response to genotoxic stress
Abdel-Halim Mahfouz, H.I.
Citation
Abdel-Halim Mahfouz, H. I. (2009, February 24). Changes in chromatin organization of human cells in response to genotoxic stress. Retrieved from https://hdl.handle.net/1887/13517
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Chapter 5
Impact of DNA repair on the DNA damage-induced heterochromatin pairing in human cells
Manuscript in preparation
Impact of DNA repair on DNA damage induced heterochromatin pairing in human cells
H.I. Abdel-Halim1, 2, J. J.W. A. Boei1, B.C. Godthelp1, L.H.F. Mullenders1
1Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands. 2Department of Zoology, Faculty of Sciences, Suez Canal University, Ismailia, Egypt.
Abstract
In our previous studies we have shown that cellular stress generated by mitomycin C (MMC), UV-irradiation, X-rays or heat shock treatment induces pairing of the heterochromatic region 9q12-13 in a subset of confluent human cells. The protein encoded by Xerodema pigmentosum group F (XPF) gene was required for pairing induced by MMC or UV. In the present study, we show that FANCD1 (BRCA2) implicated in homologous recombination (HR) is required for pairing after MMC treatment but not after UV irradiation. Moreover, we provide evidence for initiation of DNA damage processing of MMC-induced interstrand cross-links (ICLs) in confluent cells using the comet assay and phosphorylation of H2AX. The pairing of heterochromatin appeared to be correlated with the induction of DNA strand breaks.
XPF and FA-D1/BRCA2 cells that did not undergo pairing of heterochromatin after MMC treatment also lacked phosphorylation of H2AX. Taken together, the results indicate that processing of ICLs involves XPF and BRCA2 and can be initiated in non-dividing cells. Furthermore, the pairing of homologous heterochromatic regions after ICL induction requires components from nucleotide excision repair as well as HR.
Introduction
The induction and the subsequent repair of DNA damage have been shown to affect nuclear organization (Figgitt and Savage, 1999; Aten et al., 2004). During recent years, there is growing evidence that different types of cellular stress change the positioning of homologous chromosomes within the nucleus. Treatments with DNA damaging agents including ionizing radiation (Dolling et al., 1997; Spitkovsky et al., 2002; Abdel-Halim et al., 2004), the cross-linking agent mitomycin C (MMC) (Abdel-Halim et al., 2005), hydrogen peroxide H2O2 (Monajembashi et al., 2005) and
ultraviolet (UV) irradiation (Abdel-Halim et al., 2006) induce pairing or repositioning of homologous heterochromatic regions of the human genome. Moreover, other types of cellular stress such as heat shock treatment also induce pairing of the heterochromatic regions in replicating and G0/G1 human cells (Abdel-Halim et al., 2006). Pairing or positional changes of homologous chromosomes after infliction of DNA damage have been suggested to be pre-requisites for or consequences of homologous recombinational (HR) repair of DNA damage (Dolling et al., 1997;
Spitkovsky et al., 2002; Abdel-Halim et al., 2004, 2005). This hypothesis was supported by the fact that treatment with MMC or exposure to UV irradiation did not induce pairing of homologous chromosomes in cells derived from xeroderma pigmentosum group F (XPF) patients (Abdel-Halim et al., 2005; 2006). The heterodimeric XPF/ERCC1 protein is known to play a key role in the repair of DNA interstrand cross links (ICLs) (De Silva et al., 2000; 2002; Clingen et al., 2007) and is required for targeted HR (Adair et al., 2000; Sargent et al., 2000; Niedernhofer et al., 2001) in mammalian cells, suggesting that the pairing induced by DNA damaging agents might be dependent on recombinational DNA repair.
