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Mol Cancer Ther. 2006;5:1602-1609
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Time-dependent cytotoxicity induced by SJG-136 (NSC 694501): influence of the rate of interstrand cross-link formation on DNA damage signaling

Stéphanie Arnould1, Victoria J. Spanswick2, Janet S. Macpherson1, John A. Hartley2, David E. Thurston3, Duncan I. Jodrell1 and Sylvie M. Guichard1

1 Pharmacology and Drug Development Group, Cancer Research UK Centre, The University of Edinburgh, Edinburgh; 2 Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School; and 3 Cancer Research UK Gene Targeted Drug Design Research Group, The School of Pharmacy, University of London, London, United Kingdom

Requests for reprints: Sylvie Guichard, Cancer Research UK Centre, The University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, United Kingdom. Phone: 44-131-777-3556; Fax: 44-131-777-3520. E-mail: Sylvie.Guichard{at}cancer.org.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SJG-136 is a new pyrrolobenzodiazepine dimer inducing time-dependent cytotoxicity. HCT 116 cells were exposed to 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours to achieve similar levels of interstrand cross-links (ICL). The short exposure led to a rapid formation of ICLs (1 hour), early H2AX foci formation (4 hours), prominent S phase arrest, and greater phosphorylation of Nbs1 (on serine 343) and Chk1 (on serine 317) than a 24-hour exposure. The prolonged exposure at low concentrations of SJG-136 induced a gradual formation of ICLs (up to 24 hours) which was associated with a limited S phase arrest and delayed Nbs1 phosphorylation. Prolonged exposure was also associated with a reduced phosphorylation of p53 on serines 15 and 20, a limited and delayed phosphorylation on serine 392, and a less prominent increase in p21 levels. These data suggest that the 24-hour exposure to a low concentration of SJG-136 led to delayed and reduced DNA damage signaling compared with a higher concentration of SJG-136 for 1 hour, resulting in greater cytotoxicity and contributing to the time-dependent cytotoxic effect of SJG-136. [Mol Cancer Ther 2006;5(6):1602–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SJG-136 (NSC 694501) is a pyrrolo[2,1-c]benzodiazepine dimer that binds covalently to purine-GATC-pyrimidine sequences in the minor groove of DNA, inducing little or no distortion of the double helix (1). In vitro studies have shown that it exerts a significant cytotoxic activity (2, 3) and its evaluation in the NCI 60 cell line panel has confirmed significant antitumor activity in a range of cell lines and tumor xenografts (4, 5). In vivo, SJG-136 is thought to show greater potency and less toxicity than bizelesin, another minor-groove-DNA alkylating agent (5). In in vitro studies in HCT 116 cells, bizelesin induced a transient S phase arrest, followed by a G2-M accumulation, associated with an increase in p53 and p21 levels (6).

