Introduction

Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends (Blackburn and Greider, 1995). Telomeres are highly conserved in organisms ranging from unicellular eukaryotes to mammals, indicating the widespread utility of their protective mechanisms for preventing chromosomal ends from undergoing degradation and ligation with other chromosomes (Blackburn and Greider, 1995). Without telomeric caps, human chromosomes would undergo end-to-end fusions with the formation of dicentric and multicentric chromosomes (Harley, 1991; van Steensel et al., 1998). These abnormal chromosomes would break during mitosis, resulting in severe damage to the genome and the activation of DNA damage checkpoints, leading to cell senescence or the initiation of apoptotic cell death pathways (de Lange and Jacks, 1999). Indeed, it has been proposed that telomere length specifies the number of cell divisions that a cell can undergo prior to senescence (Harley, 1991).

Genetic experiments using a dominant-negative form of human telomerase have demonstrated that telomerase inhibition can result in telomere shortening followed by proliferation arrest and cell death by apoptosis (Hahn et al., 1999; Zhang et al., 1999; Delhommeau et al., 2002; Tauchi et al., 2002; Nakajima et al., 2003). This makes telomerase a target not only for cancer diagnosis but also for the development of novel therapeutic agents (Bearss et al., 2000). One effective strategy for the design of telomerase inhibitors targets telomerase indirectly, via the telomeric substrate, and aims to block the interaction between the enzyme and the telomere (Shi et al., 2001). At the extreme 3'-termini of the telomeres, there are regions of single-stranded DNA, formed because of a limitation of the DNA polymerization mechanism, the end replication problem. A property of G-rich, single-stranded structure assembled around a core stack of guanines arranged in almost-planar, hydrogen-bonded tetrads. Ionic conditions that favor quadruplex formation have been shown to inhibit telomerase (Zahler et al., 1991), while small molecules that stabilize or promote the formation of quadruplex also show inhibitory activity (Sun et al., 1997; Wheelhouse et al., 1998; Izbicka et al., 1999; Perry and Jenkins, 1999). Thus, quadruplex DNA presents a target of considerable importance in DNA-directed drug design.

Telomestatin (SOT-095) is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor (Shin-ya et al., 2001). The structural similarity between telomestatin and a G-quadruplex suggested that the telomerase inhibition may be due to its ability either to facilitate the formation of, or trap out preformed, G-quadruplex structures, and thereby sequester single-stranded d[T₂AG₃]_n primer molecules required for telomerase activity (Kim et al., 2002). In fact, telomestatin selectively facilitates the formation of, or stabilizes, intramolecular G-quadruplexes, including that produced from the human telomeric sequence d[T₂AG₃]₄ (Kim et al., 2002). The current investigation was undertaken to determine whether the telomestatin disrupts the telomere maintenance, and therefore activates the DNA damage-responsible pathways. We observed that telomere dysfunction caused by telomestatin induces chemosensitivity in leukemic cells associated with the activation of ATM and Chk2.

Results

Effects of a G-quadruplex-interactive telomerase inhibitor, telomestatin, on telomerase activity and telomere length in human leukemia cell lines

