Biochimica et Biophysica Acta 1853 (2015) 1195–1204

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Selective killing of G2 decatenation checkpoint defective colon cancer cells by catalytic topoisomerase II inhibitor Chetan Kumar Jain a,b, Susanta Roychoudhury b,⁎, Hemanta Kumar Majumder a,⁎⁎ a b

Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India

a r t i c l e

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Article history: Received 8 September 2014 Received in revised form 9 February 2015 Accepted 25 February 2015 Available online 4 March 2015 Keywords: G2 DNA decatenation checkpoint Topoisomerase IIα Colorectal cancer ICRF193 Mitotic catastrophe

a b s t r a c t Cancer cells with defective DNA decatenation checkpoint can be selectively targeted by the catalytic inhibitors of DNA topoisomerase IIα (topo IIα) enzyme. Upon treatment with catalytic topo IIα inhibitors, cells with defective decatenation checkpoint fail to arrest their cell cycle in G2 phase and enter into M phase with catenated and under-condensed chromosomes resulting into impaired mitosis and eventually cell death. In the present work we analyzed decatenation checkpoint in five different colon cancer cell lines (HCT116, HT-29, Caco2, COLO 205 and SW480) and in one non-cancerous cell line (HEK293T). Four out of the five colon cancer cell lines i.e. HCT116, HT-29, Caco2, and COLO 205 were found to be compromised for the decatenation checkpoint function at different extents, whereas SW480 and HEK293T cell lines were found to be proficient for the checkpoint function. Upon treatment with ICRF193, decatenation checkpoint defective cell lines failed to arrest the cell cycle in G2 phase and entered into M phase without proper chromosomal decatenation, resulting into the formation of tangled mass of catenated and under-condensed chromosomes. Such cells underwent mitotic catastrophe and rapid apoptosis like cell death and showed higher sensitivity for ICRF193. Our study suggests that catalytic inhibitors of topoisomerase IIα are promising therapeutic agents for the treatment of colon cancers with defective DNA decatenation checkpoint. © 2015 Elsevier B.V. All rights reserved.

1. Introduction As a consequence of the genome replication in S phase, DNA catenations are formed between sister chromatids [1]. These catenations physically link sister DNAs and thus must be resolved to allow proper chromosome condensation and segregation in prophase and anaphase, respectively. In G2 phase of the cell cycle these catenations are resolved by the action of a highly conserved eukaryotic enzyme topoisomerase IIα (topo IIα). Chromatid catenations are actively monitored in human cells by a G2 phase decatenation checkpoint which delays entry to mitosis until all the catenations in the genome have been removed. However Bower et al. [2], have shown that the checkpoint does not sense catenations but rather it senses the catalytically inactive form of topo IIα tethered to DNA. Although complete signaling pathway for the

Abbreviations: Topo IIα, topoisomerase IIα; ATM, ataxia telangiectasia mutated; Chk2, checkpoint kinase 2 ⁎ Correspondence to: S. Roychoudhury, Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India. Tel.: +91 33 24995823; fax: +91 33 2473 5197. ⁎⁎ Correspondence to: H. K. Majumder, Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India. Te: +91 33 2412 3207; fax: +91 33 473 5197. E-mail addresses: [email protected], [email protected] (S. Roychoudhury), [email protected], [email protected] (H.K. Majumder).

http://dx.doi.org/10.1016/j.bbamcr.2015.02.021 0167-4889/© 2015 Elsevier B.V. All rights reserved.

decatenation checkpoint is not well understood, this checkpoint has been widely studied during last decade in different types of cancers including lung cancer, leukemia, bladder cancer and melanoma [3–6]. Failure to properly resolve sister chromatids has been proposed to lead nondisjunction, loss of heterozygosity, aneuploidy and translocation, which are causes of genomic instability [5,7–9]. Topoisomerase inhibitors are widely used in anticancer chemotherapy. Topoisomerase inhibitors are classified into two different categories: class I topoisomerase inhibitors or topoisomerase poisons inhibit strand rotation or re-ligation step of the enzymatic reaction and thereby stabilize topoisomerase-DNA covalent complexes, whereas class II topoisomerase inhibitors or catalytic topoisomerase inhibitors inhibit either DNA binding or DNA cleavage step of the enzymatic reaction and thereby do not generate DNA breaks. Etoposide is a class I inhibitor of topo IIα. It stabilizes covalent topo II-DNA complexes and thereby forms double strand DNA breaks. ICRF193, a bisdioxopiperazine, is a class II or catalytic inhibitor of topo IIα. ICRF193 inhibits ATPase activity of the enzyme and stabilizes the enzyme in closed clamp position thereby the enzyme cannot decatenate sister chromatid DNA and induce decatenation checkpoint pathway which arrests cells in G2 phase [10, 11]. Studies have shown that ATM (ataxia telangiectasia mutated) kinase and Rad9A are required for the activation of decatenation checkpoint and upon treatment with ICRF193, ATM is autophosphorylated at serine-1981 [12,13]. Phosphorylated ATM subsequently phosphorylates