Both bizelesin and SJG-136 induce DNA interstrand cross-links (ICL) which, as for other cross-linking agents (mitomycin C, cisplatin, and nitrogen mustards), are thought to be the main cause of the cytotoxic effect (7). Although ICLs usually represent a small fraction of the adducts formed by cross-linking agents (7), they are the main cause of cytotoxicity due to the inhibition of strand separation, which prevents replication, transcription, and segregation. ICLs also induce the formation of double-strand breaks (DSB) when a replication fork collides with a cross-link or by repair intermediates during homologous recombination (8, 9). The repair of ICLs takes place in S phase and the activation of the S phase checkpoint to allow repair and prevent genetic instability is essential (7, 9). Checkpoint-deficient cells undergo DNA replication, leading to chromosome breakage and mitotic catastrophe (10, 11). The activation of cell cycle checkpoints following the generation of ICLs is part of a coordinated stress response: DSBs and ICLs recruit the MRN (Mre11/Rad50/Nbs1) complex, whereas replication blocks are sensed by the Rad17 complex (12, 13). ATM (ataxia telangiectasia–mutated) and ATR (ATM-RAD3–related) are then recruited onto DNA and activated. ATM responds to DSBs, whereas ATR is involved in the response to stalled replication forks, UV light, and hypoxia. In turn, adaptors such as H2AX and Nbs1, transducer kinases such as Chk1 and Chk2, and effector proteins such as Cdc25A, Cdc25C, and p53 are activated (for review, see ref. 14). Nbs1 is particularly important in DNA damage response following ICLs because of its role in sensing DNA damage as part of the MRN complex, in S phase arrest via direct ATM activation, and in homologous recombination (15). Chk1 and Chk2 play an important role in the S phase checkpoint: Chk2 is phosphorylated on threonine 68 in response to DSBs by ATM, whereas both ATM and ATR phosphorylate Chk1 on serines 317 and 345 (16). Chk1 seems to play a crucial role in response to genotoxic stress, whereas the contribution of Chk2 is only significant when DSBs are generated during the S phase (17). Chk2 phosphorylates p53 on serine 20, whereas serine 15 is phosphorylated via ATM- and ATR-dependent pathways (18). NH2-terminal modifications of p53 in response to DNA damage inhibit its interaction with Mdm2, increasing the protein levels and its transcriptional activity (19). Chk1 and Chk2 also play a role in G2 arrest by phosphorylating cdc25c independently of p53. However, the maintenance in G2 arrest is p53-dependent and involves both p21 and 14-3-3{sigma}, both transcriptional targets of p53 (20). Moreover, p21 was shown to interact with the cyclin B1-cdk1 complex, maintaining the inactive complex in the nucleus and preventing its interaction with cdc25, preventing progression from G2 to mitosis (20).

SJG-136 showed its superiority to bizelesin in vivo, suggesting potential differences in their respective mechanisms of cell death. In particular, recent studies have shown that the cytotoxicity of SJG-136 is time-dependent (3, 5). We hypothesized that different rates of formation of DNA ICLs after a short (1 hour) or a long (24 hours) exposure may influence the activation of DNA damage signaling and result in differential cytotoxicity. We therefore investigated the kinetics of generation of DNA ICLs, cell cycle perturbations, and the activation of the DNA damage stress response in HCT 116 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culture Conditions
SW620 and HCT 116 colon cell lines were obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 containing 2 mmol/L of glutamine supplemented with antibiotics (100 units/mL penicillin G and 100 µg/mL streptomycin) and 5% fetal bovine serum. Cell cultures were incubated in the presence of 5% CO2 at 37°C in a humidified atmosphere and regularly checked for Mycoplasma contamination. SJG-136 was dissolved in DMSO and a 2 mmol/L stock solution was stored at –70°C.

Determination of DNA Interstrand Cross-Linking
The level of interstrand cross-linking was determined using the single-cell gel electrophoresis (comet) assay (21). Exponentially growing HCT 116 and SW620 cells (2 x 105 cells were plated in six-well plates and grown overnight) were treated with SJG-136 for either 1 or 24 hours at 37°C, and then washed and incubated in drug-free medium. Samples were collected at 1, 4, 8, 24, 30, and 48 hours after the beginning of each exposure and stored in FCS containing 10% DMSO at –80°C until analysis (21). Images were analyzed using Komet assay 4.02 software (Kinetic Imaging, Belfast, Ireland). Twenty-five images were analyzed for each duplicate slide. The tail moment was calculated using the definition by Olive et al. (22). Cross-linking was expressed as the percentage of decrease in tail moment calculated by the formula:

Formula
where TMdi, tail moment of drug-treated irradiated sample; TMcu, tail moment of untreated, unirradiated control; and TMci, tail moment of untreated, irradiated control.

Cell Cycle Distribution
After drug exposure, HCT 116 cells were washed with PBS, trypsinized, counted, and an aliquot of 5 x 105 cells was fixed in 70% ethanol on ice for 30 minutes and stored at 4°C until the end of the time course (4, 8, 24, 30, 48, and 72 hours). On the day of the analysis, they were washed with PBS, suspended at a concentration of 5 x 105 cells/mL in a solution of PBS containing 25 µg/mL of propidium iodide and 100 µg/mL of RNase A, and incubated for 30 minutes at 37°C. Analysis of 10,000 events was done on a Becton Dickinson FACScalibur flow cytometer (measurement wavelength, 564–607 nm) using CellQuest software and ungated data was gathered within 1 hour. Data was analyzed using ModFit 2.0 program (Verity Software, Topsham, ME). Histograms were gated (FSC versus SSC, FL2-A versus FL2-W) to exclude debris and doublets for analysis.

