Telomerase activity transiently increases when HL60 cells are treated with the topoisomerase II inhibitor etoposide. A quantitative assessment revealed that telomerase is activated by etoposide treatment in a number of cell lines and that the increase is reversible after withdrawal of etoposide from the cell culture. Telomerase activation correlated with the occurrence of DNA damage but not with cell cycle arrest. We did not detect any transcriptional upregulation of hTERT mRNA, suggesting a post-transcriptional mechanism of telomerase activation. Furthermore, the mRNA expression of the telomere binding protein TRF2 was upregulated early and reversibly after etoposide treatment. TRF1 mRNA expression levels were unchanged after DNA damage, but increased when the cells accumulated in the G2/M phase. The data show that the telosome reacts after DNA damage by upregulating telomerase activity and TRF2 expression in malignant cells. It has previously been shown that overexpression of TRF2 can repress senescence signals arising from critically shortened telomeres. We show here that TRF2 is upregulated by undirected DNA damage that also affects the telomeric DNA. These data suggest that upregulation of telomerase activity and TRF2 expression might act as antiapoptotic mechanisms in the DNA-damage response of malignant cells.
The chromosomal ends are composed of short repetitive DNA sequences and specialized telomere binding proteins.1 The function of this ‘telosome’ is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks and to mediate chromosomal pairing and movement during cell division.2 Immortal cells, such as germ line cells, stem cells and cancer cells, express the ribonucleoprotein telomerase, a reverse transcriptase, that synthesizes new telomeric repeats onto the chromosome ends and compensates for the loss occurring during cell division.3 In most conditions analyzed so far, the limiting component of an active telomerase enzyme is the catalytic subunit hTERT. Upregulation of telomerase activity by enhanced proliferation of tumor cell lines or of sporadic tumors with high proliferation in vivo is virtually always mediated by a transcriptional overexpression of hTERT mRNA.4 Although the predominant function of telomerase is maintenance of telomere length, some data indicate that the catalytic subunit hTERT additionally mediates a survival promoting function that is independent of its catalytic activity.5,6 Inhibition of telomerase by targeting the catalytic subunit hTERT induces slow, proliferation-associated telomere shortening and finally senescence.7,8 However, even before a critical telomere length is reached, the telomerase-inhibited cell lines show an increased sensitivity to DNA-damaging agents like etoposide.8,9 Therefore, targeting telomerase simultaneously with DNA-damage-inducing agents might potentiate the effect of both therapeutic approaches.
The telomere binding proteins TRF1 and TRF2 are specialized DNA-binding proteins that are exclusively found at the telomere.10 The expression levels of telomere binding proteins have only been examined in a limited number of tumors and cell culture experiments. The expression of TRF1 and TRF2 has been reported to be increased, decreased or unchanged in tumors.11,12,13,14,15 Increased TRF2 expression was observed in human fibroblasts during in vitro senescence16 and TRF1 and TRF2 expressions were reported to increase during in vitro differentiation of HL60 cells.17 So far no defined common conditions or signals for differential expression of telomere binding proteins are known. On the other hand, there have been reports of manipulation of telomere binding protein expression by transfection of cell lines and primary cells. In these experiments, it was shown that TRF1 mediates the interaction of the telomere with the mitotic spindle and negatively regulates telomere length by limiting the access of telomerase to the telomere.18,19 Overexpression of TRF1 can induce entry into mitosis and enhance apoptosis.20 Following DNA damage, ATM phosphorylates TRF1 and suppresses its ability to induce abortive mitosis and apoptosis.21,22 On the other hand, TRF2 overexpression also negatively regulates telomere length, probably by an active degradation process.19,23 TRF2 protects telomeres from end-to-end fusion,24 presumably because it mediates the proper folding of the telomere.25 Overexpression of a dominant-negative TRF2 induces apoptosis.26 The apoptotic signal from telomeres devoid of TRF2 is independent of telomere length. Interestingly, overexpression of functional TRF2 can overcome the senescence-inducing signals from critically short telomeres and thus allow cell growth in the presence of short and therefore ‘damaged’ telomeres.27
Telomerase activity can be induced by treating cell lines with etoposide, as it has been shown by one group of investigators for HL60 cells28 and by another for five pancreatic cell lines.29 The induction of activity was abolished with the onset of apoptosis, and the early time at which telomerase was upregulated suggested an association with DNA damage rather than cell cycle arrest, although direct evidence was lacking.28 Detection of hTERT mRNA or telomere binding protein mRNA levels and direct measurement of DNA damage, for example by the comet assay, were not performed. HTERT protein levels were reported to be unchanged.28 Conflicting results were reported by another group who observed downregulation of telomerase activity by etoposide treatment that occurred despite increased hTERT expression in Jurkat, Raji and CEM-6 cells.30
To gain insights into the effect of etoposide on telomerase regulation, we treated HL60 cells with various concentrations of etoposide and measured telomerase activity, telomere binding protein expression, DNA damage and cell cycle phase distribution.