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checkpoint kinase 2 (Chk2) at Thr-68 and Rad9A at Ser-272 [13,14]. Phosphorylated Chk2 phosphorylates Cdc25C which prevents mitotic entry [15]. Luo et al. [16], have shown that during the activation of decatenation checkpoint topo IIα is phosphorylated at Ser-1524, facilitating interaction of topo IIα with MDC1 (mediator of DNA damage checkpoint protein-1). Adjuvant 5-fluorouracil (5-FU) based chemotherapy in patients with resected stage III colorectal cancer improves recovery of the disease and overall survival rate by 35% and 22%, respectively [17]. But in advance stage CRC response rates for 5-FU based monotherapy is only 10–15% [18]. Combinations of 5-FU with CPT-11 and oxaliplatin improves response rates to 40–50% and prolonged progression-free survival [19,20]. Although these improvements have been made but more than half of the patients with advanced stage colorectal cancer do not show any measurable shrinkage in their tumors [21], thus improvement in the current chemotherapy for colorectal cancer and identification of novel chemotherapeutic targets for colorectal cancer are justified. In this study we analyzed G2 phase decatenation checkpoint in five different colon cancer cell lines (HCT116, HT-20, Caco2, COLO 205 and SW480) and in one non-cancerous cell line (HEK293T). Four out of the five cell lines i.e. HCT116, HT-20, Caco2 and COLO 205 were found to be defective for the checkpoint function at different extents while SW480 and HEK293T cell lines efficiently blocked their cell cycle in G2 phase after ICRF193 treatment. We found that such decatenation checkpoint defective cells undergo mitotic catastrophe and rapid cell death and show higher sensitivity for catalytic topo II inhibitor, ICRF193. Mitotic catastrophe describes a form of cell death affecting mammalian cells during mitosis. Mitotic catastrophe is characterized by the formation of large cells containing large and multiple nuclei (fragmented nuclei). Such cells are morphologically distinguishable from apoptotic cells [22].

2. Materials and methods 2.1. Cell culture and cell viability assay All the cell lines were cultured in the ATCC recommended culture media supplemented with 10% fetal bovine serum (GIBCO, Invitrogen, Carlsbad, CA, US), 1× PSN (GIBCO) and gentamicin (GIBCO). Cells were incubated in a humidified CO2 incubator at 37 °C. ICRF193 and etoposide were purchased from Sigma (St. Louis, MO, US). For cell viability assay, cells were seeded in 96 well plates and were treated with vehicle or ICRF193 or etoposide for 48 h. After 48 h cells were treated with MTT and formation of formazan was quantified on Thermo MULTISKAN EX plate reader at 595 nm.

2.2. Colony formation assay Approximately 103 cells were seeded in 6 cm culture dishes and treated with vehicle or ICRF193 or etoposide for 24 h. After the treatment cells were allowed to form colonies for 15–20 days. Colonies were stained with 0.2% methylene blue. Colonies were counted in each plate and percentage of colonies formed was calculated.

2.3. Cell synchronization One day prior to start of synchronization, cells were seeded on coverslips in 35 mm dishes. Next day 2.5 mM thymidine (Calbiochem, San Diego, CA, US) was added to the dishes (first thymidine block). After 16 h of thymidine treatment cells were replenished with fresh media. After 8 h again 2.5 mM thymidine was added to the cells (second thymidine block). After 22 h cells were replenished with fresh media. To obtain G2 phase synchronized cells, cells were harvested/collected after 6 h of thymidine release.

2.4. Measurement of mitotic indices Immunostaining for phospho-histone H3 (serine-10) was performed to identify the cells undergoing mitosis. G2 phase synchronized cells were treated with vehicle or ICRF193 or etoposide at IC50 concentrations and were fixed with acetone–methanol mixture (1:1) at 0, 2, 4 and 6 h post-treatment. Cells were washed with 1 × PBS thrice and permeabilized with 0.03% saponin for 30 min. Cells were washed with 1 × PBS thrice and blocked in 3% BSA for 2 h on rocker. One hundred microliters of p-histone H3 (serine-10) antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) (1:50 dilution) was directly added on to the cells and incubated overnight at 4 °C. Cells were washed with 1× PBS thrice. One hundred microliters of FITC-tagged secondary antibody (1:100 dilution) was directly added on to the cells and incubated at room temperature for 2 h in the dark. Cells were mounted on glass slides with 4′-6-diamidino-2-phenylindole (DAPI) and sealed with nail enamel. Approximately 200 cells per slide were observed for phosphorylation of histone H3 and percentage of mitotic cells (mitotic index) was calculated. 2.5. Flow cytometry analysis of cell cycle and apoptosis For cell cycle analysis, cells were fixed in 70% ethanol for overnight at 4 °C. Next day cells were stained with propidium iodide/RNase staining buffer (BD Biosciences, San Diego, CA, USA) and flow cytometry was performed. For detection of apoptosis, cells were stained with FITC-annexin V and propidium iodide solution using apoptosis detection kit (BD Biosciences). 2.6. Immunoblotting Cells were lysed in NP-40 lysis buffer and total cellular proteins were measured by Bradford's assay. Equal amounts of proteins were electrophoresed on SDS-poly acryl amide gel, separated proteins were transferred on to nitrocellulose membrane and immunoblotting was performed using antibodies specific to the desired proteins. 2.7. Quantification of G2 decatenation checkpoint function in colon cancer cell lines Synchronized cells were treated with vehicle or ICRF193 or etoposide in G2 phase (6 h post-thymidine release). Cells were fixed after 6 h of the treatment (12 h post-thymidine release) and immunostained using cyclin A2 antibody and FITC-tagged secondary antibody. Approximately 200 cells per slide were observed for cyclin A2 expression and extent of G2 checkpoint function was quantified by the following equation: Percentage of G2 checkpoint functional cells = percentage of cyclin A2 positive cells in ICRF193 or etoposide treated condition − percentage of cyclin A2 positive cells in vehicle treated condition. 2.8. Nuclear extracts preparation and topoisomerase enzyme activity assay Synchronized HCT116 and HEK293T cells were treated with vehicle or 4 μM ICRF193 or 4 μM etoposide in G2 phase for 2 h and were suspended in cytoplasmic extraction buffer containing 10 mM Tris–HCl (pH 7.9), 340 mM sucrose, 3 mM CaCl2, 2 mM MgCl2, 100 μM EDTA, 1 mM DTT, 0.1% Nonidet P-40 and 1× protease inhibitor cocktail. Cells were mixed with the buffer and centrifuged at 4000 rpm for 15 min at 4 °C. Supernatant was discarded and pellet was dissolved in nuclear lysis buffer containing 20 mM HEPES (pH 7.9), 3 mM EDTA, 10% glycerol, 150 mM potassium acetate, 1.5 mM MgCl2, 1 mM DTT, 0.1% Nonidet P-40 and 1× protease inhibitor cocktail. Suspension was cleared by centrifugation at 13,000 rpm for 10 min at 4 °C. Supernatant (nuclear extract) was collected in fresh pre-chilled tubes and stored at −80 °C. DNA relaxation assay for topoisomerases was performed using 50 μg of nuclear extract per reaction. Briefly 100 fmol of supercoiled pBlueScript (SK+) (pBS)

