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[Cancer Research 66, 7203-7210, July 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Generation of Replication-Dependent Double-Strand Breaks by the Novel N2-G-Alkylator S23906-1

Stéphane Léonce1, Laurence Kraus-Berthier1, Roy M. Golsteyn1, Marie-Hélène David-Cordonnier2, Christelle Tardy2, Amélie Lansiaux2, Virginie Poindessous3, Annette K. Larsen3 and Alain Pierré1

1 Institut de Recherches Servier, Cancer Drug Discovery, Croissy sur Seine, France; 2 Institut National de la Santé et de la Recherche Médicale (INSERM) U-524 and Centre Oscar Lambret, Institut de Recherche sur le Cancer de Lille, Lille, France; and 3 INSERM U-673 and Université Pierre et Marie Curie Paris 6, Hôpital Saint-Antoine, Paris, France

Requests for reprints: Stéphane Léonce, Institut de Recherches Servier, Division de Recherches Cancerologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France. Phone: 33-1-55-72-22-84; E-mail: stephane.leonce{at}fr.netgrs.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
S23906-1, a new DNA alkylating agent that reacts with the exocyclic 2-NH2 group of guanine residues yielding monofunctional adducts, is currently under clinical evaluation in phase I trials. To investigate the mechanism of action of S23906-1, we compared parental KB-3-1 cells and KB/S23-500 cells that are 15-fold resistant to S23906-1. Cell death induced by 1 µmol/L S23906-1 in KB-3-1 cells was associated with their irreversible arrest in the G2-M phases of the cell cycle followed by apoptosis, whereas a proportion of the resistant KB/S23-500 cells were able to exit from the G2 arrest and divide, leading to a significantly lower rate of apoptosis. The attenuated apoptotic response was associated with decreased Chk2 protein phosphorylation, indicating that the DNA damage signaling pathways are more potently activated in the sensitive cells. However, similar rates of adduct formation and repair were measured in both cell lines. Exposure to S23906-1 induced a higher formation of DNA breaks, measured by the comet assay, in sensitive cells. In agreement, a histone H2AX phosphorylation assay revealed that S23906-1 induced double-strand breaks (DSB) in a dose- and time-dependent manner and that these were more persistent in the parental cells. These DSBs were found mainly in S-phase cells and inhibited by aphidicolin, suggesting that they are DNA replication-mediated DSBs. These results suggest that secondary DNA lesions play an important role in the cytotoxicity of this compound and make histone H2AX phosphorylation an attractive marker for monitoring the efficacy of S23906-1. (Cancer Res 2006; 66(14): 7203-10)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acronycine, an alkaloid that was first isolated from the Australian shrub Sarcomelicope simplicifolia, was shown to exhibit a broad spectrum of antitumor activity in experimental models (1). However, the phase I/II clinical trials of acronycine were not conclusive due to both a low response rate as well as neurologic and gastrointestinal toxicities (2).

The unstable acronycine epoxide, which was later isolated from Sarcomelicope argyrophylla, was assumed to be the active metabolite of the parent acronycine molecule (3). More stable prodrugs of this electrophilic epoxide, obtained by grafting two esters at the C1-C2 position of the pyran ring, were synthesized (4), leading to a family of diesters of benzo[b]acronycine, which were markedly more potent than acronycine (5). From this series, the diacetate S23906-1 was identified as one of the most potent derivatives both in vitro (6) and in vivo in orthotopic models of human solid tumors (5, 7). S23906-1 is currently in phase I clinical trials.

DNA was identified as an important target for this alkylating agent. We have previously shown, using DNA relaxation experiments done with supercoiled DNA, that S23906-1 neither affects the topoisomerase I–mediated relaxation of plasmid DNA nor intercalates into DNA, as shown by circular and linear dichroism measurements, but specifically reacts with the exocyclic 2-NH2 group of guanine residues exposed in the minor groove of DNA to form a covalent adduct (8, 9).

This compound is one of very few antitumor agents that induce monofunctional DNA adducts at the N2 position of guanine: ecteinascidin 743, a marine product with a very different structure, being the most recent one (10). However, ecteinascidin 743 potently stabilizes duplex DNA against heat denaturation, whereas S23906-1 markedly destabilizes the duplex (11).

To date, all the structure-activity relationships support DNA alkylation as the main molecular mechanism of action in the benzo[b]acronycine series (12). However, the reactivity of S23906-1 to glutathione (13) suggests that additional nucleophilic targets other than DNA might be involved in its mechanism of action.

