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[Cancer Research 65, 5337-5343, June 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

DNA-Protein Cross-links and Replication-Dependent Histone H2AX Phosphorylation Induced by Aminoflavone (NSC 686288), a Novel Anticancer Agent Active against Human Breast Cancer Cells

Ling-hua Meng1, Glenda Kohlhagen1, Zhi-yong Liao1, Smitha Antony1, Edward Sausville2 and Yves Pommier1

1 Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and 2 Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland

Requests for reprints: Yves Pommier, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Building 37, Room 5068, 37 Convent Drive, Bethesda, MD 20892-4255. Phone: 301-496-5944; Fax: 301-401-0752; pommier{at}nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose- and time-dependent histone H2AX phosphorylation ({gamma}-H2AX). {gamma}-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented {gamma}-H2AX formation, suggesting that {gamma}-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no {gamma}-H2AX foci were found in proliferating cell nuclear antigen–negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21Waf1/Cip1 and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. {gamma}-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan; Fig. 1) is one of a series of diaminoflavone analogues with potent growth inhibitory activity against human breast and renal cancer cells in vitro and in mice bearing human tumor xenografts (1–4). Aminoflavone displays a unique COMPARE pattern of antiproliferative activity in the U.S. National Cancer Institute tumor cell line screen with no statistically significant correlation to patterns of known classes of antitumor agents.3 Based on its demonstrable potent in vitro and in vivo antitumor activity against a number of human tumor cell lines, aminoflavone has been selected as a candidate for possible advancement to phase I clinical trials.



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  		Figure 1. Production of DPC in MCF-7 breast cancer cells treated with aminoflavone (AF). DPC were measured by alkaline elution. A, time dependence (3, 6, or 18 hours) for DPC formation in cells treated with 1 µmol/L aminoflavone. B, concentration dependence: DPC were measured after treatment with 0.1, 0.3, 1, or 3 µmol/L aminoflavone for 18 hours. Bars, SE for at least three independent experiments. C, persistence of DPC after aminoflavone removal. Cells were treated with 1 µmol/L aminoflavone for 3 hours at 37°C and were rinsed with fresh medium and incubated in drug-free medium for 1 or 3 hours at 37°C. Cells were lysed and assayed by alkaline elution.

 
A unique pattern of activity for a new agent in the NCI cell screen suggests a novel mechanism of drug action. Previous studies showed that aminoflavone treatment resulted in elevated CYP1A1 and CYP1A2 in human breast cancer MCF-7 cells and that cellular sensitivity to aminoflavone is accompanied by its conversion to a series of yet to be identified metabolites by CYP1A1 and CYP1A2 (5). These aminoflavone metabolites covalently bind cellular proteins and DNA (4). Further studies found that aminoflavone activates the aryl hydrocarbon receptor signaling pathway, which in turn induces expression of CYP1A1 in sensitive MCF-7 cells but not in resistant MDA-MB-435, PC-3, and AHR100 cells (6). Preliminary experiments show that sulfotransferase 1A1 is required for the antitumor activity of aminoflavone, suggesting this enzyme might also be involved in the metabolism of aminoflavone.4 How aminoflavone and its metabolites exert their antitumor activity remains poorly understood. A recent study (5) found that exposure of MCF-7 cells to aminoflavone results in phosphorylation and stabilization of p53 with induction of the p53 downstream target p21Waf1/Cip1, suggesting DNA damage might be induced in aminoflavone-treated cells.

The present studies were done to elucidate the cellular and molecular effect of aminoflavone. We report the induction of DNA-protein cross-links (DPC) and replication-mediated DNA damage by aminoflavone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human breast cancer MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and were grown at 37°C in the presence of 5% CO2 in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 100 units/mL penicillin, and 100 µg/mL streptomycin.