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DNA was incubated with 50 μg of nuclear extract in the reaction buffer containing 25 mM Tris–HCl (pH 7.5), 5% glycerol, 0.5 mM DTT, 10 mM MgCl2, 50 mM KCl, 25 mM EDTA and 150 μg/ml BSA. Reaction mixtures were incubated at 37 °C for 30 min and electrophoresed in 1% agarose gel at 1.0 V/cm for 10–12 h. Gels were stained with 0.5 μg/ml ethidium bromide (EtBr) and viewed under UV illumination.

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were stained with DAPI and mitotic catastrophe was assessed in approximately 100 nuclei per slide.

3. Results 3.1. Majority of colon cancer cell lines show higher sensitivity for ICRF193

2.9. Chromosome preparation and Giemsa staining G2 phase synchronized cells were treated with vehicle or 4 μM ICRF193 for 2 h. Cells were re-suspended in 10 ml pre-warmed 0.56% KCl (hypotonic solution) and kept at 37 °C for 12 min. About 3–4 drops of fixative (acetic acid and methanol, 1:3) were added to the suspension and centrifuged at 1500 rpm for 5 min. The hypotonic solution was completely discarded. Ten milliliters of fresh fixative was added to the pellet and incubated at room temperature for 15 min to fix the cells. This step was repeated 3 times with fresh fixative and the resulting pellet was dissolved in appropriate amount of fresh fixative. For Giemsa staining clean glass slides were wiped with methanol and 2–3 drops of cell suspension were added on to glass slides from 20 cm height. Slides were dried at 37 °C for 2 h and Giemsa stained for 5–6 min. Slides were washed with distilled water twice and mounted in DPX mounting reagent with cover glass. Chromosomes were visualized under bright field microscope using 100× oil immersion objective lens. 2.10. Assessment of mitotic catastrophe HCT116 cells were treated with vehicle or 1 or 2 or 3 μM ICRF193 for 48 and 72 h. Cells were fixed using methanol and acetic acid mixture (1:1) and permeabilized using 0.01% saponin. Nuclei of fixed cells

Effects of ICRF193 and etoposide on viability of the five colon cancer cell lines (HCT116, HT-29, Caco2, COLO 205 and SW480) and one noncancerous cell line (HEK293T) were assessed by MTT assay (Fig. 1A–F). In ICRF193 treated condition IC50 values for HCT116, HT-29, Caco2, COLO 205, SW480 and HEK293T cell lines were found to be 1.29 ± 0.04 μM, 1.92 ± 0.05 μM, 2.42 ± 0.12 μM, 1.24 ± 0.05 μM, 3.29 ± 0.06 μM and 4.24 ± 0.19 μM, respectively (Fig. 1G). While in etoposide treated condition IC50 values for HCT116, HT-29, Caco2, COLO 205, SW480 and HEK293T cell lines were found to be 1.73 ± 0.21 μM, 7.2 ± 1.04 μM, 7.26 ± 1.68 μM, 1.61 ± 0.02 μM, 4.92 ± 0.33 μM and 2.42 ± 0.05 μM, respectively. For ICRF193; HCT116, HT-29, Caco2 and COLO 205 cell lines showed higher sensitivity than SW480 and HEK293 cell lines. While for etoposide; HCT116, COLO 205 and HEK293T cell lines were found to be more sensitive than HT-29, Caco2 and SW480 cell lines. In addition to MTT assay, colony formation assay was also performed to assess sensitivity of the six cell lines for ICRF193 and etoposide (Fig. 2A–F). Colony formation assay corroborated the results of MTT assay. For ICRF193; HCT116, HT-29, Caco2 and COLO 205 cell lines showed approximately three fold higher sensitivities than SW480 cell line and approximately four fold higher sensitivities than HEK293T cell line (Fig. 2G). While for etoposide; HCT116, COLO 205 and HEK293T cell lines were found to be more sensitive than HT-29, Caco2 and SW480 cell

Fig. 1. MTT cell viability assay. (A–F) Plots for MTT cell viability assay. The six cell lines were treated with ICRF193 or etoposide at indicated concentrations for 48 h and cell viability was assessed by MTT assay. Each error bar represents mean ± SDM for the three independent experiments. (G) IC50 values of ICRF193 and etoposide for the six cell lines. Each IC50 value is mean ± SDM for the three independent experiments with three technical repeats in each independent experiment. *P b 0.05, t-test (vehicle versus ICRF193 or vehicle versus etoposide).

