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The radiosensitizer 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide induces DNA damage in EMT-6 mammary carcinoma cells

The radiosensitizer 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide induces DNA damage in... Background: DCQ (2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide), a synthetic quinoxaline 1,4-dioxide, enhances the cytotoxic effect of ionizing radiation (IR) in vivo and in vitro. We sought to clarify whether increased radiation-induced DNA damage, decreased rate of damage repair, and the generation of reactive oxygen species (ROS) contribute to DCQ enhancement of IR. Methods: Murine mammary adenocarcinoma EMT-6 cells were treated with DCQ for 4 h before exposure to 10 Gy IR. Treated cells were monitored for modulations in cell cycle, induction of DNA damage, and generation of ROS. Results: Combined DCQ and IR treatments (DCQ+IR) induced rapid cell-cycle arrests in EMT-6 cells, particularly in S and G /M phases. Alkaline comet assays revealed high levels of DNA damage in cells after exposure to DCQ+IR, consistent with damage-induced arrest. Unlike IR-only and DCQ-only treated cells, the damage induced by combined DCQ+IR was repaired at a slower rate. Combined treatment, compared to separate DCQ and IR treatments, activated DNA-protein kinase and induced more p-ATM, supporting a role for double strand breaks (DSBs), which are more toxic and difficult to repair than single strand breaks (SSBs). Contributing factors to DCQ radiosensitization appear to be the induction of ROS and DSBs. Conclusion: Collectively, our findings indicate that radiosensitization by DCQ is mediated by DNA damage and decreased repair and that ROS are at least partially responsible. hence, DNA synthesis and replication may proceed Background Eukaryotic cells have evolved DNA damage checkpoints despite the presence of unrepaired DNA damage, leading that control the fate of an insulted cell by inducing cell- eventually to unviable daughter cells [1,2]. Thus, such cycle arrest, repair of the damage, and cell death. Many malignant cells are sensitive to therapies that induce DNA malignant cells have incompetent cell-cycle controls, and damage [3]. Page 1 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 Some aromatic N-oxides such as quinoxalines induce Cell Culture, Drug and Irradiation Treatment DNA damage in cancer cells. The hypoxic cytotoxin 7- The murine mammary adenocarcinoma cell line EMT-6 chloro-3-[(N, N-dimethylamino) propyl]amino]-2-qui- was cultured in growth media containing RPMI 1640 with noxalinecarbonitrile 1,4-dioxide hydrochloride (Q-85 L-glutamine and 25 mm HEPES, supplemented with 10% HCl) has been shown to induce DNA damage under FBS and 1% penicillin-streptomycin (50 μg/mL), and hypoxic conditions in CaCo-2 cells by producing reactive incubated in a humidified incubator (95% air 5% CO ) at oxygen species (ROS) [4,5]. The mechanism of action of 37°C (Forma Scientific Inc. Ohio, US). such compounds is not yet clear. However, studies on qui- noxaline 1,4-dioxide has shown that it is reduced enzy- DCQ was dissolved in DMSO at a concentration of 10 matically into an active, oxygen-sensitive radical mg/mL. Prior to treatment, it was diluted in media con- responsible for DNA cleavage [6]. taining FBS. EMT-6 cells were plated at a density of 16 × 3 2 10 cells/cm . At 50% confluency, they were incubated A similar quinoxaline, 2-benzoyl-3-phenyl 6,7-dichloro- with DCQ (0–10 μM) for 4 h prior to irradiation (0–10 quinoxaline 1,4-dioxide (DCQ) has been shown to be Gy). cytotoxic and a radiosensitizer on several cancer cell lines, including colon cancer cells. The radiosensitization effect Cells were irradiated at room temperature using a high was also shown in vivo, using C57BL/6 mouse model [7]. dose rate Cesium-137 Laboratory Irradiator (JL Shepherd) Combined treatment with DCQ and radiation delayed the that delivers gamma-irradiation at a dose rate of 174 cGy/ growth of LLC tumors injected in the mice and reduced min. After irradiation, cells were replenished with fresh the mean tumor volume by 80% [7]. Recent results have media containing no drug and incubated for different shown that DCQ causes DNA damage in DLD-1 colon times. cancer cells [8]. Despite data in vitro and in vivo confirming that DCQ is a radiosensitizer little is known about its The murine mammary epithelial cell line SCp2 (kindly mechanism of action. In this study, we first assessed the provided by R. Talhouk, Biology Department, American effects of DCQ ± IR on cell cycle progression at early time- University of Beirut, Lebanon) was used as a model for points. Then, we tested whether DCQ radiosensitization normal, slowly proliferating cells [10]. SCp2 cells were is associated with an enhancement in radiation-induced grown in normal growth media composed of DMEM: F12 DNA damage or with a decrease in the rate of damage supplemented with 5% FBS, 1% Penicillin-Streptomycin, repair. Finally, we investigated the possible involvement and 0.1% insulin (5 μg/mL, Sigma, St. Louis). To induce of ROS in the mechanism of DCQ toxicity. differentiation of the SCp2 cells, the cells were plated in growth media and 12 later the media was replaced with differentiation medium lacking FBS [10]. The differentia- Methods Chemicals tion medium consisted of DMEM: F12 supplemented RPMI 1640 with 25 mm HEPES and L-glutamine, Dul- with 0.1% insulin (5 μg/mL), 0.1% hydrocortisone (1 μg/ becco's modified eagle medium nutrient mixture F12, mL), and 0.1% prolactin (3 μg/mL). For a more differen- fetal bovine serum, trypsin, penicillin-streptomycin and tiated state, a growth factor reduced basement membrane Dulbecco's Phosphate Buffered Saline (PBS) were pur- derived from Engelbreth-Holm-Swarm tumor was added chased from Gibco BRL Life Technologies (Gaithersburg, 12 h after plating. A basement membrane is known to Maryland, US). The Cytotox non-radioactive cytotoxicity induce differentiation in SCp2 cells by making their envi- assay kit and the Cell Titer 96 non-radioactive cell prolif- ronment more similar to that of normal cells [10]. eration assay kit were purchased from Promega Corp (Madison, Wisconsin, US). Propidium iodide (PI), Proliferation and Cytotoxicity Assay YOYO-1 dye, fluorescein isothiocyanate (FITC) goat anti- Cells were plated at a density of 10 cells/mL in 96-well mouse IgG (H+L), and 5-(and-6)-chloromethyl-2',7'- plates. After 24 h, cells were treated in triplicates with dif- dichlordihydrofluorescein diacetate, acetyl ester (CM- ferent DCQ concentrations. In some experiments, EMT-6 H DCFDA) were purchased from Molecular Probes cells were pre-treated with either NAC (5 mM) or Tiron (1 (Eugene, Oregon, US). RNase A, dimethylsulfoxide mM) for 2 h prior to DCQ treatment. (DMSO) and N-acetyl cysteine (NAC) were obtained from Sigma Chemical Company (St. Louis, Missouri, US). ATM Cytotoxicity was performed after 4 h of DCQ treatment kinase phosphoser1981 antibody was obtained from using the Cell Titer 96 non-radioactive cytotoxicity kit. Chemicon International (California, US). DCQ was syn- Briefly, supernatants were mixed with a substrate mix con- thesized from 5,6-dichlorobenzofurazan oxide and taining tetrazolium salt that interacts with lactate dehy- dibenzoylmethane by the Beirut Reaction [9]. drogenase, a stable cytosolic enzyme that is released into the supernatant upon cell lysis. The interaction results in Page 2 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 the conversion of the tetrazolium salt into a red formazan microscope (AXIOVERT 200, ZEISS Fluorescence and product, the absorbance of which is recorded at 492 nm. optical microscope with ZEISS AXIOCAM HRC (Ger- many) and KS 300 V3 image analysis software) illumi- As for the proliferation assays, cells were replenished with nated with blue light (490 nm). Images of a minimum of drug-free media after the 4 h-DCQ treatment, and were 50 cells per treatment were analyzed using the Comet- incubated for 20 h before the assay was performed using Score™ software. In the present study, percentage of DNA the Cell Titer 96 non-radioactive cell proliferation. This in the tail region, and tail moment (%DNA in tail × by tail assay measures the ability of metabolically-active cells to length (μm)) were used as parameters to assess DNA dam- convert tetrazolium salt into a blue formazan product that age. can be measured by its absorbance at 595 nm. Immunocytochemistry Detected by Flow Cytometry Flow Cytometry Ser-1981-phosphorylated ATM (p-ATM) was detected Cells were either treated with 0.1% DMSO (control), immunocytochemically by multiparameter cytometry DCQ (0–10 μM) for 4 