Drugs and chemicals. Aminoflavone was obtained from Kyowa Hakko Kogyo. Aphidicolin, nocodazole, bromodeoxyuridine (BrdUrd), camptothecin, and etoposide (VP-16) were purchased from Sigma Chemical Co. (St. Louis, MO). Aminoflavone, aphidicolin, and camptothecin were dissolved at a concentration of 10 mmol/L and nocodazole was dissolved at 2 mg/mL in DMSO. Aliquots were stored at –20°C. All drugs were diluted to desired concentrations in medium immediately before each experiment. The final DMSO concentration did not exceed 0.1%.

Alkaline elution assays. Alkaline elutions were done to assess DNA damage by detecting DPC and DNA single-strand breaks (SSB) as described (7, 8). Cellular DNA was radio-labeled with 1 µCi/mL [3H]thymidine (Perkin-Elmer Life Science Co., Boston, MA) for 48 hours at 37°C and chased in nonradioactive medium overnight. After drug treatments, cells were scraped in HBSS, counted, and aliquots were placed in drug-containing ice-cold HBSS. After alkaline elution, filters were incubated at 65°C with 1 mol/L HCl for 45 minutes and 0.04 mol/L NaCl was added for an additional 45 minutes. Radioactivity in all fractions was measured with a liquid scintillation analyzer (Packard Instruments, Meriden, CT). DPC were analyzed under nondeproteinizing, DNA-denaturing conditions using protein-adsorbing filters. DPC frequencies were calculated according to the bound to one terminus model formula (7):

SSB were assessed by alkaline elution under deproteinizing, DNA denaturing conditions. SSB frequencies were calculated with the formula (7):

where RT, R0, and R3 correspond to the DNA selections for drug-treated cells, untreated cells, and cells treated with 3 Gy, respectively.

Detection covalent topoisomerase I-DNA and topoisomerase II-DNA complex. Topoisomerase I-DNA or topoisomerase II-DNA complexes were detected using the in vivo complex of enzyme bioassay (9). Topoisomerase I was detected by immunoblotting using the C21 topoisomerase I mouse monoclonal antibody (a kind gift from Dr. Yung-Chi Cheng, Yale University, New Haven, CT), and topoisomerase II was detected with topoisomerase II monoclonal antibody (clone Ki-S1) from Chemicon International (Temecula, CA).

Topoisomerase I–mediated DNA cleavage assay. Topoisomerase I–mediated DNA Cleavage Assays were done as described (10). Imaging was done using a phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Laser scanning confocal microscopy. Laser scanning confocal microscopy was done as described (11). Cells were grown in culture medium on chamber slides. After drug treatment, cells were fixed in 2% paraformaldehyde in PBS for 5 minutes, washed in PBS, permeabilized in 100% methanol at –20°C for 20 minutes, and washed with PBS. Slides were blocked with PBS containing 1% bovine serum albumin (BSA) and 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour, incubated with one or two first antibodies at 800-fold dilution for 2 hours, washed, incubated with a AlexaFluor 488, or AlexaFluor 546-conjugated goat anti-rabbit or anti-mouse immunoglobulin G second antibody (Molecular Probes, Eugene, OR) at 200-fold dilution for 1 hour, and washed in PBS. Slides to be stained with propidium iodide (PI) were pretreated with 10 units/mL RNase (Sigma Chemical). Slides were mounted with mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA) and viewed with a PCM2000 laser scanning confocal microscopy (Nikon Co., Tokyo, Japan) using x40 objectives. Projections were saved as a BMP file.

Western blot analysis for phosphorylated H2AX ({gamma}-H2AX). Cells treated at 50% to 80% confluence were scraped and pelleted by centrifugation at 1,000 x g for 15 minutes at 4°C. Pellets were washed twice in PBS, homogenized in 0.2 mol/L H2SO4, and centrifuged at 13,000 x g. Histones were precipitated by adding 0.25 volume of 100% (w/v) trichloroacetic acid. Pellets were suspended in 100% ethanol overnight and centrifuged again at 13,000 x g. Histones were dissolved in Ultrapure water, and evaluated for protein concentration (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA). Aliquots corresponding to 10 µg of protein were boiled in Tris-glycine SDS sample buffer (Invitrogen, San Diego, CA) and loaded onto 4-20% Tris-glycine precast gels (Novex, San Diego, CA). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membrane were blocked with PBST (PBS containing 0.05% Tween 20) containing 5% nonfat milk for 60 minutes before incubation with 50 ng/mL anti-{gamma}-H2AX antibody (Upstate, Waltham, MA) for 2 hours. Blots were washed in PBST and then incubated with horseradish peroxidase-conjugated anti-mouse antibody (1/10,000 dilution) and visualized by chemiluminescence using the Supersignal kit (SuperSignal West Pico chemiluminescent substrate, Pierce, Rockford, IL). All of the presented data were confirmed in independent experiments.