At the cellular level, S23906-1 also presents uncommon properties, such as a selective increase in cyclin E, and a dual effect on cell cycle depending on the concentration (14).

The aim of the present work was to investigate the cellular responses to S23906-1 to delineate some determinants for sensitivity to S23906-1. For this purpose, we used a sensitive parental cell line, KB-3-1, and a variant cell line, KB/S23-500, which was made resistant by stepwise exposure of KB-3-1 cells to S23906-1. Due to the fact that a similar number of primary DNA adducts were found in sensitive and resistant cells, we searched for secondary DNA lesions and investigated the relationship with induction of apoptosis. We provide evidence, using the comet and histone H2AX phosphorylation assays, supporting the conversion of the primary adducts into DNA double-strand breaks (DSB).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Compounds. Acronycine and S23906-1 (Fig. 1 ) were synthesized as described (5). Both compounds were solubilized at 10–2 mol/L in DMSO, aliquoted, and stored at –20°C. Cytarabine was provided by Pharmacia & Upjohn SA (St-Quentin-en-Yvelines, France); vincristine and gemcitabine by Eli Lilly (Saint-Cloud, France); cisplatin by Bellon-Aventis (Montrouge, France); methotrexate by Wyeth-Lederle (Puteaux, France); 5-fluorouracil by Roche (Neuilly-sur-Seine, France); paclitaxel and carmustine by Bristol-Myers Squibb (La Défense, France); doxorubicin by Carlo-Erba (Paris, France); and etoposide, camptothecin, mitomycin C, aphidicolin, and staurosporine by Sigma Chemical (St. Louis, MO).


Figure 1
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  		Figure 1. Chemical structure of S23906-1.

 
Cell lines. The parental human KB-3-1 epidermoid carcinoma (15) and its multidrug resistant KB-A1 subline were kindly provided by Dr. M. Gottesman (NIH, Bethesda, MD). The KB/S23-500 cells were made resistant to S23906-1 by stepwise exposure to the drug up to 500 nmol/L. Cells were maintained in DMEM supplemented with 10% decomplemented FCS, 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L HEPES (pH 7.4). Cells were grown at 37°C in 5% CO2/95% air. All media and supplements were from Life Technologies (Cergy-Pontoise, France), except FCS, which was purchased from Sigma Chemical.

Standard proliferation assay. Cell proliferation was measured by the microculture tetrazolium assay essentially as described (16). Results are expressed as IC50, the drug concentration that reduced by 50% the absorbance at 540 nm. All measurements were done in triplicate.

Detection of apoptosis by Annexin V labeling. Cells were exposed to S23906-1 for 96 hours, rinsed, and labeled with Annexin V-FITC as described previously (14). Samples were analyzed by flow cytometry on an Epics XL-MCL flow cytometer (Beckman Coulter, Roissy, France). For each sample, 104 cells were analyzed. FITC and propidium iodide fluorescences were collected through 520 and 630 nm bandpass filters, respectively.

Mice and tumor models. Nude female congenic athymic BALB/c mice homozygous for the nude gene (nu/nu) were purchased from Charles River (Lyon, France). Mice weighed 20 to 22 g at the start of the experiments, and they received proper care and maintenance in accordance with institutional guidelines. The KB-3-1 and KB/S23-500 cells were cultured in vitro, and 107 cells were grafted s.c. into nude mice to give the generation 1. Tumor fragments of 2 to 3 mm3 were then transplanted into the flanks of nude mice and used for the chemotherapy experiment (generation 2). S23906-1 was prepared as a microemulsion in a mixture of 10% labrasol/labrafil/transcutol (2/2/1) at 0.625 mg/mL and diluted in the same vehicle. Tumors were measured twice weekly, and tumor volumes (Vt) were calculated using the following formula: length (mm) x width2 (mm2) / 2. Tumor volumes in treated mice were compared with those of control animals using Student's t test. A one-way ANOVA analysis was carried out followed, in case of significance of the overall analysis, by a Newman-Keuls' test for pairwise comparisons. All significance thresholds were fixed at 5% (17).

Cell cycle analysis. Cells were exposed to S23906-1, then washed, fixed by 70% ethanol, and incubated for 30 minutes in PBS containing 100 µg/mL RNase and 50 µg/mL propidium iodide. For each sample, 104 cells were analyzed by flow cytometry. To quantify cells in mitosis, samples were fixed by ethanol, washed, and incubated for 2 hours with 2 µg of anti-phosphorylated histone H3 rabbit polyclonal antibody (Euromedex, Mundolsheim, France). Cells were then washed with PBS and incubated for 1 hour with 4 µg of FITC-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were washed and incubated for 30 minutes with 100 µg/mL RNase and 10 µg/mL propidium iodide and analyzed by flow cytometry. FITC and propidium iodide fluorescences were collected through 520 and 630 nm bandpass filters, respectively. Results are displayed as bivariate distribution of phosphorylated histone H3 level versus DNA content.

