Chaetocin induced chromatin condensation: effect on DNA repair signaling and survival

A. Sakti , K. Bannikti , M. Groneberg, and M. Stuschke
Department of Radiotherapy, Universit€atsklinikum Essen, Essen, Germany

 

ABSTRACT
Purpose: The aim of the present study was to evaluate the effect of the histone lysine-methyl- transferase (HKMT) inhibitor chaetocin on chromatin structure and its effect on ionizing radiation (IR) induced DNA damage response.
Methods: Concentration and time-dependent effects of chaetocin on chromatin clustering and its reversibility were analyzed by immunofluorescent assays in the non-small cell lung carcinoma (NSCLC) cell lines H460 and H1299Q4 and in human skin fibroblasts. In addition, IR induced dam- age response (cH2AX, 53BP1, and pATM foci formation) was studied by immunofluorescent assays. The effect on survival was determined by performing single-cell clonogenic assays.
Results: Chaetocin significantly increased the radiation sensitivity of H460 (F test on nonlinear regression, p < .0011) and of H1299 (p ¼ .0201). In addition, treatment with 15 nM chaetocin also decreased the total radiation doses that control 50% of the plaque monolayers (TCD50) from 17.2 ± 0.3 Gy to 7.3 ± 0.4 Gy (p < .0001) in H1299 cells and from 11.6 ± 0.1 Gy to 6.5 ± 0.3 Gy (p < .0001). Phenotypically, chaetocin led to a time and concentration-dependent clustering of the chromatin in H1299 as well as in fibroblasts, but not in H460 cells. This phenotype of chaetocin induced chromatin clustering (CICC) was reversible and depended on the expression of the HKMTs SUV39H1 and G9a. Treatment with siRNA for SUV39h1 and G9a significantly reduced the CICC phenotype. Immunofluorescent assay results showed that the CICC phenotype was enriched for the heterochromatic marker proteins H3K9me3 and HP1a. cH2AX foci formation was not affected, neither in cells with normal nor with CICC phenotype. In contrast, repair signaling with 53BP1 and pATM foci formation was significantly reduced in the CICC phenotype.
Conclusions: Treatment with chaetocin increased the radiation sensitivity of cells in vitro and DNA damage response, especially of 53BP1 and ATM-dependent repair by affecting chromatin structure. The obtained results support the potential use of natural HKMT inhibitors such as chae- tocin or other bioactive compounds in improving radiosensitivity of cancer cells.
ARTICLE HISTORY Received 23 June 2020 Revised 7 December 2020 Accepted 28 December 2020

KEYWORDS
Radiation sensitivity; chaetocin; histone methyl transferases; DNA repair; chromatin structure; ATM

 

 

Introduction
Chromatin structure is an important issue for the mainten- ance of genomic stability which is mainly regulated by epigenetic modification of core histones. Covalent post- translational modifications of the chromatin, i.e. acetylation, methylation, and phosphorylation, can impact diverse bio- logical processes, e.g. transcription, chromosome condensa- tion, and processing of DNA damage (Misteli and Soutoglou 2009; Li et al. 2007). Histone lysine methyltransferases (HKMTs) and histone lysine demethylases (HKDMs) play an important role in specifying the functional status of the chromatin. Genetic alterations in the HKDMs and HKMTs, e.g. translocation of chromosomes, mutations, protein fusions, and resultant aberrant expression of the genes are commonly found in cancer (Kubicek et al. 2012).
Recent studies have indicated that HKMTs and HKDMs can be a target for the modulation of the expression of selected transcripts which are specific to particular cancers

 
cells (Kubicek et al. 2012). Thus, chromatin modifiers could be interesting targets for the design and development of therapeutic drugs. HKMT enzymes transfer one to three methyl groups from S-adenosylmethionine (SAM) to lysine residues on histone molecules. Thus, targeting SAM binding sites of protein methyltransferases seems to be the first step for the inhibition of HKMTs (Barbties et al. 1990).
The fungal metabolite chaetocin, a member of epipoly- thiodiketopiperazines (EPTs) which targets histone methyl- transferases (HMTs), was used in the present study to modulate the chromatin structure. Chaetocin has been found to be an inhibitor of the HKMT Suv39 family, includ- ing SUV39h1 with an IC50 of 0.8 lM, and did not inhibit other HKMTs such as EZH2 or SET7/9 at concentrations below 90 lM, suggesting its potential selectivity (Greiner et al. 2005). Chaetocin has also been reported to inhibit SUV39h1 activity in acute myeloid leukemia cells with hypermethylated tumor suppressor genes and thus exhibited anti-myeloma activity (Lakshmikuttyamma et al. 2010).
CONTACT Ali Sak [email protected] Department of Radiotherapy, Universit€atsklinikum Essen, Essen 45122, Germany *These authors contributed equally to this paper.
Copyright ti 2021 Taylor & Francis Group LLC.
Although the human HKMT SUV39h1 was shown to be altered in various types of human cancers (Pandey et al. 2014), its functional role in the radiation response of human lung cancer cell lines remains unclear. Thus, the present study explores the effect of chaetocin on the radiation response with respect to cell cycle progression, repair, apop- tosis, and radiation sensitivity by measuring clonogenic sur- vival as well as growth control of plaque-monolayers of non-small cell lung cancer (NSCLC) cell lines.