h, irradiation (10 Gy), or combina- with respect to the cell cycle phases, using the method tions. Immediately after radiation or drug treatment, cells developed by Huang and Darzynkiewicz [12]. Cells were were replenished with fresh media containing no drug collected by trypsinization, centrifuged, washed with PBS, and incubated for 0 h, 2 h, and 4 h. Subsequently, cells and fixed with ice-cold 70% ethanol for a minimum of 2 were harvested and fixed in ice-cold 70% ethanol and h at -20°C. Ethanol was discarded by centrifugation at a stored at -20°C. On the day of DNA staining, cells were speed of 10000 rpm for 5 min, and the pellets were incubated in 0.2 mg/mL RNase A at 37°C, and stained washed with BSA-T-PBS containing 1% BSA and 0.2% Tri- with 6.25 μg/mL PI for 30 min in the dark at room tem- ton X-100 dissolved in PBS. The pellets were blocked in perature. Finally, cell cycle analysis was performed using a BSA-T-PBS for 5 min at room temperature. After removal Fluorescence Activated Cell Sorter (Becton Dickinson, of the 1% BSA solution by centrifugation, the cells were Research Triangle, NC), and the percentages of cells in incubated with the primary antibody Ser-1981-p-ATM at sub-G (< 2n), G /G , S and G /M phases were deter- a dilution of 1:100 overnight at 4°C. The cells were 1 0 1 2 mined using the Cell Quest program (BD Biosciences, washed twice with BSA-T-PBS, and the pellets were then California, USA). incubated in the dark with fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse antibody (1:30) DNA Damage Detection by the Alkaline Comet Assay for 1 h at room temperature. A volume of 5 mL of BSA-T- The alkaline comet assay used is a modification of the PBS was added to the cell suspension and kept for 2 min method developed by Singh that detects the frequency of before centrifugation at 12000 rpm for 4 min. Finally, the SSBs and alkaline-labile lesions in DNA [11]. Microscope cells were counterstained with PI (5 μg/mL) solution con- slides were coated with 1% normal melting agarose, and taining RNase A (0.1 mg/mL) for 30 min at room temper- left overnight to dry. Cells suspended in media were ature in the dark. Both the fluorescence of PI and FITC of mixed with 75 μL of 0.5% low-melting-point agarose 10 cells/treatment were measured using the FACS cytom- (LMPA) and were distributed on the coated slide. The eter, and analyzed using Cell Quest. slides were left to gel for 10 min at 4°C, before a third layer of 80 μL 0.5% LMPA was added to the slide and left Detection of ROS by DCFDA assay 3 2 Cells were plated at a density of 16 × 10 cells/cm and for 10 min at 4°C. The slides were then dipped in cold lys- ing solution (1.25 M NaCl, 50 mM EDTA, 100 mM Tris treated at 50% confluency with 10 μM DCQ for 30 min. base and 0.01% sodium lauroyl sarcosine; pH 10) for a Control and treated cells were collected by trypsinization, minimum of 2 h at 4°C. Before proceeding, the slides centrifuged, washed with PBS, and incubated in 500 μl of were incubated in pre-warmed lysing buffer containing media (with 2% FBS) containing 10 μM of DCFDA for DNAse-free proteinase K for 1 h at 37°C. The slides were 20–30 min at 37°C. DCFDA is a chemically-stable, non- transferred to an electrophoresis unit filled with electro- fluorescent molecule that is hydrolyzed to DCFH inside phoresis buffer (300 mM NaOH, 1 mM EDTA, 0.2% the cell. DCFH interacts with ROS to form a fluorescent DMSO, and 0.1% 8-hydroxyquinoline; pH ~12.3), and complex. Samples were then centrifuged, washed with were left immersed in the solution for 20 min, before PBS, and then resuspended in 500 μl of PBS. The fluores- being subjected to electrophoresis. Electrophoresis was cence of DCF was immediately measured by flow cytome- carried out for 20 min at a voltage of 0.5 V/cm and a cur- try. rent of 250 mA. Next, the slides were rinsed with neutral- ization buffer (20 mM Tris, 1 mg/mL spermine, and 50% Chromatin Immunoprecipitation Followed by Western ethanol; pH 7.4) for 10 min. Finally, each slide was Blot stained with 50 μL of YOYO-1 stain (0.25 μM YOYO-1, Chromatin immunoprecipitaion was performed using 2.5% DMSO and 0.5% sucrose). YOYO-stained nuclei Chromatin Immunoprecipitation (CHIP) Assay Kit were observed and photographed using a fluorescence (Upstate, New York, USA) according to the manufac- Page 3 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 turer's protocol. Briefly, EMT-6 cells were treated at 70% diately after drug treatment, and this accumulation confluency. DNA-binding proteins were cross-linked to increased to 26% after 2 h. Although IR alone and DCQ /M DNA by adding 1% formaldehyde for 10 min at 37°C. alone caused similar level of arrests at the S + G Cells were washed twice with ice-cold PBS containing pro- phases, they induced distinct cell distribution profiles tease inhibitors (1 mM PMSF, 1 μg/mL aprotinin and 1 where IR caused more intra-S phase arrest, while DCQ μg/mL pepstatin A). Cells were collected and centrifuged induced more G /M arrest suggesting differences in their at a speed of 2000 rpm for 4 min at 4°C. Pellets (of 10 mechanisms of action. The combination treatment of cells) were lysed with SDS Lysis buffer (provided by the DCQ+IR resulted in a strong arrest at 4 h where 61% of kit) containing protease inhibitors. The chromatin, the population accumulated in the S+G /M phases. More- including bound proteins, was sonicated into smaller over, a significant increase in cell death represented by the fragments (200–1000 base pairs) using Misonix Sonicator sub-G population was associated with DCQ+IR (12.1% 3000 at 10% power (3 W) for seven 10-second pulses sep- at 4 h versus 2.5% in non-treated cells). Even at early time- arated by a 5 second-pause. Samples were centrifuged for points this cytotoxic effect of the combination treatment 10 min at 13000 rpm at 4°C, and the supernatants were appears to be at least additive. These results corroborate diluted 10 fold in CHIP dilution buffer (provided by the previous results that DCQ is anti-proliferative. We kit) and pre-cleared with protein A agarose/salmon sperm hypothesize that DCQ and IR act via different mecha- DNA (50% slurry). DNA-PK antibody (1.5 mg/mL) was nisms. DCQ may cause DNA damage such as double- used to co-immunoprecipitate the protein-DNA complex strand breaks (DSBs) or bulky adducts, which are known which was then washed with different buffers: low salt to induce S and G /M arrest [14]. immune complex, high salt immune complex, LiCl immune complex wash buffers, as well as two washes with DCQ Induces DNA Damage in EMT-6 Cells TE (Tris-EDTA) buffer. Proteins were dissolved in 25 μL 1× Since DNA damage is the primary cause of arrest at S or sample buffer, boiled for 10 min, and resolved on a 5% G /M phases, we tested whether DCQ induces DSBs in acrylamide gel to detect the level of DNA-PK by western EMT-6 cells by using neutral comet assay. Although both blotting. treatments were observed to induce DSBs, the fluores- cence intensity was too low to detect significant difference Western Blot in the level of DSBs between DCQ and IR treatments (data Proteins were resolved by sodium dodecyl sulfate-polyacr- not shown). The alkaline comet assay detects SSBs and ylamide gel electrophoresis (SDS-PAGE) on a 5% polyacr- alkaline-labile DNA damage, such as abasic sites. Using ylamide gel, and transferred onto an activated the alkaline comet assay, we detected the level of damage polyvinylidene difluoride (PVDF) membrane in cold induced by DCQ ± IR in exponentially growing EMT-6 transfer buffer (14.4 g of glycine, 3 g Tris base, and 1 g SDS (Figure 2A, B). Cells were treated at 50% confluency with dissolved in 1 L of 20% methanol) at 30 volts overnight. 