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Fig. 2. Colony formation assay. (A–F) Plots for colony formation assay. The six cell lines were treated with ICRF193 and etoposide at indicated concentrations for 24 h and were allowed to form colonies for 15–20 days. Colonies were stained with 0.2% methylene blue and percentages of colonies formed were counted. Each error bar represents mean ± SDM for the three independent experiments. (G) Concentrations of ICRF193 and etoposide where 50% inhibition of colony formation was achieved for the six cell lines. The concentrations are mean ± SDM for the three independent experiments. *P b 0.05, t-test (vehicle versus ICRF193 or vehicle versus etoposide).

lines. These results suggest that the majority of colon cancer cell lines show higher sensitivity for ICRF193. 3.2. G2 decatenation checkpoint function is compromised in the ICRF193 sensitive colon cancer cell lines Cell viability assays and colony formation assays suggested that four out of the five colon cancer cell lines show higher sensitivity for ICRF193. Previous studies have shown that cell lines with defective G2 decatenation checkpoint pathway show higher sensitivity for the catalytic topo II inhibitors [3]. Therefore we suspected that the ICRF193 sensitive colon cancer cell lines might be defective for the decatenation checkpoint function. To assess the decatenation checkpoint function in the six cell lines, mitotic indices were measured. In mammalian cells histone H3 is phosphorylated at serine-10 during mitosis. Histone H3 phosphorylation starts in prophase and reaches to peak level in metaphase, while dephosphorylation of histone H3 starts in anaphase and in telophase it is completely dephosphorylated [23]. HEK293T cell line was used as control cell line as it does not express metnase and has functional decatenation checkpoint [24]. Metnase enhances Topo IIα mediated decatenation and thereby inhibits activation of the decatenation checkpoint pathway [24]. To measure mitotic indices, G2 phase synchronized cells were treated with vehicle or ICRF193 or etoposide at IC50 concentrations and phosphorylation of histone H3 was measured at 0, 2, 4 and 6 h post-treatment. Upon ICRF193 treatment mitotic indices of HCT116, HT-29, Caco2 and COLO 205 cell lines gradually increased at 2, 4 and 6 h suggesting that the cells are not arrested in G2 phase but enter into M phase and accumulate at metaphase stage (Fig. 3A–D). While in vehicle treated conditions mitotic indices reached to peak levels at 4 h and declined at 6 h. Contrary to these results, SW480 and HEK293T cell lines upon treatment with ICRF193 remained arrested in G2 phase

and did not enter into M phase as the mitotic indices were not increased at 2, 4 and 6 h (Fig. 3E and F). These results suggest that the ICRF193 sensitive colon cancer cell lines, HCT116, HT-29, Caco2 and COLO 205 have compromised/impaired decatenation checkpoint function, while SW480 and HEK293T cell lines have functional decatenation checkpoint pathway. On the other hand, upon etoposide treatment all the six cell lines remained arrested in the G2 phase and did not enter into M phase as the mitotic indices were not increased at 2, 4 and 6 h, suggesting that these cell lines efficiently block cell cycle progression in G2 phase by activating G2 phase DNA damage checkpoint (Fig. 3A–F). Cell cycle arrest in synchronized HCT116 cells was further analyzed by flow cytometry analysis. G2 phase synchronized HCT116 cells (i.e. 6 h post-thymidine release) were treated with vehicle or ICRF193 at the IC50 concentration or at 10 times above the IC50 concentration to study more accurate comparison of the effect of ICRF193 on cell cycle arrest. Cells were fixed at 6, 8, 10, 12 and 14 h post-thymidine release and flow cytometry was performed. ICRF193 treated cells were found to be arrested in G2/M phase at 10, 12 and 14 h whereas the vehicle treated cells gradually crossed G2/M phase from 8 to 14 h (Fig. 4A and B, Table 1, Supplementary Figs. 1 and 2). Cell cycle arrest was also analyzed in asynchronously growing HCT116 cells. Cells were treated with vehicle or ICRF193 at the IC50 concentration or at 10 times above the IC50 concentration for 12, 24, 36 and 48 h and flow cytometry was performed (Fig. 4C and D, Table 2, Supplementary Figs. 3 and 4). ICRF193 treated cells were found to be arrested in G2/M phase in a time dependent manner and the percentage of G2/M population increased from 12 to 48 h. Similar results were observed with the synchronized HEK293T cells treated with vehicle or ICRF193 at the IC50 concentration or at 10 times above the IC50 concentration (Supplementary Fig. 5A–D). G2/M arrest in response to ICRF193 and etoposide treatments was further analyzed by determining cyclin A2 and cyclin B1 level. G2

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Fig. 3. Mitotic indices for the six cell lines. (A–F) G2 phase synchronized cells were treated with vehicle or ICRF193 or etoposide at the IC50 concentrations and fixed at 0, 2, 4 and 6 h posttreatment. Cells were immunostained for phospho-histone H3 (ser-10) and mitotic indices were determined by calculating the percentage of phospho-histone H3 positive cells. Each error bar represents mean ± SDM for the three independent experiments. *P b 0.05, t-test (vehicle 4 h versus vehicle 0 h). #P b 0.05, t-test (ICRF193 6 h versus vehicle 6 h).