MMC is a bifunctional alkylating agent that induces ICLs. The compound is an important chemotherapeutic agent widely used in the treatment of cancer (Verweij and Pinedo, 1990; Pow-Sang and Seigne, 2000) and exerts its cytotoxic effects by preventing the separation of DNA strands and therefore blocks essential cellular processes, such as DNA replication and transcription (Iyer and Szybalski, 1963;
Keyes et al., 1991). The repair of ICLs is a complex process involving proteins belonging to nucleotide excision repair (NER), HR and translesion synthesis (TLS) pathways (for review see Dronkert and Kanaar, 2001). Most of the models for ICL repair in mammalian cells propose that repair events are restricted to the S phase of the cell cycle when a replication fork stalls after encountering the cross-link and suggest a central role of XPF/ERCC1 in the repair process (Kuraoka et al., 2000; De Silva et al., 2000, 2002; Clingen et al., 2007). In these models, incisions mediated by XPF/ERCC1 lead to unhooking of the ICL and the generation of double strand breaks (DSBs). ICL repair in non-dividing cells is still a matter of debate and also the link between ICL incision and DSB formation remains largely unclear. It has been shown recently that cross-link repair can occur in yeast cells in G1 phase via components from both NER and (TLS) pathways (Sarkar et al., 2006). Moreover, a refined model has been proposed for human cells in which ICLs are recognized and rapidly incised
by XPF/ERCC1 independent of DNA replication; subsequently, the incised ICLs are processed in the S phase giving rise to DSBs (Rothfuss and Grompe 2004).
In this study we used a genetic approach to identify additional genes involved in the pairing process. Cells of Fanconi anemia (FA) patients are sensitive to cross- linking agents such as MMC and cisplatin (Grompe and D’Andrea, 2001; Levitus et al., 2006) and the proteins encoded by FA genes have been linked to ICL repair. Only cells derived from FA patients belonging to complementation group D1 with mutations in BRCA2 gene, are impaired in homologous recombination repair (Moynahan et al., 2001; Howlett et al., 2002; Jasin, 2002). We assessed the induction of pairing of homologous chromosomes in MMC-treated confluent cells derived from FA-A and FA-D1 patients. For comparison we used UV irradiation to which FA cells are generally not sensitive (Kalb et al., 2004; Godthelp et al., 2006). We found that cells derived from a FA-D1 patient are deficient in pairing of heterochromatin of chromosome 9 homologues after MMC treatment but not after UV, whereas FA-A cells showed normal pairing.
Furthermore, we investigated whether pairing of homologous regions observed in MMC-treated confluent cells can be attributed to ICL repair independent of S phase. DNA break induction was assessed in MMC-treated confluent cells by comet assay and by examining the phosphorylation of histone H2AX. We found that the unhooking of the ICLs is evident in confluent normal human cells indicating the initiation of ICL repair independent of DNA replication. Moreover, XPF and FA- D1/BRCA2 cells that lack heterochromatin pairing were also deficient in H2AX phosphorylation suggesting that pairing of homologous heterochromatic regions observed after treatment with DNA damaging agents is regulated and/or controlled by DNA repair genes.
Materials and Methods
Cell culture
Primary human skin fibroblasts derived from healthy individuals (VH16 and VH25), FA patients (EUFA432, complementation group A; EUFA423, group D1) and XP patients (XP25RO, complementation group A; XP7NE, group F) were grown in Ham’s F10 medium, supplemented with 10% FBS and antibiotics (100 U/ml
penicillin and 0.1 mg/ml streptomycin). The cells were incubated in a humidified 5%
CO2 atmosphere at 37qC. Cells were grown to confluency either in Petri dishes for comet assay or on glass slides for FISH or immunofluorescence of H2AX prior to MMC treatment or UV irradiation. Confluent cells were obtained by seeding and growing cells for at least 1 week before MMC treatment or irradiation.
MMC treatment and UV irradiation
For FISH experiment confluent cells on slides were treated with 4 μM MMC (Kyowa) for 1 h then washed with Ham’s F10 medium and followed by fixation. Prior to UV irradiation, confluent cells on slides were rinsed in PBS and exposed to a dose of 30 J/m2 UVC light using a Philips TUV lamp (predominantly 254 nm) at a dose rate of a0.41 J/m2/s. Subsequently, the cells were incubated at 37qC in the preserved medium for 1 hour prior to fixation.
