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A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells
© Lee et al.; licensee BioMed Central Ltd. 2013
Received: 14 January 2013
Accepted: 14 May 2013
Published: 22 May 2013
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory.
We have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry.
Using the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A.
Thus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.
An abnormal chromosome number (aneuploidy) is a feature of most solid tumors and is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN) . The gain or loss of entire chromosomes leads to large-scale changes in gene copy number and expression levels. The molecular mechanisms underlying CIN include defects in chromosome cohesion, mitotic checkpoint function, centrosome copy number, kinetochore-microtubule attachment dynamics, and cell-cycle regulation. While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth, and therefore, it can be exploited therapeutically. Janssen and co-authors  have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in tumor cell lines caused mild chromosome mis-segregation, but no lethality. These cells were, however, much more sensitive to low doses of taxol, which enhances the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing the levels of Mps1 or BubR1, proteins with dual roles in checkpoint activation and chromosome alignment. Importantly, untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sub-lethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells. In another study , a set of genes was identified that are repressed in response to taxol treatment and over-expressed in tumors exhibiting CIN. The silencing of these genes caused cancer cell death, suggesting that these genes might be involved in the survival of aneuploid cells. In diploid cells, but not in chromosomally unstable cells, taxol causes the repression of CIN-survival genes, followed by cell death. Taking into account the fact that aneuploidization per se seems to be a very inefficient path towards cancer and additional hits are necessary for the generation of a cancer cell ( and references therein), these and other studies [5, 6] indicate that increased destabilization of chromosomes might push genetically unstable cancer cells towards death, whereas more stable normal cells would be able to tolerate such insults.
Elevation of CIN as an approach to cancer therapy is attracting considerable attention [2–5]. However, none of the methods used to study CIN and its induction by environmental agents is entirely satisfactory. Karyotype analysis is bedeviled by the karyotypic variation already often present in cancer cell lines. Micronucleus assays (MNi) are widely used to detect broken or lagging chromosomes, but fail to detect non-balanced chromosome segregation .
In this study, we developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of the human artificial chromosome (HAC) constructed in our lab earlier as a gene therapy tool for the efficient and regulated expression of genes of interest [8–10]. The HAC contains centromeric repeats that form a functional centromere/kinetochore, allowing its stable inheritance as a nonessential chromosome, albeit with a loss rate roughly 10× that of the native chromosomes [11, 12]. To adopt this HAC for CIN studies, an EGFP transgene was inserted into the HAC. This allowed the measurement of the HAC loss rate by routine flow cytometry. Thus, the HAC offers a sensitized and simple system to measure CIN, particularly after drug treatment. In this study, the HAC-based CIN assay has been verified using a set of well-known aneugens and clastogens. This new assay has the potential to be developed for high-through put screening methods to identify new compounds that elevate chromosome mis-segregation and drive lethal aneuploidy. New and potentially less toxic agents that selectively elevate CIN in cancer cells to promote cancer cell death identified with this new screening tool could lay the foundation for new treatment strategies for cancer.
Human fibrosarcoma HT1080 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% (v/v) tet system-approved fetal bovine serum (Clontech Laboratories, Inc.) at 37°C in 5% CO2. Hypoxanthine phosphoribosyltransferase (HPRT)-deficient Chinese hamster ovary (CHO) cells (JCRB0218) carrying the alphoidtetO-HAC were maintained in Ham's F-12 nutrient mixture (Invitrogen) plus 10% FBS with 8 μg/ml of BS (Funakoshi). After loading of the EGFP transgene cassette into the alphoidtetO-HAC, the CHO cells were cultured in 1× HAT supplemented medium.
Loading of the EGFP transgene cassette into the loxP site of alphoidtetO-HAC in CHO cells
A total of 3 to 5 μg of a EGFP transgene plasmid (or X3.1-I-EGFP-I described previously ) and 1 to 2 μg of the Cre expression pCpG-iCre vector DNA were co-transformed into HPRT-deficient CHO cells containing the alphoidtetO-HAC by lipofection with FuGENERHD transfection reagent (Roche) or Lipofectamine 2000 (Invitrogen). HPRT-positive colonies were selected after 2 to 3 weeks growth in HAT medium. For each experiment, from 5 to 7 clones were usually selected. Correct loading of the EGFP transgene cassette into the HAC was confirmed by genomic PCR with a specific pair of primers that diagnose reconstitution of the HPRT gene .