Material and methods
Cell lines and cell culture
The NSCLC cell lines H1299 (with mutant p53) and H460 (with wild type p53) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and were usually re-thawed at 3 months interval to reduce the risk of cross-contamination and mycoplasma contamination. Although the cell lines have not routinely been authenticated prior to the experiments, we have done mutation analysis within the time line of the experiments. Panel sequencing was performed for a specific set of genes and compared the obtained results with the data from the Broad Cancer Institute (https://portals.broadinstitute.org). The location and type of mutation for KRAS, STK11 and ARID1A in H460 and NRAS for H1299 were the same as reported in the data set of the Broad Institute for these cell lines. Human fibro- blast cells obtained from a cancer patient (HF9) by skin punch biopsy were used as a control for normal cells. Cell lines were cultured in PRMI 1640 medium with 10% fetal calf serum (FBS), Fibroblast cells were maintained in MEM with 15% fetal calf serum (FBS). Both media were supple- mented with penicillin/streptomycin (100 units ml-1, Gibco- BRL, Paisley, UK) and plasmocin (PAA Laboratories, Pasching, Austria) against mycoplasma. A Co-60 source was used to irradiate cells at a dose rate of 0.9 Gy/min.

CICC phenotype
Cells were seeded in glass chamber slides, treated 4 h later with chaetocin and fixed at selected times thereafter. The nucleus with the CICC structure was determined after stain- ing the cells with DAPI by using a fluorescence microscope (Apotome, Zeiss) with Plan-APOCHROMAT 63x/1.4 oil DC objective. Cells were counted manually and the percentages of cells showing characteristic chromatin changes were determined.

Proliferation assay
The proliferation assay was used to estimate the half-max- imal inhibitory concentrations (IC50). Cells in the plateau phase were plated into 6-well tissue culture plates at a dens-
ity of 0.2 ti 106 cells per well and treated with the respective concentrations of 0–200 nM Chaetocin (Cayman Chemical, USA) at 24 h after plating. The number of cells was deter- mined at the respective times after treatment.

Clonogenic survival assay
For clonogenic survival assay, cells in the plateau phase were harvested and plated into T25 flasks with a density of
0.5 ti 106 cells/flask. At about 4 h later, cells were treated with Chaetocin for 24 h or 4 h, respectively. The further pro- cedure depended on the experimental design. For clonogenic survival, cells were reseeded at the appropriate number of
cells and irradiated 4 ti 6 h later and incubated for further 10–14 days for colony formation. Cell death after exposure to radiation after a dose D of radiation often follows the lin- ear-quadratic model (Chadwick and Leenhouts 1973) with, fraction of surviving cells ¼ e-(A ti D þ B ti D2).

Plaque monolayer assay
The plaque monolayer assay was previously described (Sak et al. 2012). Briefly, 1500 cells were seeded in 5 ll medium as a small plaque into each well of a 24-well culture plates. Cells were treated with chaetocin (7.5 nM, 15 nM) at 24 h after seeding and irradiated about 2 h later with single doses. Chaetocin was washed out at 72 h thereafter. The number of plaque monolayers reaching the survival criteria were deter- mined weekly and monitored for up to 6 weeks after irradi- ation. A plaque monolayer was designated as surviving if cells reached >50% confluency or with >10-fold doubling of the initial cell number. The monolayer control rate was calculated as the ratio of non-proliferating cultures and the total amount of seeded monolayers/treatment. The propor- tions of plaque-monolayer control in the different treatment groups were analyzed by logistic regression.

Histone extraction
Cells were harvested, washed twice with ice-cold PBS and resuspended in extraction buffer with triton (TEB, PBS con- taining 0.5% TritonX 100 (v/v), 2 mM phenylmethylsulfonyl fluoride, 0.02% (w/v) sodium azide, protease and phosphat- ase inhibitors). After cell lysis for 5 min and centrifugation
(300ti g, 5 min, 4 ti C), cells were resuspended in half the vol- ume of TEB. After centrifugation, cells were suspended in cold 0.2 N HCl and histones were extracted overnight at
4 ti C. After centrifugation (600ti g, 10 min, 4 ti C) of the cell extract, the supernatant, which mainly contains the histone proteins, was collected and stored at ti 20 ti C.