Confluent cells in petri dishes were treated with 4 μM MMC for 1 h at 37ºC then washed (in medium) followed by different recovery time intervals in fresh medium to allow repair of DNA damage prior to processing of comet assay. Parallel cultures were included as controls for all experiments.
Fixation
The slides with confluent cells were washed twice with ice-cold phosphate buffered saline (PBS) prior to a 5-min in situ fixation step with ice-cold PBS containing 2%
paraformaldehyde. To permeabilize the nuclear membrane the cells were treated with methanol (-20ºC) for 10 min after which the slides were air-dried prior to in situ hybridization. After 1-3 days, the slides were processed for interphase FISH.
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) was performed using human DNA probes specific for the bands 8p11.2 (paracentromeric euchromatic band of chromosome 8) and 9q12-13 (paracentromeric heterochromatic region of chromosome 9) as previously described (Abdel-Halim et al., 2004). Briefly, the probes were labeled with FITC-12-dUTP or biotin-16-dUTP by PCR using a protocol supplied by the manufacturers (Research Genetics). The probes were mixed and precipitated together with human Cot1 DNA (Roche) and dissolved in hybridization mixture (50%
deionized formamide, 2x SSC, 50 mM phosphate buffer (pH 7.0) and 10% dextran sulphate). Probe denaturation was performed by incubation for 7 min at 80ºC followed by competition for 1 hour at 37ºC. Prior to in situ hybridization, slides were treated with RNAse, pepsin, MgCl2 and formaldehyde as previously described (Boei et al., 1996) followed by denaturation in 60% deionized formamide, 2x SSC and 50 mM phosphate buffer (pH 7.0) for 2.5 min at 80ºC. Finally the probes were added to the slides and hybridized overnight at 37ºC. Post-hybridization, slides were washed with 50% formamide in 2XSSC (pH 7.0) at 42ºC and treated with 10% blocking protein (Cambio). For immunofluorescent detection of FITC-labeled probes, rabbit anti-FITC and fluorescein conjugated goat anti-rabbit IgG were used (Cambio), whereas Texas-Red avidin and biotinylated goat anti-avidin (Cambio) were used to detect biotin labeled probes. Subsequently, the slides were counter-stained with 10 ng/ml DAPI/PBS solution for 10 min, and embedded with Citifluor mounting medium (Agar Scientific).
Fluorescence microscopy was performed with a Zeiss Axioplan microscope equipped with filters for observation of DAPI, FITC and TRITC. Interphase nuclei hybridized with band specific probes were analyzed manually to assess the colocalization of homologous chromosome regions (Abdel-Halim et al., 2004; 2005).
For each chromosome pair a distinction was made between cells which displayed entirely separated hybridization signals and cells in which only a single (usually larger) hybridization signal or two touching signals were observed (both are called paired or colocalized signals. The data are expressed as the induced colocalization, i.e.
the values of colocalization in control samples (ranging from 4.6 to 10.2% for chromosome 9) were subtracted from that in MMC-treated or UV-irradiated samples to give the induced percentage of colocalization in each cell line examined. Student’s t-test was used to check for significant differences between groups.
Analysis of DNA strand breaks by comet assay:
Both neutral and alkaline comet assays, which measure DNA repair in individual cells (Collins et al., 2008) were carried out as described previously (Singh et al., 1988) with some modifications (Cramer et al., 2005). After treatment of cells with MMC and allowing recovery time, cells were trypsinized, collected by centrifugation and re- suspended at the appropriate density in 0.5% low melting point gelling agarose in phosphate buffer saline (PBS). 120 ȝl of this cell-agarose suspension was spread over
a precoated slide (slides were coated with 1.5% D-1 agarose solution and allowed to be solidified) covered with a coverslip and the agarose was allowed to solidify for at least 10 min at 4ºC. Coverslips were removed and slides were incubated for 1.5 h in freshly prepared and pre-cooled lysis buffer (100 m M disodium EDTA, 2.5 M NaCl, 10 mM Tris-HCl [pH-8]) containing 1% sodium-N-lauryl sarcocine and 1% Triton X- 100 at 4ºC. For analysis by the alkaline comet assay, slides were first incubated for 20 min in cold alkaline electrophoresis solution (300mM NaOH, 1mM EDTA) at 4ºC and subsequently, the electrophoresis was performed for 30 min at 25V and 300 mA.