A G-quadruplex-interactive telomerase inhibitor, telomestatin, was isolated from Streptomyces anulatus 3533-SV4 (Figure 1a). In order to study the effects of telomestatin on telomerase activity, BCR-ABL-positive K562 cells and OM9;22 cells were cultured with telomestatin for 48 h. Telomerase activity from each cell line was analysed by TRAP assay (Figure 1b). Telomestatin appears to be a potent telomerase inhibitor, with 50% inhibition at 2 M (Figure 1b). Telomestatin also reduced hTERT mRNA in a dose-dependent manner (Figure 1b). To examine the long-term effect of telomestatin on K562 and OM9;22 cells, it was necessary to identify the drug concentration window in which telomerase could be inhibited without extensive inhibition of cell proliferation. Telomestatin (2 M) had no effect on short-term cell viability or proliferation, as determined in a 7-day cytotoxicity assay (data not shown). We used 2 M as the treatment concentration in long-term cultivation experiments. As a control, untreated cells or cells treated with solvent alone were grown under the same culture conditions. Periodically, total DNA samples were prepared from treated and control cells, and digested with frequently cutting restriction enzymes, and the telomere length was examined by Southern blotting (Figure 1c). K562 cells exhibited a heterogeneous size distribution of telomeres, with an average telomere length of 5.2 kb and a predominant range of 3–7 kb (Figure 1c). As the cells were propagated in the presence of telomestatin, steady telomere shortening occurred (Figure 1c). The average telomere restriction fragment (TRF) size from K562 cells shortened progressively from 5.2 to 2.5 kb at population doubling (PD) 20 (Figure 1c). TRF size from telomestatin-treated OM9;22 cells also shortened from 3.2 to 2.7 kb (Figure 1c).

Figure 1.

Effects of telomestatin on telomerase activity and telomere length in leukemia cells. (a) Structure of telomestatin. Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4. The structure of telomestatin is very similar to that of the G-tetrad. (b) The effect of telomestatin on telomerase activity in K562 and OM9;22 cells. Telomerase activity was examined by a TRAP assay using a TRAP_EzE telomerase detection kit (Oncor, Gaithersburg, MD, USA). Telomerase activity in the indicated cell lines is expressed as relative value. Values are shown as means.d. of triplicates. Quantitation of hTERT mRNA was described previously (Hisatomi et al., 1999). Similar results were obtained in two independent experiments. (c) Total genomic DNA from K562 cells and OM9;22 cells was assessed for telomere restriction fragment size by Southern blot analysis with a telomeric probe. PD, population doubling; left margin, molecular size markers (kb)

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Effects of telomestatin on cell proliferation and apoptosis

We characterized the growth properties of telomestatin-treated cells (Figure 2a). The growth kinetics of telomestatin-treated cells initially did not differ from those of untreated control cells, regardless of the cell line used (Figure 2a). K562 cell cultures in the absence or presence of 2 M of telomestatin exhibited no or only minor differences in proliferation during 20 days of treatment (Figure 2a). However, after 30 days, telomestatin-treated K562 cells showed an almost complete inhibition of proliferation (Figure 2a). Telomestatin-treated OM9;22 cells also ceased to proliferate after 15 days (Figure 2a). Telomestatin-treated cells showed distinctive morphological features associated with apoptosis (data not shown). To determine whether telomestatin-treated cells underwent apoptosis, we employed flow cytometry analysis of living OM9;22 cells with APO2.7 monoclonal antibody and also determined the cell cycle phases (Figure 2b). Cultivation with 2 M of telomestatin for 10 days increased the population of cells in G1 phase from 43.3 to 67.9%, with unchanged APO2.7-positive cells (Figure 2b). Consistent with the cellular proliferation assay, telomestatin significantly increased the number of cells in the G1 phase of the cell cycle with a concomitant decrease in the S phase (Figure 2b). These increased populations in the G1 phase in telomestatin-treated cells represent G1-arrested cells (Figure 2b). Cultivation with telomestatin for 15 days markedly increased the population of APO2.7-positive cells from 1.5 to 77.8% (Figure 2b). These results demonstrate that telomestatin treatment of OM9;22 cells inhibits telomerase activity, resulting in further telomere shortening, which may lead to chromatin damage and the subsequent induction of apoptosis. To determine whether the telomere loss induced by telomestatin led to telomere dysfunction, we analysed chromosomal metaphase spreads derived from telomestatin-treated OM9;22 cells. Due to the reduced proliferative capacity and the correspondingly low mitotic index, only six metaphases were obtained from the telomestatin-treated cells, and these were compared qualitatively with 20 metaphases from control cultures. We identified the presence of chromosome end fusions and chromosomes with no telomere signal at both sister cosmids in telomestatin-treated cells (data not shown).