Immunostaining
Exponentially growing HCT 116 cells were seeded in chamber slides, exposed to either 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours and fixed at 1, 4, 8, 24, 30, and 48 hours after the beginning of each exposure. Cells were incubated in 2% paraformaldehyde in PBS for 5 minutes, washed in PBS, permeabilized in 100% methanol at –20°C for 20 minutes, washed, blocked with PBS containing 1% bovine serum albumin and 5% goat serum (Jackson Immunolaboratories, West Grove, PA), and incubated overnight at 4°C with an anti-phosphohistone H2AX antibody (Upstate Biotechnology, Dundee, United Kingdom), washed, incubated with a FITC, AlexaFluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, Paisley, United Kingdom) for 1 hour at room temperature, and washed in PBS. Slides were mounted and visualized with a Leitz Laborlux UV microscope (Wetzlar, Germany) using a 100x objective fitted with a Spot Insight 4 camera (Diagnostic Instruments, Sterling Heights, MI). Images were processed using Adobe Photoshop software.

Immunoblot Analysis
HCT 116 cells were incubated with either 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours. Cell pellets were collected over time and suspended in lysis buffer [62.5 mmol/L Tris (pH 6.8), 6 mol/L urea, 10% glycerol, and 2% SDS] and sonicated on ice. Protein concentration was determined using the bicinchoninic acid method (Sigma-Aldrich Company, Ltd., Gillingham, United Kingdom). Proteins were incubated with denaturing buffer for 5 minutes at 95°C, loaded onto 7.5% SDS-polyacrylamide gels for electrophoresis, transferred onto polyvinylidene fluoride membranes, blocked in 5% nonfat milk TBS-Tween 20 for 1 hour at room temperature, and incubated overnight at 4°C in a primary antibody solution. The following antibodies were used: rabbit anti-p95 Nbs1, rabbit anti-phospho-Ser343 p95 Nbs1 (Abcam Ltd., Cambridge, United Kingdom), mouse anti-Chk1 (G-4), mouse monoclonal anti-Chk2 (A-12; Autogen Bioclear UK, Ltd., Calne, United Kingdom), rabbit anti-phospho-Ser317Chk1, rabbit anti-phospho-Thr68Chk2, rabbit anti-pSer15p53, and rabbit anti-pSer20p53 antibody (Cell Signaling Technology, Inc., Beverly, MA), mouse anti-p53 antibody (Ab-6), mouse anti-p21 antibody (Ab-1), mouse ß-actin antibody (Merck Biosciences Ltd., Nottingham, United Kingdom), mouse monoclonal anti-cyclin B1 (Ab-3, clone GNS1; Neomarkers, Fremont, CA). The mouse monoclonal anti-pSer392p53 antibody was a gift from Prof. T.R. Hupp (University of Edinburgh). Membranes were then incubated for 90 minutes at room temperature in solutions of horseradish peroxidase–conjugated secondary antibody (Autogen Bioclear UK; except for actin, Merck Biosciences) in 2.5% nonfat milk TBS-Tween 20. Membranes were washed and immunoreactivity was detected using the Enhanced ChemiLuminescence Plus detection reagent (GE Healthcare, Little Chalfont, United Kingdom) and Hyperfilm enhanced chemiluminescence film (Amersham, Buckinghamshire, United Kingdom).