Material and methods
HL60, MOLT4, Jurkat and K562 cells (American Type Culture Collection) were grown in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 2 mM L-glutamine (Biochrom AG, Berlin, Germany) and 10% heat-inactivated fetal calf serum (PAA Laboratories GmbH, Austria). Etoposide (Sigma, Hannover, Germany) was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the medium was less than 0.1%, and it had no effect on cell growth. All experiments were performed in triplicate. Logarithmically growing cells were seeded at a density of 5 × 105/ml and exposed to etoposide at various concentrations and exposure intervals. For the recovery experiments, logarithmically growing cells were treated with etoposide for 6 h. Subsequently, the cells were washed in PBS and resuspended in fresh medium without etoposide. Aliquots of cells were collected, washed with PBS and stored at −80°C until further processing.
Frozen samples were resuspended in ice-cold lysis buffer (CHAPS 0.5%, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 5 mM β-mercaptoethanol, 0.01 mM AEBSF, 1 U/μl RNAsin (Promega, Mannheim, Germany), 10% glycerol). After incubation for 30 min on ice, the samples were centrifuged for 30 min at 4°C at 20 000 g. The supernatant was snap frozen in liquid nitrogen and stored at −80°C. Protein concentration was determined by an assay using reagent from Pierce Chemicals, Rockford, IL, USA.
The extracts were diluted appropriately and 100 ng of protein (in 2 μl) was mixed with 48 μl reaction mix containing 20 mM Tris-HCl (pH 8.0), 1 mM EGTA, 0.0005% Tween 20, 1.5 mM MgCl2, 63 mM KCl, 50 μM each dNTP, 2 U Taq, 0.001 attomol ITAS, 10 pmol TS primer (5′-TAMRA labeled) and 10 pmol CXext primer.31
Following a 30 min incubation period at 30°C, the PCR conditions were as follows: 95°C for 3 min and 36 cycles at 95°C for 30 s, 50°C for 30 s and 72°C for 30 s. The PCR products were analyzed on an ABI Prism 310 capillary electrophoresis unit, as described.32 The areas under peaks 2–5 after the primer dimer were added and divided by the area under the ITAS peak (internal amplification standard).32 For semiquantitative measurement, a dilution series of HL60 extract was analyzed in parallel. A linear regression analysis of the telomerase peak/ITAS ratios of the dilution series was performed (GraphPad Prim). The experimental samples were expressed as corresponding nanograms of HL60 protein. This quantification method takes the slope of the standard curve into account. We then averaged the values of untreated controls in one experiment and defined the value as 100%. Experimental values were expressed as percent of the untreated control. The generated values can be compared with each other easily and directly, because an increase to 200% truly reflects a doubling of the activity.