Flow cytometry analysis of DNA content. Cell cycle analyses were done with a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) as described (12). Cell cycle distributions were calculated using ModFit LT software (Verity Software House, Inc., Topsham, ME).

DNA synthesis assays. Thymidine incorporation assays were done as described previously (13). Briefly, cells were prelabeled with 0.005 µCi/mL of [14C]thymidine (53.6 mCi/mmol) for 48 hours at 37°C. DNA synthesis was measured by 10-minute pulses with 1 µCi/mL of [methyl-3H]thymidine (80.9 Ci/mmol). 3H incorporation was stopped by washing cell cultures twice in ice-cold HBSS and by scraping cells into 4 mL of ice-cold HBSS. One-milliliter aliquots were precipitated after addition of 100 µL trichloroacetic acid in triplicate. Samples were vortexed, mixed, and centrifuged for 10 minutes at 12,000 x g at 4°C. Precipitates were dissolved overnight at 37°C in 0.5 mL of 0.4 mol/L NaOH. Samples were counted by dual label liquid scintillation and [3H] values were normalized using [14C] counts. Inhibition of DNA synthesis was calculated as the ratio of [3H]/[14C] in the treated samples over that in the untreated control samples.

Two-dimensional flow cytometry analysis: DNA content and BrdUrd incorporation. Cells were pulse-labeled with 50 µmol/L BrdUrd during the last 30 minutes of aminoflavone treatments. Cells were collected, fixed in 70% ethanol at 4°C, washed with PBS and resuspended in 1 mL cold 0.1 mol/L HCl/0.5% Triton X-100 and left on ice for 10 minutes. Cells were spun down after adding 5 mL H2O, resuspended in 2 mL H2O and boiled for 10 minutes. Cells were left on ice for 10 minutes and 5 mL PBS/0.5% Triton X-100 was added. Nuclei were pelleted by centrifugation and resuspended in 20 µL of FITC-conjuncted anti-BrdUrd antibody (Becton Dickinson, Franklin Lakes, NJ). After incubation with the anti-BrdUrd antibody at room temperature for 30 minutes, nuclei pellets were washed once with PBS, and resuspend in 400 µL of PI solution (50 µg/mL PI and 50 µg/mL RNase). Analyses were done with a FACScan flow cytometer.

Western blot analyses for RPA2, p53, phosphorylated p53, p21Waf1/Cip1, and MDM2. Western blots analysis for RPA2, phosphorylated p53, p53, p21, and MDM2 were done as described (12) using antibodies to RPA2 (Oncogene Research products, San Diego, CA), phosphorylated p53 at Ser15 (Cell Signaling, Beverly, MA), p53 (SC-99, Santa Cruz Biotechnology, Santa Cruz, CA), p21 (SC-817, Santa Cruz Biotechnology), MDM2 (Ab-1, EMD Biosciences, San Diego, CA), and tubulin (NeoMarkers, Fremont, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aminoflavone induces DNA-protein cross-links in MCF-7 cells. Alkaline elution assays were done to detect DNA damage. Time course experiments in cells treated with 1 µmol/L aminoflavone (Fig. 1A) showed induction of DPC, which increased with time of aminoflavone exposure during the first 6 hours and remained at the same level up to 18 hours. Induction of DPC by aminoflavone was also concentration-dependent. DPC were detectable after 18 hours of exposure to submicromolar concentrations of aminoflavone and tended to reach a plateau above 1 µmol/L (Fig. 1B). To detect whether aminoflavone-induced DPC were reversible, MCF-7 cells were first treated with 1 µmol/L of aminoflavone for 3 hours and were rinsed in fresh medium and further incubated in drug-free medium for 1 or 3 hours. DPC stayed at the same level after aminoflavone removal (Fig. 1C), indicating the persistence of aminoflavone-induced DPC.