DNA alkylation by [3H]S23906. S23906-1 was tritiated by Amersham Biosciences (Whitchurch, United Kingdom) at the metabolically stable 5 and 13 positions. The labeled compound (851 GBq/mmol, 37 MBq/mL) was dissolved in acetonitrile and stored at 4°C. Under these conditions, [3H]S23906 was stable for >3 months as shown by high-performance liquid chromatography analysis. For the measurement of DNA alkylation, 5 x 105 cells were seeded in 12-well plates and incubated for 24 hours at 37°C. The culture medium was then removed, and 0.3 mL of 1 µmol/L [3H]S23906 (2 µCi/well) was added. At the indicated times, cells were washed with PBS and the DNA was extracted by phenol/chloroform/isoamylic alcohol (25:24:1) followed by precipitation by 100% ethanol at –20°C. After centrifugation, the DNA pellet was dissolved in 0.2 mL H2O and 0.1 mL was counted in 5 mL Ultima Gold in a liquid scintillation analyzer 1600 TR (Packard, Meriden, CT). DNA concentration was estimated by measuring the absorbance at 260 nm of the other 0.1 mL to express the results as the number of cpm of [3H]S23906 per 1 µmol/L of base pair. To estimate the nonspecific trapping of [3H]S23906 during DNA purification, [3H]S23906 was added to the culture medium and then immediately discarded. The cells were immediately washed and processed as above. The nonspecific trapping of [3H]S23906 was subtracted from each value. All the points were done in triplicate.

Alkaline and neutral comet assay. Cells for comet analysis were exposed to the indicated drug concentrations at 37°C in the dark and analyzed immediately. The alkaline comet assay was carried out as described previously (18, 19), whereas the neutral comet assay was done as described by Wojewodzka et al. (20). Cells were stained by ethidium bromide (2 µg/mL), and the slides were examined at x400 magnification using a fluorescent microscope (Nikon TS100, Nikon, Champigny sur Marne, France).

Coded slides were examined without any knowledge of the treatment. Image analysis was done by using the Komet 5.5 software developed by Kinetic Imaging Ltd. (Nottingham, United Kingdom). At least 100 cells were analyzed per sample. Figures represent typical experiments selected from at least two independent experiments.

Histone H2AX phosphorylation. Histone H2AX phosphorylation was measured by flow cytometry (21). Cells were exposed to S23906-1, then harvested, centrifuged, and fixed by 70% ethanol at –20°C. Samples were washed with PBS and incubated for 5 minutes in PBS containing 0.5% Triton X-100 at 0°C. Cells were washed and incubated for 2 hours with 0.5 µg of anti-phosphorylated histone H2AX (Ser139) murine monoclonal antibody (mAb; 05-636, Upstate Biotechnology, Charlottesville, VA). Cells were then washed and incubated for 1 hour with 4 µg of FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). Cells were washed and incubated for 30 minutes with 100 µg/mL RNase and 10 µg/mL propidium iodide and analyzed by flow cytometry. Results are displayed as bivariate distribution of phosphorylated histone H2AX ({gamma}H2AX) levels versus DNA content and expressed as percentages of {gamma}H2AX-positive cells.

For immunofluorescence analysis, 2.5 x 104 cells were cultured on glass coverslips for 24 hours before treatment with 1 µmol/L S23906-1 for an additional 24 hours. After one wash with PBS, cells were fixed in 3% formaldehyde for 20 minutes, blocked briefly with 50 mmol/L ammonium acetate, and permeabilized for 5 minutes in 0.2% Triton X-100. Cells were incubated with 1:400 anti-phosphorylated histone H2AX antibody (Upstate Biotechnology) for 2 hours. After three washes with PBS, the coverslips were incubated with 1:2,000 Alexa Fluor 488 secondary antibodies (Molecular Probes, Eugene, OR). Nuclei were marked with 4',6-diamidino-2-phenylindole. Cells were observed on a Zeiss Axioplan 2 microscope driven by Axiovision 4.0 software. Images were collected with a Zeiss HR camera (Zeiss SAS, Le Pecq, France) within the linear dynamic range and treated in an identical manner with Photoshop 7.0 software.

