Background
Brain tumours comprise of a wide variety of subtypes of which Glioblastoma multiforme and medulloblastoma are the most common primary malignant brain tumours in adults and children, respectively. Although current clinical data show evidence that molecular targeting has improved the management of brain tumours, prognosis still remains poor [
1,
2]. Therefore, the need to discover alternative approach to improve therapeutic response in brain tumour patient remains important.
The two most common hallmarks of tumour cells are their limitless proliferation capacity and sustained massive genome instability [
3]. In normal cells, induction of DNA damage either by exogenous or endogenous agents leads to the activation of appropriate DNA damage response (DDR) pathway to ensure immediate repair and to prevent propagation of cells with highly unstable genomes. DNA double-strand breaks (DSB) are the most lethal form of DNA damage and if unrepaired, DSBs severely threaten not only the integrity of the genome but also the survival of the organism [
4]. The DDR to DSBs involves activation of homologous recombination (HR) or non-homologous end joining (NHEJ) pathway while the latter is the more dominant DSB repair mechanism compared to HR in mammalian cells [
5‐
7]. NHEJ pathway is facilitated by the DNA-dependent protein kinase (DNA-PK), composed of a catalytic subunit, DNA-PKcs, and the heterodimeric DNA binding regulatory Ku complex, Ku70 and Ku86. The Ku heterodimer binds free DSB ends and recruits DNA-PKcs, which then is activated by the DNA-bound Ku complex. Artemis, the XRCC4/ligase IV complex is also recruited to the complex and serves to catalyse re-ligation of the DNA broken ends. Studies have shown that inhibition of DNA-PKcs sensitise tumour cells to radiation, suggesting its potential as a molecular target in cancer therapy [
8].
Besides its role in NHEJ pathway, DNA-PKcs also plays a crucial role in maintaining telomeres [
9,
10]. Telomeres are nucleoprotein complexes that function to distinguish the ends of the chromosome from DSBs and to maintain chromosomal integrity [
11]. In normal cells, telomere length regulates its replication potential. As cells divide, telomere shortening due to end-replication problem of conventional DNA polymerase, coupled with low or absence of telomerase activity, eventually leads to telomere uncapping, triggering DDR or cell death, if damage is irreparable [
12,
13]. In contrast, majority of tumour cells continue to proliferate, despite harbouring relatively shorter telomeres compared to normal tissues due to re-activation of telomerase enzyme [
14,
15]. Telomerase complex is made up of the reverse transcriptase catalytic subunit (TERT) and RNA template subunit (TR) and other associated proteins [
16]. Inhibition of telomerase via genetic alteration using dominant negative hTERT, mutant hTR template [
17,
18] or pharmacological inhibitors slows tumour cell growth by triggering telomere shortening and cell death [
19,
20]. Given the high level of telomerase activity in brain tumour progression [
21,
22], telomerase provides a molecular target in brain tumour cells.
One major drawback of targeting telomerase is the lag time needed for telomere erosion-mediated cell death leading to possible emergence of adaptive response and resistance mechanisms such as alternative lengthening of telomeres (ALT) mechanism which allows telomere maintenances via HR [
23,
24]. Previously, we have shown that inhibition of telomerase and DNA repair protein in mouse embryonic fibroblasts sensitises cells to DNA damaging agents [
25]. Here, we provide evidence that efficient telomerase inhibition was achieved using MST-312 in brain tumour cells inducing telomere shortening. Acute telomerase inhibition in brain tumour cells triggered DSBs, cell cycle arrest and subsequent DDR coupled with activation of DNA-PKcs. Inhibition of DNA-PKcs following MST-312 mediated telomere dysfunction leads to increase cell death in brain tumour cells demonstrating the potential therapeutic combinations in enhancing telomerase-mediated therapy.