Comparison of DNA single-strand breaks and DNA-protein cross-links induced by aminoflavone in MCF-7 cells. Camptothecin, an inhibitor of topoisomerase I, induces SSB due to the trapping topoisomerase I cleavage complexes (14, 15). The ratio of camptothecin-induced SSB and DPC is close to 1 because each break is associated with the covalent linkage of topoisomerase I at the 3' end of the broken DNA (refs. 15–17).5 To investigate whether DPC induced by aminoflavone were associated with SSB, we simultaneously measured DPC and SSB in aminoflavone-treated cells. Camptothecin-treated cells were used as a comparison. As shown in Fig. 2A, 1 µmol/L aminoflavone induced detectable SSB. However, the SSB frequency was <300 rad-equivalents after treatment for 6 hours. Camptothecin (1 µmol/L) induced much more SSB than 300 rad-equivalents (Fig. 2A). Under the same conditions, aminoflavone induced markedly more DPC than camptothecin (Fig. 2B). These results indicate that aminoflavone-induced DNA damage consists of much higher frequency of DPC than SSB, which suggests that aminoflavone produces DNA lesion unrelated to topoisomerase.



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  		Figure 2. Relationship between DNA SSB and DPC induced by aminoflavone (AF) in MCF-7 cells. Cells were treated with 1 µmol/L aminoflavone for 6 hours or with 1 µmol/L camptothecin (CPT) for 3 hours. SSB (A) and DPC (B) were detected by alkaline elution. C, immunoblot analysis of drug-stabilized topoisomerase I (Top 1) or topoisomerase II (Top 2)-DNA complexes. MCF-7 cells were treated with 1 µmol/L camptothecin or 100 µmol/L VP-16 for 1 hour or aminoflavone for 6 hours. The lysates were applied to CsCl gradients and centrifuged overnight. DNA-containing fractions were immunobloted with topoisomerase I or topoisomerase II antibodies. D, topoisomerase I–mediated DNA cleavage assay. A 3'-32 p-end-labeled 161-bp DNA fragment was incubated with recombinant human topoisomerase I in the absence (+Top1) or presence of camptothecin or different aminoflavone concentrations (10). DNA control without topoisomerase I was also included (DNA).

 
To further investigate whether DPC induced by aminoflavone were due to topoisomerases, cellular topoisomerase-DNA complexes were analyzed using cesium chloride gradient centrifugation of cellular DNA and immunoblotting (9). As expected, in camptothecin-treated cells, immunoblotting revealed the presence of topoisomerase I in the DNA-containing fractions (fractions 7-10, Fig. 2C). No topoisomerase II was detected in the same fractions. Conversely, topoisomerase II-DNA complexes were only observed in cells treated with VP-16 (Fig. 2C). Aminoflavone treatment induced neither topoisomerase I nor topoisomerase II complexes in MCF-7 cells even up to 10 µmol/L. Similarly, aminoflavone was not able to trap topoisomerase I-DNA cleavage complex in normal DNA in the presence of recombinant human topoisomerase I (Fig. 2D). These experiments show that aminoflavone is not able to trap topoisomerase I or topoisomerase II cleavage complexes and that the protein(s) forming the DNA complexes in aminoflavone-treated cells are not topoisomerases.