Statistical Analysis
Statistical analyses were done using StatView 4.5 software (Abacus Concepts, Berkeley, CA). Data were expressed as mean ± SD from three independent experiments. The influence of the tested schedules was analyzed by means of ANOVA. A post hoc Fisher's protected least significant difference test was used to correct for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Rate of Accumulation of SJG-136-Induced ICLs Is Concentration- and Time-Dependent
To test whether the cytotoxic effect of SJG-136 was related to the rate of formation of ICLs, the accumulation of DNA ICLs was measured over 48 hours in HCT 116 and SW620 cells using the comet assay. Cells were exposed to 50 nmol/L of SJG-136 for 1 hour and 1 nmol/L of SJG-136 for 24 hours [concentration (C) x time (T) values of 50 and 24, respectively]. Maximal formation of ICLs was achieved at the end of 1 hour of exposure. During the 24-hour exposure, the level of cross-links increased gradually from 7.3 ± 11.6% at 1 hour to 44.2 ± 4.7% at 24 hours in HCT 116 (Fig. 1A ) and from 13.9 ± 7.7% at 1 hour to 46.4 ± 5.4% at 24 hours in SW620 (Fig. 1B). The amount of DNA cross-links persisted up to 48 hours whatever the duration of exposure. Because kinetic profiles were similar for HCT 116 and SW620, subsequent experiments were only done in the HCT 116 cell line.


Figure 1
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  		Figure 1. Kinetics of formation of DNA ICLs upon exposure to SJG-136. Cross-linking was determined as the percentage of decrease in tail moment using the single-cell gel electrophoresis (comet) assay. HCT 116 (A) and SW620 (B) cell lines were exposed to SJG-136 for either 1 h at 50 nmol/L ({blacktriangleup}) or 24 h at 1 nmol/L ({blacksquare}). Points, mean of three independent experiments; bars, ±SD; *, determined using Fisher's protected least significant difference test (P < 0.05).

 
SJG-136 Induces Exposure-Dependent Cell Cycle Perturbations in the HCT 116 Cell Line
The cell cycle perturbations induced by 50 nmol/L of SJG-136 for 1 hour and 1 nmol/L of SJG-136 for 24 hours were analyzed over 72 hours by flow cytometry (Fig. 2 ). A short exposure to high concentrations of SJG-136 led to S phase arrest 8 hours after the start of drug exposure. The cells subsequently progressed to G2-M at 24 hours (68% versus 24% in control cells) and to G0/G1 at 48 hours. In contrast, a long exposure at low concentrations of SJG-136 induced a limited S phase arrest followed by a delayed accumulation to G2-M peaking at 48 hours and reentry into G0/G1 phase at 72 hours.


Figure 2
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  		Figure 2. Time-dependent cell cycle distribution upon exposure to SJG-136 in HCT 116 cell line. HCT 116 cells were exposed to either 50 nmol/L of SJG-136 for 1 h or 1 nmol/L of SJG-136 for 24 h, and were harvested at 0, 4, 8, 24, 30, 48, and 72 h after the beginning of each time course. Flow cytometry was then done for cell cycle distribution.

 
The Phosphorylation of Histone H2AX in Response to SJG-136 Is Concentration- and Time-Dependent
In order to investigate whether the rate of formation of ICLs might influence the appearance of {gamma}-H2AX foci, the phosphorylation of serine 139 on histone H2AX was visualized over 24 hours in HCT 116 cells exposed to 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours (Fig. 3 ). A significant phosphorylation signal was detectable at 4 hours in cells treated with 50 nmol/L of SJG-136 for 1 hour, whereas a 24-hour exposure to the compound at 1 nmol/L resulted in the appearance of {gamma}-H2AX foci 8 hours after the start of incubation. However, the levels of histone H2AX phosphorylation were equivalent at 24 hours.


Figure 3
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  		Figure 3. The phosphorylation of histone H2AX in response to SJG-136 is concentration- and time-dependent. HCT 116 cells were exposed to SJG-136 for either 1 h at 50 nmol/L or 24 h at 1 nmol/L, fixed at 1, 4, 8, and 24 h after the start of the time course and immunostained with an anti-{gamma}-H2AX antibody.

