a phosphoimager (Storm 820, Molecular Dynamics, Amersham). Average fragment length per lane was calculated as weighted mean of the optical density using the AIDA densitometry software (Raytek, Sheffield, UK).
The length of single stranded terminal overhangs in telomeres was measured by in-gel hybridisation of telomeric probes onto nondenatured DNA as described29 with the following modifications: In-gel hybridisation with 32P-γ-ATP (CCCTAA)4 was performed on nondenatured gels at 37°C for 16 hr. Gels were then washed as for telomeres though at 30°C and visualised. Under these conditions, only single stranded G-rich DNA is available as target for hybridisation. Afterwards, DNA was denatured and gels were hybridised as above to obtain the total telomeric signal.
Telomeric single strand breaks
Digested DNA was pre-incubated for 2 hr in 1× alkaline buffer (50 mM NaOH, 1 mM EDTA) and then electrophoresed at 26 V for 20 hr for SHSY5Y cells and 40 V for 24 hr for 1301 cells in 0.7% agarose in alkaline buffer. Gels were neutralised (1.5 M Nacl, 0.5 M Tris-HCl, pH 7.4) for 1 hr, dried at room temperature, hybridised with 32P-γ-ATP (AATCCC)4 at 43°C for 16 hr and further processed as indicated above.
Telomeric DNA was purified as described30 by annealing of the telomeric overhangs to 5′ biotinylated oligonucleotides with the sequence (CCTAA)6 followed by capture with streptavidin-coated magnetic beads. The bound telomeres were eluted from the beads by melting the oligonucleotide/telomere interaction at 65°C for 2 min in 10 mM EDTA/95% formamide. Purified telomeric DNA was analysed by pulsed field gel electrophoresis and Southern hybridisation using 32P-γ-ATP (TTAGGG)4 as described above.
Telomerase PCR ELISA and Telo TAGGG Telomerase PCR ELISAplus (Roche) were used for analysis of telomerase activity in SHSY5Y cells using 2 μg of protein diluted in lysis buffer according to the manufacturer's recommendations.
Cisplatin induces apoptosis in SHSY5Y cells
Two different cisplatin treatment regimes were analysed: a short treatment for 2 hr and a continuous treatment for at least 48 hr. Both treatments led to concentration-dependent inhibition of net growth with IC50 (growth inhibitory concentration at 50% threshold) values of 23 μM for the short treatment and 0.72 μM for the continuous treatment. Apoptosis was also induced in a concentration-dependent manner (Fig. 1). Measurement of the cellular DNA content and BrdU (2-bromo-5-deoxuridine) incorporation revealed that most surviving cells were arrested with an S phase DNA content (data not shown). We conclude that cisplatin treatment induces both apoptosis and S phase arrest in SHSY5Y cells within 24 hr following a short exposure and within 24–48 hr of a continuous exposure.
Cisplatin induces delayed DNA strand breaks and DNA damage response
A major signal transduction pathway from DNA damage to cell cycle arrest and apoptosis is via formation of DNA damage foci containing the activated form of the histone variant H2A.X (γ-H2A.X).31 γ-H2A.X immunostaining is observable within minutes after DNA strand break induction. However, DNA damage foci did not form immediately after a short 100 μM cisplatin treatment. Rather, foci formation was delayed by at least 12 hr and peaked only after about 24 hr (Fig. 2a). Even a treatment with 500 μM cisplatin induced only delayed foci formation (not shown).
To understand this delayed DNA damage response, we next measured the kinetics of DNA strand break formation using an automated version of the FADU (fluorescence-activated DNA unwinding) assay (Fig. 2b). Measurements were performed at 0, 14 and 24 hr after a short (2 hr) exposure to either 7 μM or 100 μM cisplatin. No DNA strand breaks were found immediately after treatment, but DNA damage was significant at 14 hr after the treatment for both low and high cisplatin concentrations. It increased even further at 24 hr, which parallels the data on H2A.X phosphorylation. Similar results were found following treatment with 500 μM cisplatin, although induction of apoptosis at this high concentration did prevent full quantification of the results. It appears that it is this DNA strand breakage, which might occur as an intermediate step during attempted repair of DNA cross-links by nucleotide excision repair or as result of a replication fork being stalled at cross-linked DNA that leads to DNA damage foci formation and damage response.