Slides were immersed in 1M ammonium acetate in 90% ethanol and air dried. In the neutral comet assay, slides were subsequently transferred to an electrophoresis tank containing ice-cold neutral buffer solution (TBE: 90 mM Tris-Borate, 2mM bisodium EDTA) and electrophoresis was carried out for 15 min at 25V at 4ºC. Slides were removed, rinsed with distilled water, dehydrated in ethanol for 5 min and air dried.
For experiments aimed at examining the uncoupling of MMC-induced ICLs, a modified comet assay was used as described previously (De Silva et al., 2000). This modification allows the measurement of DNA cross-links and consequently their repair by assessing the relative reduction in DNA migration induced by X-rays. After treatment with MMC and subsequent repair intervals, cells were trypsinized and kept on ice. Subsequently, all drug treated samples and a control sample were exposed to a dose of 12 Gy X-ray on ice and cells omitting X-irradiation served as a control.
Irradiation was performed using an Andrex SMART 255 X-ray machine, operating at 200 kV and 4 mA with a dose rate of 4 Gy/min; following irradiation, the alkaline comet assay was performed as described.
Prior to analysis, DNA on slides was stained with 10 ȝg/ml ethidium bromide and comets were analyzed by a DM RXA Leica microscope equipped with a Photometrics camera using a HQ-Tritc filter. The analysis was done at 40X for 50 comets randomly chosen per slide using Scil comet software (Noz et al., 1996). The mean tail moment was calculated for each slide and the average of mean tail moments obtained from 2 experiments was used to compare the MMC treated and control samples.
In the experiment with combined MMC treatment and X-irradiation, the degree of DNA interstrand cross-linking present in MMC-treated sample was determined by comparing the tail moment of the irradiated MMC-treated samples with irradiated and unirradiated control samples (De Silva et al., 2000). The level of
interstrand cross-linking is proportional to the decrease in the tail moment in the X- irradiated and MMC-treated sample compared to cells exposed to X-rays only. The decrease in tail (DTM) is calculated by the formula % DTM = [1-(Tmdi- Tmcu)/(Tmci-Tmcu)] x 100, where Tmdi is the mean tail moment of the MMC- treated, irradiated sample, TMci and Tmcu are the mean tail moments of the irradiated and unirradiated control samples respectively. The unhooking (uncoupling) of DNA ICLs was expressed as percent unhooking, which was calculated using the formula: % unhooking at T1 = [(% DTM at T0-%DTM at T1)/% DTM at T0] x 100, where T0 is the time immediately following MMC treatment and T1 is the post incubation time in MMC-free medium.
Immunofluorescent labeling of H2AX
To examine the kinetics of phosphorylation of H2AX, confluent normal (VH10 and VH16) XPF (XP24KY [XPF1] and XP51ROht [XPF2]), ERCC1 (165TAZ) and FA- D1 (EUFA423) cells grown on glass slides were treated with 4 μM MMC for 1 h, washed in PBS and kept in low-serum growth medium for different recovery time intervals before fixation. Measurement and quantification of H2AX foci was performed as described previously (van der Burg et al., 2006). Cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS followed by blocking step with 20mM glycine 0.5% wt/vol BSA in PBS. Slides were incubated with monoclonal mouse anti-J-H2AX (Upstate) and subsequently with Alexa 488-conjugated goat anti mouse IgG (Invitrogen Corp). Cell nuclei were counterstained with DAPI (Sigma-Aldrich) and H2AX foci and signal intensity were analyzed under a Zeiss Axioplan fluorescence microscope. For each time point, the number of cells having more than 5 foci was analyzed per 200 normal cells.
Moreover, the signal intensity was measured in all cell lines at 24 h fixation time.