Figure 2.

Effects of telomestatin on cell proliferation, cell cycle and induction of apoptosis. (a) K562 cells and OM9;22 cells were plated in 24-well plates in the presence of 2 M of telomestatin in 0.1% methanol. Control cells were treated with 0.1% methanol. Cultures were replated every 3–4 days to maintain log-phase growth and to calculate the growth rate. (b) OM9;22 cells were incubated with 2 M of telomestatin for the indicated days. DNA was stained with propidium iodide, and analysis was immediately performed using the CELLFIT program. Apoptosis was examined by the cell surface expression of APO2.7, as determined by flow cytometry. The percentages of APO2.7-positive cells are shown at the top right of each panel

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Telomestatin inhibits proliferation of leukemic cell line but not of normal bone marrow progenitors

In order to compare the effects of telomestatin on leukemic cells and its effects on normal hematopoietic cells, we examined the plating efficacy of K562 cells and the standard progenitor colony assay of normal CD34-positive bone marrow cells treated with telomestatin (Figure 3a, b). Treatment with 0.2 M of telomestatin suppressed the plating efficacy of K562 cells by 30% (Figure 3a). Further, treatment with 1 M of telomestatin completely suppressed the plating efficacy of K562 cells (Figure 3a). Treatment with 1 M of telomestatin did not significantly reduce CFU-GMs, but suppressed BFU-Es by 50% of normal bone marrow CD34-positive cells (Figure 3b). These data suggest a selective elimination of leukemia cells by telomestatin.

Figure 3.

The plating efficacy of K562 cells and the standard progenitor colony assay of normal CD34 positive bone marrow cells treated with telomestatin. In all, 1000 (K562) cells (a) or 1000 (bone marrow CD34-positive cells (b) were treated with telomestatin and seeded in triplicate in conditioned medium MethCult GF H4434 (Stem Cell Technologies, Vancouver, Canada). The leukemic colonies (>50 cells) of K562 were scored on day 14. Progenitor cell-derived colonies were scored on day 14 and classified as either BFU-Es or CFU-GMs

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Enhancement of apoptosis in telomestatin-treated K562 cells by chemotherapeutic agents

Since early passaged telomestatin-treated K562 cells did not show induction of apoptosis, we examined the impact of telomerase inhibition on chemotherapeutic responses (Figure 4a–d). Mechanistically distinct classes of reagents were selected for analysis, including imatinib, daunorubicin (DNR), mitoxantrone (MIT) and vincristine (VCR). To assess the effects of telomerase inhibition in modulating responses to these reagents, experiments focused on early passaged telomestatin-treated K562 cells (PD10). In this series of experiments, K562 cells were cultured with telomestatin for 10 days; subsequently, the telomestatin-treated K562 cells were incubated with the agents for 72 h, and the incidence of apoptosis was determined by flow cytometric analysis with APO2.7 mAb (Figure 4a–d). The telomestatin-treated K562 cells showed enhanced induction of apoptosis compared with control cells after exposure to imatinib, DNR, MIT and VCR (Figure 4a–d), whereas significant chemosensitivity was not observed in cells exposed to VP-16, 6-MP and MTX (data not shown). These results, demonstrating enhanced sensitivity to some classes of chemotherapeutic agents, imply cytotoxic synergy between telomere dysfunction and these agents.

Figure 4.

Enhancement of apoptosis in telomestatin-treated K562 cells by chemotherapeutic agents. K562 cells were cultured with 2 M of telomestatin for 10 days, and subsequently these cells were incubated with the indicated concentrations of imatinib (a), DNR (b), MIT (c) and VCR for 72 h. The incidence of apoptosis was determined by flow cytometric analysis with APO2.7 mAb

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Colony-forming assay

It was also confirmed that telomestatin and imatinib have at least an additive combination effect on bone marrow progenitor cells from chronic myelogenous leukemia (CML) patients (Figure 5). The combination of telomestatin and imatinib produced a substantial decrease (normalized colony numbers of about 30%) in colony formation compared with each agent alone (normalized colony numbers of about 60%) (Figure 5). This increased inhibition of colony formation was seen in both BFU-E and CFU-GM (Figure 5).