10 μM DCQ and the assay was directly performed after a The membrane was then blocked for 1 h with 5% non-fat 4 h-incubation with DCQ, IR treatment, or combination milk dissolved in Tris-buffered saline (TBS) containing treatment. Treatment with DCQ alone induced significant 0.1% Tween-20, and probed with DNA-PK antibody levels of damage, similar to that induced by 10 Gy IR. In diluted in 1% blocking buffer overnight at 4°C. The mem- response to combined DCQ and IR treatment, higher lev- brane was incubated with horseradish peroxidase-conju- els of damage were observed: tail moment (%DNA in tail gated secondary antibody 1 h at room temperature. The × tail length) increased by 19.6-fold in comparison with membrane was exposed to X-ray film (Hyperfilm ECL) untreated cells. using chemiluminescent substrate (Amersham). DCQ Activates ATM and DNA-PK in Irradiated EMT-6 Cells Results DCQ Induces S Phase and G2/M Arrest in EMT-6 Cells The nuclear kinase ATM is rapidly phosphorylated in the Previous work has shown that DCQ, in combination with presence of low levels of DSBs [15]. The immunocyto- IR, induces apoptosis in EMT-6 cells 24 h post-treatment, chemical detection of p-ATM thus provides a sensitive and decreases their clonogenic survival [13]. To determine approach to detect double-strand breaks (DSBs) gener- the direct effects of DCQ ± IR on cell cycle progression of ated following drug treatment in cells [16]. Cells were EMT-6, cells were treated with 10 μM DCQ for 4 h fol- treated with DCQ (10 μM), IR (10 Gy) or combinations lowed by irradiation with 10 Gy IR, or separately treated. followed by replenishment with drug-free media. After 2 Treated cells were collected for flow cytometry either h, cells were collected and the level of p-ATM in relation directly (0 h), or at 2 h or 4 h after IR exposure (Figure 1). to the cell cycle was assayed in EMT-6 cells for each treat- IR induced cell-cycle arrest in the S phase at 2 h post-expo- ment by subjecting the samples to immunocytochemistry sure and this arrest increased at 4 h with 27% of the pop- (Figure 3A, B). As expected, control cells showed the basal ulation accumulated in the S phase. DCQ alone also level of p-ATM expression was higher in G /M population caused an accumulation of cells in the late S phase imme- due to the role of ATM in mitosis. Exposure of EMT-6 cells Page 4 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 S and G2/M arr Figure 1 est induced by DCQ ± IR in EMT-6 cells at 0, 2, and 4 h post-treatment S and G2/M arrest induced by DCQ ± IR in EMT-6 cells at 0, 2, and 4 h post-treatment. EMT-6 cells were treated at 50% confluency with DCQ (10 μM) for 4 h, and/or irradiated (10 Gy). Immediately after the 4 h-drug incubation or IR expo- sure, cells were replenished with media containing no drug. Cells were collected at 0 h, 2 h, and 4 h after the refreshment of media and subjected to flow cytometry. Percentages of cells in the sub-G1 (A), S (B), and G2/M (C) phases of the cell cycle were determined by CellQuest and the averages ± SD are plotted for each treatment. Dashed lines represent the % of control cells in each phase of the cell cycle. SD: standard deviation. to 10 μM DCQ triggered the activation of ATM by phos- significant increase in the active DNA-PK level was phorylation at Ser-1981; this phosphorylation level was induced in response to DCQ+IR (Figure 3C). higher than that of 10 Gy-treated cells, reflecting higher amounts of DSBs generated by DCQ than IR. The combi- Slow Repair of Damage Observed in EMT-6 Exposed to nation treatment had the highest levels of p-ATM over DCQ+IR untreated cells reaching 2 fold only in the G /M phase. The time required to repair DNA depends on the type of DCQ and DCQ+IR induced the activation of ATM in all damage. SSBs are usually repaired much faster than DSB phases of the cell cycle similar to the Topoisomerase II after induction [19]. To assess whether DCQ toxicity is inhibitor mitoxantrone [17]. due to the extent of damage induced or to slow repair fol- lowing treatment, the extent of damage was assessed by Another major kinase activated by DNA damage is DNA- the alkaline comet assay at 0 h, 4 h and 16 h post-treat- PK, which is activated by binding to the damaged sites on ments. Although a large extent of the damage induced by DNA [18]. The binding of DNA-PK to DNA was evaluated IR alone and DCQ alone is repaired in less than 4 h, we by DNA-PK chromatin immunoprecipitation followed by observed dramatically slowed repair of the damage western blotting with an antibody against DNA-PK. We induced by DCQ+IR. Even at 16 h, significant DNA dam- observed that untreated cells had no significant DNA-PK age remained unrepaired as evidenced by tail moments. bound to the DNA, but a moderate signal was detected in Damage was significantly higher (P-value < 0.01) in EMT-6 cells after 10 μM DCQ or 10 Gy IR, and a highly response to DCQ+IR as compared to untreated and singly- treated cells (Figure 4). Page 5 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 DNA dama Figure 2 ge induced by DCQ+IR in EMT-6 cells DNA damage induced by DCQ+IR in EMT-6 cells. A. Representative images of comets induced by DCQ± IR in EMT-6 cells subjected to the alkaline comet assay. EMT-6 cells were treated with IR (10 Gy), 4 h of DCQ (10 μM), or in combination. Cells were directly collected after the treatment and subjected to the alkaline comet assay. Images were taken using a fluores- cent microscope at 40× (oil immersion) magnification. The tail lengths of the comets observed by each treatment are propor- tional to the amount of DNA damage induced. B. The mean of the parameters (% DNA in comet's tail and tail moment) are shown in the graphs above. The histograms summarize the averages of two independent experiments ± SE and show the mean of the %DNA in comet's tail and tail moments. More than 50 cells per treatment were photographed and quantified using TriTek CometScore software. SE: standard error. DCQ Generates Reactive Oxygen Species in EMT-6 cells DCQ Targets Rapidly-Proliferating Cells N-oxides undergo redox-cycling producing reactive oxy- Because DCQ appears to slow repair, we expected that tox- gen species (ROS) [20]. We hypothesized that DCQ may icity would depend on proliferation rate. We assessed cause DNA damage by ROS induction due to its redox- whether reducing proliferation would decrease DCQ tox- cycling. Indeed, DCQ treatment alone, either directly or icity by culturing murine mammary epithelial cell line indirectly, induced the generation of ROS in EMT-6 cells SCp2 under conditions to induce differentiation and after 30 min of treatment as measured by the DCFH assay thereby slow proliferation. When cultured in differentia- (Figure 5A). To determine if ROS play a role in the radio- tion media, SCp2 proliferation rate was reduced to sensitizing effect of DCQ in EMT-6 cells, strong anti-oxi- approximately 50% compared to cells cultured in normal dants such as Tiron and NAC were added before treatment growth media, and an even stronger decrease in prolifera- with DCQ alone or in combination with IR, to scavenge tion was observed with cells supplemented with basement any DCQ-generated ROS. Cells pretreated with anti-oxi- membrane (Figure 6A). After 4 h of DCQ treatment, dants were more resistant to the anti-proliferative effect of slowly proliferating SCp2 cells were more resistant to DCQ. However, the anti-oxidants did not completely toxic concentrations of 10 μM DCQ, suggesting selective abolish the anti-neoplastic effect of DCQ whether alone toxicity to proliferating cells (Figure 6B). or in combination with IR (Figure 5B, C). These results indicate that ROS play at least a partial role in the radio- Discussion sensitizing effect of DCQ in EMT-6 cells. Several mechanisms of radiosensitization are known, including redox modulators [21], inhibitors of DNA dam- Page 6 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 Figure 3 Phosphorylation of ATM and activation of DNA-PK by DCQ ± IR in EMT-6 cells at 2 h post-treatment Phosphorylation of ATM and activation of DNA-PK by DCQ ± IR in EMT-6 cells at 2 h post-treatment. A. EMT- 6 cells were treated with 10 μM DCQ, 10 Gy IR, or combination treatments, fixed and subjected to immunocytochemical detection of ATM phosphorylated on Ser1981, and stained with PI to detect at the same time p-ATM in each phase of the cell cycle. B. The mean of the FL-1 intensity (reflecting the level of p-ATM expression) at each phase of the cell cycle are plotted. C. Anti-DNA-PK was immunoprecipitated with DNA from lysates of 10 EMT-6 cells treated with 10 μM DCQ, 10 Gy IR, or combination treatments using CHIP assay. The immunoprecipitate was resolved on a 5% gel by electrophoresis, transferred to nitrocellulose and probed with anti-DNA-PK. The bands were quantified using LabWorks 4.0 software. CHIP: chromatin immunoprecipitation. age repair [22], and regulators of growth factor receptors Here, we show for the first time that DCQ induces DSBs and other signaling molecules [23,24]. Misrepair of DNA in EMT-6 cells, in addition to SSBs and alkaline-labile damage causes mutation, and extensive damage may lesions detected by the alkaline comet assay. DCQ causes cause cell cycle arrest, or death if irreparable or too slowly more G2/M arrest than IR. Exposure of EMT-6 cells to 10 repaired. The role ROS can play in cellular response to μM DCQ produced damage detected by the alkaline radiation has been well established [25]. comet assay, and DSBs evaluated by p-ATM level, almost equivalent to that produced by 10 Gy IR. The combina- Sl Figure 4 ow repair of DNA damage in DCQ+IR-treated cells Slow repair of DNA