 
The Timing of Phosphorylation of Nbs1 on Serine 343 Is Dependent on the Concentration and Duration of Exposure to SJG-136
Nbs1 is a component of the MRN complex and plays a major role in the activation of the intra-S phase checkpoint. The level of phosphorylation of Nbs1 on serine 343 was evaluated after 1- and 24-hour exposures to SJG-136 over 48 hours. When HCT 116 cells were exposed to 50 nmol/L of SJG-136 for 1 hour, a rapid phosphorylation of Nbs1 was detected at 4 hours and the maximum effect was observed at 8 hours (Fig. 4A ). In contrast, a significant phosphorylation of Nbs1 was only detectable at the end of drug exposure when cells were exposed to 1 nmol/L of SJG-136 for 24 hours (Fig. 4B).


Figure 4
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  		Figure 4. The timing of phosphorylation of Nbs1 on serine 343 is dependent on the exposure to SJG-136. HCT 116 cells were exposed to either 50 nmol/L of SJG-136 for 1 h (A) or 1 nmol/L of SJG-136 for 24 h (B) and harvested at 0, 4, 8, 24, 30, and 48 h after the beginning of each time course. Total cell lysates were subjected to SDS-PAGE followed by immunoblotting. Membranes were probed with anti-pSer343Nbs1, Nbs1, and actin antibodies followed by appropriate peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence detection system.

 
The Pattern of Phosphorylation of Chk1 on Serine 317 and Chk2 on Threonine 68 Depends on the Exposure to SJG-136
Checkpoint kinases Chk1 and Chk2 have been implicated in the activation of the S phase checkpoint. The short, high concentration exposure of HCT 116 cells to SJG-136 resulted in the phosphorylation of Chk1 on serine 317 as early as 4 hours after the start of the time course, and levels increased up to 30 hours (Fig. 5A ). This phosphorylation was observed late (8 hours) when cells were exposed to SJG-136 for 24 hours (Fig. 5B). The phosphorylation of Chk2 on threonine 68 was transiently detected (Fig. 5C and D) but appeared earlier (8 hours) when cells were exposed to low concentrations of SJG-136 over 24 hours (Fig. 5D).


Figure 5
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  		Figure 5. SJG-136 induces the phosphorylation of Chk1 on serine 317 and Chk2 on threonine 68. HCT 116 cells were exposed to either 50 nmol/L of SJG-136 for 1 h (A and C) or 1 nmol/L of SJG-136 for 24 h (B and D) and harvested at 0, 4, 8, 24, 30, 48, and 72 h after the beginning of each time course. Total cell lysates were subjected to SDS-PAGE followed by immunoblotting. Membranes were probed with anti-pSer317Chk1, Chk1 (A and B), anti-pThr68Chk2, Chk2 (C and D), and actin antibodies followed by appropriate peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence detection system.

 
p53 Is Phosphorylated on Serines 15, 20, and 392 upon Exposure to SJG-136
The phosphorylation of p53 on serines 15, 20, and 392 was examined by Western blotting after either a 1- or a 24-hour exposure to SJG-136 in the HCT 116 cell line (Fig. 6 ). An increase in the phosphorylation level of serine 15 was observed 8 hours after the beginning of a short exposure to the compound (Fig. 6A), but it only increased after 30 hours, following the longer exposure to the drug. Phosphorylation of serines 20 and 392 occurred later and the signal became significantly higher than the matched control 48 hours after the beginning of the time course (Fig. 6B). p53 levels increased earlier and to a larger extent when cells were exposed to SJG-136 for 1 hour. p21CIP/WAF1 levels increased from the 24-hour time point to the end of both time courses and followed the same trend, albeit to a lesser extent after a 24-hour exposure. These results show that the exposure to SJG-136 induces the phosphorylation of p53 on serines 15, 20, and 392 and results in the gradual accumulation of p53 and p21.


Figure 6
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  		Figure 6. The phosphorylation of p53 is dependent on the concentration and duration of exposure to SJG-136 in HCT 116 cell line. HCT 116 cells were exposed to either 50 nmol/L of SJG-136 for 1 h (A) or 1 nmol/L of SJG-136 for 24 h (B) and harvested at 0, 4, 8, 24, 30, 48, and 72 h after the beginning of each time course. Total cell lysates were subjected to SDS-PAGE followed by immunoblotting. Membranes were probed with anti-pSer15p53, pSer392p53, pSer20p53, p53, p21CIP/WAF1, cyclin B1, and actin antibodies followed by appropriate peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence detection system.

