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Inhibition of poly (ADP-Ribose) polymerase-1 in telomerase deficient mouse embryonic fibroblasts increases arsenite-induced genome instability
© Gurung et al; licensee BioMed Central Ltd. 2010
Received: 29 January 2010
Accepted: 26 May 2010
Published: 26 May 2010
The telomerase enzyme is a viable target for anti-cancer therapy given the innate differences in telomerase activity between tumour cells and normal somatic cells. However, the time lag between telomerase inhibition and telomeres becoming critically short to trigger cell death, allows cancer cells to acquire drug resistance. Inhibition of DNA repair pathways along with telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. Poly (ADP-Ribose) Polymerase-1 (PARP-1), an important DNA damage sensor and a DNA repair factor, has important roles in maintaining telomeres and chromosomal stability. In this study, the effects of combined inhibition of PARP-1 and telomerase in mouse embryonic fibroblasts (MEFs) following sodium arsenite exposure (a carcinogen and potent DNA damaging agent), were evaluated.
Inhibition of PARP in telomerase deficient MEFs induced an increase in arsenite-induced DNA damage as compared to control cells. Combined inhibition also resulted in enhanced genomic instability, demonstrated by elevated micronuclei induction and chromosomal aberrations with decreased cell survival. In addition, telomerase inhibition in PARP-1 deficient MEFs led to greater telomere shortening and increased genomic instability.
Our study demonstrated that the co-inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of combined inhibition of PARP-1 and telomerase as a strategy to minimise the problems associated with long-term telomerase inhibition in cancer therapeutics.
Telomeres are specialised dynamic structures at the ends of linear eukaryotic chromosomes consisting of non-coding DNA repeats (TTAGGG)n and associated proteins [1, 2]. These terminal DNA-protein complexes function as protective caps preventing chromosomal end-to-end fusions and the recognition of chromosomal ends as damaged DNA . Telomeres shorten with each cell division, eventually triggering senescence [4, 5]. In contrast, majority of tumour cells overcome telomere-mediated senescence via the activation of telomerase enzyme .
Telomerase contains two core components, an RNA subunit (hTERC and mTERC in human and mouse respectively), which provides the template for replenishment of telomeres  and a catalytic protein subunit, telomerase reverse transcriptase (hTERT or mTERT) that adds telomeric repeats to existing telomeres . Deletion of mTERC in mice resulted in the shortening of telomeres leading to increased genomic instability and reduction in growth rate [9–11]. In addition, these studies have also demonstrated that no phenotypic differences occur in the first generation mice lacking mTERC component. The abrogation of telomerase results in the reduction in cell proliferation only after telomeres are critically short.
Numerous DNA repair proteins along with the telomerase complex have been shown to have pivotal roles in the maintenance of telomere homoeostasis without any effect on telomerase activity. Dysfunctional telomeres, resulting from the loss of telomeric repeats or the loss of function of telomere-associated proteins, trigger DNA damage responses similar to that observed for DNA breaks [12–14].
Poly (ADP-ribose) polymerase-1 (PARP-1), a member of the PARP family, is a DNA damage sensor [15, 16] that allows for DNA repair upon binding to DNA strand breaks. This is effected by the post-translation mediation of downstream proteins in the base excision repair pathway [17–19]. Poly (ADP-ribose) polymerases (PARPs) mediates addition of ribose moiety using NAD+ as substrate in post translational modification of histones and other nuclear proteins that contributes to the survival of cells following DNA damage[20, 21]. PARP-1 also plays a role in telomere maintenance [22, 23]. PARP-1-/- mouse embryonic fibroblasts (MEFs) exhibited heightened genomic instability and telomere dysfunction following exposure to DNA damaging agents [24, 25]. Recently, it was shown that in the absence of telomere dysfunction, PARP-1 appears sporadically at telomeres but following DNA damage, PARP-1 localised to the damaged telomeres through its interaction with the telomere repeat binding factor 2, TRF2 .
Apart from the complex protein network involved in telomere homeostasis, telomerase enzyme plays a dominant role in telomere maintenance in tumour cells. Telomerase inhibition has become an attractive target for cancer therapeutics due to specific targeting of tumour cells [27–29]. However, a potential drawback of telomerase inhibition as a chemotherapeutic agent is that telomeres must become critically short before cytotoxic effects are observed in cancer cells. This lag phase may allow cancer cells to adapt using mechanisms such as alternative lengthening of telomeres, to counteract telomere shortening triggered by the absence of telomerase.