Total cellular RNA was collected from samples using TRIzol reagent (GibcoBRL). RNA yields and purity were determined spectrophotometrically. First-strand synthesis was performed using the ‘Superscript™ first-strand synthesis system’ (Invitrogen) according to the manufacturer's instructions for random hexamer primer. A volume of 2 μl of the first-strand cDNA reaction was used for real-time PCR. All PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems) and reagents as recommended by the manufacturer. For each PCR run, a master mix was prepared containing 1 × TaqMan buffer, 3.5 mM MgCl2, 200 μM dATP, dCTP, dGTP, and 400 μM dUTP, 200 nM each primer, 80 nM probe, and 0.625 U AmpliTaq Gold DNA polymerase (all reagents except primer and probe from Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised 10 min hold at 95°C for AmpliTaq Gold® DNA Polymerase activation and 40 cycles at 95°C for 15 s and at 60°C for 1 min.
Standard curves were constructed with four-fold serial dilutions of pure gel-purified DNA. The DNA was obtained by RT-PCR from HeLa cell line cDNA using the same primers as for the TaqMan PCR. The standard curve used for real-time PCR is composed of six points (equivalent to 0.2, 0.005, 0.0125, 0.003125, 0.00078125 and 0.0001953125 attomol of pure DNA). The values in attomol of the target gene were divided by the value of b-actin. The ratios from all untreated samples of one experiment were averaged and defined as 100%. All other experimental samples were expressed as percentage of the untreated control.
Primers and probes
Table 1 shows the primers and TaqMan probes used in the experiments. All TaqMan probes were 5′-FAM and 3′-TAMRA labeled.
Cells were washed in PBS and resuspended for 30 min in ice-cold ethanol (70%). After two washes with PBS, the cells were resuspended in 250 μl PBS. For DNA staining, 300 μl stock propidium iodide (100 μg/ml H2O stock solution, Sigma) and 50 μl RNAse (10 mg/ml PBS; Type 1-AS; Sigma) were added and incubated at room temperature for 15 min. Cells were analyzed with a commercial Coulter Epics Altra flow cytometer (Beckman Coulter, Fullerton, CA, USA) equipped with an INNOVA Enterprise II laser (Coherent, Santa Clara, CA, USA). The 488 nm light beam was used. It was regulated at 150 mW and dimmed by an ND filter approximately to the intensity of a 15 mW laser. Propidium iodide fluorescence was deflected by a 640LP filter and detected at the PMT 4 through a 610BP filter on a linear scale. A total of 10 000 events per sample were collected and the data were stored as list mode files (FCS 2.0). For acquiring and analysis, the software package EXPO32 (Applied Cytometry Systems, Sheffield) was used. For doublet discrimination, the data were displayed as two-parameter dot-plot diagrams of peak height and peak area signal and gated with the strategy described for example by Wersto et al.33
Briefly, after etoposide treatment, HL60 cells were harvested, embedded in 1% low-melting agarose gel (Sigma) at a density of 1 × 105/ml and spread onto a comet™ slide (Trevigen). The slides were immersed in ice-cold lysis buffer (2.5 M sodium chloride, 100 mM EDTA, pH 10, 10 mM Tris-HCL, 1% sodium lauryl sarcosinate and 1% Triton X-100) for 30 min at 4°C. Cellular DNA was denatured in alkaline solution (300 mM NaOH and 200 mM EDTA) at room temperature in the dark for 20 min and then electrophoresed at 25 V and 300 mA for 10 min. Afterwards, the slides were stained with SYBR green and examined under a fluorescence microscope (Zeiss).
Etoposide treatment of HL60 cells transiently induces telomerase activity
To test whether telomerase activation can be induced by DNA damage, we treated HL60 cell with 1, 4 or 9 μM of etoposide. Telomerase activity was measured by a semiquantitative assay after 3, 6, 12, 24 and 36 h. Between 3 and 12 h of treatment, telomerase activity increased up to 200% of the activity found in untreated controls; then the effect was lost and telomerase activity dropped (Figure 1a). Owing to the high standard deviation, the increase of telomerase activity was not statistically significant in the experiment shown (one-way analysis of variance, ANOVA). However, in several repeated experiments, the activity after 6 h of treatment with 1 μM etoposide was significantly higher than the activity of untreated cells (P<0.05, t-test, data in figure 2c).