Results
Differential effects of MMC and UV irradiation on the colocalization of homologous heterochromatin in FA cells
The effect of 1 h MMC treatment (4 μM) on heterochromatin pairing was investigated in confluent primary FA-A, FA-D1 and normal human fibroblasts. The percentage
colocalization of the heterochromatic regions (9q12-13) of chromosome 9 homologues in the three cell lines corrected for the background level is shown in Figure 1a. The pairing in FA-A and VH25 cells was clearly enhanced after MMC treatment (4.8% and 7.4% respectively) whereas only a slight enhancement of colocalization (0.5%) was observed in FA-D1 cells. Incubation of FA-D1 cells up to 8 h after MMC treatment did not increase the percentage of pairing of chromosome 9 heterochromatic regions (data not shown). Chromosome 8 euchromatic regions did not show any change in the colocalization after treatment of the examined cell lines with MMC (data not shown).
In contrast to the effect of MMC, UV irradiation enhanced the colocalization of chromosome 9 heterochromatic regions significantly in both FA cell lines and to the same extent as in normal human cells after exposure to 30J/m2 UV (Figure 1b).
Both MMC and UV irradiation (Abdel-Halim et al., 2005, 2006) did not induce pairing of heterochromatic regions in XPF cells (Figure 1a and b).
Figure 1: Percentage of colocalization of the heterochromatic regions of chromosome 9 homologues in FA fibroblasts (groups FA-A and FA-D1) treated with 4 μM MMC for 1 h (a), or exposed to 30J/m2 UV irradiation, and incubated 1 h before fixation (b). For comparison data for normal human and XPF fibroblasts (Abdel-Halim et al., 2005, 2006 for MMC and UV effects respectively) are included. For each graph, the average percentage of the induced pairing (subtraction of the background level was done as mentioned in the Material and Methods section) of 3 independent experiments is presented together with the standard deviation of the mean values. In each experiment, 500 cells were scored for each data point.
Unhooking of ICLs in non-dividing human cells
The unhooking of MMC-induced ICLs was measured at the single-cell level using a modified version of the comet assay (De Silva et al., 2000). Prior to cell lysis, samples received X-rays to induce random DNA strand breaks. The presence of ICLs retards
0 1 2 3 4 5 6 7 8 9 10
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the migration of the irradiated DNA during electrophoresis, resulting in a reduced tail moment compared to the irradiated control. The ability of cells to unhook cross-links can therefore be monitored as an increase in tail moment following a repair period in MMC-free medium (Figure 2). The efficiency of unhooking ICLs in confluent normal human fibroblasts (VH25) is presented in Figure 2 (insert). The cells were able to unhook approximately 50% of the ICLs during 3 hours post incubation and there was no further increase in the percentage of unhooking with increasing recovery time.
Figure 2: Efficiency of unhooking of MMC induced ICLs in normal (VH25) human cells measured by alkaline comet assay after treatment with 4 μM MMC plus 12 Gy X-rays and allowing recovery up to 20 h. Data from a single experiment are presented. For comparison data for unirradiated control, irradiated control and MMC treated unirradiated control are included (the first 3 bars to the left).
Percent unhooking of MMC-induced ICLs within time after treatment (insert) was calculated according to the formula described in the materials and method section (De Silva et al., 2000).
Direct induction of DNA strand breaks following treatment of non-dividing cells with MMC
To examine whether DNA strand breaks are generated as intermediates during the processing of MMC-induced cross-links in non-dividing cells, we performed alkaline and neutral comet assays using confluent fibroblasts derived from a normal individual and XPA and XPF patients. H2AX phosphorylation as indication of DSB was also quantified in confluent cells after MMC treatment.
Alkaline comet experiments revealed the presence of DNA breaks which were observed after 1h treatment with 4μM MMC in all cell lines examined as assessed by the mean tail moment (Figure 3a). The increase of the mean tail moment above the control values was significant at p<0.01 and did not change by prolongation of
0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180
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recovery time. Interestingly, the response of XPA and XPF fibroblasts (Figure 3a), known to be deficient in nucleotide excision repair, was not different from normal cells (VH25).
Figure 3: Kinetics of the induction of DNA single and double strand breaks after MMC treatment.