Analysis of protein expression

Protein extracts were mixed with 4ti LDS sample buffer (Invitrogen) heated to 95 ti C and separated using 12% bis- tris NuPAGE gels. After electrophoresis, proteins were trans- ferred to InvitrolonTM PVDF blotting membranes (Invitrogen) and subjected to the respective primary anti- bodies H3K9me1 (ab9045, abcam), H3K9me2 (ab1220, abcam), H3K9me3 (ab8898, abcam), SUV39h1 (ab38637, abcam). Bound antibodies were detected by incubation with secondary antibody for actin (AM4302, Ambion), GAPDH (ab9485, abcam) and H3 (ab10799, abcam) detection
conjugated with Alexa Fluor 488 or horseradish peroxidase (HRP). Chemiluminescence signals were detected by enhanced (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and fluorescent light emission by the aid of Chemidoc MP Imaging system (Bio Rad, Munich, Germany) allowing multiplex fluorescent detection.

Foci analysis
Cells were fixed for immunofluorescence analysis at 0.5 h and 4 h after irradiation as previously described (13). The following primary antibodies were used: rabbit anti-53BP1 (abcam), mouse anti-cH2AX (Millipore), and rabbit anti–Rad51 (Ab-1, Oncogene Research Products), all at a dilution of 1:500 and probed with the respective Alexa 488- labelled secondary antibodies at a dilution of 1:500. At least 40 nuclei per sample were counted for each treatment.

Statistical analysis
Clonogenic survival curves were created with the help of the software GraphPad Prism 8. The significance of the effect was assessed by an ANOVA F-test.

Results
Radiosensitising effect of chaetocin on NSCLC cell lines
Initially, the effect of chaetocin on proliferation was deter- mined with the half-maximal inhibitory concentration
(IC50) of chaetocin which amounted to 26.3 þ 6.6 nM and 30.3 þ 6.6 nM in the NSCLC cell lines H1299 and H460, respectively, showing no significant difference between the cell lines.
To investigate the effect of chaetocin on radiation sensi- tivity, cells were treated with 15 nM, 30 nM, and 60 nM
chaetocin, corresponding to about 0.5, 1, and 2 ti IC50. Single cells were plated out at 24 h after chaetocin treatment,
irradiated 2 ti 4 h thereafter with 0–6 Gy and incubated for further 10–14 days for colony formation. Survival curves as fitted with the linear-quadratic model after treatment of H1299 and H460 cells with chaetocin (15, 30, and 60 nM) and ionizing radiation (0 Gy, 2 Gy, 4 Gy, and 6 Gy) are shown in Figure 1(A,C). Overall irradiation dose points, chaetocin significantly increased the radiation sensitivity of
H460 at 15 nM (p ¼ .0027, F test), 30 nM (p < .0001) and 60 nM (p < .0001). In comparison, chaetocin also signifi- cantly radiosensitised H1299 cells at 15 nM (p < .0204), 30 nM (p < .0001) and 60 nM (p < .0001).
In addition to clonogenic survival, which measures single cell survival, we also measured the effect of chaetocin on the survival of cell plaques containing 1500 cells (Figure 1(B,D)). The data demonstrate a significant shift of the curves toward lower irradiation doses upon chaetocin treat- ment. The total radiation doses that control 50% of the pla- que monolayers (TCD50 values) was 17.2 ± 0.3 Gy for H1299 cells not treated with chaetocin. The respective TCD50 val- ues for 7.5 nM and 15 nM chaetocin were 14.9 ± 0.1 Gy
(p ¼ .0311) and 7.3 ± 0.4 Gy (p < .0001). In comparison, the TCD50 for non-treated H460 cells was 11.6 ± 0.1 Gy, which was significantly decreased to 6.5 ± 0.3 Gy (p < .0001) upon treatment with 15 nM chaetocin.

Chaetocin induced chromatin clustering (CICC) phenotype
Chaetocin has been shown to be an inhibitor of histone methyl transferases (HMT) and therefore its effect on chro- matin was of interest. The data demonstrated global chro- matin remodeling after chaetocin treatment only in H1299 (Figure 2(A,B)) and fibroblast cells (Figure 2(C)), but not in H460 cells (Figure 2(D)) and was clearly visible in fluores- cent microscopy (Figure 2(A,C)). H1299 cells were in add- ition treated with gliotoxin, which is also a member of ETPs in order to explore if hyper-condensed chromatin clustering is a result of the unique chemical structure of EPTs. In add- ition, UNC0638 and BIX01294 with different chemical struc- tures (Decarlo and Hadden 2012; Gardiner et al. 2005) were also used. Condensed chromatin clustering was found only after chaetocin and UNC0638 treatment (Bannik 2016), both with structurally distinct classes of HMT inhibitors. Overall, the chemical structure of chaetocin was obviously not crit- ical for the formation of CICC. In order to understand the principles and mechanism of the CICC, time kinetics, its reversibility, association with cell cycle and chromatin markers as well as the effect of ionizing radiation were fur- ther evaluated.