phase synchronized HCT116 and HEK293T cell lines were treated with vehicle or ICRF193 or etoposide at IC50 concentrations and harvested at every one hour interval from 6 to 12 h post-thymidine release. Immunoblotting was performed for cyclin A2, cyclin B1 and β-actin proteins. In vehicle treated HCT116 cells, cyclin A2 degradation started at 9 h and its level completely abolished at 11 h whereas cyclin B1 expression peaked from 10 to 11 h and its level completely abolished at 12 h (Fig. 4E and Supplementary Fig. 6A–C). Upon ICRF193 treatment cyclin A2 was completely degraded at 10 h and cyclin B1 expression persisted from 8 to 12 h, indicating that the cells are not arrested in G2 phase and enter M phase. Contrary to these, upon etoposide treatment, cyclin A2 expression persisted till 12 h and cyclin B1 expression was not significant, indicating that the cells activate DNA damage checkpoint which arrest cell cycle in G2 phase [25]. In vehicle treated HEK293T cells, cyclin A2 degraded at 10 h and cyclin B1 expression peaked from 8 to 11 h (Fig. 4E and Supplementary Fig. 6D). Upon ICRF193 and etoposide treatments, cyclin A2 expressions persisted till 12 h while cyclin B1 expressions were not significant, indicating G2 phase arrest (Fig. 4E and Supplementary Fig. 6E and F). Cyclin A2 and cyclin B1 levels were also analyzed in the asynchronously growing HCT116 cells treated with vehicle or ICRF193 at the IC50 concentration for 12, 24, 36 and 48 h. Upon ICRF193 treatment cyclin A2 levels were decreased after 36 and 48 h whereas cyclin B1 levels increased in the time dependent manner from 12 to 48 h, suggesting accumulation of the cells in mitotic phase (Supplementary Fig. 6G). According to Chang et al. [26], cyclin B1 degradation starts in early metaphase and its level completely vanishes before the onset of anaphase. Considering the results of mitotic indices and cyclin levels it is apparent that upon ICRF193 treatment decatenation checkpoint defective HCT116 cells are accumulated at early metaphase stage as cyclin B1 levels are stabilized and histone H3 remains phosphorylated in the cells. Together these results suggest that the ICRF193 sensitive colon cancer cell lines have defects in the G 2 decatenation checkpoint function and upon treatment with ICRF193 they do not arrest cell cycle in G2 phase but enter into M phase and accumulate at early metaphase stage.

3.3. Decatenation checkpoint function is compromised at different extents among different colon cancer cell lines Extent of the G2 decatenation/damage checkpoint function in the six cell lines was calculated as described in the Materials and methods section. Table 3 describes extents of the G2 decatenation/damage checkpoint function in the six cell lines. For HCT1116 cell line, only 4.52 ± 3.38% cells were found to be efficient for the G2 decatenation checkpoint function, suggesting that the cell line is severely compromised for the checkpoint function. For HT-29, Caco2 and COLO 205 cell lines, 10.77 ± 4.45, 13.41 ± 5.61 and 29.02 ± 5.37% cells were found to be efficient for the G2 decatenation checkpoint function, respectively. Contrary to these results, for SW480 and HEK293T cell lines, 79.35 ± 2.55 and 81.4 ± 3.5% cells were found to be efficient for the G2 decatenation checkpoint function, respectively. Whereas the G2 DNA damage checkpoint function was quite high in all the cell lines. For HCT116, HT-29, Caco2, COLO 205, SW480 and HEK293T cell lines; 70.18 ± 16.14, 67.6 ± 8.25, 48.96 ± 10.62, 59.67 ± 17.35, 63.74 ± 8.83 and 66.86 ± 4.27% cells were found to be G2 DNA damage checkpoint functional, respectively. These results corroborate the results of mitotic indices and suggest that the extent of G2 decatenation checkpoint function varies among the five colon cancer cell lines.

3.4. Decatenation checkpoint pathway is not activated in HCT116 cells upon inhibition of DNA decatenation To check activation of the decatenation checkpoint pathway we examined the phosphorylations of ATM at serine-1981 and Chk2 at threonine-68. G2 phase synchronized HCT116 and HEK293T cells were treated with vehicle or ICRF193 or etoposide at IC50 concentrations. Cells were harvested after 2 h of the treatment and immunoblotting was performed. In ICRF193 treated HCT116 cells, the phosphorylations of ATM and Chk2 were not significant, while treatment with etoposide resulted in 37.74 ± 1.87 and 14.12 ± 0.91 fold increase in the

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Fig. 4. Cell cycle arrest analysis in HCT116 cell line and activation of decatenation checkpoint pathway in HCT116 and HEK293T cell lines. (A) G2 phase synchronized HCT116 cells (6 h post thymidine release) were treated with vehicle or ICRF193 at the IC50 concentration and were fixed at the indicated time points. Percentage of G2/M population was calculated by flow cytometry analysis. (B) Percentage of G2/M population in synchronized HCT116 cells treated with vehicle or ICRF193 at 10 times above the IC50 concentration for the indicated time points. (C) Percentage of G2/M population in asynchronously growing HCT116 cells treated with vehicle or ICRF193 at the IC50 concentration for the indicated time points. (D) Percentage of G2/M population in asynchronously growing HCT116 cells treated with vehicle or ICRF193 at 10 times above the IC50 concentration for the indicated time points. Each error bar represents mean ± SDM for the three independent experiments. *P b 0.05, t-test (ICRF193 versus vehicle). (E) Cyclin A2 and cyclin B1 levels in HCT116 and HEK293T cells. G2 phase synchronized cells (6 h post-thymidine release) were treated with vehicle or ICRF193 or etoposide at the IC50 concentrations and harvested at 6, 7, 8, 9, 10, 11 and 12 h post-thymidine release. Immunoblotting was performed for cyclin A2, cyclin B1 and β-actin proteins. (F) ATM and Chk-2 phosphorylations in G2 phase synchronized HCT116 and HEK293T cell lines. G2 phase cells were treated with vehicle or ICRF193 or etoposide at the IC50 concentrations for 2 h and immunoblotting was performed for p-ATM, p-Chk2 and β-actin proteins. V = vehicle, I = ICRF193, E = etoposide.