Chk2 phosphorylation. Cells were exposed to S23906-1 for 6 hours, then collected and lysed in a solution of 100 mmol/L DTT in 2x Laemmli sample buffer (Bio-Rad Laboratories, Richmond, CA), denaturated at 95°C for 5 minutes, and resolved on 4% to 20% Tris-glycine gels. After gel transfer, anti-phosphorylated Chk2 antibody (Thr68; Cell Signaling Technology, Beverly, MA) was added for 2 hours and membranes were blotted for 1 hour with a horseradish peroxidase–linked secondary antibody (Cell Signaling Technology). Proteins were visualized with a chemiluminescence assay system (Amersham Biosciences). Equal protein loading (~15 µg) for each concentration of S23906-1 was confirmed using an anti-ß-actin antibody (Sigma Chemical).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of the resistant KB/S23-500 cell line. Sensitive KB-3-1 cells were cultured in the presence of increasing concentrations of S23906-1, starting at 200 nmol/L. After 6 months and three steps, cells were able to grow in the presence of 500 nmol/L S23906-1, with a stable doubling time of ~27 hours versus 24 hours for the parental line.

The resistant KB/S23-500 line was ~15-fold resistant to S23906-1, IC50 of 1,670 nmol/L ± 111 versus 109 nmol/L ± 4 (n = 27) for KB-3-1 cells (Fig. 2A ). The resistance of KB/S23-500 to S23906-1 was maintained after 2 months of culture in drug-free medium (data not shown).


Figure 2
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  		Figure 2. In vitro and in vivo characterization of the resistant KB/S23-500 cell line. A, sensitive KB-3-1 and resistant KB/S23-500 cells were incubated for 96 hours with various concentrations of S23906-1, and the cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results are expressed as the % proliferation compared with untreated cells. B, induction of apoptosis by S23906-1. Cells were exposed to the indicated concentrations of S23906-1 for 96 hours, washed, and resuspended in binding buffer containing Annexin V-FITC and propidium iodide. Annexin V–positive cells were measured by flow cytometry, and the corresponding % was reported as a function of S23906-1 concentration. C and D, mice bearing s.c. implanted KB-3-1 or KB/S23-500 tumors were treated orally with at 6.25, 3.12, and 1.56 mg/kg S23906-1 on days 11 and 22 after the graft. Tumors were measured twice weekly, and the results are expressed as mean tumor volumes. **, P < 0.01; ns, P > 0.05.

 
To investigate whether the resistance of KB/S23-500 cells could be due to a reduced propensity to undergo apoptosis, cells were incubated for 96 hours with various concentrations of S23906-1 and labeled with Annexin V-FITC and propidium iodide. Figure 2B shows that ~10-fold higher concentrations of S23906-1 were required to induce an apoptotic response of KB/S23-500 comparable with that of KB-3-1 cells. The strong correlation between cytotoxicity and apoptosis strongly suggests apoptosis as the main mechanism of cell death induced by S23906-1.

To determine whether the in vitro resistance could be translated in vivo, sensitive and resistant cells were grafted s.c. to nude mice. S23906-1 was given orally to mice on days 12 and 22. The KB-3-1 tumor proved very sensitive in vivo to S23906-1 the tumor growth inhibition being statistically significant (P < 0.01) at the three doses used (Fig. 2C). At the maximal tolerated dose (6.25 mg/kg), which is devoid of toxicity (no toxic death and maximal weight loss of 17%), S23906-1 induced tumor regression, lasting >2 weeks after the last treatment. The compound was significantly less active against KB/S23-500 tumors because only the 6.25 mg/kg dose significantly reduced tumor growth (P < 0.01) without any tumor regression (Fig. 2D).

Cross-resistance profile and lack of P-glycoprotein overexpression. We then investigated the sensitivity of the resistant cells to various antitumor agents to determine whether patterns of cross-resistance would suggest mechanistic similarities with other agents. A variety of drugs with different mechanisms of action were used (see Materials and Methods). Among these drugs, cytarabine shows a 7-fold resistance level versus 15-fold for S23906-1. Cisplatin, camptothecin, and mitomycin C were 3- to 5-fold less potent toward the resistant line. The lack of cross-resistance to drugs, such as doxorubicin, etoposide, and paclitaxel, a priori ruled out a classic multidrug resistance (MDR) phenotype associated with overexpression of ATP-binding cassette (ABC) transporters. To further confirm this, the level of P-glycoprotein (P-gp) expression was measured by flow cytometry with the MRK16 mAb. P-gp was not overexpressed in KB/S23-500 cells compared with KB-3-1 cells (data not shown). In comparison, the fluorescence intensity was ~55-fold higher in the P-gp overexpressing MDR line KB-A1 than in KB-3-1 as shown previously (22).