Results from alkaline comet assay (a) and neutral comet assay (b) in normal (VH25), XPA and XPF cells showing that DNA breaks (as measured by the increase in the tail moment) are induced in all cell lines immediately after 1h treatment (0 h recovery) and persist up to 24 h recovery. In these graphs, control refers to untreated cells. Data for normal cells exposed to 12 Gy X-rays are included in graph (b) for comparison. c) Kinetics of JH2AX foci formation in confluent normal (WT) cells treated with MMC for 1h (top). Representative images of nuclei displaying H2AX foci (bottom) in control (left) and MMC treated (right) cells are included. (d) Signal intensity of JH2AX after treatment of normal (WT) and XPF (2 cell lines) cells with MMC. In each graph data from at least 2 experiments are presented. The error bars represent standard deviation of the mean values.
Similar results were obtained using the neutral comet assay (Figure 3b). After MMC treatment, a significant increase in the mean tail moment was observed in normal cells as well as repair deficient XP cells when compared with their control values. Increasing the recovery time up to 24 h after MMC treatment did not lead to significant change in the mean tail moment when compared with the response of cells at 0h (Figure 3b). The effect of 12 Gy X-rays is included as a positive control. The
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mean tail moment of cells exposed to 12 Gy was 6-fold higher than in cells treated with 4μM MMC (Figure 3b).
Analysis of H2AX foci in confluent normal human cells treated with MMC showed a significant increase of JH2AX positive cells after 1 h treatment. The percentage of normal cells with JH2AX foci (Figure 3c) increased significantly with increasing recovery time up to 24h after treatment (from 22.5% at 1h to 67.5% at 24 h recovery; p<0.01). In confluent cultures the percentage of S-phase was less than 2%.
Moreover, upon MMC treatment, the morphology of cells did not change and the cells remained attached up to 72 h (unpublished data) indicating that the increase of cells with JH2AX foci is unlikely due to apoptosis. Measurement of the nuclear immunofluorescent signal of JH2AX also revealed a gradual increase of the signal intensity (data not shown) and a maximal response was observed 24 h after MMC treatment in normal cells (Figure 3d). In contrast, in cells derived from two XPF patients the signal intensity was not increased above the control level (Figure 3d).
Preliminary results with FA-D1 cells revealed impaired phosphorylation of JH2AX after MMC treatment (unpublished data).
Discussion
Previous studies (Abdel-Halim et al., 2005; 2006) suggested that pairing of heterochromatin represents a cellular stress response based on two findings. Firstly, different genotoxic and non-genotoxic agents induced similar frequencies of heterochromatin pairing. Secondly, pairing can be induced by DNA damage distal to the heterochromatic regions or in unexposed neighboring cells. The absence of pairing in certain DNA repair deficient cells however indicates that this stress response is a genetically controlled process. Primary fibroblasts derived from XPF patients but not XPA patients were deficient in pairing of chromosome 9 heterochromatin after exposure to MMC or UV irradiation (Abdel-Halim et al., 2005, 2006). Taking into account that XPF/ERCC1 is essential for ICL repair and recombinational processes such as gene targeting and single strand annealing (Adair et al., 2000; De Silva et al., 2000; 2002; Mu et al., 2000; Sargent et al., 2000; Niedernhofer et al., 2001; Al- Minawi et al., 2007), these results indicated that recombination dependent pathways involving the XPF protein might play a role in the induction of heterochromatin pairing and/or maintenance of the paired status of heterochromatin.