Time course and reversibility of chaetocin induced chromatin clustering
To investigate the CICC time course, cells were treated with chaetocin for up to 40 h. The data showed that the percent- age of cells with CICC increased in a concentration- and time-dependent manner. The first chromatin clustering was detected at about 8 h and the maximum effect was achieved at about 24 h after treatment (Figure 2(E)). In addition, the data revealed that ionizing radiation did not change the per- centage of CICC cells (Figure 2(F)).
In order to investigate the reversibility of the CICC, cells were treated with chaetocin for 24 h and washed out there- after. The fraction of cells with CICC significantly decreased within the first 24 h after washout (Figure 3). Several control experiments were performed to exclude the possibility that the proliferation of normal non-affected cells or death of CICC cells within this time window distorted the results.
First, to exclude that the fraction of CICC cells could be overgrown by proliferation of normal, i.e. non-affected cells, Aphidicolin (Aph) was used to stop the proliferation of the cells. Aph is a reversible inhibitor of eukaryotic DNA repli- cation and arrests cell cycle transition of the cells at the early S phase. The data showed that treatment of cells enriched in the G1 phase of the cell cycle for 20 h with 2 lM Aph arrested more than 80% of the cells in the late G1 early S phase, while untreated cells went through the cell cycle (Figure 3(A)). Proliferation of cells with and without

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 1. Effect of chaetocin on survival in the clonogenic (A, C) and plaque- monolayer assays (B, D) in H460 (A, B) and H1299 (C, D). For clonogenic survival (A, C), cells were treated for 24 h with different concentrations of chaetocin (0, 15, 30, and 60 nM), single cells were reseeded for colony formation without chaetocin and irradiated at about 4 h thereafter. Surviving colonies were counted at about 10 days after irradiation. Means ± sem from three independent experiments are shown. For the plaque-monolayer survival (B, D), 1500 cells were seeded in as a small plaque, treated with chaetocin (7.5 and 15 nM) and irradiated with single doses between 0 and 24 Gy at 24 h after seeding. The number of plaque monolayers reaching the survival criteria was determined 6 weeks after irradiation. The data represent means ± sem of 3 (H1299 and 6 (H460) experiments.
chaetocin treatment significantly ceased after treatment of the cells with Aph (Figure 3(B)). These data clearly showed a significant reduction in the percentage of CICC cells, even in non-proliferating Aph treated cells (Figure 3(C)). Furthermore, the data with human Fibroblasts (hFbs) which have naturally a low proliferation capacity clearly demon- strated that the fraction of cells with CICC also increased in a concentration-dependent manner in hFbs and decreased significantly within 24 h after washout of chaetocin (Figure 3(D)).
Second, to preclude preferential death of CICC cells which may falsify the results regarding reversibility, live cell imaging was performed to monitor the fate of the cells. For this purpose, chaetocin treated cells were stained with Hoechst 33342 and monitored in real time up to 16 h after washout. The data showed that about 33.5% ± 9.9% of the cells with CICC phenotype were reversible. For a more detailed quantification, the chaetocin treated cells were div- ided in cells without CICC (normal) and cells with CICC phenotype, either reversible or nonreversible. These cells were then characterized by Hoechst 33342 staining with respect to apoptotic cell death. The results showed that only 18% ± 6% of CICC reversible cells died. Sham treated cells
did not show any apoptotic features during the observa- tion time.