We recently reported that cells with dysfunctional telomeres are susceptible to DNA damage induced by sodium arsenite . Although this study highlights the protective role of telomeres in the event of DNA damage, it also draws attention to the requirement of critically short telomeres for the onset of cytotoxicity. Interestingly, microarray analysis showed differential expression of PARP-1 in wild type and G1-mTERC-/- MEFs . Thus, we wanted to test if the inhibition of PARP-1 in mTERC-/- MEFs could sensitise cells to DNA damaging agents and whether this combinatorial approach can be utilised to sensitise cells to chemotherapeutic agents.
PARP inhibition in MEFs lacking telomerase RNA component (mTERC-/-) induced elevated arsenite induced DNA damage
In our recent study, we observed an up-regulation of PARP-1 expression following arsenite treatment, which was higher in MEFs deficient in TERC component as compared to wild type . We sought to evaluate whether the inhibition of PARP sensitises mTERC-/- MEFs to sodium arsenite-induced DNA-damage. Wild type and mTERC-/- MEFs, pre-incubated with 3-aminobenzoamide (3-AB; A competitive inhibitor of PARP) [31, 32], were treated with different doses of sodium arsenite (As3+). Single cell gel electrophoresis assay under alkaline conditions, which allows for the analysis of all types of DNA damage, including double strand breaks, single strand breaks, and alkali labile sites, was carried out to estimate the extent of DNA damage induced by arsenite treatment.
Inhibition of PARP in mTERC-/-MEFs resulted in elevated arsenite-induced chromosomal instability
The presence of MN is a result of the exclusion of chromosomes or chromosomal fragments from the daughter nuclei. Consequently, we undertook fluorescence in situ hybridisation (FISH) analysis using telomeric PNA probes to analyse structural chromosome aberrations, particularly end to end fusions and chromosomal breaks. Chromosome preparations were carried out only for the 1.5 μg/ml dose of arsenite as it was not possible to obtain metaphases at the higher dose of arsenite. 3-AB treated mTERC-/- MEFs displayed greatest incidences of chromosomal fusions and chromosomal breaks following As3+ treatment (Fig. 2D and 2E). Moreover, the highest percentages of aberrant cells were also observed in PARP inhibited mTERC-/- MEFs following arsenite treatment (56%).
PARP-1 inhibition sensitised telomerase deficient mouse cells to arsenite induced cell death
Combined inhibition of telomerase and PARP reduced telomere length and increased genomic instability in mouse cells
Differential gene expression patterns in PARP-1-/- and mTERC-/-MEFs compared to normal MEFs
Functional categorisation of differentially expressed genes in wild type, mTERC -/- and PARP-1-/- MEFs following arsenite treatment.
DNA damage response
Several lines of evidence indicate that the activation of telomerase and the subsequent stabilisation of the telomeres are vital for the growth for majority of tumour cells. While most tumour cells express telomerase, somatic cells generally do not express this enzyme . Telomerase thus serves as a potential target for cancer therapy. The main drawback is the duration required for telomere length to reach a critical level before triggering senescence and/or apoptosis, which allow tumour cells time to develop resistance to anti-telomerase agents. Mice lacking telomerase RNA component survived but exhibited telomere shortening and increased chromosome instability . Telomerase deficient mouse cells were also viable in culture and found to employ telomerase independent mechanisms to maintain their telomeres [11, 34]. We have demonstrated in earlier studies that MEFs with dysfunctional telomeres exhibited sensitivity towards arsenite induced genomic instability treatment . Similar observations were made in the present study whereby mTERC-/- MEFs sustained shorter basal telomere length and exhibited significantly higher arsenite induced genomic instability as compared to mTERC+/+ MEFs. However to counteract the limitations of using telomerase inhibition alone, we explored the effects of a combinatorial approach of targeting telomerase activity and PARP-1 using PARP-1-/- and mTERC-/- MEFs with arsenite exposure.