Production of {gamma}-H2AX foci in MCF-7 cells treated with aminoflavone. To investigate whether aminoflavone induces DNA double-strand breaks (DSB), we looked for {gamma}-H2AX focus formation (18) in aminoflavone-treated MCF-7 cells. H2AX phosphorylation (at its COOH terminus on Ser139) is induced at DSB sites in chromosomal DNA (19). Because {gamma}-H2AX appears within minutes after ionized radiation, {gamma}-H2AX focus formation is considered to be a sensitive and selective signal for the existence of DSB (18, 20). Figure 3 shows {gamma}-H2AX foci in cells treated with aminoflavone. In cells treated with aminoflavone for 6 hours, {gamma}-H2AX focus formation increased with aminoflavone concentration and also occurred differently in different cells. The cells can be classified into three classes according to the patterns of {gamma}-H2AX focus formation: negative cells, which fail to form {gamma}-H2AX; discrete foci forming cells, in which discrete and punctuated foci can be observed; and cells with diffuse staining, in which {gamma}-H2AX staining appears bright and diffuse (Fig. 3A). Quantification of the different classes of cells showed that cells forming discrete foci (blue lines and circles) were prominent after exposure to low concentration (0.3 µmol/L) of aminoflavone or short time (3 hours in Fig. 3B). Diffuse {gamma}-H2AX staining (green lines and triangles) increased as a function of dose and time. These observations suggest that DSB accumulate in cells with longer treatment (up to 6 hours). Furthermore, production of {gamma}-H2AX was not due to apoptosis induced by aminoflavone because very few cells were Annexin V positive after exposure to 1 µmol/L aminoflavone for 6 hours (data not shown).



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  		Figure 3. Generation of {gamma}-H2AX foci in MCF-7 cells treated with aminoflavone. A, dose-dependent {gamma}-H2AX formation. Cells were treated with the indicated concentrations of aminoflavone for 6 hours. B, time-dependent induction of {gamma}-H2AX in cells treated with 1 µmol/L aminoflavone for 3 or 6 hours. Three fields and at least 30 cells were counted and classified as {gamma}-H2AX–negative cells (negative cells), cells forming discrete foci (discrete foci), and cells with bright and diffuse staining (diffuse staining). Plots (right). C, irreversibility of x-H2AX foci induced by aminoflavone. After treatment with 1 µmol/L aminoflavone for 4 hours, cells were further incubated in drug-free medium for the indicated times. Cells were stained with mouse anti-{gamma}-H2AX antibody and goat anti-mouse antibody conjugated with AlexaFluor 488 (green). Nuclei were stained with PI (red).

 
To investigate the reversibility of {gamma}-H2AX generated by aminoflavone, MCF-7 cells were first treated with aminoflavone (1 µmol/L for 4 hours). After aminoflavone removal, cells were further incubated in drug-free medium for up to 4 hours. As shown in Fig. 3C, {gamma}-H2AX foci did not reverse and even increased after aminoflavone removal. These results indicate that the dose and time dependencies of the {gamma}-H2AX foci induced by aminoflavone are correlated with the DPC induction.

Aminoflavone-induced {gamma}-H2AX foci are related to replication-mediated DNA damage. {gamma}-H2AX forms in response to DSB both in nonreplicating (18, 19) and replicating DNA (11). To investigate whether aminoflavone-induced {gamma}-H2AX was initiated by replication associated DSB, MCF-7 cells were pretreated with 1 µmol/L of aphidicolin (a specific inhibitor of replication polymerases; ref. 11) 15 minutes before treatment with aminoflavone, and aphidicolin was kept throughout the aminoflavone treatment. As shown in Fig. 4A, aphidicolin markedly reduced the level of {gamma}-H2AX in aminoflavone-treated cells. This inhibition was confirmed by Western blot analysis (Fig. 4B) against phosphorylated H2AX ({gamma}-H2AX). To further test whether aminoflavone-induced {gamma}-H2AX foci are dependent on DNA synthesis, we did double staining for both {gamma}-H2AX and proliferating cell nuclear antigen (PCNA) because PCNA levels are highest in replicating cells and undetectable outside of S phase (11, 21). As shown in Fig. 4C, positive cells for {gamma}-H2AX foci were also positive for PCNA, and cells negative in {gamma}-H2AX were also negative for PCNA (white circles). Mitotic cells failed to form aminoflavone-induced {gamma}-H2AX foci (Fig. 4D, white arrow). These results show that aminoflavone induces {gamma}-H2AX in replicating cells and that {gamma}-H2AX formation is replication dependent.