The lack of an active efflux pump in the KB/S23-500 cells was confirmed by measuring a similar rhodamine 123 uptake by KB-3-1 and KB/S23-500 cells, in clear contrast to the KB-A1 cells where the rhodamine 123 uptake was significantly lower (data not shown). These results show that KB/S23-500 cells do not overexpress ABC transporters known to efflux rhodamine 123, such as P-gp, MDR-associated protein, and breast cancer resistance protein (23, 24), and suggest that the resistance of KB/S23-500 is not associated with a decreased intracellular concentration of S23906-1.

Cell cycle analysis. We have previously shown that S23906-1 induced an accumulation of HT29 cells in the G2-M phases of the cell cycle at relatively low concentrations (from 0.1 to 1 µmol/L) and in S phase at higher concentrations (14). To further investigate the effect of S23906-1 on cell cycle progression, sensitive and resistant cells were exposed to various concentrations of S23906-1 and analyzed 24 and 48 hours later. Figure 3A shows that this apparent dual effect was due to both a delay of S-phase progression and an arrest in the G2-M phases. Similar profiles were observed for the sensitive and resistant cell lines when ~10-fold higher concentrations were applied to the latter. At 1 µmol/L S23906-1, 86% of sensitive KB-3-1 cells were arrested in the G2-M phases after 48-hour drug exposure versus only 35% of resistant cells. Interestingly, 38% of the resistant cells were in the G1 phase. To determine if these G1 cells were arrested after 24 hours (irreversible G1 arrest) or if they result from the mitosis of cells previously arrested in the G2-M phases, nocodazole was added for 24 hours, from 24 to 48 hours (Fig. 3B). Nocodazole had no effect on the perturbation of the KB-3-1 cell cycle induced by S23906-1, showing that the G2-M arrest was irreversible. In contrast, nocodazole induced an accumulation of KB/S23-500 cells in M phase, as revealed by expression of phosphorylated histone H3, with only 5% of cells in G1 phase. The G1 cells observed after 48 hours of exposure of KB/S23-500 cells to S23906-1 alone are thus cells that have exited from the G2 phase and completed mitosis. These results show that 1 µmol/L S23906-1 induces a delay of S-phase progression in both sensitive and resistant cells (although more marked in sensitive cells) followed by a complete G2-M arrest in the sensitive cells, whereas a proportion of the resistant cells were able to exit from the G2 phase and divide.


Figure 3
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  		Figure 3. Cell cycle analysis. A, KB-3-1 and KB/S23-500 cells were exposed to the indicated concentrations of S23906-1 for 24 or 48 hours, fixed, and labeled with propidium iodide before flow cytometric analysis. Results are monoparametric histograms of propidium iodide fluorescence (DNA content). B, cells were incubated with 1 µmol/L S23906-1 for 48 hours in the presence or absence of nocodazole (Noco) for the last 24 hours. At 24 and 48 hours, cells were fixed and labeled with an anti-phosphorylated histone H3 antibody. Propidium iodide was added, and cells were analyzed by flow cytometry.

 
DNA alkylation by [3H]S23906. To quantify S23906-1 DNA adducts, sensitive and resistant cells were incubated with 1 µmol/L [3H]S23906 for various times and their DNA was extracted and the associated radioactivity was counted. Figure 4A shows the kinetics of DNA alkylation in the two cell lines: radiolabeled DNA was detected as early as 5 minutes after the addition of [3H]S23906 to the culture medium and the plateau was reached after ~1 hour. The extent of alkylation was similar in the two cell lines, with about three molecules of S23906-1 bound per million of base pairs. To measure the repair of the adducts, cells were incubated for 2 hours with 1 µmol/L [3H]S23906 and then washed and incubated in drug-free medium for various times before extraction of their DNA. Figure 4B shows that the DNA-associated radioactivity decreased slowly and similarly in the two cell lines, with ~40% of the initial radioactivity still associated with DNA 24 hours after incubation in drug-free medium. Taken together, these results suggest that the 15-fold resistance of KB/S23-500 cells is not due to an important decrease in either the formation or the removal of S23906-1 DNA adducts.