In the present study, the pairing of heterochromatin was assessed in FA cells that are known to be deficient in the repair of DNA ICLs (Levitus et al., 2006). FA is a rare autosomal recessive cancer susceptibility syndrome characterized by developmental abnormalities, progressive bone marrow failure, and cellular hypersensitivity to DNA cross-linking agents (Bagby, 2003; Levitus et al., 2006). So far, thirteen FA complementation groups have been identified and the genes (A-N) have been cloned (Joenje et al., 1997; Timmers et al., 2001; Meetei et al., 2003;
Levitus et al., 2006). The FANCD1 gene is identical to the breast cancer susceptibility gene BRCA2, which has been implicated in HR (Howlett et al., 2002). In response to DNA damage FANCD1 (BRCA2) forms nuclear foci with FANCN/PALB2, FANCD2 and RAD51 (Taniguchi et al., 2002; Xia et al., 2007) representing sites of HR-mediated DNA repair (Moynahan et al., 2001; Jasin, 2002). The current finding that heterochromatin pairing was not induced in FA-D1/BRCA2 cells after treatment with MMC provides evidence that BRCA2 has a role in the pairing of the homologues after ICL induction and gives additional prove that HR might play a role in the pairing process. FA-A cells are capable of normal pairing indicating that FANCA is not required for this process. On the basis of these results we can speculate that FANCN/PALB2 might also be involved in the pairing process since the protein is also involved in HR similarly to BRCA2. Similarly, FANCJ and the core complex members of the FA pathway are likely not involved in pairing since they are not directly linked to HR. In line with our observations, positional changes were reported for pericentromeric regions of chromosome 1 in human lymphocytes exposed to low- doses of ionizing radiation (Spitkovsky et al., 2002); these changes were absent in lymphocytes of patients with hereditary BRCA2 mutations. Our observation that UV irradiation induces pairing of heterochromatin in confluent FA-D1/BRCA2 deficient cells reveals that these cells have the intrinsic capacity to carry out heterochromatic pairing and that HR involving BRCA2 is not the mechanism underlying UV induced pairing.
Next, we addressed the question whether processing and/or repair of MMC- induced ICLs occurs in confluent cells and whether these events are the trigger for pairing of homologous heterochromatic regions. Using a modified alkaline comet approach (De Silva et al., 2000, 2002), we found that the unhooking of MMC-induced ICLs is initiated in confluent normal human cells after treatment with MMC. This result is consistent with a recent model in which ICLs are recognized and rapidly
incised in G1 human cells independent of DNA replication (Rothfuss and Grompe, 2004) but contrasts other studies (De Silva et al. 2000, 2002). Also, an earlier study (Fujiwara, 1982) showed that confluent human cells were not able to unhook ICLs induced by MMC, whereas cycling cells appear to repair the ICLs.
Genetic and biochemical evidence suggests that repair of ICLs induced by various cross-linking agents including MMC yields double strand breaks (DSBs) as an intermediate lesion (De Silva et al., 2000; Pichierri et al., 2002; Niedernhofer et al., 2004; Rothfuss and Grompe, 2004; Franenberg-Schwager et al., 2005; Clingen et al., 2007). In these studies DSBs appear to be exclusively formed during DNA replication after the incision of the cross-linked DNA. Our results obtained with neutral comet assay and phosphorylation of H2AX suggest that additional processing of MMC- induced ICLs occurs in non-dividing cells, i.e. formation of DSBs independent of DNA replication. Yet, the kinetics of H2AX phosphorylation differed from DNA break induction detected by comet as the maximum level of H2AX phosphorylation was observed after 24 h compared to an immediate formation of DNA breaks after MMC treatment. This induction of DNA breaks in confluent normal cells is consistent with chromosome breaks in the heterochromatin of human chromosome 9 in G1 cells (Abdel-Halim et al., 2005). The similar response of confluent NER deficient XPA cells (completely deficient in monoadduct repair) excludes that NER is the process responsible for the induction of DSBs. The same conclusion has been drawn from results obtained for nitrogen mustard and MMC treatments of dividing XPA deficient Chinese hamster, mouse and human cells (De Silva et al. 2000; Niedernhofer et al., 2004; Clingen et al., 2007).