Association of CICC phenotype with chromatin markers
The histone markers H3K9me3 and H3K27me3 are hall- marks of condensed heterochromatin. In order to confirm that the CICC phenotype is associated with these hetero- chromatin markers, the cells were treated with chaetocin for 24 h, stained and analyzed for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3H4me3 and H3K9ac) specific histone markers (Figure 4). The data showed that the CICC phenotype positively correlated with each of these heterochromatin markers. In comparison, the whole nucleus was evenly stained for the euchromatin markers (H3H4me3 and H3K9ac) indicating no preferential association of these markers with CICC structures. Collectively, the presented data showed that the CICC clusters were mainly enriched for heterochromatin markers.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Chromatin remodeling after chaetocin treatment. Fluorescence pictures represent nuclei with CICC phenotype after treatment with 30 nM chaetocin for 24 h in H1299 (A) and irradiated nuclei without chaetocin (B). (C) Human fibroblasts at 24 h after treatment with 15 nM chaetocin. (D) H460 cells after treatment with chaetocin. (E) Concentration- and time-dependent formation of CICC in H1299 cells after treatment with 30, 60, and 300 nM chaetocin for 4 h, 8 h, 24 h, and 40 h. The results from at least 500 scored cells per point are shown. (F) Effect of IR on CICC formation at 24 h after combined treatment with chaetocin and 4 Gy. Cells were treated with 30, 60, 120, and 300 nM chaetocin and irradiated with 4 Gy at about 20 h after seeding, and stained with DAPI (blue) at about 24 h after treatment. Bars represent the mean ± sd of n ¼ 2 independent experiments.
Chaetocin has no effect on global histone methylation
Chaetocin was reported to be a potential inhibitor of SUV39h1 which tri-methylates histone H3 at K9 (H3K9me3). Thus, we explore the effect of chaetocin on methylation of H3K9 as a measure for SUV39h1 activity. For this purpose, cells were treated for 48 h with chaetocin, histone proteins were isolated and the level of the histone
variants was studied by immunoblotting assay. At

concentrations of ti 300 nM, corresponding to about 10x IC50 for proliferation, there was no effect of chaetocin on global trimethylation of H3K9 (Figure 5). To explore directly the effect of the HKMT SUV39h1 on the global methylation pattern of H3K9, cells were treated with shRNA and expres- sion of SUV39h1 protein was determined (Figure 6). Treatment with SUV39h1 shRNA significantly downregu- lated its expression to about 50% of the non-treated cells

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3. The reversibility of CICC after treatment with aphidicolin (Aph) in H1299 and HFbs. (A) Cell cycle distribution in G1, S and G2/M phases at 4 h and 20 h after treatment with and without 2 mM Aph. (B) Proliferation of H1299 cells with and without 2 mM Aph treatment in the presence and absence of chaetocin (60 nM). (C) Decrease in the percentage of CICC cells with and without Aph (2 mM) after chaetocin (60 nM) washout in H1299 and in hFbs (D). Representative results from n ¼ 3 independent experiments are shown. Bars represent the mean ± sd.

 
and thereby reduced global di- and tri-methylation of H3K9, both used as a measure for heterochromatin. On the other hand, the fraction of mono-methylated H3K9 as a measure for euchromatin, increased significantly after downregulating SUV39h1 expression. These data showed that H3K9 di- and tri-methylation can be used as a measure for SUV39h1 activity.
In order to test if the observed CICC phenotype after chaetocin treatment depends on SUV39h1 activity, SUV39h1 was downregulated by siRNA. Unexpectedly, the percentage of H1299 cells with the CICC phenotype reduced signifi- cantly upon chaetocin treatment (30 nM) in H1299 cells transfected with G9a and SUV39h1 specific siRNA (Figure 7). Thus, it was concluded that histone methyl transferases G9a and SUV39h1 were necessary for the formation of CICC.

Radiation induced repair foci upon CICC formation
The p53-binding protein 1 (53BP1) as well as phospho- H2AX (cH2AX) are both important regulators of DSB repair signaling, especially of non-homologous recombin- ation if measured at early times after irradiation. In order to analyze the influence of chaetocin on radiation induced 53BP1 and cH2AX foci formation, cells were pretreated for 24 h with chaetocin at a low concentration of about 15 nM, which is equal to half the IC50 concentration for prolifer- ation of H1299 cells. For comparison, human fibroblast cells
were pretreated for 24 h with 30 nM chaetocin. Both pre- treated cell types were fixed at 0.5 h, 1 h, and 24 h after irradiation with 0.5 Gy. 53BP1 and cH2AX foci were ana- lyzed in chaetocin treated cells with ‘normal’ and CICC phenotype (Figure 8). In contrast, cells that had the ‘normal’ phenotype showed no effect on 53BP1 foci upon chaetocin treatment. In comparison, chaetocin had no effect on cH2AX foci formation in CICC cells as well as in ‘normal’ cells.
In order to explore if the abrogated 53BP1 foci forma- tion relies on the formation of foci or if protein expres- sion is also downregulated, cells were pretreated with chaetocin for 24 h, irradiated with 0 Gy and 10 Gy and proteins were isolated at 1 h after IR. The data showed a reduced expression of the 53BP1 protein only at a high
concentration of about 2ti IC50. In contrast, chaetocin had no effect on the expression of other proteins involved in the DNA damage response of the cells, such as Rad51 and cH2AX, even at high concentration levels of about
10ti IC50 (Figure 9). Overall, the present data showed that 53BP1 foci were mostly diminished in CICC cells and thus depended on the chromatin structure. Treatment with UNC0638, which also shows the CICC phenotype in H1299 cells, also reduced 53BP1 foci formation as chaeto- cin did. Other repair signaling proteins, e.g. cH2AX and Rad51, were not affected in cells with the CICC phenotype and thus the observed effect seems to be specific to the 53BP1 protein.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Association of heterochromatin markers and CICC. The H1299 cells were treated with chaetocin for 24 h. (A) H3K9me3 (DAPI, H3K9me3, overlap) stained CICC nucleus. (B) H3K27me3 (DAPI, H3K27m3, overlap) stained CICC cell; (C) Association of CICC with euchromatin markers H3K9ac (green) and H3K4me3 (red). DAPI (blue) was used to stain the overall DNA. The respective overlaps are shown. Scale bar ¼ 10 mm.
Effect of chaetocin on radiation induced pATM foci formation
Based on the results in the previous paragraphs demon- strating a significant reduction of radiation-induced 53BP1 foci formation in CICC cells, we were also interested in the effect of chaetocin on pATM foci formation, which was shown to require 53BP1 protein (Noon and Goodarzi 2011). Again, H1299 cells were pretreated with chaetocin (30 nM) for 24 h, irradiated (0.5 Gy) and fixed at the respective times thereafter and pATM foci were scored in cells with the CICC and ‘normal’ phenotype. Formation of
radiation induced pATM foci were reduced in CICC as well as in ‘normal’ cells, however with a higher effect in CICC cells (Figure 10). In contrast to the observed effect on 53BP1 expression, treatment with chaetocin (60 nM and 300 nM) for 24 h had no effect on pATM and pKAP1 pro- tein expression at 1 h after irradiation with 10 Gy (Figure 10(C)).