Discussion
Despite harbouring massive genome instability, majority of tumour cells continue to proliferate due to activation of telomerase and subsequent telomere stabilisation [
14], In this study, we demonstrated the involvement of DNA-PKcs in DDR pathway following telomerase inhibition in brain tumour cells. Inhibition of telomerase using MST-312 has been shown to induce telomere shortening and impairment of cell proliferation (19). In addition, a study on lung cancer cells revealed that acute telomerase inhibition with MST-312 induced DNA damage [
37]. Similarly, we observed DNA damage and cell cycle arrest (Figure
3A-D) following acute telomerase inhibition. Continual exposure of brain tumour cells to telomerase inhibition led to gradual telomere shortening (Figure
2A). However, shortened telomeres were more resistant to telomerase inhibition mediated growth reduction (Figure
6B-D). It has been reported that short telomeres initiate telomere recombination in tumour cells [
38] and such recombination can occur in telomerase-positive cells following telomere dysfunction [
39]. Although we did not evaluate the mode of telomere maintenance in brain tumour cells after 6 weeks of MST-312 treatment, it is possible that telomere maintenance could also be mediated via ALT pathway as telomere maintenance by telomerase and recombination can co-exist in cells [
40].
More importantly, we uncover a link between telomere dysfunction and chemosensitivity specifically towards agents that inhibit DNA repair pathway, providing alternatives to overcome problems associated with long-term exposure to telomerase inhibitors. The sensitizing effects of DNA repair protein are more pronounced in rapidly dividing cells like tumour cells [
41] and a recent study has also shown that DNA-PKcs inhibition sensitises breast cancer cells to radiation via telomere capping disruption [
42]. In this study, telomerase inhibition in brain tumour cells showed impairment of HR pathway (Table
1) suggesting that DSBs could potentially be repaired by NHEJ. Consistent with this, we observed phosphorylation of DNA-PKcs in brain tumour cells following telomerase inhibition (Figure
6A) and genetic ablation or pharmacological inhibition of DNA-PKcs in glioblastoma cells led to compromised DSBs repair, increased telomere dysfunction and greater cell death (Figures
4 and
5). Dual inhibition of DNA-PKcs and telomerase in glioblastoma, medulloblastoma cells and telomerase-negative osteosarcoma cells, U2OS showed that telomerase-positive brain tumour cells were sensitive to such combinatorial approach as compared to U2OS cells. These data suggest that cells with impaired DNA-PKcs activity appear to enhance a pre-existing telomerase inhibitory effect in brain tumour cells treated with MST-312, leading to accumulation of unrepaired DSBs and reduced cell proliferation (Figures
3 and
6). In addition, MST-312 had relatively minimal effect in non-cancerous, cell lines, MCF10A and MRC-5 (data not shown).
It is important to note that in medulloblastoma cells, ONS76, we did not detect activation of DNA-PKcs following acute telomerase inhibition. In comparison to glioblastoma cells, the relative decrease in telomerase activity was lower in medulloblastoma cells. This could possibly explain the lower level of DSBs induced (Figure
3B). Differential gene expression detected between glioblastoma and medulloblastoma cells clearly suggest some variation in the downstream effects of telomerase inhibition in these brain tumour cells. The genetic background of tumour cells also needs to be assessed as this possibility influences the outcome of treatments. For example, different status of p53 might have influenced the opposite expression profiles of mitochondrial function regulators genes
PGC-1α and
PGC-1β in our study.
Methods
Cell types and cell culture
Human glioblastoma multiforme cells MO59K (CRL-2365) and MO59J (CRL-2366) (American Type Culture Collection, USA) while glioblastoma multiforme cells KNS60, and medulloblastoma cells ONS76 (Institute for Fermentation, IF050357, and IF050355, respectively) were obtained from Dr. Masao Suzuki, National Institute of Radiological Sciences, Chiba, Japan. The cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% heat inactivated foetal bovine serum (Hyclone, USA) and 100 U/ml of penicillin/streptomycin (Gibco, USA). U2OS, ALT-positive osteosarcoma cells (ATCC) were grown in 5A McCoy’s medium, supplemented with 10% foetal bovine serum and L-glutamine (Gibco, USA). All the cells were maintained in a humidified 5% CO2 incubator at 37°C. Fresh medium was added after every 2 days and the cell density was kept below 80% confluence.
Drug treatment
Stock solution of MST-312 (Sigma, USA) [
19] and DNA-PKcs inhibitor, NU7026 (Calbiochem, USA) were prepared in dimethyl sulfoxide (DMSO) and suitable working concentrations were made from the stock using complete medium. For short- term experiments, cells were treated with MST-312 and NU7026 for 48 hours. 10 μM of NU7026 was used in this study as previous report has shown that DNA-PKcs activity is completely inhibited at this dose [
43].