The induction of DNA breaks in confluent XPF cells detected by the comet assay in the current study appears to be contradictory to the absence of H2AX phosphorylation in these cells even up to 24 h after exposure to MMC. Also levels of DNA strand breaks were found in MMC exposed confluent FA-A and FA- D1/BRCA2 cells similarly to that in normal cells, yet impaired H2AX phosphorylation in FA-D1 cells was observed (unpublished data). H2AX phosphorylation has been used as a marker for DSBs (Niedernhofer et al., 2004;
Rothfuss and Grompe, 2004; Pilch et al., 2003) and also for ssDNA (Marti et al., 2006; Matsumoto et al., 2007). Normal human cells have been shown to form distinctive ssDNA foci detected by BrdU following treatment with MMC or with both psoralen and UVA, but not either alone (Lee et al., 2006). These foci were not formed
by treating cells with agents inducing alkylation, oxidative damage or DNA strand breaks, and were dependent on XPF and XPG, suggesting that MMC-induced ssDNA foci represent ssDNA patches during cross-link repair (Lee et al., 2006). Moreover, neutral as well as alkaline comet assays detect both SSBs and DSBs (for review see Collins et al., 2008). Indeed, in both neutral and alkaline comet experiments we noted that the mean tail moment was nearly the same up to 24 h incubation after 1 h treatment with MMC. Similarly, the number of cells displaying phosphorylated H2AX remained constant up to 48 h after MMC treatment of G1 cells. The persistence of DNA breaks or H2AX phosphorylation suggests that further processing is required to complete the resolution of DNA breaks and ICL repair. Alternatively, if the induction of DSBs during ICL repair is restricted to S-phase, it is tempting to speculate that XPF/ERCC1 dependent H2AX phosphorylation in the present study represents ssDNA regions formed by incision of ICLs in G1. These ssDNA regions may persist in the nucleus until they are completely processed in S-phase. The DNA breaks measured by comet assay might represent a mixture of SSBs (resulting from initiation of ICL processing) and DSBs induced by MMC (as shown by PCC experiments; Abdel-Halim et al., 2005) independent of functional NER and FA proteins. The exchange-like figures observed in PCC preparations (Abdel-Halim et al., 2005) likely represent stabilized paired homologues that are initiated in G1 and completed in S-phase. If the model of G1 ICL repair in yeast (Sarkar et al., 2006) exists in mammalian cells it implicates that SSBs are formed after incisions of ICLs followed by TLS-mediated gap filling. Also a role for TLS in ICL repair throughout the cell cycle including G1 has been recently suggested based on studies in human cells (Mogi et al., 2008).
To summarize, the results suggest that the DNA breaks detected by comet assay directly after 1 h MMC treatment represent the initial DNA damage induced by MMC including ICLs. The induction of these breaks is not dependent on repair activity. We speculate that a low level of ICL unhooking might occur which is not sufficient for H2AX signaling but can provoke the pairing of homologous heterochromatic regions in a sub-fraction of cells. Subsequently, increase of ICL unhooking and/or additional processing of ICLs (possibly generating DSBs) with time might induce phosphorylation of H2AX, which requires XPF and FANCD1 (BRCA2). The pairing of heterochromatin observed after MMC (Abdel-Halim et al., 2005) and also UV radiation (Abdel-Halim et al., 2006) was stable up to 24 h post
exposure indicating that the paired status of the homologues is stabilized for further processing of ICL and UV induced DNA damage. In contrast, pairing induced after X-rays (Abdel-Halim et al., 2004) disappeared in time. This reduction of pairing correlates with the rejoining kinetics of X-rays-induced DSB in heterochromatin of primary human fibroblasts (Rief and Lobrich, 2002) and indicates a possible link between heterochromatin pairing and repair of IR-induced DNA DSBs. In the case of MMC exposure, the lack of heterochromatic pairing and H2AX phosphorylation in mutant cells (XPF and FA-D1/BRCA2) supports a correlation between pairing and processing of ICLs. We propose that the processing of MMC induced ICLs involving at least XPF and BRCA2 is functional in G1 cells independent of DNA replication.
This processing induces and/or stabilizes the repositioning and pairing of homologous chromosomal regions.
Acknowledgements
We thank Patricia Cramers for help with the comet assay, Hans Vrolijk for allowing using Scil comet software, Mischa Vrouwe for help with the analysis of ȖH2AX signal measurements and Hans Joenje for providing Fanconi anemia cells.
This work was supported by the Euratom contracts FIGH-CT-1999-00011 and F16R-CT-2003-508842.
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