phosphorylations of ATM and Chk2, respectively, (P b 0.05, vehicle versus etoposide) (Fig. 4F and Supplementary Fig. 7A). In HEK293T cells, ICRF193 treatment induced 13.92 ± 1.11 and 11.77 ± 0.55 fold increase in the phosphorylations of ATM and Chk2, respectively, (P b 0.05, vehicle versus ICRF193) and etoposide treatment induced 23.18 ± 0.85 and 7.99 ± 0.84 fold increase in the phosphorylations of ATM and

Chk2, respectively, (P b 0.05, vehicle versus etoposide) (Fig. 4F and Supplementary Fig. 7B). These results suggest that in HCT116 cells upon ICRF193 treatment, decatenation checkpoint pathway is not activated while upon etoposide treatment, DNA damage pathway is activated and as a consequence ATM and Chk2 are phosphorylated. Whereas in HEK293T cells upon ICRF193 treatment, decatenation

Table. 1 Percentage of G2/M population in synchronized HCT116 cells treated with vehicle or ICRF193 at the IC50 concentration or at 10 times above the IC50 concentration. Values are shown as mean percentage G2/M population ± SD, for the three independent experiments.

Treated at the IC50 concentration

Treated at 10 times above the IC50 concentration

Time (post-thymidine release)

Vehicle treated cells (mean % G2/M population ± SD)

ICRF193 treated cells (mean % G2/M population ± SD)

G1/S Synchronized cells (0 h) 6h 8h 10 h 12 h 14 h G1/S Synchronized cells (0 h) 6h 8h 10 h 12 h 14 h

3.45 ± 0.25 59.66 ± 0.72 61.66 ± 3.58 50 ± 4.7 28.46 ± 0.4 22.93 ± 0.82 5.6 ± 0.5 68.7 ± 3.23 64.2 ± 5.51 53.43 ± 5.65 31.26 ± 2.18 25.2 ± 1.24

3.45 ± 0.25 66.26 ± 1.92 66.4 ± 4.36 71.2 ± 2.43 75.63 ± 1.23 78.9 ± 0.57 5.6 ± 0.5 67.9 ± 2.28 70.3 ± 5.66 76.46 ± 4.23 80.16 ± 1.94 80.83 ± 1.93

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Table. 2 Percentage of G2/M population in asynchronously growing HCT116 cells treated with vehicle or ICRF193 at the IC50 concentration or at 10 times above the IC50 concentration for the indicated time points. Values are shown as mean percentage G2/M population ± SD, for the three independent experiments. Treatment time Vehicle treated cells (mean % G2/M population ± SD) ICRF193 treated cells (mean % G2/M population ± SD) Treated at the IC50 concentration

12 h 24 h 36 h 48 h Treated at 10 times above the IC50 concentration 12 h 24 h 36 h 48 h

16.76 ± 0.40 8.13 ± 0.61 8.33 ± 0.66 7.36 ± 0.80 18.1 ± 0.96 12.83 ± 0.45 11.9 ± 1.01 10.53 ± 1.32

checkpoint pathway is activated and upon etoposide treatment, DNA damage checkpoint pathway is activated and as a consequence ATM and Chk2 are phosphorylated in both the conditions. 3.5. ICRF193 and etoposide both inhibit DNA topoisomerase relaxation activity in HCT116 and HEK293T cell lines To understand whether ICRF193 and etoposide treatments inhibit topoisomerase II enzyme in HCT116 and HEK293T cell lines, in vitro DNA relaxation assays were performed using nuclear extracts from ICRF193 and etoposide treated HCT116 and HEK293T cell lines. G2 phase synchronized HCT116 and HEK293T cell lines were treated with vehicle or 4 μM ICRF193 or 4 μM etoposide for 2 h and nuclear extracts were prepared as described in the Materials and methods section. Nuclear extracts from vehicle treated cells efficiently relaxed supercoiled DNA while nuclear extracts from ICRF193 and etoposide treated cells were partially inefficient in relaxing supercoiled DNA (Fig. 5A and B), suggesting that both ICRF193 and etoposide inhibit topo II enzyme activity in these cells. Residual relaxation activities are due to the other forms of topoisomerases present in the nuclear extracts. 3.6. ICRF193 treatment generates under-condensed and entangled mass of chromosomes in HCT116 cells To examine chromosomal morphology Giemsa staining was performed. G2 phase synchronized HCT116 cells were treated with vehicle or 4 μM ICRF193 for 2 h. Cells were lysed by hypotonic treatment, nuclei were fixed in acetic acid methanol mixture and Giemsa staining was performed. In vehicle treated cells perfectly condensed and decatenated chromosomes were observed whereas in ICRF193 treated cells undercondensed and entangled masses of chromosomes were observed (Fig. 5C), suggesting that ICRF193 inhibits topo IIα mediated chromosomal decatenation in the decatenation checkpoint defective HCT116 cell line. 3.7. ICRF193 treatment induces mitotic catastrophe and apoptotic cell death in HCT116 cells Cells undergoing mitotic catastrophe can be distinguish by their large fragmented multinuclei [22]. To assess mitotic catastrophe induction in response to ICRF193 treatment, HCT116 cells were treated with vehicle or 1 or 2 or 3 μM ICRF193 for 48 h and 72 h and nuclei were stained with DAPI for microscopic observation. In cells treated for 48 h with vehicle, 1, 2 and 3 μM ICRF193; 1.06 ± 0.48%, 24.6 ± 7.34%,