Pharmacological inhibition of PARP in telomerase null MEFs increased the sensitivity to arsenite-induced cytotoxicity and genotoxicity. The inhibition of Tankyrase 1, a member of PARP family, in human cancer cells, has been shown to enhance telomere shortening and hasten cell death . As observed in this study, the inhibition of PARP-1 activity in telomerase deficient MEFs led to reduced cell survival, which were accompanied by elevated genomic instability. Concurrently, we treated PARP-1-/- MEFs with a synthetic telomerase inhibitor MST-312. Interestingly, the telomere length analysis by Q-FISH indicated that the cells deficient in PARP-1 activity with pharmacological reduction in telomerase activity displayed the shortest average telomere length. Telomerase inhibition in PARP-1-/- MEFs enhanced arsenite-induced genomic instability as well. Mammalian telomeric ends are protected by proteins such as TRF2 which stabilise the loop structure of the telomeres [36, 37]. A recent report has shown that the localisation of PARP-1 to damaged telomeric ends is partly attributed to its interaction with the TRF2 protein . Arsenite-induced damage at the chromosome ends may trigger the recruitment of PARP-1 to the telomeres to mediate repair. Thus, the increased sensitivity of telomerase deficient MEFs with PARP-1 inhibition to arsenite damage may be due to the lack of repair at both non-telomeric DNA and telomeric ends. The present study thus demonstrates the potential of combined inhibition of PARP-1 and telomerase for cancer therapy.
Gene expression studies revealed differential gene expression patterns in PARP-1-/- and mTERC-/- compared to wild type MEFs. Genes that are differentially regulated in both the cell types include genes involved in the biological processes such as cell growth and/or maintenance, DNA damage response, repair and telomere maintenance. More importantly, about 42 genes which are important for DNA damage signalling and repair and cell death were shown to be altered in their expression patterns. The gene expression level of BRCA2 was reduced in PARP-1-/- and mTERC-/- compared to wild type MEFs suggesting potential interplay of these genetic factors. It was recently found that the targeting of PARP-1 in cells defective in homologous recombination due to BRCA1 or BRCA2 dysfunction results in chromosomal instability, cell cycle arrest and subsequent apoptosis [38, 39]. Some of the PARP inhibitions are also currently used in combination with chemotherapy in clinical trials . Hence, understanding the changes in gene expression profiles may provide potential insights into selecting appropriate candidates for further combinatorial studies.
Integrating the findings from both models used in this study, it is possible to infer that telomerase inhibition and the consequent telomere shortening sensitises MEFs to DNA damaging agents. Our study demonstrated that the combined inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of co-inhibition of PARP-1 and telomerase as a strategy to overcome the limitations associated with telomerase inhibition alone for cancer therapy.
Materials and methods
Cell culture and drug treatment
Wild type, PARP-1-/- and mTERC-/- MEFs (Kindly provided by Dr. Zhao-Qi Wang, Germany and Dr. Han-Woong Lee, South Korea respectively) were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum (Hyclone, USA) and 100 U/ml of penicillin/streptomycin (Gibco, USA). To evaluate the response of PARP-1 and telomerase deficiency to arsenite-induced damage, cells in exponential growth phase (at about 70% confluence) were exposed to two different doses of sodium m-arsenite [(As3+; Sigma, USA) 1.5 μg/ml (11.5 μM) and 3.0 μg/ml (23 μM)] for 24 hours. PARP-1-/- MEFs were pre-treated with 1 μM telomerase inhibitor, MST-312 for 48 hours and mTERC-/- MEFs were treated with 3 mM 3-Aminobenzamide (3-AB; Sigma, USA) for 24 hours prior to exposure to As3+. All cells were maintained in a humidified 5% CO2 incubator at 37°C.
Assay for cell viability
Following drug treatment, cells were washed with phosphate buffered saline (PBS). Crystal violet solution (0.75% crystal violet in 50% ethanol: distilled water with 1.75% formaldehyde and 0.25% NaCl), which stains DNA by binding to nuclear proteins, was added to the culture wells and incubated for 20 minutes at room temperature. Following successive PBS washes to remove excess crystal violet solution, wells were air dried and 1% sodium dodecyl sulphate in PBS was added to lyse the cells and solubilise the dye. Cell viability was measured at 590 nm absorbance and expressed as the percentage of controls.