Cisplatin treatment does not shorten telomeres in SHSY5Y cells
Telomere restriction fragment lengths were examined over 48 hr after a short exposure to cisplatin using a wide range of concentrations in the SHSY5Y cells (Fig. 3a). The average fragment length was calculated for each lane as weighted mean of the optical density (white bars in Fig. 3a). There was only random variation between lanes, and the averages from 4 independent experiments did not significantly depend on either cisplatin dose or time after treatment (Fig. 3b). Similarly, no telomere shortening was detected in the SHSY5Y cells after a continuous cisplatin treatment lasting for up to 72 hr (Fig. 3c). Moreover, electrophoresis under denaturing conditions revealed no evidence of DNA single strand break induction in the telomeric G-rich strand (Fig. 3d). These data show that even after more than 24 hr the induction of strand breaks in telomeres in SHSY5Y cells is not frequent enough to lead to measurable telomere shortening, not even in the strand containing 50% guanines.
Cisplatin had no effect on telomeric overhangs in SHSY5Y cells
Telomeric single stranded overhangs have been implicated in telomere structural maintenance,8 shortening32 and generation of a DNA damage/growth arrest response.9, 10, 11 To test whether cisplatin treatment might interfere with the integrity of the overhangs, we first assessed overhang length by measuring the hybridisation signal intensity with a telomere repeat probe in a nondenatured gel. Exposure of cells to 500 μM cisplatin for 2 hr did not diminish the amount of single stranded telomeric overhang DNA available for hybridisation (Fig. 4a). To further investigate the effect of cisplatin on telomeric overhangs, telomeres from cisplatin-treated DNA were captured by hybridisation of the single stranded overhangs to oligonucleotides coupled to magnetic beads.30 This technique requires the presence of functional single stranded overhangs at telomeres in solution. The isolated telomeres were then run on a pulsed field gel and hybridised with a telomere-specific probe. Cisplatin treatment did not reduce the efficiency of telomere capture (Fig. 4b). We conclude that cisplatin treatment even at high concentrations does not induce degradation of the G-rich telomeric overhang.
Cisplatin at high concentrations decreases telomerase activity in SHSY5Y cells
Telomerase activity was measured by the TRAP assay after a 2 hr cisplatin treatment at various time points and cisplatin concentrations (Fig. 5). A decrease in telomerase activity occurred after treatment with high cisplatin concentrations (i.e. ≥100 μM). This decrease became only significant at late time points and concentrations where 70–90% of the cells were apoptotic (compare Figs. 1a and 5). This suggests that the loss of telomerase activity is a consequence of cisplatin-induced apoptosis rather than a possible cause.
Response of 1301 and HeLa 229 cells to cisplatin treatment
SHSY5Y cells have short telomeres with an average telomere restriction fragment length of about 4 kbp. This is similar to the majority of tumour cells, but has the disadvantage that telomere length changes might not be easy to detect. Moreover, longer telomeres are expected to be more sensitive to treatment with DNA-damaging drugs. Thus, we exposed the acute lymphoblastic T-cell line 1301, which has telomere lengths of ∼80 kbp, to the same cisplatin treatments as the SHSY5Y cells (Fig. 6). Net cell counts (data not shown) and apoptosis measurements (Figs. 6a and b) indicated that the sensitivity of this cell line to cisplatin exposure was similar to that of SHSY5Y cells. Despite these cells having long telomeres, telomere degradation was not apparent after either a short (Fig. 6c) or continuous exposure (Fig. 6d). In fact, telomeres remained at full length even after induction of apoptosis in more than 60% of the cells (compare Figs. 6a and 6c or Figs. 6b and 6d). There was no detectable degradation of single stranded G-rich overhangs for up to 72 hr under treatment (Fig. 6e). Moreover, even after 72 hr of continuous treatment, there were not sufficient single strand breaks in the G-rich telomeric strand to be detectable by denaturing gel electrophoresis and in-gel hybridisation (Fig. 6f).
Telomere shortening has been reported before in a HeLa cell strain with a telomere length of around 20 kbp following continuous treatment with low concentrations of cisplatin.33 We repeated these experiments in HeLa 229 cells and found a very similar time- and concentration-dependent induction of apoptosis (Fig. 7a). However, in 2 independent experiments, we were not able to see any significant change in telomere length (Fig. 7b).
DNA damage foci are not localised at telomeres
Uncapped, dysfunctional telomeres signal growth arrest and/or apoptosis by triggering the formation of DNA damage foci containing γ-H2A.X at telomeric sites.34, 35, 36 We therefore examined whether the foci formed after cisplatin treatment would co-localise with telomeres by using immunoFISH with antibodies against γ-H2A.X and a telomeric PNA oligonucleotide probe. As in SHSY5Y cells, γ-H2A.X-containing foci were formed at 14 hr after a 7 μM short exposure treatment in both 1301 and HeLa 229 cells. However, co-localisation with the telomeric signal did not appear more often as would be expected by chance, indicating that telomeres are not a major site of DNA damage foci formation following cisplatin treatment (Fig. 8).