The highest concentration used here (9 μM) failed to induce telomerase activity, and the decrease in activity was earliest and strongest in these samples (Figure 1a). The failure of the highest concentration to induce telomerase activity might be due to the rapid induction of cell death in these samples, as indicated by the number of cells with sub-G1 DNA content in the FACS analysis (Figure 1b). However, at 3 h treatment with 9 μM etoposide, no increase of telomerase activity could be detected although no increase in the number of sub-G1 cells was apparent. We believe that the highest concentration of etoposide (9 μM) induces an immediate apoptotic signaling, which prevents the upregulation of telomerase activity even if cell death (cell with sub-G1 DNA content) is not detectable yet. However, more detailed studies of very short incubation are necessary to clarify this observation.
When telomerase was activated during the early times and low etoposide concentrations 1 μM), there was only a small number of cells with sub-G1 DNA content. The increase in sub-G1 cells paralleled the decrease in telomerase activity (Figure 1b). A cell cycle arrest in G2/M-phase occurred later about 24 h after treatment (Figure 1b). A positive comet assay performed with cells treated with 1 μM etoposide for 6 h indicated that DNA damage was present at 6 h of treatment with 1 μM etoposide (Figure 1c), although no increase in cells with sub-G1 DNA content was observed under these conditions (Figure 1b). We therefore reasoned that the induction of telomerase activity occurs early during treatment with etoposide in HL60 cells, when DNA damage is present but cell death or apoptosis does not occur as detected by the number of cells with a sub-G1 DNA content. Since the induction of telomerase by low concentrations of etoposide (1 μM) ended after 12 h without a significant increase of cells with a sub-G1 DNA content, an association with cell cycle progression in these cells seems unlikely.
Telomerase activity might be upregulated by a post-transcriptional mechanism
To gain insights into the mechanism of telomerase activation by etoposide, we performed real-time RT-PCR to quantify the mRNA level of hTERT, the catalytic subunit of the enzyme. As shown in Figure 2a, hTERT mRNA varied but did not increase significantly after etoposide treatment. To test whether the upregulation of telomerase activity was independent of protein synthesis, we incubated HL60 cells with cycloheximide, an inhibitor of protein synthesis, and etoposide simultaneously. An upregulation of telomerase activity was still clearly evident in the presence of cycloheximide, and it was even more pronounced than in the previous experiments (Figure 2b). Thus, the induction of telomerase activity by etoposide is independent of transcription of hTERT mRNA and probably based on post-transcriptional modification, for example of hTERT.
To rule out a cell line specific effect, we treated several cell lines with etoposide under conditions under which we expected an upregulation of activity. Telomerase activation was observed in three of four cell lines tested (Figure 2c).