58.66 ± 3.00 71.03 ± 6.10 78.16 ± 3.25 85.66 ± 3.00 85.86 ± 0.76 87.86 ± 0.37 91.93 ± 1.24 93.1 ± 0.26

39.72 ± 0.55% and 51.16 ± 5.02% multinuclei were observed, respectively (Fig. 6A and B, Supplementary Fig. 8). In cells treated for 72 h with vehicle, 1, 2 and 3 μM ICRF193; 0.64 ± 0.27, 43.56 ± 0.96%, 54.98 ± 2.66% and 69.04 ± 3.46% multinuclei were observed, respectively. These observations suggest that ICRF193 induces mitotic catastrophe in the decatenation checkpoint defective HCT116 cells. Since mitotic catastrophe shares some characteristics of apoptotic cell death therefore we analyzed the induction of early and late apoptosis in ICRF193 and etoposide treated HCT116 cells. Asynchronously growing HCT116 cells were treated with vehicle or ICRF193 or etoposide at IC50 concentrations or at 10 times above the IC50 concentrations for 48 and 72 h and were stained with annexin V-propidium iodide solution for flow cytometry analysis. The aim of using ICRF193 or etoposide concentrations at 10 times above the IC50 concentrations was to study more accurate comparison of effects of ICRF193 and etoposide on the cell death. Both ICRF193 and etoposide induced apoptosis after 48 and 72 h of treatments and the apoptosis was comparatively higher in ICRF193 treated conditions than etoposide treated conditions (Fig. 6C and D, Supplementary Fig. 9A and B). After 48 h treatment with vehicle, ICRF193 and etoposide at IC50 concentrations, 5.2 ± 1.53%, 39.83 ± 4.80% and 24.06 ± 5.43% cells were apoptotic, respectively, which after 72 h increased to 7.16 ± 2.10%, 45.83 ± 4.1% and 41.7 ± 6.26%, respectively. After 48 h treatment with vehicle, ICRF193 and etoposide at 10 times above the IC50 concentrations, 8.56 ± 5.4%, 47.9 ± 5.99% and 47.06 ± 2.05% were apoptotic, respectively, which after 72 h increased to 0.76 ± 0.46%, 74.86 ± 4.70% and 56.2 ± 3.95%, respectively. Apoptosis induction was also analyzed in synchronously growing HCT116 cells. G2 phase synchronized cells were treated with vehicle or ICRF193 or etoposide at IC50 concentrations or at 4 μM concentrations for 20, 24, 28 and 32 h (Fig. 6E and F, Supplementary Fig. 9C and D, Supplementary Fig. 10). Percentages of apoptotic cells were very high in 4 μM ICRF193 and 4 μM etoposide treated conditions. This elevated apoptosis could be due to the effect of cell synchronization, making the cells more vulnerable for ICRF193 and etoposide treatments.

4. Discussion Our study demonstrates that the ICRF193 sensitive colon cancer cell lines have defects in the decatenation checkpoint function and such checkpoint defective cells upon inhibition of DNA decatenation are not arrested in G2 phase but enter into M phase and are accumulated at early metaphase stage. Our study also demonstrates that extent of the G2 decatenation checkpoint function varies among the ICRF193 sensitive colon cancer cell lines. Our findings are in support with a previous

Table. 3 Extents of G2 decatenation/damage checkpoint function in the six cell lines. Values are shown as mean percentage checkpoint function ± SD, for the three independent experiments.

G2 decatenation checkpoint function (%) G2 DNA damage checkpoint function (%)

HCT116

HT-29

Caco2

Colo205

SW480

HEK293T

4.52 ± 3.38 70.18 ± 16.14

10.77 ± 4.45 67.6 ± 8.25

13.41 ± 5.61 48.96 ± 10.62

29.02 ± 5.37 59.67 ± 17.35

79.35 ± 2.55 63.74 ± 8.83

81.4 ± 3.5 66.86 ± 4.27

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Fig. 5. DNA topoisomerase relaxation assays and Giemsa staining of chromosomes. (A) DNA relaxation assay for nuclear extracts from HCT116 cells. G2 phase cells were treated with vehicle or 4 μM ICRF193 or 4 μM etoposide for 2 h and nuclear extracts were prepared. (B) DNA relaxation assay for nuclear extracts from HEK293T cells. V = vehicle, I = ICRF193, E = etoposide. (C) Giemsa stained metaphase chromosomes from vehicle and 4 μM ICRF193 treated HCT116 cells. The G2 phase cells were treated with vehicle or ICRF193 for 2 h and Giemsa staining was performed.