Discussion
Changes in chromatin structure play an important role dur- ing essential functional mechanisms such as transcription,
replication, and DNA repair (Groth et al. 2007; Li et al. 2007; Misteli and Soutoglou 2009). Here, we explored the role of chromatin remodeling in the DNA damage response of irradiated cells, especially on repair mechanisms. It was shown that chromatin undergoes rapid local and global decondensation after ionizing irradiation ( €Unal et al. 2004;

 

 

 

 

 

 

 

 

 

 

 

Figure 5. Effect chaetocin on H3K9me3 protein expression in the H1299 cell line. A: Representative western blot showing the expression of H3K9me3 after treatment with 0, 30, 60 and 300 nM chaetocin. H3 was used as an internal con- trol. B: The respective results with means ± sd from three independent experi- ments are shown.
Figure 7. Effect of downregulation of SUV39h1 and G9a on CICC formation. Knockdown of siRNA mediated SUV39h1 and G9a decreased CICC at 48 h after chaetocin (30 nM). The cells were fixed at 24, 28 and 48 h after chaetocin treat- ment and analyzed for CICC cells. Bars represent the mean ± sd from three inde- pendent experiments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Effect of downregulating SUV39h1 expression with shRNA on H3K9 methylation. (A) Representative western blot showing the expression of SUV39h1 protein after transfection with shRNA and the respective results from three independent experiments with bars representing mean ± sd. GAPDH was used as internal control. (B) Effect of SUV39h1 downregulation on H3K9 methylation pattern. Representative western blots were shown for mono-, di-, and tri-methylated H3K9 and H3 as an internal control. The histogram represents the relative expression level of the different histone variants. Representative results of at least three independ- ent experiments are shown. Bars represent means ± sd.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 8. Effect of chaetocin on 53BP1 and cH2AX foci formation in H1299 and hFbs. (A) Selective inhibition of 53BP1 foci formation in H1299 cells with the CICC phenotype after chaetocin pretreatment (15 nM) for 24 h. Cells were fixed at 0.5 h after IR with 0.5 Gy (DAPI, blue; 53BP1 foci, red; cH2AX foci, green). (B) Kinetics of cH2AX foci repair in H1299 (left) and hFb (right) after chaetocin treatment for 24 h (15 nM for H1299 and 30 nM for hFb) and irradiation with 0.5 Gy at 0.5 h, 4 h and 24 h thereafter. ‘Normal’ cells without chromatin changings after chaetocin treatment and ‘CICC’, chaetocin induced chromatin clustering. (C) Kinetics of 53BP1 foci repair in H1299 (left) and in hFb (right) after chaetocin treatment for 24 h (15 nM for H1299 and 30 nM for hFb) and irradiation with 0.5 Gy at 0.5 h, 4 h and 24 h thereafter. Representative results of at least two independent experiments are shown. Bars represent the mean ± sd.