Telomere repeat amplification protocol (TRAP)
Brain tumour cells were treated with 0–5 μM MST-312 for 48 hours and telomerase activity detection was performed with the commercially available TRAPeze® XL Telomerase Detection Kit (Chemicon International, USA). All steps were done according to the manufacturer’s instructions. Total protein was extracted from the cell pellet by incubating in CHAPS lysis buffer for 30 minutes on ice. Samples were spun at maximum speed at 4°C for 20 minutes to collect the supernatant. Protein quantification was carried out using Bradford method and 1.5 μg protein was treated with 1 ml/ml RNase inhibitor to eliminate RNase before performing PCR reaction.
Telomere restriction fragment (TRF) length analysis
Following treatment of brain tumour cells with 0.5 μM MST-312, DNA extraction was performed according to the manufacturers’ protocol using DNeasy Tissue Kit (Qiagen, USA). The telomere restriction fragment length analysis assay was performed using Telo-TAGGG Length Assay Kit (Roche Applied Science, USA). Kodak Gel imaging system and the Kodak imaging software was used to calculate the quantitative measurements of the mean TRF length.
Alkaline single cell Gel electrophoresis assay
Following treatments with 1.0 μM MST-312, cells were harvested and resuspended in Hank’s Balanced Salt Solution (Sigma) with 10% DMSO and 0.5 M EDTA. The cell suspension was then suspended in 0.7% low melting agarose at 37°C (Conda, Spain), and layered on to comet slides (Trevigen, USA). The cells were then lysed in lysis solution containing 2.5 M NaCl, 100 mM pH 8.0 EDTA, 10 mM Tris–HCl, 1% Triton–X at 4°C for 1 hour. Denaturation was carried out for 40 minutes, in chilled alkaline electrophoresis buffer (pH 13.0-13.7). Electrophoresis was subsequently carried out for 20 minutes. Slides were immersed in neutralization buffer (500 mM Tris–HCl, pH 7.4), dehydrated, dried and stained with SYBR Green dye (Trevigen) and scored with Comet Analysis Software (Metasystems, Germany). The images were captured using Zeiss Axioplan 2 imaging fluorescence microscope (Carl Zeiss, Germany) equipped with triple band filter. One hundred cells were randomly selected and analysed. The extent of DNA damage was expressed as tail moment, which corresponded to the fraction of the DNA in the tail of the comet.
Immunofluorescence
Briefly, 5 × 104 brain tumour cells were plated in cover slips in six well plates and grown for 48 hours in the presence of 1.0 μM MST-312. Cells were fixed in 4% paraformaldehyde and permeabilise in 0.1% Triton-X-100. Following incubation with anti-phospho-H2AX (Ser139) (Upstate, biotechnology) diluted in PBS with 4% FCS and 0.1% Triton X-100. Cells were washed and incubated with FITC-conjugated anti-mouse secondary antibody (1:500) secondary antibodies at room temperature in the dark for one hour. Subsequent washes were also conducted in the dark. The cover slips were sufficiently dried prior to mounting them onto slides containing Vectashield mounting media with DAPI (Vector laboratories). One hundred cells were randomly selected and screen for the present of foci in each experiments. For the detection of telomere dysfunction induced foci (TIF), cells were fixed as described above with the following changes. Incubation with mouse monoclonal anti- phosphor-H2AX (Millipore)(1:300) was carried out overnight at 4°C, followed by incubation with goat poly anti-TRF2 (Santa Cruz)(1:300) for 1 hour incubation at room temperature. Goat anti-mouse Texas Red (Invitrogen) (1:600) and rabbit anti-goat Fluorescein (Vector Laboratories) were added and incubated for 1 hour at room temperature. Images were captured using a Zeiss Axioplan Imaging fluorescent microscope with 63 × objective and are processed using Adobe Photoshop CS2 (Adobe Systems Incorporated, USA) for clarity and illustration.
Cell viability assay
A total of 1 × 104 cells per well were plated in transparent 24-well plates (Corning, Costar, USA) and treated with various concentration of MST-312 (0–5 μM) and/or 10 μM NU7026 for 48 hours. Cell viability was measured using CellTiter-Glo® luminescent cell viability assay (Promega, USA) according to the manufacturer’s instructions with some modifications. Briefly, the CellTiter-Glo® reagent was added to cell culture and the mixture were then transferred to white opaque walled 96-well plates (Corning, Costar, USA) for measurement of luminescence using plate reader (Tecan, Switzerland). Cell viability is expressed as percentage of untreated controls.