 
To confirm the induction of a G2-M checkpoint suggested by cell cycle analysis, cyclin B1 levels were measured in HCT 116 cells (Fig. 6). Western blots showed increased intracellular levels of cyclin B1 as compared with matched controls between 24 and 48 hours, whatever the exposure, with a higher increase when cells were exposed to SJG-136 for 1 hour (Fig. 6A). Thus, SJG-136 induced an accumulation at the G2-M transition that depended on the concentration and the duration of exposure in the HCT116 cell line.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study investigates the potential elements responsible for the time-dependent cytotoxic effect of SJG-136. Therefore, HCT 116 cells were exposed to the drug for 1 or 24 hours at concentrations generating equivalent levels of ICLs. SJG-136 concentrations were determined based on the results of the comet assay: 50 nmol/L delivered over 1 hour led to a similar decrease in tail moment as a 24-hour exposure to 1 nmol/L. The rate of formation of ICLs was markedly different between the two exposures: a maximum level of ICLs were observed at the end of the 1-hour exposure, whereas a gradual increase in ICLs over 24 hours was observed during a 24-hour exposure at lower concentrations of SJG-136. This is consistent with DNA thermal denaturation studies on calf thymus DNA showing that although there is significant adduct formation immediately upon exposure of the DNA to SJG-136, further significant adduct formation occurs up to 18 hours and possibly beyond (1). Moreover, the two modalities tested generated equivalent levels of ICLs whereas the "drug exposure over time" or C x T = 50 for the short exposure, and only 24 for the long exposure. This suggests that there were twice as many DNA ICLs formed per unit of concentration when cells were exposed to the drug for 24 hours as opposed to 1 hour. The persistence of ICLs over 48 hours has previously been reported with SJG-136 (23) but is in contrast with ICLs induced by melphalan, which are efficiently repaired through a nucleotide excision repair–dependent mechanism (24).

The kinetics of histone H2AX foci formation showed a delay between the formation of ICLs (maximal at the end of 1 hour exposure) and foci formation (increased at 4 hours) when cells were exposed to the drug for 1 hour. This delay in formation was more apparent when cells were exposed to the drug for 24 hours, with foci appearing 8 hours after the start of drug exposure. This suggests that the damage is processed prior to H2AX detection. Moreover when cells were exposed to SJG-136 for 24 hours, there was a gradual increase in H2AX foci formation, which paralleled the increase in ICLs, suggesting that ICLs (or their sequelae) were being sensed. The phosphorylation of H2AX, which reflects dynamic changes in chromatin structure, has been described following irradiation (25), with nuclear foci forming at sites of DSBs (26) but has also been reported in response to replication blocks induced by hydroxyurea or UV radiation (27), both events being potentially induced by SJG-136.