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  		Figure 4. Aminoflavone (AF)–induced {gamma}-H2AX is dependent on DNA synthesis and restricted to the S phase. A, aphidicolin (APH) inhibits {gamma}-H2AX formation (green) in MCF-7 cells treated with aminoflavone. MCF-7 cells were treated with aminoflavone (1 µmol/L, for 6 hours) in the absence or presence of aphidicolin. Aphidicolin was added 15 minutes before and kept during the aminoflavone treatment. B, Western blot analysis for the effects of aphidicolin on {gamma}-H2AX induction by aminoflavone. Ponceau S staining of the same samples was used as loading control. Treatment schedules were the same as in (A). C, {gamma}-H2AX focus formation in relation to cellular PCNA after treatment with 1 µmol/L aminoflavone for 6 hours. Cells were stained with anti-{gamma}-H2AX (blue) or anti-PCNA antibodies (green). D, {gamma}-H2AX foci (green) were not observed in a mitotic cell (white arrow) treated with aminoflavone (1 µmol/L).

 
Aminoflavone arrests S phase progression and inhibits DNA synthesis. To investigate cell cycle progression in cells treated with aminoflavone, MCF-7 cells were exposed to aminoflavone in the absence or presence of nocodazole (0.2 µg/mL) for 18 hours. As shown in Fig. 5A, increasing concentrations of aminoflavone arrested cells in mid-S phase at 0.1 and 0.3 µmol/L, and in early S phase at 1 µmol/L. Nocodazole failed to arrest aminoflavone-treated cells, indicating aminoflavone-induced S-phase arrest and lack of cell cycle progression to mitosis.



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  		Figure 5. Aminoflavone (AF) arrests S-phase progression and inhibits DNA synthesis. A, left, MCF-7 cells were treated with the indicated concentrations of aminoflavone alone or in association with nocodazole (NOC, 0.2 µg/ml) for 18 hours. Right, MCF-7 cells were treated with aminoflavone for 18 hours (T18) and further incubated in the absence of aminoflavone with or without of nocodazole for 24 hours (R24). DNA content distribution histograms were measured by flow cytometry. B, DNA synthesis was measured by 10-minute pulses with 1 µCi/ml of [methyl-3H] thymidine. Concentration response for cells treated with aminoflavone for 18 hours (left) and time course with 1 µmol/L aminoflavone (right). C, cell cycle distribution and DNA synthesis were measured by BrdUrd labeling and PI staining. Cells were pulse labeled with 50 µmol/L BrdUrd for 30 minutes and incubated with anti-BrdUrd antibody and PI. Scatter plots depict BrdUrd labeling (y axis, log scale) as a function of cell cycle distribution (x axis, PI content). Top, dose-response with aminoflavone for 18 hours; bottom, time course with 1 µmol/L aminoflavone. D, effect of aminoflavone on BrdUrd incorporation patterns. Cells were treated with the indicated concentrations of aminoflavone for indicated times, pulse labeled with BrdUrd during the last 30 minutes of the aminoflavone exposure. Cells were fixed, stained with PI (red) and anti-BrdUrd antibodies (green), and examined by confocal microscopy.

 
To examine whether the aminoflavone-induced S phase was reversible, MCF-7 cells were treated with aminoflavone for 18 hours (T18) to induce early S arrest (Fig. 5A, right). Then aminoflavone was washed out and cells were further incubated without aminoflavone in the absence or presence of nocodazole for an additional 24 hours (R24). Aminoflavone-treated cells remained in early S phase irrespective of in the presence of nocodazole, demonstrating irreversible S phase arrest by aminoflavone.