Figure 5.

Methylcellulose colony-forming assay of CML primary cells grown in telomestatin and imatinib. Bone marrow samples were obtained from two patients in untreated CML-chronic phase. Bone marrow cells were grown in methylcellulose containing the indicated concentrations of telomestatin and imatinib. Colony counts were assessed on each individual sample at least twice, and results are presented as averages.d. for colonies counted from triplicate plates under each condition

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Telomere dysfunction caused by telomestatin activates ATM, Chk2 and induces the expression of p21^CIP1 and p27^KIP1

Inhibition of telomerase activity in OM9;22 cells causes loss of viability through the activation of apoptotic pathways. Although the signals that activate apoptosis are still unknown, the occurrence of chromosomal breakage suggests that the signals may involve sensors associated with DNA damage. To test this hypothesis, OM9;22 cells were cultured with 2 M of telomestatin for indicated days. Cell lysates from telomestatin-treated cells or untreated cells were immunoprecipitated with anti-ATM Ab, and these immune complexes were subjected to in vitro kinase assay with [-³²P]ATP and recombinant p53 protein (Figure 6, left). The immunoprecipitates from anti-Chk2 were also subjected to in vitro kinase assay with [-³²P]ATP (Figure 6, right). Cultivation with 2 M of telomestatin for 10 days markedly increased the ATM and Chk2 kinase activity (Figure 6, left). These results demonstrate that ATM-dependent DNA damage response pathways are activated by telomere dysfunction. Since telomestatin significantly increased the G1 phase of the cell cycle with a concomitant decrease in the S phase, we examined the expression of cdk inhibitors in OM9;22 cells by immunoblotting (Figure 7). p21^CIP1 and p27^KIP1 proteins were induced in cell lysates from telomestatin-treated OM9;22 cells and DN-hTERT-transfected OM9;22 cells (PD10), but not in lysates from control cells; however, p15^INK4b and p16^INK4a were not induced under these conditions (data not shown). Taken together, G1 arrest in OM9;22 cells induced by telomestatin was mediated by the expressions of p21^CIP1 and p27^KIP1 proteins.

Figure 6.

Telomestatin induces the activation of ATM and Chk2. ATM and Chk2 were immunoprecipitated (IP) from untreated OM9;22 cells and telomestatin-treated OM9;22 cells for indicated days. Activation of ATM was examined by in vitro kinase assays with recombinant p53 proteins as a substrate (left). Autophosphorylation of Chk2 was examined by in vitro kinase assays without target substrate

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Figure 7.

Immunoblot analysis of p21^CIP1 and p27^KIP1 protein. Total cellular protein (10 mg per lane) from untreated OM9;22 cells, DN-hTERT-transfected OM9;22 cells (PD10), and telomestatin-treated OM9;22 cells (10 days) was separated on 12.5% SDS–PAGE. p21^CIP1 and p27^KIP1 protein levels were detected by immunoblotting with antibodies directed against p21^CIP1 and p27^KIP1