Microcell-mediated chromosome transfer
The alphoidtetO-HAC containing the EGFP transgene cassette (EGFP-HAC) was transferred from CHO cells to HT1080 cells using a standard microcell-mediated chromosome transfer (MMCT) protocol [13, 14]. Blasticidin (BS) was used to select resistant colonies containing the HAC. Typically, three to ten BSR colonies were obtained in one MMCT experiment. BSR colonies were analyzed by FISH for the presence of the autonomous form of the HAC. The co-transfer of CHO chromosomes was examined using a sensitive PCR test for rodent-specific SINE elements .
Analysis of EGFP expression was performed on a FACS Calibur instrument (BD Biosciences) using CellQuest acquisition software and analyzed statistically with FlowJo software [15, 16]. The cells were harvested by trypsin-treatment. Intensities of fluorescence were determined by flow cytometry. A minimum of 4 x 104 cells was analyzed for each cell sample.
Drugs used in this study
Concentration/ time treatment
Fold increase of HAC loss per cell division
10 nM-2 hrs
Aurora B, MPS1
1 μM-3 days
1 μM-3 days
Calculation of the rate of HAC loss after drug treatment
Once (RNormal) was obtained, the rate of HAC loss induced by drug treatment (RDrug) is then determined using the formula, . Justification of this algorithm is presented in Figure 1. As before, P0 represents the percentage of EGFP(+) cells at the start of the experiment, cultured under HAC selection condition. PTreated is the percentage of EGFP(+) cells at the end of a drug treatment experiment with a duration of (t2 + t3), where t2 is the duration of drug treatment and t3 is the duration of culturing after the drug is removed. (t2 + t3) was 14 days in this study. n2 is the number of cell doublings that occurs during drug treatment, while n3 is the number of cell doublings that occurs during the culturing without selection after drug treatment.
In the present study, the duration of most drug treatments were less than the duration of a single cell cycle of HT1080 (t2 <18 hr). We made the assumption that any significant increase in HAC loss occurs only during the first mitotic division after washing off a drug (n2 = 1). Thus n3 = (14 d / 18 hr - 1). The exceptions, reversine and ZM-447439 both severely inhibits cell growth, thus despite the 3 day drug treatment only one cell division is assumed to have occurred. We also assumed that HAC stability in subsequent cell divisions is no different from that of untreated cells.
The algorithm we used is valid between the ranges of 0 to 1. R values large than 1 indicate that the assumptions made in this model are incorrect. The assumption of synchronous growth in the model means that the estimated mis-segregation rate is lower than real values. As the spontaneous rate of HAC mis-segregation (RNormal) was found to be low, this algorithm is relatively insensitive to the number of cell divisions that occurs post drug treatment. It is worth noting that the frequency of HAC loss in the clones carrying two copies of the HAC is indistinguishable from those containing a single copy of the HAC (data not shown). Therefore, the model is applicable when a cell inherits two copies of the HAC due to non-disjunction.
Cell viability test
MTS tetrazolium cell viability assays were done according to the manufacturer’s instructions (CellTiter 96 AQueous Assay reagent; Promega). Briefly, the CellTiter 96 AQueous One Solution Reagent was added to each well and incubated at 37°C for 3 h. Cell proliferation was determined by measuring the absorbance at 490 nm using a microtiter plate reader (Molecular Devices, Sunnyvale, CA). The half-maximal inhibitory concentration (IC50) was obtained from the MTS viability curves using GraphPad Prism 5. Experiments were carried out in triplicate.