There is published evidence to suggest a preferential role for telomeres as signal transducers between drug-mediated DNA damage and cellular response, i.e. growth arrest and/or apoptosis. Telomeres are specifically sensitive to DNA damage induced by UV,37 oxidative stress6, 38 and possibly chemotherapeutic drugs.39 Dysfunctional telomeres trigger a growth arrest and/or apoptosis via telomere-specific induction of DNA damage foci, also termed senescence-associated DNA damage foci.35 Inhibition of telomerase sensitises mice cells40 and human cells25, 41 towards cytotoxic drugs. However, other results are contradictory. For instance, telomeres were shown to be degraded after etoposide treatment in a HeLa cell line,39 though this was not detected in other tumour cell lines.42
More specifically, there are indications to suggest a relationship between cisplatin and telomeres. A DNA repair yeast mutant exhibited a gradual shortening of telomeres in the presence of cisplatin.43 In HeLa cells telomeres shortened after treatment with low cisplatin concentrations.33 Some telomere loss was also reported in cisplatin-treated BEL-7404 human hepatoma cells.17 In contrast, long-term cultivation of colorectal cancer cells with cisplatin resulted in telomere elongation.44
Published data on the relationship between effects of cisplatin and changes in telomerase activity are also conflicting. Inhibition of telomerase activity together with decreased hTR expression has been shown in testicular tumour cells after cisplatin treatment,16 while no changes in expression levels of either hTR or hTERT mRNA were found in cisplatin-treated BEL-7404 human hepatoma cells.17 Akeshima et al. demonstrated that hTERT expression did not change with time after cisplatin treatment in ovarian cancer cells.45 In human nasopharyngeal cells telomerase activity was not diminished after cisplatin treatment,46 and an increase in hTERT mRNA and protein during cisplatin treatment has also been reported.47 In testicular teratoma and haematopoietic cell lines the decline in telomerase activity following cisplatin treatment was shown to be a consequence of, rather than a cause for, apoptosis.23, 24
We analysed the effect of cisplatin on the telomere/telomerase complex using 2 different treatment regimens and wide ranges of drug concentrations in 3 tumour cell lines that differed by more than 1 order of magnitude in their telomere length. Cisplatin treatments induced loss of telomerase activity only after the majority of cells were in apoptosis, suggesting down-regulation of telomerase activity as a possible consequence but not a cause of cisplatin-induced apoptosis or growth arrest in accordance with others.23, 24
We found no significant effect of cisplatin on telomere length. This is in disagreement with some published data.17, 33 We cannot formally exclude the possibility that telomeres in some cell lines might be more sensitive to cisplatin than those in others. However, we note that this disagreement cannot be due to differences in telomere lengths, because the cell lines used here varied more widely in terms of telomere length than any others examined previously under cisplatin treatment. We also note that the reported telomere loss in cisplatin-treated BEL-7404 cells was independent of time or dose and did not correlate with the induction of apoptosis.17 Importantly, in directly repeated experiments we could not confirm the telomere shortening reported by others in HeLa cells.33 We tested for unspecific DNA degradation in all our experiments by observing the integrity of microsatellite bands. There was no observable DNA degradation, probably because we excluded late-apoptotic cells as much as possible from the DNA sampling. It is possible that at least some of the apparent telomere shortening seen in earlier reports might have been due to unspecific, genomwide DNA degradation.
Further, we found no effect of cisplatin on the length or functional integrity of the single stranded G-rich telomeric overhangs, and we could not detect any accumulation of single strand breaks in the telomeric strand that is most sensitive to intrastrand cross-linking by cisplatin.
In contrast, the formation of DNA strand breaks in the bulk of the genome could be measured directly using the sensitive FADU assay and indirectly by immunostaining for γ-H2A.X. Both techniques reveal that DNA strand breaks are formed after a delay following cisplatin treatment, probably as a result of attempted but incomplete DNA replication or repair. Importantly, γ-H2A.X foci did not co-localise with telomeres, indicating that telomeres are not major triggers of a DNA damage response after cisplatin treatment. Our results do not support the idea that telomeric damage and uncapping is essential for cisplatin-induced apoptosis. Rather, they suggest that signalling from interstitial sites of DNA damage via DNA damage foci formation is the major trigger for tumour cell growth arrest and death following treatment with cisplatin.