Figure 4
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  		Figure 4. A, DNA alkylation by [3H]S23906. Cells were incubated with 1 µmol/L [3H]S23906 for the indicated times and washed. DNA was extracted, and the associated radioactivity was counted. B, for measurement of the release of DNA adducts, cells were incubated for 2 hours with 1 µmol/L [3H]S23906 and then washed and incubated in drug-free medium for various times before extraction and radioactivity measurement of their DNA. Results are expressed as cpm/µM bp.

 
These findings prompted us to search for secondary DNA lesions that might be more predictive for the cytotoxicity of S23906-1. Because the resistant KB/S23-500 cells show some degree of cross-resistance to camptothecin, a drug that indirectly induces DSB, we decided to investigate whether S23906-1 could induce DSB.

Induction of DNA strand breaks. The alkaline comet assay was carried out to determine if exposure to S23906-1 was accompanied by induction of DNA strand breaks. The results show that exposure to S23906-1 for 4 hours was associated with a dose-dependent induction of DNA strand breaks (Fig. 5A ). A similar dose-dependent response was also obtained for the resistant KB/S23-500 cells, although the overall levels of DNA strand breaks were less than for the parental cells. For example, the fraction of cells with highly damaged DNA (defined as >20% of the nuclear DNA in the comet) after exposure to 2, 5, and 10 µmol/L S23906-1 corresponded to 13%, 21%, and 34% for the sensitive KB-3-1 cells compared with 1%, 4%, and 13%, respectively, for the resistant KB/S23-500 cells. Essentially similar results were obtained when the parental and resistant KB-3-1 cells were exposed to S23906-1 for 1 hour (data not shown), indicating that the induction of DNA strand breaks is an early event.


Figure 5
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  		Figure 5. Induction of DNA strand breaks: comet assay. Cells were exposed to the indicated concentrations of S23906-1 for 4 hours and subjected to single-cell electrophoresis under alkaline (A) or neutral (B) conditions. The extent of DNA damage was determined by image analysis of at least 100 cells per concentration.

 
The alkaline comet assay detects both single-strand breaks and DSBs. To distinguish between the two types of DNA damage, parental and resistant KB cells were exposed to different concentrations of S23906-1 and the induction of DNA damage was determined by the neutral comet assay, which only detects DSBs. The results show that S23906-1 exposure was associated with the formation of DSBs, which became more pronounced over time (Fig. 5B). Although the results from the alkaline and the neutral comet assay are not directly comparable, it is noticeable that the fraction of cells with damaged DNA is lower for the neutral comet assay than for the alkaline comet assay. Together, our results clearly show that S23906-1 exposure is associated with formation of DNA strand breaks, of which some correspond to DSBs.

Histone H2AX phosphorylation. The phosphorylation of histone H2AX by the ataxia telangiectasia mutated (ATM) kinase is an early event observed after the introduction of DSB into DNA by ionizing radiation or antitumor drugs (25, 26). The high number of phosphorylated residues covering each DSB confers to this assay a high sensitivity, allowing the detection of a few DSB per cell. As shown in Fig. 6A , S23906-1 induced a concentration-dependent increase of {gamma}H2AX, the percentage of positive cells being as high as 90% for the parental cells after 24-hour exposure to 1 µmol/L. In the resistant cells, phosphorylation of H2AX was also concentration dependent, but higher concentrations of S23906-1 were required to achieve the same response. Biparameter flow cytometric analysis ({gamma}H2AX and cell cycle distribution) shows that the S23906-1-induced expression of {gamma}H2AX was restricted to S-phase cells (Fig. 6B). Representative images of KB-3-1 cells show an intense {gamma}H2AX staining in nearly all nuclei, whereas in KB/S23-500 cells {gamma}H2AX staining is weaker with some unstained nuclei. We next studied the effect of aphidicolin, a specific inhibitor of replicative DNA polymerases, on {gamma}H2AX formation after S23906-1. Figure 6C shows that phosphorylation of histone H2AX was markedly reduced when cells were pretreated by aphidicolin before S23906-1 exposure. The kinetics of {gamma}H2AX formation was also examined (Fig. 6D). Sensitive cells were strongly {gamma}H2AX positive at 1 µmol/L S23906-1 after 24 hours and remained at the same levels for the next 48 hours. In contrast, although the initial rate of DSB formation was similar in resistant and sensitive cells (28% and 29% at 1 hour, respectively), we observed, from 28 to 72 hours, a decrease of {gamma}H2AX-positive cells with 22% at 72 hours in the resistant line, whereas 92% to 93% of KB-3-1 cells remained positive, suggesting a higher repair of DSB in resistant cells.