 

Ziv et al. 2006). Local perturbations in chromatin structure and histone modifications enable the access of proteins to damaged DNA sites. Heterochromatic regions with con- densed chromatin were shown to be less prone to radiation induced DSB and with slower repair kinetics than those in euchromatic regions (Goodarzi et al. 2008; Sak et al. 2015).
In the present work, we could show that the HMT inhibi- tor chaetocin significantly reduced the proliferation of NSCLC cell lines H460 and H1299. In addition, a significant
radiosensitising effect was observed in both cell lines. However, visible changes of global nuclear chromatin struc- ture by chaetocin, i.e. the CICC phenotype were observed only in H1299 but not in H460. In addition, CICC were clearly visible and detectable in fibroblasts with the same kinetics as shown in H1299 cells with a maximum at about 24 h after treatment. Late passages of primary human fibro- blasts with low S phase and low mitotic activity also show the CICC phenotype. Thus, chromatin condensation upon

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 9. Effect of chaetocin on the expression of repair proteins after IR. (A) Western blot showing the expression of repair proteins (53BP1, Rad51, cH2AX) and the respective loading controls (Actin, H3) after chaetocin (2xIC50, 10xIC50) and IR (10 Gy). (B) The respective results from three independent experiments are shown for 53BP1 (B), cH2AX (C) and Rad51. Bars represent the mean ± sd. C, control; IR, 10 Gy, 2IC50 (60 nM chaetocin), 10IC50 (300 nM chaetocin).
chaetocin treatment seems not to be essential for the radio- sensitisation effect of chaetocin. Previous studies reported that the chaetocin induced chromatin condensation (CICC) could be induced in human fibroblasts, immortalized fibro- blast and in immortalized cells derived from retina epithe- lium, but not in tumor cell lines (Illner et al. 2010). In comparison, the present study shows that chaetocin also induces visible chromatin reorganization in the human tumor cell line H1299. The observed CICC phenotype becomes visible at about 8 h after chaetocin treatment, with a maximum at about 24 h. The percentage of CICC reached its maximum with about 80% of the cells showing CICC phenotype at 24 h after treatment with 300 nM chaetocin, the highest concentration used in our study. Illner et al. (2010) reported that human fibroblast cells form irreversible CICC phenotype after 48 h treatment with 10 nM chaetocin, irrespective of whether chaetocin was then released from the medium or not. In comparison, our data showed that the formation of CICC after 30 nM chaetocin for 24 h is a reversible process, as studied in human H1299 cells. Same results were observed in H1299 cells after blocking the cells in late G1, early S phase after treatment with aphidicolin as well as in high passage primary human fibroblasts. Thus, reversibility of the CICC phenotype was not dependent on the proliferation status of the cells. The difference between our results and that of Illner et al. (2010) may be due to the different forms of CICC described. Illner et al. (2010) described three stages of CICC, an early stage (CICC 0) that is phenotypically not different from untreated control cells; CICC I with nuclei containing small foci of condensed chro- matin surrounded by areas of decondensed chromatin, with
a maximum at about 24 h after chaetocin treatment. In add- ition, CICC II stage nuclei was described with distinct chro- matin clusters interconnected by sparse chromatin bridges and surrounded by chromatin free regions, which reached its maximum at about 48 h after chaetocin treatment. In contrast, the CICC phenotype as described in the present study mainly represents the CICC type I described by Illner et al. (2010). Therefore, the reversibility of both types may differ.
Chaetocin was shown to modulate chromatin structure by reorganization of the genome on the sub-chromosomal level. Preferentially, chromatin with high gene density was located in the nuclear interior, whereas chromatin regions with less gene density were located toward the nuclear per- iphery. This radial distribution of the nuclear material, which was shown to be correlated to gene density, was lost in CICC nuclei (Illner et al. 2010). Chaetocin as a member of the ETPs class of fungal metabolites was shown to inhibit the histone methyl transferases G9a and SUV39H1. Thus, we were interested in whether the chemical structure of the ETPs has an impact on CICC induction and tested add- itional small molecules, e.g. Gliotoxin and UNC0638, for their effect on CICC formation. Gliotoxin, as the structurally simplest member of ETPs and also an inhibitor of SUV39h1 and G9a (Takahashi et al. 2012), does not induce chromatin condensation. UNC0638, which belongs to a different chem- ical class, induces CICC. The effect of chaetocin on chroma- tin condensation seems to be dependent on the presence of the SUV39H1 and G9a proteins because transfection with specific siRNA targeting these genes mostly abrogated CICC formation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 10. Effect of chaetocin on pATM and pKAP1 in H1299 cells. (A) The immunofluorescence images of pATM foci (red) with or without chaetocin treatment (30 nM) in combination with radiation (0.5 Gy) at 1 h after IR. (B) Quantification of pATM foci after treatment with 30 nM chaetocin (30 nM) for 24 h and 1 h, 4 h and 24 h after irradiation with 0.5 Gy. A cell is classified as ‘Normal’ if it does not have visible morphological changes in nucleus after chaetocin treatment. Representative results from three independent experiments are shown. Bars represent the mean ± sd. Statistical analysis: 2-way ANOVA analysis with Bonferroni posttest, p < .01 (titi), p < .001 (tititi). Scale bar ¼ 10 mm. (C) Western blot results of pATM and pKAP1 protein expression with and without chaetocin treatment (0, 60, 300 nM) after at 1 h after irradiation with 10 Gy.