Telomerase activity is reversibly induced by DNA damage but not by cell cycle arrest
To test the reversibility of the telomerase activation, we treated HL60 cells with 1 or 4 μM of etoposide for 6 h and replaced the etoposide-containing medium with normal medium for 48 h. As shown before, no cell cycle arrest was detected after 6 h of treatment (Figure 3a). However, a substantial number of cells were arrested in the G2/M phase after 48 h of recovery in fresh medium although the number of cells with a sub-G1 DNA content did not increase significantly (Figure 3a). We performed a comet assay with the cells treated with 1 μM etoposide for 6 h immediately after treatment and with 21 and 48 h of recovery in etoposide-free medium. Figure 3b demonstrates that the DNA damage detected by the comet assay declined substantially already after 21 h of recovery (compare also Figure 1c). Figure 3c shows the telomerase activity levels in cells treated for 6 h and in cells treated for 6 h with 48 h of recovery (upper panel). Telomerase activation was reversible after removal of etoposide from the medium. However, after 21 h of recovery, telomerase activity was still slightly above the level of untreated cells (data not shown). Thus the disappearance of DNA damage in the comet assay seems to precede the decrease of telomerase activity. Further studies are necessary to determine the exact timing of telomerase activation and telomerase recovery in correlation to the induction of DNA damage as well as the recovery from the damage. HTERT mRNA levels dropped after etoposide treatment, as shown above, and did not recover to normal levels within 48 h. There was no upregulation of hTERT mRNA in cells arrested in the G2/M phase (Figure 3c, lower panel). The data show that telomerase activation is correlated with the presence of DNA damage. In agreement with the results of other authors, no association between high telomerase activity and the G2/M phase was detected.28
TRF2 expression but not TRF1 is induced by DNA damage
As the effects of DNA damage on telomerase activity occurred within hours of treatment, changes in the telomere length cannot be assumed to be detectable with the available methods.28 To further characterize changes at the telomere associated with etoposide treatment, we quantified the expression levels of the best-characterized telomere binding proteins, TRF1 and TRF2, after etoposide treatment by real-time RT-PCR. We found significant upregulation of TRF2 mRNA expression immediately after etoposide treatment, whereas TRF1 mRNA levels remained unchanged. After 12 h, TRF1 mRNA levels increased in the samples in which a G2/M-phase arrest became more pronounced (Figure 4a, compare also Figure 1b). Treating HL60 cells for 6 h with etoposide, including a recovery for 48 h in normal medium, showed that, similar to the telomerase activation described above, the overexpression of TRF2 mRNA was reversible when DNA damage disappeared (Figure 4b, compare also Figure 3b). However, TRF1 mRNA expression was strikingly higher in cells arrested in the G2/M phase of the cell cycle. These data indicate that TRF2 mRNA overexpression is correlated with DNA damage and is not cell cycle associated. TRF1 expression seems to be upregulated in the G2/M phase. Immunohistochemical staining of the cells using a monoclonal antibody against TRF2 (Upstate Biotechnology) showed a nuclear staining pattern that was comparable to untreated cells although a slight increase in staining intensity and a nucleolar accumulation was observed (data not shown).
Telomeres protect the chromosome ends from degradation by the DNA double-strand-break machinery.2 The integrity of the telomere has long been ascribed solely to the length of the telomeric DNA. Recent findings indicate that a functional telomere has to be considered as a ‘telosome’, a structure composed of telomeric DNA and telomere binding proteins.1 Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome and, dependent on the kind of alteration and the cell type, induce senescence or apoptosis.34
Telomerase adds new telomeric repeats to the telomere and compensates the loss that occurs with each cell division.2 Furthermore, telomerase is able to initiate de novo telomere formation on artificially generated chromosome ends, although most of the data regarding this function were obtained from experiments with non-human cells.35,36,37,38,39 Nevertheless, human telomerase can be considered to be an enzyme with DNA-repair function. Consequently, telomerase-inhibited cancer cells show two alterations: (i) cell proliferation dependent, slow telomere shortening that finally leads to senescence,7,40 and (ii) increased sensitivity to DNA-damaging agents and to apoptosis.8,9
Etoposide is a potent inhibitor of topoisomerase II. Treatment with etoposide induces undirected DNA damage in vitro.41 However, since topoisomerase uses telomeric DNA as a substrate, DNA damage caused by topoisomerase inhibition can also be assumed to occur at the telomere.42 Two groups of investigators reported that telomerase activity is upregulated in cell lines treated with the topoisomerase II inhibitor etoposide,28,29 whereas a third group observed downregulation of telomerase activity and upregulation of hTERT mRNA expression.30 DNA-damaging conditions like radiation or other cytotoxic agents have been shown to downregulate telomerase activity.43,44 In these studies, the longer times of exposure (starting at 24 h) might provide an explanation for the conflicting results.43,44 In this work, an upregulation of telomerase activity could also be observed in Jurkat and Molt4 cells, while K562 cells did show unchanged telomerase activity after etoposide treatment. The data confirm previous findings that demonstrated unchanged telomerase activity after 12 h of etoposide treatment in K562 cells.45 Why some cell lines do not show telomerase activation after etoposide treatment remains unclear. Further studies are needed that interfere with cell signaling pathways during etoposide treatment to find possible differences in the signaling between DNA damage and telomerase in these cell lines.