Cell proliferation assay
About 1 × 104 cells per well were plated in transparent 12-well plates (Corning, Costar, USA) and 0.5 μM or 1.0 μM of MST-312 and/or 10 μM of NU7026 were added. Fresh drug was added every 72 hours and cell proliferation was determined with trypan blue exclusion assay after indicated number of days.
Cell cycle analysis
Following 0–2.0 μM MST-312 treatment, cells were harvested, washed in 0.1% BSA: PBS, fixed in 70% ethanol: 1 × PBS, and stained with propidium iodide (Sigma, USA): RNase A (Roche, USA) (2 mg propidium iodide and 2 mg RNaseA/100 mL 0.1% BSA in 1 × PBS). Samples were analysed by flow cytometry (FACSCalibur™, Becton Dickinson, USA) at 488 nm excitation λ and 610 nm emission λ. A total of 10,000 events were captured and data obtained was analysed using Summit 4.3 software. The proportion of cells in different stages of cell cycle is expressed in terms of percentage of total cells analysed.
Western blot analysis
Total cellular proteins were isolated following a 48 hour-treatment with 1.0 μM of MST-312 and/or 10 μM of NU7026, using RIPA (radio-immunoprecipitation assay) buffer (1% nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate and 100 U/ml aprotinin, pH 7.2) from control and treated cells. The whole cell lysate was recovered by centrifugation at 14,000 rpm for 10 minutes. Protein concentration was determined by the bicinchoninic acid method using an assay kit (Pierce Biotechnology, USA) with bovine serum albumin as a standard. Western blot analyses of, DNA-PKcs, p-DNA-PKcs(Ser2056), (phospho DNA-PKcs, Abcam, UK,1:1000) hTERT (Epitomics, USA), and β-actin were performed using specific antibodies from Santa Cruz Biotechnology, USA unless otherwise stated.
Peptide nucleic acid fluorescence in situ hybridisation (PNA-FISH) analysis
For cytogenetic analysis, MO59K cells were treated with 1.0 μM of MST-312 and/or 10 μM of NU7026 for 1 week and cells were arrested at mitosis by treatment with colcemid (0.1 mg/ml). Cells were subsequently incubated with a hypotonic solution of potassium chloride at 37°C for 15 minutes followed by fixation in Carnoy’s fixative. Fluorescence
in situ hybridisation (FISH) was performed using telomere sequence-specific peptide nucleic acid (PNA) probe labelled with Cy3 as described [
44,
45]. Metaphase spreads were captured using Zeiss Axioplan 2 Imaging fluorescence microscope and analysed using ISIS Software (Metasystems, Germany) for telomere-mediated chromosome alterations.
Gene expression analysis
Following treatment with 1.0 μM of MST-312 for 48 hours, the total RNA was extracted from KNS60 and ONS76 cells using QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany). The extracted RNA was quantified using NanoDrop 1000 (Thermo Scientific, USA). RNA integrity was checked using Bio-Analyzer (Agilent Technologies, Inc., USA). Five hundred nanograms of extracted RNA from each sample were used for gene expression study. TotalPrep RNA Amplification Kit (Ambion Inc., TX, USA) was used for cRNA amplification process. The biotinylated amplified RNA thus generated was used for hybridization with HumanRef8 V3.0, Human Whole-Genome Expression BeadChips (Illumina Inc., USA) for 16 hours at 58°C. After the incubation period, the arrays were washed and stained with Streptavidin-Cy3 (GE Healthcare, Bio-Sciences, UK). Illumina Bead Array Reader was used to scan the arrays. The array data thus obtained after scanning was imported and analysed using Partek® Genomics Suite™ (Partek GS) (Partek Incorporated, MO, USA).
Statistical analysis
Statistical significance in the data sets was assessed by Student’s t-test (Microsoft Corporation, USA) and two-way ANOVA using Graphpad Prism. The difference was considered to be statistically significant when p < 0.05.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RLG and MPH designed the experiments. RLG, PSWL, HKL and SV performed the experiments. RLG and MPH analysed the data. RLG wrote the manuscript and MPH edited it. All authors read and approved the final manuscript.