Alkaline single cell gel electrophoresis (Comet) assay
Cells were treated with As3+ for 24 hours with the doses mentioned earlier. The treated cells were harvested by trypsinisation and washed in ice-cold PBS. The cells were then suspended in Hank's balanced salt solution (Sigma, USA) and mixed with 0.7% low melting point agarose (at 37°C). The cells were then applied on Comet slides (Trevigen, USA), and subjected to lysis (2.5 M NaCl, 0.1 M pH 8 EDTA, 10 mM Tris base, 1% Triton X) at 4°C for 1 hour. The slides were loaded into a gel electrophoresis tank in 0.3 M NaOH, pH 13 with EDTA, allowed to denature for 40 minutes, and electrophoresis was done as per vendor's suggestions. After electrophoresis, slides were briefly rinsed in neutralization buffer (500 mmol/L Tris-HCl, pH 7.5), air-dried, and stained with SYBR green dye (Trevigen, USA). One hundred randomly selected nuclei were examined per sample using Comet Imager Software (Metasystems, Germany). Extent of DNA damage was expressed as a measure of comet tail moments, which corresponds to the fraction of DNA in the comet tail.
Cytokinesis-blocked micronucleus analysis (CBMN)
Following arsenite treatment, cells were incubated with 4 μg/ml cytochalasin B (Sigma, USA) for 22 hours and processed as described earlier [41, 42]. One thousand binucleated cells for each sample were scored for the presence of MN under a Zeiss Axioplan 2 imaging fluorescence microscope (Carl Zeiss, Germany) with appropriate triple band filter.
Telomeric Repeat Amplification Protocol (TRAP) Assay
Telomerase activity was measured by the commercially available TRAPeze® XL Telomerase Detection Kit (Chemicon International) according to the manufacturer's instructions.
Peptide nucleic acid-fluorescence in situ hybridisation (PNA-FISH)
Following arsenite treatment, cells were released from the treatment and allowed to grow for 24 hours in fresh media. Cells were arrested at metaphase with 0.1 μg/ml colcemid (Gibco, USA). The cells were harvested and subjected to hypotonic treatment of 0.03 M sodium citrate buffer at 37°C for 20 minutes followed by fixation in Carnoy's fixative. Fluorescence in situ hybridisation (FISH) was performed using telomere specific PNA probe labelled with Cy3 and the cells were counterstained with 4', 6-Diamidino-2-phenylindole (DAPI, Vectashield) . Fifty metaphases per sample were captured using the Zeiss Axioplan 2 imaging fluorescence microscope and analysed using the in situ imaging software (Metasystems, Germany) for chromosomal aberration analysis. Ten metaphase spreads per sample were analysed for telomere length measurement using the ISIS imaging software (Metasystems, Germany).
Gene expression analysis
Gene expression profiles were generated for Wild type, PARP-1-/- and mTERC-/- MEFs by microarray gene chip assay. Total RNA was extracted (RNeasy kit, Qiagen, Germany), and double-stranded cDNA was synthesized from 5 μg of total RNA using Superscript system (Invitrogen, USA) primed with T7-(dT)-24 primer. For biotin-labelled cRNA synthesis, in vitro transcription reaction was done in the presence of T7 RNA polymerase and biotinylated ribonucleotides (Enzo Diagnostics, USA). The cRNA product was purified (RNeasy kit, Qiagen, Germany), fragmented, and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 in a Gene chip hybridization oven 640 (Affymetrix Inc., USA) as per the Gene Chip Expression Analysis manual (Affymetrix Inc., USA). After 16 hours of hybridisation, the gene chips were washed and stained using the Affymetrix Fluidic station and scanned by Gene Array Scanner (Affymetrix Inc., USA). Image data were normalized and statistically analysed using Gene Spring 7.2 (Silicon Genetics, USA). Microarray experiments were repeated twice in order to confirm the differential gene expression. More than 300 genes with P < 0.05 (one-way ANOVA) were differentially expressed and they were annotated according to GO-biological process. Subsequent data analysis involved Agglomerative average-linkage hierarchical clustering for finding different patterns and levels of gene expression.
Statistical significance in the data sets was assessed using Student's t-test using Microsoft Excel 2003 (Microsoft Corp., USA) and two-way ANOVA, using Graphpad Prism. The difference was considered to be statistically significant when the p values are < 0.05.
This is supported by grants from Academic Research Fund, Ministry of Education, Singapore (T206B3108) to MPH.
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