damage in DCQ+IR-treated cells. Treated cells were either collected directly after treatment (0 h) or refreshed with drug-free media and incubated for another 4 or 16 h. The mean of the parameters (% DNA in comet's tail and tail moment) are shown in the graphs above. Page 7 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 ROS g Figure 5 eneration by DCQ in EMT-6 cells ROS generation by DCQ in EMT-6 cells. A. Cells were treated with 10 μM DCQ for 30 min before measuring ROS release by the DCFH-DA assay as described in the materials and methods. B & C. EMT-6 cells were pretreated for 2 h with the strong anti-oxidants Tiron and NAC, then with DCQ (2.5, 5 and 10 μM), and later subjected to 0 Gy (-IR) or 10 Gy (+IR) radiation. Afterwards, cells were replenished with drug-free media and incubated for an additional 20 h before the determina- tion of cell proliferation. The values plotted represent an average (± SD) of two independent experiments. ROS: Reactive oxy- gen species. tion of DCQ+IR induced significantly higher SSBs than phase arrest. Activated by DSBs, ATM becomes phosphor- each treatment alone. Radiosensitization of DCQ not ylated at Ser-1981 [15]. We show that ATM was activated only correlates with higher induction of DNA damage, but in all phases of the cell cycle in response to the damage also with slower repair of this damage. Alkaline comet induced by all treatments. In the combination treatment assays 4-hours post treatment revealed dramatically the expression of p-ATM in G /M phase was twice that of slowed repair of damage in DCQ+IR treated cells com- untreated cells. pared to separate IR or DCQ treatments. Little damage remained 4 h after separate treatments with DCQ or IR, Following IR treatment, EMT-6 cells arrest in S phase. supporting a model in which radiosensitization involves Such an arrest is mainly caused by the activation of the the generation of more difficult-to-repair DSBs. These intra-S-phase checkpoint due to significant amount of results suggest combination treatment may have thera- DSBs [28]. It is responsible for inhibition of DNA replica- peutic value. tion at late origins of replication. In addition to cell cycle arrest, the intra-S-phase checkpoint induces a cascade of DNA damage, in particular DSBs, imposes a critical threat reactions, that either attempt to repair the damage, mainly to the survival of cells if left unrepaired [26]. As a response by homologous recombination, or induce cell death, to the damage, cells activate the DNA damage checkpoint. depending on the extent of the damage induced. If dam- DSBs are detected by two main players in the DNA dam- age is not repaired before the end of the S phase, cells age checkpoint: ATM and DNA-PK. Signal transduction, would arrest at the G /M DNA-damage checkpoint [28]. induced by the activation of these two signals, can cause The G /M arrest induced by DCQ and the S-phase accu- cell-cycle arrest, repair, and cell death. Moreover, both are mulation induced by IR appear together in combination activated at very early stages of the DNA damage response, treatments. Despite our intriguing findings that the com- and are involved in DNA repair [27]. DNA-PK was acti- bination treatment DCQ+IR induces DNA damage, vated in response to DCQ alone more than IR alone. The including DSBs, and slows repair, the precise mechanisms combination treatment induced the highest amount of are still not clear. active DNA-PK. ATM plays a critical role in S and G /M Page 8 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 with increased levels of difficult-to-repair DSBs could overwhelm cellular DNA repair pathways. The proposed mechanism was observed to be selective for rapidly prolif- erating cells, presumably because slowly dividing cells have more time to repair DNA damage. This finding sug- gests clinical potential. Conclusion This study presents evidence that the radiosensitizing effects of DCQ are associated with an increase in DNA damage, including DSBs, the activation of the key DNA damage markers, p-ATM and DNA-PK, and the generation of ROS. The significant levels of unrepaired damage detected by alkaline comet assay in EMT-6 cells following treatment by DCQ+IR indicate that decreased DNA repair contributes to the mechanism of DCQ radiosensitization. Competing interests The authors declare that they have no competing interests. Authors' contributions JH carried out the experiments in the study and drafted Resi Figure 6 stance of slow proliferating cells to DCQ the manuscript. FG and CAS were involved in revising the Resistance of slow proliferating cells to DCQ. A. After manuscript critically for important intellectual content plating SCp2 cells in growth media (GM), they were sub- and CAS helped in preparing the final draft of the manu- jected to different conditions. Cells were either kept in GM, script. MH provided the compound and reviewed the or shifted 12 h after plating to differentiation media (DM) or manuscript. HGM conceived of the study, and partici- DM+BM (basement membrane) for a more differentiated pated in its design and coordination and drafting of the state. SCp2 cells were incubated for one day then assayed for manuscript. All authors read and approved the final man- proliferation using Cell Titer 96 non-radioactive cell prolifer- uscript. ation. The proliferation rate of cells grown in GM was con- sidered as 100%. B. The viability of SCp2 cells at different differentiating states were measured by Cytotox 96 non- Acknowledgements radioactive cytotoxicity assay directly after 4 h of DCQ This study was supported by the University Research Board of the Ameri- treatment. can University of Beirut and the Lebanese National Council for Scientific Research. References The slow repair of DNA damage caused by DCQ+IR may 1. Shapiro GI, Harper JW: Anticancer drug targets: cell cycle and have multiple contributing factors. DCQ appears to cause checkpoint control. J Clin Investig 1999, 104:1645-1653. 2. Hartwell LH, Kastan MB: Cell cycle control and cancer. Science more DSBs than IR, as evidenced by the increase in p-ATM 1994, 266:1821-1828. and DNA-PK levels. DCQ could create DSBs by generating 3. Gasser S: DNA damage response and development of tar- closely opposed SSBs via ROS. Our observation of ROS geted cancer treatments. Ann Med 2007, 39:457-464. 4. Azqueta A, Pachon G, Cascante M, Creppy EE, Lopez de Cerain A: generation upon DCQ treatment and the decrease in the DNA damage induced by a quinoxaline 1,4-di-N-oxide deriv- sensitivity of cells to DCQ upon addition of anti-oxidants, ative (hypoxic selective agent) in Caco-2 cells evaluated by support a role for redox cycling of DCQ. Although, the the comet assay. Mutagenesis 2005, 20:165-171. 5. Azqueta A, Arbillaga L, Pachon G, Cascante M, Creppy EE, López de ROS scavengers did not completely reverse the effect of Cerain A: A quinoxaline 1,4-di-N-oxide derivative induces DCQ alone or in combination with IR, this does not elim- DNA oxidative damage not attenuated by vitamin C and E treatment. Chem Biol Interact 2007, 168:95-105. inate the possibility that the radiosensitizing effect of 6. Ganley B, Chowdhury G, Bhansali J, Daniels JS, Gates KS: Redox DCQ may only involve ROS, because they may be mainly activated, hypoxia-selective DNA cleavage by quinoxaline short-lived hydroxyl radicals that are not quenched by the 1,4-di-N-oxide. Bioorg Med Chem 2001, 9:2395-2401. 7. Gali-Muhtasib H, Sidani M, Geara F, Assaf-Diab M, Al-Hmaira J, Hadd- anti-oxidants. adin M, Zaatari G: Quinoxaline 1,4-dioxides are novel angio- genesis inhibitors that potentiate antitumor effects of ionizing radiation. Int J Oncol 2004, 24:1121-1131. One possible mechanism of DCQ radiosensitization is 8. Itani W, Geara F, Haykal J, Haddadin M, Gali-Muhtasib H: Radiosen- that IR induces a higher concentration of the free radical sitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4- character of DCQ, which is translated into increased SSBs dioxide under oxia and hypoxia in human colon cancer cells. Radiat Oncol 2007, 2:1. and DSBs. The increased levels of SSBs, in combination Page 9 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 9. Haddadin M, Issidorides C: The Beirut Reaction. Heterocycles 1993, 35:1503-1525. 10. Talhouk RS, Mroue R, Mokalled M, Abi-Mosleh L, Nehme R, Ismail A, Khalil A, Zaatari M, El-Sabban ME: Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junc- tion-dependant differentiation of mammary epithelial cells. Exp Cell Res 2008, 314:3275-3291. 11. Singh NP: Sodium ascorbate induces DNA single-strand breaks in human cells in vitro. Mutat Res 1997, 375:195-203. 12. Huang X, Darzynkiewicz Z: Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage. Methods Mol Biol 2006, 314:73-80. 