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Discussion

Progressive telomere shortening is thought to be important in the regulation of cellular senescence, and upregulation of telomerase activity may be critical in the development of neoplastic cells (Blasco et al., 1999; Liu, 1999; Meyerson, 2000). One prediction of this model is that specific inhibition of telomerase function alters the growth properties of neoplastic cells and may represent a new strategy for antineoplastic therapy (Bearss et al., 2000). Genetic experiments using a dominant-negative form of human telomerase demonstrated that telomerase inhibition can result in telomere shortening, followed by proliferation arrest and cell death by apoptosis (Hahn et al., 1999; Zhang et al., 1999; Tauchi et al., 2002; Delhommeau et al., 2002; Nakajima et al., 2003). Although this approach is selective in vitro, the gene therapy method limits the proportion of completely telomerase-negative clones (Delhommeau et al., 2002). Several other strategies to inhibit telomerase activity have been reported so far. Strategies that target the RNA subunit of telomerase with antisense RNA or peptide nucleic acids diminish cellular telomerase activity and induce some changes in cellular growth (Glukhov et al., 1998; Kondo et al., 1998). These approaches, however, did not consistently lead to complete inhibition of telomerase activity, and the specific effects of such agents on both telomere shortening and cell death were difficult to assess. A recent study described non-nucleosidic small-molecule telomerase inhibitors (BIBR1532 and BIBR1591), which should display improved clinical efficacy (Damm et al., 2001).

In the current study, we observed that treatment with telomestatin reproducibly inhibited telomerase activity, resulting in telomere shortening, in the leukemic cell lines OM9;22 and K562 (Figure 1b, c). Since telomestatin selectively stabilizes intramolecular G-quadruplexes, including that produced from the human telomeric sequence d[T₂AG₃]4 (Kim et al., 2002), telomestatin might lead to more rapid onset of telomere shortening, chromosomal instability, cell crisis and apoptosis. We also observed that telomestatin completely suppressed the plating efficacy of K562 cells at 1 M (Figure 3a); however, telomestatin had less effect on BFU-Es and CFU-GMs colony formation of normal bone marrow CD34 positive cells (Figure 3b). There are some difficulties inherent in using in vitro normal cell systems to evaluate the cytotoxicities of the telomerase inhibitor. Owing to the finite life span, we could not evaluate the effect of long-term culture. The present study only provides us with a spectrum of short-term toxicity against normal hematopoietic progenitor cells (Figure 3a, b).

Previously, Lee et al. (2001) reported that neoplastic cells from telomerase RNA null (mTERC-/-) mice showed enhanced chemosensitivity to doxorubicin or related double-strand DNA break (DSB)-inducing agents. Telomere dysfunction, rather than telomere inhibition, is proposed to be the principal determinant governing chemosensitivity specifically to DBS-inducing agents (Lee et al., 2001; Nakajima et al., 2003). We observed that imatinib and VCR, in addition to DBS-inducing agents, also enhanced chemosensitivity in telomestatin-treated K562 cells (Figure 4a–d). When tested against human primary CML cells, telomestatin combined with imatinib strongly suppressed hematopoietic colonies from patients with CML (Figure 5). The 2-phenylaminopyrimidine derivative imatinib is a recently designed tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both c-ABL and BCR-ABL (Druker et al., 1996). Imatinib is specifically active both in vitro and in vivo against a variety of BCR-ABL transformed cells (Druker et al., 1996; 2001; Gorre et al., 2001; Nakajima et al., 2001). The enhanced sensitivity to imatinib suggests cytotoxic synergy between telomerase inhibition and imatinib. It has been shown that activation of the nuclear c-Abl protein can contribute to the induction of apoptosis (Sawyers et al., 1994). Since Kharbanda et al. (2000) have reported that c-Abl protein is directly associated with hTERT and inhibits telomerase activity, imatinib in combination with telomestatin may affect the dynamic regulation of telomeres. This observation suggests that combined use of telomerase inhibitors and imatinib or other chemotherapeutic agents may be a very useful approach to treatment of BCR-ABL-positive leukemia (Figures 3, 4 and 5).