FISH analysis with PNA probes
The presence of the HAC in an autonomous form was confirmed by FISH analysis as previously described [8, 9]. HT1080 cells containing the HAC were grown in DMEM medium to 70-80% confluence. Metaphase cells were obtained by adding colcemid (Gibco) to a final concentration of 0.05 μg/ml and incubating overnight. Media was aspirated, and the plate washed with 1x PBS. Cells were removed from the plate by 0.25% Typsin, washed off with DMEM, pelleted and resuspended in 10 ml of 50 mM KCl hypotonic solution for 30 min at 37°C. Cells were fixed by three washes of fixative solution (75% acetic acid, 25% methanol). Between each wash, cells were pelleted by centrifugation at 900 rpm for 4 min. Metaphase cells were evenly spread on a microscope slide and the fixative solution evaporated over boiling water. Dry slides were rehydrated with 1× PBS for 15 min, and fixed in 4% formaldehyde-1× PBS for 2 min, followed by three 5 min 1× PBS washes and ethanol series dehydration. PNA (peptide nucleic acid) labeled probes used were telomere (CCCTAA)3-Cy3) (PerSeptive Biosystems, Inc.) and tetO-alphoid array (FITC-OO-ACCACTCCCTATCAG) (Panagene, South Korea). Ten nanomol of each PNA probe was mixed with hybridization buffer and applied to the slide, followed by denaturation at 80°C for 3 min. Slides were hybridized for 2 hours at room temperature in the dark. Slides were washed twice in 70% formamide, 10 mM Tris pH 7.2, 0.1% BSA and followed by three washes with 1xTBS, 0.08% Tween-20. Slides were dehydrated gradually with a series of 70%, 90% and 100% ethanol washes and mounted (Vectorshield with DAPI). Images were captured using a Zeiss Microscope (Axiophot) equipped with a cooled-charge-coupled device (CCD) camera (Cool SNAP HQ, Photometric) and analyzed by IP lab software (Signal Analytics). The PNA-DNA hybrid probes demonstrated a high hybridization efficiency, staining intensity and adopt a stable duplex form with complementary nucleic acid.
FISH analysis with the BAC probe
HT1080 cells were processed for fluorescence in situ hybridization (FISH) after drug treatment followed by the 14 day washout. The probe used for FISH was BAC32-2-mer(tetO) DNA containing 40 kb of alphoid-tetO array cloned into a BAC vector as described previously . Specifically, a BAC32-2-mer(tetO) clone contains an amplified synthetic alphoid DNA dimer. One monomer of this dimer is an alphoid DNA consensus sequence carrying the tetO sequence; another monomer is alphoid DNA from chromosome 17. This probe is specific to the HAC but also gives a low signal with centromeric regions of several endogenous chromosomes. BAC DNA was digoxigenin-labeled using a nick-translation kit with digoxigenin-11dUTP or biotin16-dUTP (Roche Diagnostics). Images were captured as before. The probe was denatured for 5 min at 95°C and added to the slides, which were incubated at 72°C for 2 min before overnight incubation at 39°C. After washes with 0.1 × SSC at 65°C followed by a wash with 4 × SSC + 0.1% Tween 20 at room temperature, standard procedures were used to detect biotinylated probes. Slides were mounted with VectaShield and screened for the presence or absence of the HAC. Between 70–250 metaphases were analyzed for each experiment (one drug treatment).
Genomic DNA preparation and PCR analysis
Genomic DNA for PCR analysis was prepared using a QIAmp DNA Mini Kit (QIAGEN Inc., Valencia, CA, USA). Reconstitution of the HPRT gene after Cre/lox-mediated recombination was determined by specific primers, Lox137-R 5′-agccttctgtacacatttcttctc-3′ and Rev #65′-gctctactaagcagatggccacagaactag-3′. Cross contamination by hamster chromosomes was determined by specific primers detecting hamster SINEs: Cons B2 5′-ccatctgtaatgagatctgatgc-3′, Ham B2-F 5′-gctcagaggttaagagcactgac-3′ and Ham B2-R 5′-tgcttccatgtatatctgcacac-3′. PCR products for sequencing were separated by agarose gel electrophoresis and gel extracted.
Micronucleus formation assay (MNi)
Micronuclei (MNi) formation in the HAC-containing HT1080 cells treated by different drugs
Fraction of cells with MNi
Mean ± SD (%)**
2 ± 1
10 nM Taxol
40 ± 3
100 nM Peloruside A
69 ± 5
1 μM ZM447439
57 ± 1
100 nM Nocodazole
53 ± 3
Experimental design for identification of drugs that elevate CIN in cancer cells
Construction of the HAC carrying a single copy of the EGFPtransgene and its transfer to human cells
Comparison between FISH and FACS data to evaluate HAC induced by drug treatment
Rate of HAC loss
% Cells with HAC
% Cells with EGFP
per cell division**
85.1 ± 0.9
13 × 10-3
59.5 ± 0.9
597 × 10-3
68.0 ± 0.1
396 × 10-3
79.1 ± 1.8
133 × 10-3
67.4 ± 0.9
411 × 10-3
80.3 ± 0.9
107 × 10-3
84.3 ± 1.2
12 × 10-3
80.7 ± 0.9
96 × 10-3
79.0 ± 0.5
176 × 10-3
70.4 ± 0.5
374 × 10-3
Effect of aneugens and clastogens on the rate of HAC mis-segregation during mitotic divisions
We next investigated whether the EGFP/HAC-based assay could be used to detect compounds that cause chromosome loss and mis-segregation. HT1080 cells with an autonomously propagated EGFP-HAC were treated with eight known aneugens: taxol (pacilitaxel), docetaxel, peloruside A, ixabepilone, nocodazole, ZM447439, reversine, and SAHA (Table 1). Taxol, docetaxel, peloruside A, ixabepilone, and nocodazole are microtubule-targeting drugs ; ZM447439 and reversine are inhibitors of Aurora B and MPS1, respectively, and function in the spindle assembly checkpoint [20, 21]. SAHA is an inhibitor of histone deacetylases (HDAC) . Cells were also treated with the well-known clastogenic DNA topoisomerase II inhibitor VP16 (etoposide) .