13. Haykal J, Fernainy P, Itani W, Haddadin M, Geara F, Smith CA, Gali- Muhtasib H: Radiosensitization of EMT6 mammary carcinoma cells by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-diox- ide. Radiother Oncol 2008, 86:412-418. 14. Orren DK, Peterson LN, Bohr VA: Persistent DNA damage inhibits S-phase and G2 progression, and results in apoptosis. Mol Biol Cell 1997, 8:1129-1142. 15. Bakkenist CJ, Kastan MB: DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003, 421:499-506. 16. Tanaka T, Kurose A, Huang X, Dai W, Darzynkiewicz Z: ATM acti- vation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis. Cell Prolif 2006, 39:49-60. 17. Kurose A, Tanaka T, Huang X, Halicka HD, Traganos F, Dai W, Darzynkiewicz Z: Assessment of ATM phosphorylation on Ser- 1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis. Cytometry Part A 2005, 68A:1-9. 18. Collis SJ, DeWeese TL, Jeggo PA, Parker AR: The life and death of DNA-PK. Oncogene 2005, 24:949-961. 19. Olive PL: The role of DNA single- and double-strand breaks in cell killing by ionizing radiation. Radiat Res 1998, 150:S42-S51. 20. Cerecetto H, Gonzalez M: N-oxides as hypoxia selective cyto- toxins. Mini Rev Med Chem 2001, 1:219-231. 21. Rosenberg A, Knox S: Radiation sensitization with redox mod- ulators: a promising approach. Int J Radiat Oncol Biol Phys 2006, 64:343-354. 22. Lawrence TS, Blackstock AW, McGinn C: The mechanism of action of radiosensitization of conventional chemotherapeu- tic agents. Semin Radiat Oncol 2003, 13:13-21. 23. Katz D, Ito E, Liu FF: On the path to seeking novel radiosensi- tizers. Int J Radiat Oncol Biol Phys. 2009, 73(4):988-996. 24. Wardman P: Chemical Radiosensitizers for use in radiother- apy. Clin Oncol (R Coll Radiol) 2007, 19:397-417. 25. Valerie K, Yacoub A, Hagan MP, Curiel DT, Fisher PB, Grant S, Dent P: Radiation-induced cell signaling: inside-out and outside-in. Mol Cancer Ther 2007, 6:789-801. 26. Vilenchik MM, Knudson AG: Endogenous DNA double-strand breaks: production, fidelity of repair and induction of cancer. Proc Natl Acad Sci USA 2003, 100:12871-12876. 27. Yang J, Yu Y, Hamrick HE, Duerksen-Hughes PJ: ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 2003, 24:1571-1580. 28. Bartek J, Lukas C, Lukas J: Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 2004, 5:792-804. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 10 of 10 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

The radiosensitizer 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide induces DNA damage in EMT-6 mammary carcinoma cells

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Springer Journals
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Copyright © 2009 by Haykal et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-4-25
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19594955
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Abstract

Background: DCQ (2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide), a synthetic quinoxaline 1,4-dioxide, enhances the cytotoxic effect of ionizing radiation (IR) in vivo and in vitro. We sought to clarify whether increased radiation-induced DNA damage, decreased rate of damage repair, and the generation of reactive oxygen species (ROS) contribute to DCQ enhancement of IR. Methods: Murine mammary adenocarcinoma EMT-6 cells were treated with DCQ for 4 h before exposure to 10 Gy IR. Treated cells were monitored for modulations in cell cycle, induction of DNA damage, and generation of ROS. Results: Combined DCQ and IR treatments (DCQ+IR) induced rapid cell-cycle arrests in EMT-6 cells, particularly in S and G /M phases. Alkaline comet assays revealed high levels of DNA damage in cells after exposure to DCQ+IR, consistent with damage-induced arrest. Unlike IR-only and DCQ-only treated cells, the damage induced by combined DCQ+IR was repaired at a slower rate. Combined treatment, compared to separate DCQ and IR treatments, activated DNA-protein kinase and induced more p-ATM, supporting a role for double strand breaks (DSBs), which are more toxic and difficult to repair than single strand breaks (SSBs). Contributing factors to DCQ radiosensitization appear to be the induction of ROS and DSBs. Conclusion: Collectively, our findings indicate that radiosensitization by DCQ is mediated by DNA damage and decreased repair and that ROS are at least partially responsible. hence, DNA synthesis and replication may proceed Background Eukaryotic cells have evolved DNA damage checkpoints despite the presence of unrepaired DNA damage, leading that control the fate of an insulted cell by inducing cell- eventually to unviable daughter cells [1,2]. Thus, such cycle arrest, repair of the damage, and cell death. Many malignant cells are sensitive to therapies that induce DNA malignant cells have incompetent cell-cycle controls, and damage [3]. Page 1 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 Some aromatic N-oxides such as quinoxalines induce Cell Culture, Drug and Irradiation Treatment DNA damage in cancer cells. The hypoxic cytotoxin 7- The murine mammary adenocarcinoma cell line EMT-6 chloro-3-[(N, N-dimethylamino) propyl]amino]-2-qui- was cultured in growth media containing RPMI 1640 with noxalinecarbonitrile 1,4-dioxide hydrochloride (Q-85 L-glutamine and 25 mm HEPES, supplemented with 10% HCl) has been shown to induce DNA damage under FBS and 1% penicillin-streptomycin (50 μg/mL), and hypoxic conditions in CaCo-2 cells by producing reactive incubated in a humidified incubator (95% air 5% CO ) at oxygen species (ROS) [4,5]. The mechanism of action of 37°C (Forma Scientific Inc. Ohio, US). such compounds is not yet clear. However, studies on qui- noxaline 1,4-dioxide has shown that it is reduced enzy- DCQ was dissolved in DMSO at a concentration of 10 matically into an active, oxygen-sensitive radical mg/mL. Prior to treatment, it was diluted in media con- responsible for DNA cleavage [6]. taining FBS. EMT-6 cells were plated at a density of 16 × 3 2 10 cells/cm . At 50% confluency, they were incubated A similar quinoxaline, 2-benzoyl-3-phenyl 6,7-dichloro- with DCQ (0–10 μM) for 4 h prior to irradiation (0–10 quinoxaline 1,4-dioxide (DCQ) has been shown to be Gy). cytotoxic and a radiosensitizer on several cancer cell lines, including colon cancer cells. The radiosensitization effect Cells were irradiated at room temperature using a high was also shown in vivo, using C57BL/6 mouse model [7]. dose rate Cesium-137 Laboratory Irradiator (JL Shepherd) Combined treatment with DCQ and radiation delayed the that delivers gamma-irradiation at a dose rate of 174 cGy/ growth of LLC tumors injected in the mice and reduced min. After irradiation, cells were replenished with fresh the mean tumor volume by 80% [7]. Recent results have media containing no drug and incubated for different shown that DCQ causes DNA damage in DLD-1 colon times. cancer cells [8]. Despite data in vitro and in vivo confirming that DCQ is a radiosensitizer little is known about its The murine mammary epithelial cell line SCp2 (kindly mechanism of action. In this study, we first assessed the provided by R. Talhouk, Biology Department, American effects of DCQ ± IR on cell cycle progression at early time- University of Beirut, Lebanon) was used as a model for points. Then, we tested whether DCQ radiosensitization normal, slowly proliferating cells [10]. SCp2 cells were is associated with an enhancement in radiation-induced grown in normal growth media composed of DMEM: F12 DNA damage or with a decrease in the rate of damage supplemented with 5% FBS, 1% Penicillin-Streptomycin, repair. Finally, we investigated the possible involvement and 0.1% insulin (5 μg/mL, Sigma, St. Louis). To induce of ROS in the mechanism of DCQ toxicity. differentiation of the SCp2 cells, the cells were plated in growth media and 12 later the media was replaced with differentiation medium lacking FBS [10]. The differentia- Methods Chemicals tion medium consisted of DMEM: F12 supplemented RPMI 1640 with 25 mm HEPES and L-glutamine, Dul- with 0.1% insulin (5 μg/mL), 0.1% hydrocortisone (1 μg/ becco's modified eagle medium nutrient mixture F12, mL), and 0.1% prolactin (3 μg/mL). For a more differen- fetal bovine serum, trypsin, penicillin-streptomycin and tiated state, a growth factor reduced basement membrane Dulbecco's Phosphate Buffered Saline (PBS) were pur- derived from Engelbreth-Holm-Swarm tumor was added chased from Gibco BRL Life Technologies (Gaithersburg, 12 h after plating. A basement membrane is known to Maryland, US). The Cytotox non-radioactive cytotoxicity induce differentiation in SCp2 cells by making their