Pulse-labeling experiments showed dose- and time-dependent inhibition of DNA synthesis by aminoflavone (Fig. 5B). To further dissect DNA synthesis inhibition, cells were analyzed by two-dimensional flow cytometry (Fig. 5C). Control cells uniformly incorporated BrdUrd throughout S phase during the 30-minute pulse (left). Figure 5C, top shows BrdUrd incorporation after treatment with various concentrations of aminoflavone for 18 hours. BrdUrd incorporation was inhibited by aminoflavone starting at 0.1 µmol/L aminoflavone. Only a few cells had high incorporation of BrdUrd after exposure to 0.3 µmol/L aminoflavone and incorporation was completely inhibited at 1 µmol/L aminoflavone. Time course experiments showed that BrdUrd incorporation decreased gradually throughout S phase after 4 hours of treatment with 1 µmol/L aminoflavone and was almost inhibited after a 8-hour treatment (Fig. 5C, bottom). These experiments show that aminoflavone is a potent inhibitor of DNA synthesis throughout the S phase.

To further gain insight into the effect of aminoflavone on the pattern of DNA synthesis, MCF-7 cells were pulse-labeled with BrdUrd, stained with PI and anti-BrdUrd antibody, and examined by indirect immunofluorescence microscopy. The doses and treatment times of aminoflavone inducing partial DNA synthesis inhibition were determined based on results described in Fig. 5B and C. As shown in Fig. 5D (control), DNA synthesis patterns (22) can be classified into early S phase: identified by the presence of many sites of BrdUrd incorporation distributed throughout euchromatic compartments of the nucleus (white arrow, E), middle S phase: identified by peripheral and peri-nucleolar BrdUrd staining (white arrow, M), and late S phase: identified by few large sites of BrdUrd incorporation (white arrow, L). All three patterns were observed in aminoflavone-treated cells (Fig. 5D, white arrows), but the BrdUrd incorporation levels decreased. These observations are consistent with the fluorescence-activated cell sorting analyses results and confirm that aminoflavone inhibits DNA synthesis throughout the S phase.

Aminoflavone induces RPA2 and p53 phosphorylation and induces p21Waf1/Cip1 and MDM2 in MCF-7 cells. Because RPA2 is hyperphosphorylated by DNA-PK, ATM, and/or ATR in response to DNA replication damage (13, 23), we tested whether aminoflavone induces RPA2 phosphorylation. Figure 6A shows the appearance of upper-shifted bands (see arrows) of RPA2 with in aminoflavone-treated cells, indicating phosphorylation of RPA2 (13). p53 was also phosphorylated at Ser15 after 4 hours of treatment with 0.4 µmol/L aminoflavone and this phosphorylation increased as a function of exposure time (Fig. 6B). p53 protein levels were concurrently elevated and the downstream target genes, p21Waf1/Cip1 and MDM2 were induced after a 6-hour treatment. Together, these results indicate that DNA damage produced by aminoflavone activates S phase checkpoint with RPA2 and p53 phosphorylation and p21Waf1/Cip1 and MDM2 induction.



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  		Figure 6. Aminoflavone (AF) induces RPA2 and p53 phosphorylation and induces p21Waf1/Cip1 and MDM2. A, Western blot analysis for RPA in MCF-7 cells treated with 0.1 or 1 µmol/L aminoflavone for 6 hours or with 1 µmol/L aminoflavone for 1, 3, and 6 hours. Tubulin was used as loading control. B, Western blot analysis for total p53, p53 phosphorylated on Ser15, p21Waf1/Cip1 and MDM2 in MCF-7 cells treated with 0.4 µmol/L aminoflavone for the indicated times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes the production of the DNA damage in response to aminoflavone in human breast cancer MCF-7 cells. We find that aminoflavone induces high frequency of DPC. DPC are hallmarks of topoisomerase inhibitors, which trap the enzyme-DNA cleavage intermediates (24, 25). The cleavage intermediates are linked to the enzyme at the 3' end of the DNA break for topoisomerase I and at the 5' end of the breaks for topoisomerase II (25, 26). Because each topoisomerase-induced break is linked to one enzyme molecule, the observed SSB/DPC ratio is close to 1 (15, 27). Aminoflavone produced ~10-fold excess DPC over SSB (see Figs. 1 and 2), suggesting that DPC induced by aminoflavone are unrelated to topoisomerase cleavage complexes. Other chemotherapeutic agents, such as platinum derivatives or DNA alkylating agents induce DPC (27). Although the metabolites of aminoflavone is not yet fully elucidated, covalent drug adducts have been identified, suggesting that aminoflavone metabolite(s) might alkylate(s) DNA. The mechanism of DPC formation by aminoflavone remains unknown. In addition to topoisomerases, several other DNA processing enzymes act via covalent intermediates such as endonuclease III, glycosylases, tyrosyl-DNA phosphodiesterase 1, and DNA methyltransferases (28). Identification of the protein(s), forming DPC after aminoflavone exposure is ongoing using cesium chloride centrifugation (29) to purify the protein(s) involved. We have excluded topoisomerase I and topoisomerase II (Fig. 2C). It might be challenging to identify the protein(s) linked to DNA as proteins involved in the DPC induced by platinum derivatives or alkylating agents still remain(s) to be identified.