As seen, a single dose of taxol greatly increases (~50 times) the rate of HAC loss. Taxol (pacilitaxel) was isolated ~ 40 years ago and is currently administered in a large variety of indications, including solid tumors and haematological malignancies  and references therein]. While its mechanism of action remains controversial, accumulating data suggest that at clinically relevant concentrations, taxol-mediated cell death involves elevation of chromosome mis-segregation that is incompatible with cancer cell survival  and references therein]. HAC loss was also significantly increased by peloruside A, ixabepilone, which also binds to the microtubule as taxol, and by the Aurora B inhibitor ZM-447439. For the other analyzed compounds except SAHA, the rates of HAC loss were also increased compared to controls, but much lower compared to those obtained after taxol or peloruside A treatment. The lowest increase of HAC loss was obtained after treatment by the inhibitor of histone deacetylases, SAHA.
In separate experiments, the relationship of HAC loss to chemical dose was investigated. Treatment of cells with higher doses of the compounds kills more cells but does not increase the rate of HAC loss. This may be explained if cells arrest, then apoptosis in response to damage of the spindle or if the daughter cells are not viable. We also performed the experiments with lower doses of drugs, i.e. with concentrations ½, ¼, 1/8 and 1/16 of the concentration corresponding to IC50. For some drugs, the frequency of HAC loss was dose independent while for others displayed a linear dose-dependence. For example, HAC loss was decreased in an almost linear fashion after treatment by VP16 (which is inhibitor of TOP2) with lower doses. In contrast, our analysis revealed a non-linear dose-dependent decrease in HAC loss frequencies for nocodazole and taxol, which were previously reported as compounds that interact with the mitotic spindle (thresholded concentration-effect response) .
FISH analysis was used to confirm that the appearance of non-fluorescent cells detected by flow cytometry corresponds to HAC loss events and to exclude possible alternative explanations such as mutations in the EGFP gene or its epigenetic silencing. Quantitative analysis of metaphase chromosome spreads using a HAC-specific probe (see Methods) correlated with the data on HAC loss determined by FACS (Table 3). Therefore, we conclude that the appearance of non-fluorescent cells is caused by HAC loss.
An unexpected result was the different effects of microtubule-binding drugs on HAC stability. Ixabepilone, docetaxel, and peloruside A are microtubule-stabilizing agents similar to taxol . However, each drug exhibited a different effect on HAC mitotic stability, suggesting a different cell response to these compounds. Interestingly, under these conditions, all of the analyzed drugs induced micronuclei formation in the HAC-containing HT1080 cells (Table 2) similar to that reported for other cell lines . However, there was no detectable correlation between frequencies of MNi formation (that varied between 37% and 63%) and rates of HAC loss determined by FISH and FACS (Table 3). Thus, aneugens with a similar mechanism of action and cytotoxicity may differ from each other by their effect on the mitotic stability of the non-essential human artificial chromosome. It is unlikely that karyotyping or another variant of MNi tests can detect such differences between the compounds. Based on these results, we conclude that a newly developed EGFP/HAC-based assay for measuring CIN is a more sensitized system than other previously described methods to detect chromosome mis-segregation.
Targeting of CIN in cancer therapy requires measurement of the accuracy of chromosome transmission. At present, a variety of methods is used to study chromosome instability (CIN) and its induction by environmental agents [7, 28] and references therein]. Because test systems screening for numerical chromosomal effects rely on labor-intensive microscopic assessment, the micronucleus (MNi) formation test is the most widely used method for large-scale detection of broken or lagging chromosomes . However, because the origins and fates of MNi have not been completely elucidated [29, 30], intra- and inter-laboratory variability in scoring is still common , and complicates the development of a standard protocol for quantitative measurement of chromosome loss rates based on the appearance of MNi. It is also noteworthy that the MNi assay does not measure the fraction of drug-arrested cells that undergo mitosis and form viable aneuploid cells.