envi- assay kit and the Cell Titer 96 non-radioactive cell prolif- ronment more similar to that of normal cells [10]. eration assay kit were purchased from Promega Corp (Madison, Wisconsin, US). Propidium iodide (PI), Proliferation and Cytotoxicity Assay YOYO-1 dye, fluorescein isothiocyanate (FITC) goat anti- Cells were plated at a density of 10 cells/mL in 96-well mouse IgG (H+L), and 5-(and-6)-chloromethyl-2',7'- plates. After 24 h, cells were treated in triplicates with dif- dichlordihydrofluorescein diacetate, acetyl ester (CM- ferent DCQ concentrations. In some experiments, EMT-6 H DCFDA) were purchased from Molecular Probes cells were pre-treated with either NAC (5 mM) or Tiron (1 (Eugene, Oregon, US). RNase A, dimethylsulfoxide mM) for 2 h prior to DCQ treatment. (DMSO) and N-acetyl cysteine (NAC) were obtained from Sigma Chemical Company (St. Louis, Missouri, US). ATM Cytotoxicity was performed after 4 h of DCQ treatment kinase phosphoser1981 antibody was obtained from using the Cell Titer 96 non-radioactive cytotoxicity kit. Chemicon International (California, US). DCQ was syn- Briefly, supernatants were mixed with a substrate mix con- thesized from 5,6-dichlorobenzofurazan oxide and taining tetrazolium salt that interacts with lactate dehy- dibenzoylmethane by the Beirut Reaction [9]. drogenase, a stable cytosolic enzyme that is released into the supernatant upon cell lysis. The interaction results in Page 2 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 the conversion of the tetrazolium salt into a red formazan microscope (AXIOVERT 200, ZEISS Fluorescence and product, the absorbance of which is recorded at 492 nm. optical microscope with ZEISS AXIOCAM HRC (Ger- many) and KS 300 V3 image analysis software) illumi- As for the proliferation assays, cells were replenished with nated with blue light (490 nm). Images of a minimum of drug-free media after the 4 h-DCQ treatment, and were 50 cells per treatment were analyzed using the Comet- incubated for 20 h before the assay was performed using Score™ software. In the present study, percentage of DNA the Cell Titer 96 non-radioactive cell proliferation. This in the tail region, and tail moment (%DNA in tail × by tail assay measures the ability of metabolically-active cells to length (μm)) were used as parameters to assess DNA dam- convert tetrazolium salt into a blue formazan product that age. can be measured by its absorbance at 595 nm. Immunocytochemistry Detected by Flow Cytometry Flow Cytometry Ser-1981-phosphorylated ATM (p-ATM) was detected Cells were either treated with 0.1% DMSO (control), immunocytochemically by multiparameter cytometry DCQ (0–10 μM) for 4 h, irradiation (10 Gy), or combina- with respect to the cell cycle phases, using the method tions. Immediately after radiation or drug treatment, cells developed by Huang and Darzynkiewicz [12]. Cells were were replenished with fresh media containing no drug collected by trypsinization, centrifuged, washed with PBS, and incubated for 0 h, 2 h, and 4 h. Subsequently, cells and fixed with ice-cold 70% ethanol for a minimum of 2 were harvested and fixed in ice-cold 70% ethanol and h at -20°C. Ethanol was discarded by centrifugation at a stored at -20°C. On the day of DNA staining, cells were speed of 10000 rpm for 5 min, and the pellets were incubated in 0.2 mg/mL RNase A at 37°C, and stained washed with BSA-T-PBS containing 1% BSA and 0.2% Tri- with 6.25 μg/mL PI for 30 min in the dark at room tem- ton X-100 dissolved in PBS. The pellets were blocked in perature. Finally, cell cycle analysis was performed using a BSA-T-PBS for 5 min at room temperature. After removal Fluorescence Activated Cell Sorter (Becton Dickinson, of the 1% BSA solution by centrifugation, the cells were Research Triangle, NC), and the percentages of cells in incubated with the primary antibody Ser-1981-p-ATM at sub-G (< 2n), G /G , S and G /M phases were deter- a dilution of 1:100 overnight at 4°C. The cells were 1 0 1 2 mined using the Cell Quest program (BD Biosciences, washed twice with BSA-T-PBS, and the pellets were then California, USA). incubated in the dark with fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse antibody (1:30) DNA Damage Detection by the Alkaline Comet Assay for 1 h at room temperature. A volume of 5 mL of BSA-T- The alkaline comet assay used is a modification of the PBS was added to the cell suspension and kept for 2 min method developed by Singh that detects the frequency of before centrifugation at 12000 rpm for 4 min. Finally, the SSBs and alkaline-labile lesions in DNA [11]. Microscope cells were counterstained with PI (5 μg/mL) solution con- slides were coated with 1% normal melting agarose, and taining RNase A (0.1 mg/mL) for 30 min at room temper- left overnight to dry. Cells suspended in media were ature in the dark. Both the fluorescence of PI and FITC of mixed with 75 μL of 0.5% low-melting-point agarose 10 cells/treatment were measured using the FACS cytom- (LMPA) and were distributed on the coated slide. The eter, and analyzed using Cell Quest. slides were left to gel for 10 min at 4°C, before a third layer of 80 μL 0.5% LMPA was added to the slide and left Detection of ROS by DCFDA assay 3 2 Cells were plated at a density of 16 × 10 cells/cm and for 10 min at 4°C. The slides were then dipped in cold lys- ing solution (1.25 M NaCl, 50 mM EDTA, 100 mM Tris treated at 50% confluency with 10 μM DCQ for 30 min. base and 0.01% sodium lauroyl sarcosine; pH 10) for a Control and treated cells were collected by trypsinization, minimum of 2 h at 4°C. Before proceeding, the slides centrifuged, washed with PBS, and incubated in 500 μl of were incubated in pre-warmed lysing buffer containing media (with 2% FBS) containing 10 μM of DCFDA for DNAse-free proteinase K for 1 h at 37°C. The slides were 20–30 min at 37°C. DCFDA is a chemically-stable, non- transferred to an electrophoresis unit filled with electro- fluorescent molecule that is hydrolyzed to DCFH inside phoresis buffer (300 mM NaOH, 1 mM EDTA, 0.2% the cell. DCFH interacts with ROS to form a fluorescent DMSO, and 0.1% 8-hydroxyquinoline; pH ~12.3), and complex. Samples were then centrifuged, washed with were left immersed in the solution for 20 min, before PBS, and then resuspended in 500 μl of PBS. The fluores- being subjected to electrophoresis. Electrophoresis was cence of DCF was immediately measured by flow cytome- carried out for 20 min at a voltage of 0.5 V/cm and a cur- try. rent of 250 mA. Next, the slides were rinsed with neutral- ization buffer (20 mM Tris, 1 mg/mL spermine, and 50% Chromatin Immunoprecipitation Followed by Western ethanol; pH 7.4) for 10 min. Finally, each slide was Blot stained with 50 μL of YOYO-1 stain (0.25 μM YOYO-1, Chromatin immunoprecipitaion was performed using 2.5% DMSO and 0.5% sucrose). YOYO-stained nuclei Chromatin Immunoprecipitation (CHIP) Assay Kit were observed and photographed using a fluorescence (Upstate, New York, USA) according to the manufac- Page 3 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 turer's protocol. Briefly, EMT-6 cells were treated at 70% diately after drug treatment, and this accumulation confluency. DNA-binding proteins were cross-linked to increased to 26% after 2 h. Although IR alone and DCQ /M DNA by adding 1% formaldehyde for 10 min at 37°C. alone caused similar level of arrests at the S + G Cells were washed twice with ice-cold PBS containing pro- phases, they induced distinct cell distribution profiles tease inhibitors (1 mM PMSF, 1 μg/mL aprotinin and 1 where IR caused more intra-S phase arrest, while DCQ μg/mL pepstatin A). Cells were collected and centrifuged induced more G /M arrest suggesting differences in their at a speed of 2000 rpm for 4 min at 4°C. Pellets (of 10 mechanisms of action. The combination treatment of cells) were lysed with SDS Lysis buffer (provided by the DCQ+IR resulted in a strong arrest at 4 h where 61% of kit) containing protease inhibitors. The chromatin, the population accumulated in the S+G /M phases. More- including bound proteins, was sonicated into smaller over, a significant increase in cell death represented by the fragments (200–1000 base pairs) using Misonix Sonicator sub-G population was associated with DCQ+IR (12.1% 3000 at 10% power (3 W) for seven 10-second pulses sep- at 4 h versus 2.5% in non-treated cells). Even at early time- arated by a 5 second-pause. Samples were centrifuged for points this cytotoxic effect of the combination treatment 10 min at 13000 rpm at 4°C, and the supernatants were appears to be at least additive. These results corroborate diluted 10 fold in CHIP dilution buffer (provided by the previous results that DCQ is anti-proliferative. We