{gamma}-H2AX is formed in cells treated with ionizing radiation (19) but also in response to replication-induced DSB in cells treated with camptothecin (11) and hydroxyurea (30). {gamma}-H2AX foci appear minutes after ionizing radiation and within 1 hour after camptothecin treatment (11), whereas it takes time (3 hours) for aminoflavone to induce {gamma}-H2AX foci in MCF-7 cells probably because of the time required to induce DPC. Unlike ionizing radiation, DSB-induced by aminoflavone could not be detected by alkaline elution (data not shown), suggesting that DSB occur in nascent DNA. In favor of this possibility, we found that aminoflavone-induced {gamma}-H2AX foci are restricted to replicating cells (Fig. 4C). Moreover, aphidicolin pretreatment prevents the formation of {gamma}-H2AX in aminoflavone-treated cells (Fig. 4A). Both aminoflavone-induced DPC and {gamma}-H2AX display similar kinetics and dose-response. They persist after drug removal, suggesting that DNA replication forks collide with preexisting DPC produced by aminoflavone, which result in DSB in nascent DNA. We also found that aminoflavone effectively inhibits DNA synthesis. Persistence of {gamma}-H2AX is consistent with the persistent S-phase arrest and DPC.

When the genetic material is damaged, a delay in cell cycle progression facilitates DNA repair, thereby avoiding the replication and subsequent formation of potentially hazardous mutations. Aminoflavone inhibits DNA synthesis uniformly at all stages of the S phase and arrests cells in the S phase. We also observed RPA2 phosphorylation in aminoflavone-treated cells, which appeared with similar kinetics as {gamma}-H2AX. RPA2 is rapidly phosphorylated after replication-dependent DNA damages and has been proposed to regulate S-phase checkpoint (13, 31). Phosphorylation and accumulation of RPA at the stalled replication forks may signal the presence of damage and activate the DNA damage response (13, 31, 32). We also found p53 phosphorylation at Ser15, another indicator of DNA damage checkpoint activation. We confirmed the stabilization of p53 and induction of its downstream gene p21Waf1/Cip1 (5). Another p53-target gene, MDM2 is also activated, which is also involved in the DNA damage response (33).

In summary, aminoflavone induces a new type of DNA replication-dependent DNA damage associated with DPC and phosphorylation of histone H2AX, RPA2, and p53 in human breast carcinoma MCF-7 cells. The irreversibility {gamma}-H2AX is consistent with the persistent S-phase arrest induced by aminoflavone. {gamma}-H2AX might be a useful clinical marker for monitoring the efficacy of aminoflavone. The selective generation of {gamma}-H2AX in replicating cells might also provide selectivity for aminoflavone in cancer chemotherapy. Indeed, a fundamental difference between normal and cancer cells is the deregulated proliferation of cancer cells compared with normal cells.


   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.


   Footnotes
 
3 http://www.dtp.nci.nih.gov/docs/dtp_search.html. Back

4 L. Meng, U. Shankavaram, J. Weinstein, and Y. Pommier, unpublished data. Back

5 http://discover.nci.nih.gov/pommier/pommier.htm. Back

Received 1/ 4/05. Revised 3/16/05. Accepted 4/ 1/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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