Figure 6
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  		Figure 6. Histone H2AX phosphorylation. Cells were exposed to the indicated concentrations of S23906-1 for 24 hours, fixed, and labeled with an anti-phosphorylated histone H2AX mAb. Propidium iodide was added, and cells were analyzed by flow cytometry. A, % of {gamma}H2AX-positive cells were reported as a function of S23906-1 concentration. B, typical biparametric histograms of {gamma}H2AX expression as a function of DNA content and representative photos of {gamma}H2AX staining in KB-3-1 and KB/S23-500 treated with 1 µmol/L of S23906-1 for 24 hours. Arrows, unstained nuclei. Bar, 10 µm. C, effect of aphidicolin on histone H2AX phosphorylation by S23906-1. KB-3-1 cells were preincubated for 30 minutes with 2 µmol/L aphidicolin (APH) or with PBS, and then S23906-1 was added at the indicated concentrations for 4 hours. {gamma}H2AX-positive cells were reported as a function of S23906-1 concentration. D, kinetics of histone H2AX phosphorylation. Cells were incubated with 1 µmol/L S23906-1 for 1 to 72 hours, fixed, and labeled. {gamma}H2AX-positive cells were reported as a function of time.

 
Phosphorylation of Chk2. Following the generation of DSB in DNA by ionizing radiation, ATM kinase phosphorylates various substrates, including the checkpoint kinase Chk2, which, when activated, triggers cell cycle arrest and apoptosis (27). Another monofunctional alkylating agent, irofulven, was shown to activate Chk2 (28). Therefore, we examined whether S23906-1 could induce the phosphorylation of Chk2. In preliminary experiments, phosphorylated Chk2 protein was detected after 90-minute exposure of KB-3-1 cells to 1 µmol/L S23906-1 and peaked after 6 to 8 hours (data not shown). Figure 7 shows that S23906-1 induced Chk2 the phosphorylation in a concentration-dependent manner and that the levels of activated Chk2 are lower in the resistant than in the sensitive cells for all examined concentrations of S23906-1. These findings suggest that the G2-M arrest observed by flow cytometry is a DNA damage checkpoint response that is attenuated in the resistant cell line.


Figure 7
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  		Figure 7. Chk2 phosphorylation. Cells were treated with various concentrations of S23906-1 for 6 hours, and phosphorylated Chk2 was analyzed by Western blotting. Membrane was also probed with antibody to ß-actin as an internal control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
S23906-1 is a novel DNA alkylating agent with an interesting pharmacological profile (7, 9, 12–14). Because this promising compound is currently being evaluated in phase I clinical trials, it is of interest to investigate the molecular determinants for sensitivity and resistance of tumor cells. For this purpose, we have established a resistant cell line by stepwise exposure of KB-3-1 cells to S23906-1 to undertake a comparative study of the cellular responses to the compound. The resistance proved stable when cells were cultured in drug-free medium and was maintained in vivo in s.c. xenografts in the mouse, as shown by the significantly lower sensitivity to S23906-1 and lack of tumor regression of the KB/S23-500 xenografts in nude mice.

The cross-resistance profile, as well as the lack of P-gp overexpression in the resistant cells and the similar uptake of rhodamine 123 by sensitive and resistant cells, argued against a decreased uptake of S23906-1 as the resistance mechanism. This was unambiguously confirmed by the direct measurement of the number of tritiated S23906-1 molecules bound to DNA, which was similar in sensitive and resistant cells. About three molecules of S23906-1 bound per 106 bp, after exposure of the cells to 1 µmol/L S23906-1 (~7-fold the IC50 for KB-3-1 cells), were found for both the parental and the resistant cell lines. Interestingly, similar values were previously reported for cisplatin (29), with 3.18 DNA adducts per 106 bp in CEM cells exposed to 10 µmol/L (~10-fold the IC50 of cisplatin in this cell line). No major difference between the parental and the resistant lines was found with regard to both the formation and the removal of S23906-1 adducts. Despite the lack of data about the precise location of the adducts formed in the DNA of living cells, which could modulate their toxicity, these results suggest that a similar number of adducts elicit different cellular responses in the resistant cells.