kit) and pre-cleared with protein A agarose/salmon sperm hypothesize that DCQ and IR act via different mecha- DNA (50% slurry). DNA-PK antibody (1.5 mg/mL) was nisms. DCQ may cause DNA damage such as double- used to co-immunoprecipitate the protein-DNA complex strand breaks (DSBs) or bulky adducts, which are known which was then washed with different buffers: low salt to induce S and G /M arrest [14]. immune complex, high salt immune complex, LiCl immune complex wash buffers, as well as two washes with DCQ Induces DNA Damage in EMT-6 Cells TE (Tris-EDTA) buffer. Proteins were dissolved in 25 μL 1× Since DNA damage is the primary cause of arrest at S or sample buffer, boiled for 10 min, and resolved on a 5% G /M phases, we tested whether DCQ induces DSBs in acrylamide gel to detect the level of DNA-PK by western EMT-6 cells by using neutral comet assay. Although both blotting. treatments were observed to induce DSBs, the fluores- cence intensity was too low to detect significant difference Western Blot in the level of DSBs between DCQ and IR treatments (data Proteins were resolved by sodium dodecyl sulfate-polyacr- not shown). The alkaline comet assay detects SSBs and ylamide gel electrophoresis (SDS-PAGE) on a 5% polyacr- alkaline-labile DNA damage, such as abasic sites. Using ylamide gel, and transferred onto an activated the alkaline comet assay, we detected the level of damage polyvinylidene difluoride (PVDF) membrane in cold induced by DCQ ± IR in exponentially growing EMT-6 transfer buffer (14.4 g of glycine, 3 g Tris base, and 1 g SDS (Figure 2A, B). Cells were treated at 50% confluency with dissolved in 1 L of 20% methanol) at 30 volts overnight. 10 μM DCQ and the assay was directly performed after a The membrane was then blocked for 1 h with 5% non-fat 4 h-incubation with DCQ, IR treatment, or combination milk dissolved in Tris-buffered saline (TBS) containing treatment. Treatment with DCQ alone induced significant 0.1% Tween-20, and probed with DNA-PK antibody levels of damage, similar to that induced by 10 Gy IR. In diluted in 1% blocking buffer overnight at 4°C. The mem- response to combined DCQ and IR treatment, higher lev- brane was incubated with horseradish peroxidase-conju- els of damage were observed: tail moment (%DNA in tail gated secondary antibody 1 h at room temperature. The × tail length) increased by 19.6-fold in comparison with membrane was exposed to X-ray film (Hyperfilm ECL) untreated cells. using chemiluminescent substrate (Amersham). DCQ Activates ATM and DNA-PK in Irradiated EMT-6 Cells Results DCQ Induces S Phase and G2/M Arrest in EMT-6 Cells The nuclear kinase ATM is rapidly phosphorylated in the Previous work has shown that DCQ, in combination with presence of low levels of DSBs [15]. The immunocyto- IR, induces apoptosis in EMT-6 cells 24 h post-treatment, chemical detection of p-ATM thus provides a sensitive and decreases their clonogenic survival [13]. To determine approach to detect double-strand breaks (DSBs) gener- the direct effects of DCQ ± IR on cell cycle progression of ated following drug treatment in cells [16]. Cells were EMT-6, cells were treated with 10 μM DCQ for 4 h fol- treated with DCQ (10 μM), IR (10 Gy) or combinations lowed by irradiation with 10 Gy IR, or separately treated. followed by replenishment with drug-free media. After 2 Treated cells were collected for flow cytometry either h, cells were collected and the level of p-ATM in relation directly (0 h), or at 2 h or 4 h after IR exposure (Figure 1). to the cell cycle was assayed in EMT-6 cells for each treat- IR induced cell-cycle arrest in the S phase at 2 h post-expo- ment by subjecting the samples to immunocytochemistry sure and this arrest increased at 4 h with 27% of the pop- (Figure 3A, B). As expected, control cells showed the basal ulation accumulated in the S phase. DCQ alone also level of p-ATM expression was higher in G /M population caused an accumulation of cells in the late S phase imme- due to the role of ATM in mitosis. Exposure of EMT-6 cells Page 4 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 S and G2/M arr Figure 1 est induced by DCQ ± IR in EMT-6 cells at 0, 2, and 4 h post-treatment S and G2/M arrest induced by DCQ ± IR in EMT-6 cells at 0, 2, and 4 h post-treatment. EMT-6 cells were treated at 50% confluency with DCQ (10 μM) for 4 h, and/or irradiated (10 Gy). Immediately after the 4 h-drug incubation or IR expo- sure, cells were replenished with media containing no drug. Cells were collected at 0 h, 2 h, and 4 h after the refreshment of media and subjected to flow cytometry. Percentages of cells in the sub-G1 (A), S (B), and G2/M (C) phases of the cell cycle were determined by CellQuest and the averages ± SD are plotted for each treatment. Dashed lines represent the % of control cells in each phase of the cell cycle. SD: standard deviation. to 10 μM DCQ triggered the activation of ATM by phos- significant increase in the active DNA-PK level was phorylation at Ser-1981; this phosphorylation level was induced in response to DCQ+IR (Figure 3C). higher than that of 10 Gy-treated cells, reflecting higher amounts of DSBs generated by DCQ than IR. The combi- Slow Repair of Damage Observed in EMT-6 Exposed to nation treatment had the highest levels of p-ATM over DCQ+IR untreated cells reaching 2 fold only in the G /M phase. The time required to repair DNA depends on the type of DCQ and DCQ+IR induced the activation of ATM in all damage. SSBs are usually repaired much faster than DSB phases of the cell cycle similar to the Topoisomerase II after induction [19]. To assess whether DCQ toxicity is inhibitor mitoxantrone [17]. due to the extent of damage induced or to slow repair fol- lowing treatment, the extent of damage was assessed by Another major kinase activated by DNA damage is DNA- the alkaline comet assay at 0 h, 4 h and 16 h post-treat- PK, which is activated by binding to the damaged sites on ments. Although a large extent of the damage induced by DNA [18]. The binding of DNA-PK to DNA was evaluated IR alone and DCQ alone is repaired in less than 4 h, we by DNA-PK chromatin immunoprecipitation followed by observed dramatically slowed repair of the damage western blotting with an antibody against DNA-PK. We induced by DCQ+IR. Even at 16 h, significant DNA dam- observed that untreated cells had no significant DNA-PK age remained unrepaired as evidenced by tail moments. bound to the DNA, but a moderate signal was detected in Damage was significantly higher (P-value < 0.01) in EMT-6 cells after 10 μM DCQ or 10 Gy IR, and a highly response to DCQ+IR as compared to untreated and singly- treated cells (Figure 4). Page 5 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 DNA dama Figure 2 ge induced by DCQ+IR in EMT-6 cells DNA damage induced by DCQ+IR in EMT-6 cells. A. Representative images of comets induced by DCQ± IR in EMT-6 cells subjected to the alkaline comet assay. EMT-6 cells were treated with IR (10 Gy), 4 h of DCQ (10 μM), or in combination. Cells were directly collected after the treatment and subjected to the alkaline comet assay. Images were taken using a fluores- cent microscope at 40× (oil immersion) magnification. The tail lengths of the comets observed by each treatment are propor- tional to the amount of DNA damage induced. B. The mean of the parameters (% DNA in comet's tail and tail moment) are shown in the graphs above. The histograms summarize the averages of two independent experiments ± SE and show the mean of the %DNA in comet's tail and tail moments. More than 50 cells per treatment were photographed and quantified using TriTek CometScore software. SE: standard error. DCQ Generates Reactive Oxygen Species in EMT-6 cells DCQ Targets Rapidly-Proliferating Cells N-oxides undergo redox-cycling producing reactive oxy- Because DCQ appears to slow repair, we expected that tox- gen species (ROS) [20]. We hypothesized that DCQ may icity would depend on proliferation rate. We assessed cause DNA damage by ROS induction due to its redox- whether reducing proliferation would decrease DCQ tox- cycling. Indeed, DCQ treatment alone, either directly or icity by culturing murine mammary epithelial cell line indirectly, induced the generation of ROS in EMT-6 cells SCp2 under conditions to induce differentiation and after 30 min of treatment as measured by the DCFH assay thereby slow proliferation. When cultured in differentia- (Figure 5A). To determine if ROS play a role in the radio- tion media, SCp2 proliferation rate was reduced to sensitizing effect of DCQ in EMT-6 cells, strong anti-oxi- approximately 50% compared to cells cultured in normal dants such as Tiron and NAC were added before treatment