The cell cycle effects induced by a high concentration of SJG-136 for 1 hour and a low concentration for 24 hours were different: the short exposure leading to a rapid formation of ICLs induced a more prominent S phase arrest which seems correlated with early H2AX foci formation, a greater phosphorylation of Nbs1 on serine 343 and Chk1 on serine 317. The prolonged exposure at low concentrations of SJG-136 induced a gradual formation of ICLs, which seems correlated with a limited S phase arrest and delayed Nbs1 phosphorylation. H2AX is involved in the recruitment of proteins such as Nbs1 that take part in DNA repair processes at the sites of DNA damage (26), which may explain the parallel phosphorylation of the two proteins. In normal fibroblasts, the early activation of Nbs1 on serine 343 has been reported in response to DNA damage, with phosphorylation occurring as early as 30 minutes postirradiation and persisting for up to 24 hours (28). Moreover, Nbs1 was recruited in MRN subnuclear foci upon exposure of lymphoblasts to mitomycin C (12, 29). Nbs1 is also involved in homologous recombination (30), which plays a crucial role in DNA ICL repair in higher vertebrate cells (8, 9) and Nbs1-deficient cells were hypersensitive to mitomycin C due to the decrease in sister chromatid exchange and increase in chromosome breaks. A recent study has described increased sensitivity to SJG-136 in Chinese hamster ovary cell lines that were mutated for XPF/ERCC1, which is consistent with the role of a homologous recombination-dependent pathway in cross-link repair (23). The phosphorylation of Nbs1 on serine 343 might therefore contribute to the strength of the cellular response to ICLs as suggested in a previous study (31). In the presence of DSBs, the phosphorylation of Nbs1 and Chk1 was dependent on ATM (32). A gradual phosphorylation of Chk1 and Chk2 has been described following irradiation, and that phenomenon was dose-independent in the case of Chk1, whereas Chk2 was only fully activated after high doses of irradiation and more extensive DSB formation (33). In this study, the prolonged drug exposure was associated with an early but transient activation of Chk2 on threonine 68 and suggests the activation of ATM, possibly due to the formation of DSBs, either as repair intermediates or as the result of the collision of ICLs with replication forks, potentially in greater numbers compared with the short exposure.

Following the generation of cell stress, particularly DNA damage, p53 is posttranslationally stabilized and this is associated with increased levels of its target gene p21 (34). In the present study, p53 levels increased early (4 hours) in response to the short exposure to SJG-136, but later (24 hours) with the long exposure. Moreover, p53 was differentially phosphorylated on serine 15 between 1- and 24-hour exposures. This residue has been reported to be phosphorylated early in response to DNA damage (35). The difference between the two modalities of drug exposure could therefore be consistent with a lack of recognition of the damage and progression through the cell cycle during a prolonged incubation. The uncoupling of the phosphorylation of Chk2 on threonine 68 and its downstream target serine 20 on p53, makes the involvement of this checkpoint kinase in this activation unlikely. The preincubation of HCT 116 cells with wortmannin before the exposure to SJG-136 transiently abolished the phosphorylation of p53 on serine 20, which paralleled the changes in the phosphorylated state of DAPK on serine 308 (data not shown), which could therefore be a lead candidate. The increase in p21 levels was delayed when cells were exposed to SJG-136 for 24 hours and is consistent with the reduced and delayed activation of p53 as well as a delayed G2-M arrest. A concentration-dependent increase in p21 levels has been reported in HCT116 cells that were exposed to bizelesin and was linked to both G2-M arrest and induction of senescence (6). Experiments carried out in HeLa cell–free extracts showed that p21 interacts with proliferating cell nuclear antigen and inhibits the DNA resynthesis step of nucleotide excision repair at the sites of psoralen-induced ICLs (36). p21 has also been shown to interact with the cyclin B1-cdk1 complex to prevent cell progression from G2 to mitosis (37). Chk1 and Chk2 might also contribute to this effect by direct interaction with cdc25. Indeed, whereas p21 levels remained high up to 72 hours, cyclin B1 levels dropped at 48 hours after a 1-hour exposure to SJG-136, and to 72 hours after a 24-hour exposure.

In summary, the prolonged (24 hours) exposure to a low concentration (1 nmol/L) of SJG-136 was characterized by a slower accumulation of DNA ICLs that appeared less likely to be sensed, thus resulting in a greater cytotoxic effect as compared with the shorter (1 hour) exposure to 50 nmol/L. We suggest that this contributes to the apparent time-dependent cytotoxicity of SJG-136 and may have implications for the design of clinical dosing strategies.


   Acknowledgments
 
We thank Janet Hartley, Helen Lowe, and Claire Newton for their technical assistance, and Spirogen, Ltd., and Ipsen Ltd., for their permission to use SJG-136 in these studies. Stephanie Arnould was awarded a Core Skills and Training Bursary by Cancer Research UK during this study.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/11/06; revised 2/24/06; accepted 4/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Grillari, H. Katinger, and R. Voglauer
Contributions of DNA interstrand cross-links to aging of cells and organisms
Nucleic Acids Res., December 14, 2007; (2007) gkm1065v1.
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