study by Skoufias et al. [27], where they found that in HCT116, Hela and U2OS cell lines, the inhibition of DNA decatenation during mitosis arrest cell cycle in metaphase. Spindle assembly checkpoint also arrests cells in metaphase but the metaphase arrest in response to inhibition of chromosomal decatenation does not involve recruitment of Mad2 and Bub1 at kinetochores [27,28]. Damelin et al. [29], have shown that decatenation checkpoint is highly inefficient in mice embryonic stem cells, in mice neural progenitor cells and in human CD34+ hematopoietic progenitor cells. They also showed that the checkpoint efficiency is increased when embryonic stem cells are induced to differentiate, suggesting that the checkpoint deficiency is a feature of the undifferentiated state. According to Franchitto et al. [30], Werner syndrome cells have defects in G2 decatenation checkpoint function and WRN, a RecQ helicase, plays role in the activation of the checkpoint pathway. Defects in the G2 decatenation checkpoint have been shown in lung cancer [3], bladder cancer [4], Werner syndrome cells [30] and recently in melanoma [5]. It has also been shown that cancer stem cells have defects in the G2 decatenation checkpoint [7,29,31]. Recently Brooks et al. [32], have shown that melanoma cells defective in decatenation checkpoint function require phosphatidylinositol 3-kinase (PI3K) for their survival. ATM auto-phosphorylation at serine-1981 in response to ICRF193 treatment contributes to the activation of G2 decatenation checkpoint [12]. We found that in HCT116 cells ATM and Chk2 kinases are not phosphorylated in response to ICRF193 treatment. Contrary to this, in HEK293T cells both the kinases are phosphorylated in response to ICRF193 treatment. Lossaint et al. [33], have also previously shown that 12 h treatment of ICRF193 in HCT116 cell line does not induce ATM and Chk2 phopsphorylations, whereas 12 h treatment of bleomycin (a DNA damaging agent) efficiently induces ATM and Chk2 phopsphorylations in this cell line. We also found that in HCT116 cells, upon ICRF193 treatment, chromosomes become under-condensed, catenated and remain as entangled mass. Checkpoint deficient cells, particularly DNA structure checkpoint or spindle assembly checkpoint deficient cells have been shown to undergo mitotic catastrophe [34]. Failure to arrest the cell cycle before or at mitosis triggers an attempt of aberrant chromosome segregation, which may result in to chromosomal breakage or

aneuploidy and may participate in oncogenesis. Thus in order to prevent oncogenesis and/or aneuploidy such checkpoint deficient cells induce mitotic catastrophe leading to cell death [22,35]. Therefore we suspected that the decatenation checkpoint defective HCT116 cells, which are accumulated in early metaphase stage with entangled chromosomes in response to ICRF193 treatment, may undergo mitotic catastrophe. We found that treatment with ICRF193 for 48 and 72 h results in the formation of fragmented and large nuclei (multi-nuclei), which are indicative of mitotic catastrophe [22]. Mitotic catastrophe shares some characteristics of apoptosis. Therefore we studied induction of apoptosis in the decatenation checkpoint defective HCT116 cells and found that treatment with ICRF193 for 48 and 72 h results in significant increase in the early and late apoptotic cells. Nakagawa et al. [3], have previously shown that decatenation checkpoint defective cells show hypersensitivity for ICRF193 treatment. Our findings allow us to propose that mitotic catastrophe could be one reason behind the elevated sensitivity and rapid death of decatenation checkpoint defective cells in response to ICRF193 treatment. The induction of mitotic catastrophe in checkpoint defective cells is considered as an important therapeutic endpoint and can be a desirable goal in cancer treatment [36,37].

5. Conclusions Majority of colon cancer cell lines have defects in the G2 decatenation checkpoint function at different extents and such decatenation checkpoint defective cell lines are comparatively more sensitive for ICRF193 (a catalytic inhibitor of topo IIα) than checkpoint proficient cells. ICRF193 selectively kills decatenation checkpoint defective colon cancer cells by inducing mitotic catastrophe and apoptosis and thus catalytic inhibitors of topo IIα are promising therapeutic agents for the treatment of colon cancers with defective decatenation checkpoint. Our study also suggests that decatenation checkpoint defects may be a common feature in colorectal cancers and such defects may play important roles in the development and progression of colorectal cancers by gradually increasing predisposition of genetic instabilities.

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Fig. 6. Mitotic catastrophe and apoptotic cell death in ICRF193 treated HCT116 cells. (A) and (B) Assessment of mitotic catastrophe in HCT116 cells treated with vehicle or 1 or 2 or 3 μM ICRF193 for 48 and 72 h. Percentage of multinucleated cells was counted in each condition and plotted with respect to ICRF193 doses. (C) and (D) detection of apoptosis induction in HCT116 cells treated with vehicle or ICRF193 or etoposide at the IC50 or at 10 times above the IC50 concentrations for 48 and 72 h. Cells were stained with FITC-annexin V and propidium iodide solutions and flow cytometry was performed. (E) Detection of apoptosis induction in G2 phase synchronized HCT116 cells treated with vehicle, ICRF193 and etoposide at the IC50 concentrations for 20, 24, 28 and 32 h. (F) Detection of apoptosis induction in G2 phase synchronized HCT116 cells treated with vehicle, ICRF193 and etoposide at 4 μM concentrations for 20, 24, 28 and 32 h. Each error bar represents mean ± SDM for the three independent experiments. *P b 0.05, t-test (vehicle versus treated).

Author contributions

References

C.K.J. performed all the experiments. H.K.M., S.R.C. and C.K.J. analyzed and discussed the results.

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Conflict of interest The authors declare no conflict of interest. Acknowledgments We are thankful to the Director of our institute (CSIR-Indian Institute of Chemical Biology) for her interest in this work. C.K.J. acknowledges University Grants Commission, Government of India for providing UGC-Junior and Senior Research Fellowships. This research work was supported by the Network Projects from the Council of Scientific and Industrial Research (CSIR), Government of India [Grant NWP-0038] and [IAP-001] and the Department of Biotechnology (DBT), Government of India [BT/PR4456/MED/29/355/2012]. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2015.02.021.

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