ATM is a protein kinase that shares similarities with the phosphatidylinositol 3-kinase involved in signaling pathways that regulate genome stability and cell cycle check point arrest after DNA damage or incomplete DNA replication (Savitsky et al., 1995; Lavin and Shiloh, 1997). The ATM kinase is critical for the regulation of G1, S and G2/M checkpoints in response to genotoxic agents, as its activation by DNA damage leads to phosphorylation of target proteins that mediate the cell cycle (Lavin and Shiloh, 1997). The ATM kinase phosphorylates Chk2 kinase, the mammalian homolog of Saccharomyces cerevisae Rad53 (Brown et al., 1999; Chaturvedi et al., 1999). Chk2 phosphorylates p53 on Ser20, attenuating the binding of p53 to Mdm2, and allowing accumulation and subsequent activation of p21^CIP1 and G1 arrest (Chehab et al., 2000; Hirano et al., 2000; Shieh et al., 2000). To provide evidence for a direct relationship between telomere dysfunction and the activation of signaling pathways associated with DNA damage, in vitro kinase assays of ATM and Chk2 were performed in OM9;22 cells treated with telomestatin for 10 days. ATM and Chk2 were indeed activated in OM9;22 cells treated with telomestatin (Figure 6). These results did not precisely determine the mechanisms regarding telomere shortening and apoptosis; however, our results clearly show the activation of ATM-dependent DNA damage response pathways by telomere dysfunction. Although OM9;22 cells are deficient in functional p53 (data not shown), telomestatin significantly increased the number of G1-arrested cells associated with the expression of cyclin-dependent kinase inhibitors p21^CIP1 and p27^KIP1 (Figure 7). Therefore, the G1-arrest observed upon telomerase inhibition may be mediated by p53-independent pathways.

Although most human cancers express telomerase, some tumors maintain telomere length through an alternative mechanism for telomere elongation (ALT) that involves recombination (Bryan et al., 1995; Dunham et al., 2000). We have not identified cells that survive the period of crisis induced by telomestatin; however, tumor cells subjected to antitelomerase therapies may acquire resistance through the development of other mechanisms to maintain telomeres. Recent studies have shown that expression of mutant telomerase RNA template (hTR) leads to synthesis of mutated telomerase and to a reduction in growth rate and viability in telomerase-negative immortal human cells (ALT cells) (Guiducci et al., 2001; Kim et al., 2001). Since ALT cells can be reconstituted for telomerase activity and tolerate long-term expression of wild-type enzyme, combination therapy using ALT and telomerase inhibitors may help prevent the emergence of drug resistance.

There have also been concerns that inhibiting telomerase might lead to an increase in malignancy by enhancing the genomic instability of cells (Lee et al., 2001; Sachsinger et al., 2001). These concerns have arisen from the mTR-/- knockout mouse, which has an increased incidence of malignancies in both early and late generations, particularly in the setting of p53 mutant tumors (Lee et al., 2001; Sachsinger et al., 2001). It is still too early to know with certainty whether telomestatin will become a treatment option against human neoplasia, since there is concern about side effects on normal hematopoietic cells and germ cells (Ohyashiki et al., 2002). These questions will only be answered when telomestatin progresses into animal and clinical trials.

Materials and methods

Antibodies and reagents

Anti-ATM Ab (K-19), anti-Chk2 Ab (H-300), anti-p21^CIP1 Ab (F-15), anti-p27^KIP1 Ab (F-8), anti-actin Ab (H-300) and recombinant p53 protein were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Imatinib was kindly provided by Novartis Inc. (Basel, Switzerland). Daunorubicin (DNR), cytosine arabinoside (Ara C) and etoposide (VP-16) were obtained from Sigma (St Louis, MO, USA). Telomestatin was purified as previously described (Shin-ya et al., 2001).

Cells and cell culture

The Ph-positive acute lymphoblastic cell line OM9;22 has been described previously (Ohyashiki et al., 1993). K562 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). These cell lines were cultured in McCoy's 5A modified medium (Life Technology, Inc.) supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT, USA).