growth media, and an even stronger decrease in prolifera- with DCQ alone or in combination with IR, to scavenge tion was observed with cells supplemented with basement any DCQ-generated ROS. Cells pretreated with anti-oxi- membrane (Figure 6A). After 4 h of DCQ treatment, dants were more resistant to the anti-proliferative effect of slowly proliferating SCp2 cells were more resistant to DCQ. However, the anti-oxidants did not completely toxic concentrations of 10 μM DCQ, suggesting selective abolish the anti-neoplastic effect of DCQ whether alone toxicity to proliferating cells (Figure 6B). or in combination with IR (Figure 5B, C). These results indicate that ROS play at least a partial role in the radio- Discussion sensitizing effect of DCQ in EMT-6 cells. Several mechanisms of radiosensitization are known, including redox modulators [21], inhibitors of DNA dam- Page 6 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 Figure 3 Phosphorylation of ATM and activation of DNA-PK by DCQ ± IR in EMT-6 cells at 2 h post-treatment Phosphorylation of ATM and activation of DNA-PK by DCQ ± IR in EMT-6 cells at 2 h post-treatment. A. EMT- 6 cells were treated with 10 μM DCQ, 10 Gy IR, or combination treatments, fixed and subjected to immunocytochemical detection of ATM phosphorylated on Ser1981, and stained with PI to detect at the same time p-ATM in each phase of the cell cycle. B. The mean of the FL-1 intensity (reflecting the level of p-ATM expression) at each phase of the cell cycle are plotted. C. Anti-DNA-PK was immunoprecipitated with DNA from lysates of 10 EMT-6 cells treated with 10 μM DCQ, 10 Gy IR, or combination treatments using CHIP assay. The immunoprecipitate was resolved on a 5% gel by electrophoresis, transferred to nitrocellulose and probed with anti-DNA-PK. The bands were quantified using LabWorks 4.0 software. CHIP: chromatin immunoprecipitation. age repair [22], and regulators of growth factor receptors Here, we show for the first time that DCQ induces DSBs and other signaling molecules [23,24]. Misrepair of DNA in EMT-6 cells, in addition to SSBs and alkaline-labile damage causes mutation, and extensive damage may lesions detected by the alkaline comet assay. DCQ causes cause cell cycle arrest, or death if irreparable or too slowly more G2/M arrest than IR. Exposure of EMT-6 cells to 10 repaired. The role ROS can play in cellular response to μM DCQ produced damage detected by the alkaline radiation has been well established [25]. comet assay, and DSBs evaluated by p-ATM level, almost equivalent to that produced by 10 Gy IR. The combina- Sl Figure 4 ow repair of DNA damage in DCQ+IR-treated cells Slow repair of DNA damage in DCQ+IR-treated cells. Treated cells were either collected directly after treatment (0 h) or refreshed with drug-free media and incubated for another 4 or 16 h. The mean of the parameters (% DNA in comet's tail and tail moment) are shown in the graphs above. Page 7 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 ROS g Figure 5 eneration by DCQ in EMT-6 cells ROS generation by DCQ in EMT-6 cells. A. Cells were treated with 10 μM DCQ for 30 min before measuring ROS release by the DCFH-DA assay as described in the materials and methods. B & C. EMT-6 cells were pretreated for 2 h with the strong anti-oxidants Tiron and NAC, then with DCQ (2.5, 5 and 10 μM), and later subjected to 0 Gy (-IR) or 10 Gy (+IR) radiation. Afterwards, cells were replenished with drug-free media and incubated for an additional 20 h before the determina- tion of cell proliferation. The values plotted represent an average (± SD) of two independent experiments. ROS: Reactive oxy- gen species. tion of DCQ+IR induced significantly higher SSBs than phase arrest. Activated by DSBs, ATM becomes phosphor- each treatment alone. Radiosensitization of DCQ not ylated at Ser-1981 [15]. We show that ATM was activated only correlates with higher induction of DNA damage, but in all phases of the cell cycle in response to the damage also with slower repair of this damage. Alkaline comet induced by all treatments. In the combination treatment assays 4-hours post treatment revealed dramatically the expression of p-ATM in G /M phase was twice that of slowed repair of damage in DCQ+IR treated cells com- untreated cells. pared to separate IR or DCQ treatments. Little damage remained 4 h after separate treatments with DCQ or IR, Following IR treatment, EMT-6 cells arrest in S phase. supporting a model in which radiosensitization involves Such an arrest is mainly caused by the activation of the the generation of more difficult-to-repair DSBs. These intra-S-phase checkpoint due to significant amount of results suggest combination treatment may have thera- DSBs [28]. It is responsible for inhibition of DNA replica- peutic value. tion at late origins of replication. In addition to cell cycle arrest, the intra-S-phase checkpoint induces a cascade of DNA damage, in particular DSBs, imposes a critical threat reactions, that either attempt to repair the damage, mainly to the survival of cells if left unrepaired [26]. As a response by homologous recombination, or induce cell death, to the damage, cells activate the DNA damage checkpoint. depending on the extent of the damage induced. If dam- DSBs are detected by two main players in the DNA dam- age is not repaired before the end of the S phase, cells age checkpoint: ATM and DNA-PK. Signal transduction, would arrest at the G /M DNA-damage checkpoint [28]. induced by the activation of these two signals, can cause The G /M arrest induced by DCQ and the S-phase accu- cell-cycle arrest, repair, and cell death. Moreover, both are mulation induced by IR appear together in combination activated at very early stages of the DNA damage response, treatments. Despite our intriguing findings that the com- and are involved in DNA repair [27]. DNA-PK was acti- bination treatment DCQ+IR induces DNA damage, vated in response to DCQ alone more than IR alone. The including DSBs, and slows repair, the precise mechanisms combination treatment induced the highest amount of are still not clear. active DNA-PK. ATM plays a critical role in S and G /M Page 8 of 10 (page number not for citation purposes) Radiation Oncology 2009, 4:25 http://www.ro-journal.com/content/4/1/25 with increased levels of difficult-to-repair DSBs could overwhelm cellular DNA repair pathways. The proposed mechanism was observed to be selective for rapidly prolif- erating cells, presumably because slowly dividing cells have more time to repair DNA damage. This finding sug- gests clinical potential. Conclusion This study presents evidence that the radiosensitizing effects of DCQ are associated with an increase in DNA damage, including DSBs, the activation of the key DNA damage markers, p-ATM and DNA-PK, and the generation of ROS. The significant levels of unrepaired damage detected by alkaline comet assay in EMT-6 cells following treatment by DCQ+IR indicate that decreased DNA repair contributes to the mechanism of DCQ radiosensitization. Competing interests The authors declare that they have no competing interests. Authors' contributions JH carried out the experiments in the study and drafted Resi Figure 6 stance of slow proliferating cells to DCQ the manuscript. FG and CAS were involved in revising the Resistance of slow proliferating cells to DCQ. A. After manuscript critically for important intellectual content plating SCp2 cells in growth media (GM), they were sub- and CAS helped in preparing the final draft of the manu- jected to different conditions. Cells were either kept in GM, script. MH provided the compound and reviewed the or shifted 12 h after plating to differentiation media (DM) or manuscript. HGM conceived of the study, and partici- DM+BM (basement membrane) for a more differentiated pated in its design and coordination and drafting of the state. SCp2 cells were incubated for one day then assayed for manuscript. All authors read and approved the final man- proliferation using Cell Titer 96 non-radioactive cell prolifer- uscript. ation. The proliferation rate of cells grown in GM was con- sidered as 100%. B. The viability of SCp2 cells at different differentiating states were measured by Cytotox 96 non- Acknowledgements radioactive cytotoxicity assay directly after 4 h of DCQ This study was supported by the University Research Board of the Ameri- treatment. can University of Beirut and the Lebanese National Council for Scientific Research. References The slow repair of DNA damage caused by DCQ+IR may 1. 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Radiation OncologySpringer Journals

Published: Jul 14, 2009

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