We further analyzed the structural compounds of the chromatin remodeling process and could show that CICC was mostly associated with proteins specific for heterochro- matin (H3K27me3, H3K9me3).
It was already reported that heterochromatic regions are highly compacted and this might be a barrier for DNA repair (Goodarzi et al. 2010). Although it is still unclear how DNA DSBs in heterochromatic (HC) and euchromatic (EC) regions are repaired, DSB repair in HC needs relax- ation of chromatin to enable the accessibility of the DNA damage site for repair proteins (Jakob et al. 2011; Goodarzi and Jeggo 2012). Cells with the CICC phenotype can thus be used as a model system for heterochromatin to investi- gate the DSB repair in different chromatin regions. cH2AX
and 53BP1 foci assay at early time points after irradiation was used as a measure for NHEJ signaling. Formation of 53BP1 but not cH2AX foci was dramatically reduced in CICC cells.
A current model of DNA repair in heterochromatin involves the ability of 53BP1 protein to facilitate the phos- phorylation of KRAB-associated protein 1 (KAP1) by ATM kinase for DSB repair in heterochromatin (Goodarzi et al. 2008; Jakob et al. 2011; Noon and Goodarzi 2011). The pKAP1 protein is of interest because ATM phosphorylates KAP1 which is essential for global chromatin relaxation and thus for DSB repair within heterochromatic regions (Ziv et al. 2006). As expected, we observed that the abrogation of 53BP1 foci formation in CICC cells also leads to reduced
pATM foci. Frohns et al. (2014) also reported that a high chromatin density hinders the formation of 53BP1 and pATM foci after IR which strengthens the observations that heterochromatic structure negatively regulates 53BP1 and pATM foci formation. The most likely explanation of 53PB1 foci reduction in CICC cells was reported by Gonzalez- Suarez and Gonzalo (2010). They clearly showed that loss of lamin A, which is a part of the nucleoplasmic network, asso- ciates tightly with chromatin (Goldman et al. 1992) and leads to destabilization of 53BP1. As we demonstrated here, chaetocin remodeled the chromatin structure by induction of CICC, which could destroy or affect the function of lamin A and C and thereby reduces the level of 53BP1.
In conclusion, the present study shows that chaetocin remodels the nuclear chromatin structure by forming revers- ible CICC formations in primary fibroblasts as well as in H1299. CICC is mainly associated with heterochromatin markers and is primarily found in G1 cells. The results fur- ther show that CICC and radiation induced 53BP1 and pATM foci formation are mutually exclusive, which is not the case for cH2AX foci. Overall, these findings represent ‘stepping stones’ for further analysis of the role of chromatin structure in response to radiation and the development of drugs for combined radiochemotherapy. Chromatin modify- ing genes and remodelers are frequently mutated in most human tumors, especially in lung tumors (Govindan et al. 2012; Imielinski et al. 2012). Therefore, this is an innovative field for targeted therapy options.

Acknowledgements
We thank George Iliakis for valuable discussions, reagents and tech- nical advice. We thank Sabine Levegr€un for language editing and proofreading. This work is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the Graduate School (GRK1739) and is part of a PhD thesis of Kristina Bannik.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Notes on contributors
Ali Sak, PhD, is a biologist and head of the working group ‘molecular radiation oncology’ at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. He works on biological effects of radiation, in vivo and in vitro effects of combined radio-chemotherapy, modulation of epigenetic factors and their effect on the radiation response of tumor cell lines.
Kristina Bannik, PhD, was a biophysicist at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. The presented work was part of her PhD thesis pre- pared in the working group ‘molecular radiation oncology’. She was responsible for the planning and processing of the experiments and for data analysis.
Michael Groneberg is a research assistant and an expert in cell culture and microscopic analysis at the Department of Radiotherapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. He is involved in the planning and processing of the experimental settings.
Prof. Dr. med. Martin Stuschke is the Chair of the Department of Radiation Therapy, Faculty of Medicine, University of Duisburg Essen, Essen, Germany. His research focusses on clinical studies with com- bined therapy schedules involving chemotherapy and radiotherapy and preclinical studies on the role of DNA repair pathways and checkpoint activation in the response of NSCLC to IR.

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