Among the drugs tested, low but significant levels of cross-resistance were observed for camptothecin, cisplatin, and cytarabine. The cross-resistance to camptothecin, a topoisomerase I inhibitor, was unexpected because S23906-1 has no detectable activity toward purified topoisomerase I (8). Camptothecin acts through stabilization of the topoisomerase I cleavage complex due to the inhibition of the religating step (30). Because this ternary complex dissociates on removal of the drug, it is generally accepted that it can be further converted into a highly toxic DSB, resulting from the collision of the replication fork with the stabilized cleavage complex (31). The cytotoxic DNA lesion that ultimately leads to apoptosis is thus a DNA replication-mediated DSB. By using the highly sensitive H2AX phosphorylation assay as a surrogate marker for DSB formation, it was shown that the induction of {gamma}H2AX by camptothecin in HCT116 cells is restricted to S-phase cells and inhibited by the DNA replication inhibitor aphidicolin (32). Similar results were obtained with topotecan (a camptothecin derivative used for the treatment of ovarian carcinoma) in HL60 cells (33).

The nature of the lesions generated by cisplatin is more complex: in addition to intrastrand cross-links, which are the main lesions in cellular DNA, this drug can also induce interstrand cross-links and DNA-protein cross-links (34). Although none of these primary lesions are DNA DSB, their processing can lead to DSB. Recent data showed that cisplatin induces {gamma}H2AX accumulation mainly in S-phase cells and that the {gamma}H2AX foci colocalize with bromodeoxyuridine foci (35). The cisplatin-induced DSB appears thus as a consequence of DNA replication of damaged DNA.

The resistance of KB/S23-500 cells to cytarabine was unexpected. Cytarabine is a nucleoside analogue that is widely used for the treatment of leukemia. The phosphorylated cytarabine competes with dCTP for incorporation into DNA, resulting in stalling of the replication fork (36). Although the exact nature of the DNA damage generated by cytarabine in stalled replication forks has not been elucidated, DNA replication-mediated DSB may be produced following recruitment of DNA topoisomerase I or II to the replication fork (37).

Taken together, these published observations indicate that DSB formation might be a common cellular response to all three drugs. Considering the resistance profile, we hypothesized that S23906-1 could indirectly induce the formation of DNA replication-mediated DSB. We have shown using the comet assay that S23906-1 exposure is indeed associated with formation of DNA strand breaks, of which some correspond to DSBs.

Interestingly, more marked induction of {gamma}H2AX was observed for the parental cells compared with the resistant cells, suggesting that the resistance of KB/S23-500 cells is associated with reduced levels of DSB. These findings are consistent with the lower capacity of S23906-1 to activate Chk2 in the resistant cells. The fact that expression of {gamma}H2AX was detectable as soon as 1 hour after incubation of KB-3-1 cells with S23906-1 and peaked at 24 hours shows that these DSBs are early DNA lesions and not related to apoptosis-associated DNA fragmentation. Interestingly, the kinetics of the {gamma}H2AX signal suggest that the decreased levels of DSB in the resistant cells are more likely due to increased DNA repair than to a lower rate of formation. As described for camptothecin and cisplatin, these DSBs induced by S23906-1 were found mainly in S-phase cells and were inhibited by aphidicolin, an inhibitor of DNA polymerases.

The initial DNA lesions induced by S23906-1 are probably covalent monofunctional adducts, which could be eliminated by nucleotide excision repair, as recently described for irofulven (38). The fact that >40% of DNA-associated radioactivity was removed after 24 hours strongly suggests that such a DNA repair system is operating in the two KB cell lines with a similar efficiency. These primary adducts could then be converted in DSB in S-phase cells by the processing of the replication fork across the initial covalent adducts. Then, these DSBs can induce an irreversible arrest of cells in G2 phase of the cell cycle followed by apoptosis. The resistance of the KB/S23-500 cells seems to be more likely linked to DSB repair than excision repair.

In conclusion, the results obtained in this work suggest that replication-mediated DNA DSB are an important, secondary lethal lesion induced by S23906-1. This mechanism of cell killing could have important consequences for a rational use of S23906-1 in further phases of clinical trials, for example, for the choice of the combination of cytotoxic agents. In addition, these studies suggest that expression of {gamma}H2AX may be a useful clinical marker for monitoring the antitumor activity of S23906-1.


   Acknowledgments
 
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.

We thank Prof. John A. Hickman for his continuous interest in this work and for his critical reading of the article, Dr. Christian Bailly for helpful discussions, and Stéphanie Giraudet, Laétitia Marini, Valérie Pérez, Aurélie Studeny, Pascal Aumond, and Kate Wetenhall for excellent technical assistance.

Received 11/ 3/05. Revised 4/10/06. Accepted 5/12/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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