We show here that the upregulation of telomerase activity occurs early after treatment with etoposide and that the hTERT mRNA levels do not increase. The methods used in this study (TRAP assay and real-time RT-PCR) assure reliable quantification. Telomerase upregulation occurs in a number of different cell lines and is independent of protein synthesis. These results are one of the rare examples in which the regulation of telomerase activity is independent of hTERT mRNA levels.4 We further show that telomerase activation is related to the presence of DNA damage, as detected by the comet assay. The data suggest that upregulation of telomerase activity by a post-transcriptional mechanism is part of the cellular response to DNA damage. These results might explain why inhibition of telomerase activity, for example by hammerhead ribozymes directed against hTERT mRNA or in telomerase RNA-component knockout cells, increases the sensitivity of the cells to DNA-damaging agents.8,9 Both inhibition methods, although targeting different components of the enzyme, reduce the amount of active enzyme and might thus interfere with the cellular response to DNA damage. Therefore, the assumed protective effect of telomerase activation after DNA damage most likely depends on an active enzyme and is distinct from the antiapoptotic effect of hTERT protein described as being independent of its catalytic activity.5,6 However, the upregulation can only be observed if a meticulous quantitative approach is used. Our methods utilize a linear regression analysis of a dilution series of a control sample to extrapolate values obtained for the experimental samples. The increased activity measured with this method reached its maximum after 6 h of treatment with 1 μM etoposide. At this time, the activity had doubled compared to untreated controls. Such accurate methods need to be used to test other methods of inducing DNA damage, such as radiation, for their ability to induce telomerase activity. However, due to the early time of telomerase upregulation, the currently available methods fail to demonstrate telomere length changes.28
We also observed differences in the expression levels of telomere binding proteins. While TRF2 mRNA was upregulated in an obvious association with DNA damage, TRF1 mRNA levels only increased when the cells were arrested in the G2/M phase. As high levels of TRF2 protein have been shown to delay the senescence signal from shortened telomeres,27 the upregulation in response to DNA damage might represent a signal that delays apotosis in these cells. Similar results were reported for TRF2 protein levels after irradiation where an accumulation of TRF2 protein after irradiation induced DNA damage was observed. The levels of TRF2 protein decreased during the later stages of apoptotic DNA degradation.46 Others have reported a downregulation of TRF2 protein levels after treatment with chemotherapeutic agents.47,48 The discrepancies may be explained by the different cell lines tested and the later time points used in some of these studies. However, more detailed studies combining quantitative RT-PCR and Western blot using multiple cell lines and a large number of incubation times will be necessary to clearly define the expression of TRF2 after DNA damage. Nevertheless, experimental prevention of TRF2 upregulation, for example by siRNA, will be necessary to provide direct evidence for a protective role of TRF2 after DNA damage.
In summary, after DNA damage induced by etoposide treatment, changes occur in the telosome. Two possibly telomere-protecting alterations are induced: (i) telomerase activity is upregulated by a post-transcriptional mechanism, and (ii) expression of the telomere binding protein TRF2 is upregulated. The experimental settings reported here suggest a link of the two effects to the presence of DNA damage. Telomerase activation and TRF2 overexpression show a striking association with the occurrence of DNA damage, as detected by the comet assay, and may thus be part of the DNA-damage response in malignant cells. Interfering with one of these mechanisms might increase the effect of etoposide therapy.
We thank M Hauberg for her help with immunohistochemistry and E Dege for critically reading the manuscript. This work was supported in part by the ‘Kinder Krebs Initiative Buchholz-Seppensen-Holm’, Germany.
About this article
AtTBP2 and AtTRP2 in Arabidopsis encode proteins that bind plant telomeric DNA and induce DNA bending in vitro
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