This work describes a new assay for measuring CIN in response to drug treatment that overcomes the limitations of current approaches. The assay is based on quantitative measurements of mitotic loss rates of a nonessential human artificial chromosome (HAC) carrying a transgene encoding the green fluorescent protein (EGFP). Thus, cells that inherit the HAC fluoresce green, while cells that lack it do not. The proportion of cells that lose the HAC in response to drugs is measured using FACS, which allows the objective and accurate assessment of chromosome loss in a large number of cells (e.g., 106) within minutes. Because only viable cells are studied, the long-term effects of aneugen exposure on chromosome loss are readily measured. It is worth noting that a similar approach but involving natural chromosome was previously attempted , but proved not to be useful for CIN studies, as the rates of chromosome loss were too low to be measured accurately. In the present study, the use of a HAC reporter that is sensitized for chromosome loss circumvents this problem. The HAC contains a functional kinetochore and its behavior during mitotic divisions does not differ from that of normal chromosomes [8, 10], suggesting that destabilization of natural chromosomes in response to drug treatment will be increased proportionally to that observed for the HAC.
As proof of principle, the EGFP/HAC-based assay was applied to analyze a set of well-known anti-mitotic, spindle-targeting compounds previously reported to induce micronucleus formation and chromosome loss. For each drug, the rate of HAC loss was measured by flow cytometry and confirmed by FISH analysis. The most interesting result obtained from these experiments is the observation that despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. Moreover, we found that aneugens with proposed similar mechanisms of action and cytotoxicity may greatly differ from each other by their effect on the mitotic stability of the non-essential human artificial chromosome. Thus, the assay described here is sensitive enough to discriminate between drugs with similar mechanisms of action in order to reveal those with the highest activity on chromosome stability. In future drug screening studies, taxol exhibiting the highest effect on HAC stability may be used as a positive control.
In this work to compare different compounds, the cells were analyzed 14 days after drug washout. At the same time, the sampling time for HT1080 cells has a broad interval and fluorescence of the treated cells can be measured even after 5–7 days without a significant effect on the calculated rate of HAC loss (see Additional file 1). For other cell lines, where the EGFP protein is more stable, the sampling time may be exceeded up to 10–14 days. However, the speed of the assay can be significantly increased using a modified EGFP with a reduced half-life (e.g., EGFP with a degron box sequence) that would convert an assay into high-throughput screening. The transfer of EGFP-HAC into other cell lines will make possible to assess effects of inhibitors in different genetic backgrounds such as cell lines harboring mutations in genes that regulate the spindle assembly checkpoint. It is worth noting that recent innovations have allowed the generation of HACs with the EGFP transgene de novo in a broad variety of human cell lines .
This new HAC-based flow cytometry assay may be also applied for identifying genes controlling chromosome segregation in human cells because it is more sensitive to perturbations of spindle assembly and cell cycle progression than assays based on the analysis of stability of natural chromosomes . Earlier, the development of conceptually simple color colony assays in yeast provided a powerful genetic tool to assess the rates of chromosome mis-segregation and to identify mutants deficient in this process . Similarly, in humans, the EGFP/HAC-based chromosome loss assay may be used to reveal new human genes involved in chromosome transmission.
We have developed a new assay for measuring chromosome instability based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. This simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss.
This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, USA (V.L.), a Wellcome Trust Principal Research Fellowship [grant number 073915] (W.C.E.), the Grand-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology of Japan (H.M.) and the Kazusa DNA Research Institute Foundation (H.M.) and the Indiana Genomics Initiative (INGEN) (B.R.G.). INGEN is supported in part by the Lilly Endowment. Reversine and ZM-447439 were kindly provided by Dr. Alexey Arnautov (National Institute of Child Health and Human Development/ NICHHD, NIH). Ixabepilone and docetaxel were obtained from Dr. Marianne Poruchynsky (National Cancer Institute/NCI, NIH). Peloruside A was kindly provided by Dr. Dan Sackett (National Institute of Child Health and Human Development/NICHHD, NIH). The authors also would like to thank the CRC, LRBGE Fluorescence Imaging Facility (NIH) and personally Drs. Karpova and Dr. McNally for instructions, consultations and help with the usage of a DeltaVision microscopy imaging system. We also would like to thank Dr. Elisabetta Leo for help in some experiments.
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