Colony-forming assay

Clonogenic assays were performed as described elsewhere (Chaves-Dias et al., 2001). CD34-positive cells were purified as described previously (Chaves-Dias et al., 2001). In total, 1000 bone marrow CD34-positive cells or 1000 K562 cells were treated with telomestatin and seeded in triplicate in condition medium MethoCult GF H4434 (Stem Cell Technologies, Vancouver, Canada). The leukemic colonies (>50 cells) of K562 were scored on day 14. Progenitor cell-derived colonies were scored on day 14 and classified as either BFU-Es or CFU-GMs. Bone marrow samples from two untreated patients with CML-chronic phase were obtained with informed consent prior to conducting studies. The growth medium was supplemented with 0.2 M telomestatin, 0.1 M imatinib, or a combination of both. Macroscopic colonies were counted in triplicate dishes on day 14.

Generation of stable clones expressing DN-hTERT mutants

pBABE-DN-hTERT was a gift from Dr Robert Weinberg (Massachusetts Institute of Technology). OM9;22 cells were transfected with the expression vector pBABE-puro-DN-hTERT by electroporation. Beginning 48 h after electroporation, cells were selected with 2 g/ml of puromycin and cloned by limiting dilution. PD 0 was defined as the time at which cultures reached confluence in 10-cm culture dishes.

Telomerase assay and measurement of TRF

Telomerase activity was examined by a telomere repeat amplification protocol (TRAP) assay using a TRAP_EzE telomerase detection kit (Oncor, Gaithersburg, MD, USA). The PCR products were subjected to 12% acrylamide denaturing electrophoresis in an automated laser fluorescence DNA sequencer II (Pharmacia LKB Biotechnology, AB) and analysed by the Fragment Manager program (Pharmacia LKB Biotechnology, AB). Activity in the extract-based PCR TRAP assay was detected as a periodic 6-bp peak of telomerase products and, in each sample, relative telomerase activity was calculated semiquantitatively in comparison with a 36-bp internal standard. To measure TRF, genomic DNA was digested with the restriction enzymes HinfI and RsaI, fractionated on 0.7% agarose gels and transferred onto nylon membranes. Hybridization was performed by using the Telo TTAGGG telomere length assay kit (Roche Molecular Biochemicals, Mannheim, Germany).

hTERT mRNA

Quantification of hTERT mRNA was described elsewhere (Hisatomi et al., 1999).

Apoptosis assay

The incidence of apoptosis was determined by flow cytometric analysis with the FITC-conjugated APO2.7 monoclonal antibody (clone 2.7), which was raised against the 38 kDa mitochondrial membrane protein (7A6 antigen) and is expressed by cells undergoing apoptosis (Nakajima et al., 2001).

Immunoblotting and immunoprecipitation

Immunoblotting and immunoprecipitation were performed as described previously (Tauchi et al., 1994). For immune complex kinase assays, immunoprecipitated proteins were incubated with 30 l of kinase buffer (50 mM HEPES, pH 8.0, 10 mM MnCl₂, 2.5 mM EDTA, 1 mM dithiothreitol, 10 mM ATP and 30 mCi of [-³²P]ATP) at 30°C for 30 min. For immune complex kinase assays of ATM, 5 g of recombinant p53 proteins were used for a substrate. The reaction products were separated by SDS–PAGE, and transferred to the membranes for autoradiography.

Statistical analysis

Comparisons between groups were analysed by the Mann–Whitney U-test. Values of P<0.05 were considered to indicate statistical significance. The statistical tests were performed using the Microsoft Word Excel (Brain Power Inc, Calabashes, CA, USA) software package for the Macintosh personal computer.

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Acknowledgements

This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan (to TT) and NOVARTIS Foundation for the Promotion of Science (to TT) and by the Promotion and Mutual Aid Corporation for Private School of Japan (to KO) and by the high-tech research center for intractable disease of Tokyo Medical University from the Ministry of Education, Culture, Sports, Science and Technology in Japan (to KO). We thank Hisashi Hisatomi (SRL, Inc.) for analysis of telomerase activity and hTERT expression.