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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2009 Jul 7;8(7):1985–1994. doi: 10.1158/1535-7163.MCT-09-0320

Induction of glutathione-dependent DNA double-strand breaks by the novel anticancer drug brostallicin

Josée Guirouilh-Barbat 1, Yong-Wei Zhang 1, Yves Pommier 1,*
PMCID: PMC2760303  NIHMSID: NIHMS120798  PMID: 19584235

Abstract

Brostallicin is a DNA minor groove binder in Phase II clinical trials. Here we show that brostallicin induces γ-H2AX nuclear foci that co-localize with 53BP1 and are dependent on glutathione, as demonstrated by inhibition of those γ-H2AX foci by L-buthionine sulfoximine (BSO). To differentiate brostallicin from the clinically approved minor groove binder trabectedin (ecteinascidin 743), we tested whether the brostallicin-induced γ-H2AX and antiproliferative responses were dependent on nucleotide excision repair (NER) and found that, unlike trabectedin, they are not. Additionally, brostallicin retained activity in the trabectedin-resistant HCT116-ER5 cell line. Induction of γ-H2AX foci by brostallicin was partially dependent on the repair nuclease Mre11. Pretreatment with aphidicolin partially reduced brostallicin-induced γ-H2AX foci, suggesting that brostallicin induces both replication-associated and replication-independent DNA damage. Replication-associated DNA damage was further demonstrated by the co-localization of γ-H2AX foci with replication foci and by the rapid inhibition of DNA synthesis and accumulation of cells in S-phase in response to brostallicin. In addition, brostallicin was able to induce lower intensity γ-H2AX foci in human circulating lymphocytes. Together our results indicate that brostallicin induces DNA double-strand breaks and suggest ©-H2AX as a pharmacodynamic biomarker for brostallicin.

Keywords: Brostallicin, Glutathione, Mre11, Nucleotide excision repair, DNA double-strand breaks, Histone γ-H2AX, DNA minor groove binder

Introduction

Minor groove binders are an appealing class of anticancer agents as they possess high affinity and selectivity for interacting with DNA [for review see (1, 2)]. Several minor groove binders also alkylate DNA at guanine N2 or adenine N3. One such drug recently approved in Europe for the treatment of resistant or relapsed sarcomas is the marine alkaloid, trabectedin (ecteinascidin 743, Et743, Yondelis) (3–5). Although minor groove alkylating drugs generally show high anti-tumor activity in experimental systems, their activity is often hampered by dose-limiting bone marrow toxicity, which makes it difficult to reach therapeutic doses (1, 6–8).

The search for novel DNA alkylating agents has nevertheless continued (9, 10), and recently a synthetic distamycin-related compound, brostallicin (PNU-166196) was selected for clinical development because of its promising activity in experimental tumor models and of its reduced bone marrow toxicity leading to an appreciable therapeutic index (2, 11, 12). In preclinical studies, brostallicin was found active against cancer cells resistant to classical DNA alkylating agents such as cisplatin (11), and to retain activity in mismatch repair deficient cells (13). Thus, brostallicin was proposed for patients developing resistance to alkylating agents or for combination therapies with classical alkylating agents (14, 15). Currently brostallicin is under evaluation in Phase II clinical trials with promising activity in soft tissue sarcomas (16, 17).

Brostallicin also stands apart from other DNA alkylating agents based on the fact that glutathione is involved in its metabolic activation (18). Accordingly, the in vitro and in vivo activities of brostallicin are increased in tumor cells with high levels of glutathione and /or glutathione-S-transferase (18). This is in contrast with classical DNA alkylating agents that tend to be inactivated by elevated glutathione, which is a common feature of cancer cells (19, 20). Thus, the over-expression of glutathione and glutathione-S-transferase in tumor cells (21–25), confers a potential selectivity of brostallicin toward cancer cells, and sets it apart from other DNA alkylating agents. The proposed molecular mechanism for the activation of brostallicin by glutathione involves a reaction of the α-bromoacrylic moiety of brostallicin with glutathione, which is catalyzed by glutathione-S-transferase, to form a highly reactive glutathione-brostallicin complex able to alkylate the exocyclic N2-position of guanines in the minor groove of DNA (7, 18) (Fig. 1, bottom part).

Figure 1. Structure and proposed mechanism for brostallicin activation by glutathione.

Figure 1

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[Adapted from ref. (7)].

Because the induction of DNA damage by other guanine N2 minor groove alkylating agents, such as trabectedin, has recently been found readily detectable as an induction of histone γ-H2AX foci (26–28), which are landmark markers for the presence of DNA double-strand breaks (27, 29), the first aim of the present study was to determine whether brostallicin also induced γ-H2AX foci. Since we found this to be the case, the next aim of the present study was to determine the mechanism of formation of the γ-H2AX foci by testing whether those γ-H2AX foci were dependent on glutathione and on the transcription-coupled nucleotide excision repair (TC-NER), the latter being a characteristic of trabectedin (26, 30–32). Overall the results from this study differentiate brostallicin from other minor groove binding agents in development.

Materials and Methods

Cells

All cell lines were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA). Xeroderma Pigmentosum complementation group D (XPD) fibroblasts and their stably complemented counterparts XPD-c were provided by Dr. Kenneth Kraemer (Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland). Fibroblasts from a normal individual, GM00637, and Xeroderma Pigmentosum complementation group F (XPF) fibroblasts were obtained from the Coriell cell repository (Camden, NJ). Colon carcinoma cell lines HCT116 cells were obtained from the Developmental Therapeutics Program (NCI, NIH). XPG-deficient and trabectedin-resistant HCT116-ER5 cells were established in our laboratory (31). The human peripheral lymphocytes were obtained from the Blood Bank at the NIH and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum.

Drugs and antibodies

Brostallicin was kindly provided by Systems Medicine (Scottsdale, AZ), a wholly-owned subsidiary of Cell Therapeutics, Inc.. Trabectedin (Et743) was a kind gift from PharmaMar (Madrid, Spain). Ten mM stock solutions in DMSO were stored at −20°C. Aphidicolin (APD), BSO (L-buthionine sulfoximine) and N-acetylcysteine (NAC) were purchased at Sigma chemical company (St Louis, MO). The anti-γ-H2AX antibody was a mouse monoclonal antibody purchased from Upstate Biotechnology (Lake Placid, NY). The rabbit polyclonal anti-53BP1 was purchased from Novus Biologicals (Littleton, CO).

Confocal microscopy

Cells used for microscopy studies were grown one day before drug treatment in Nunc chamber slides (Nalgene, Rochester, NY). Following treatment, the medium was aspirated out and cells were washed in PBS. Cells were immediately fixed and permeabilized by a 20 minute incubation at room temperature with 2% paraformaldehyde and an overnight incubation in ice-cold 70% ethanol at 4ºC. After a 5 minute wash in phosphate buffered saline (PBS), cells were incubated with 8% bovine serum albumin for 1 h at room temperature to block non-specific binding. Cells were stained for 90 minutes with the primary antibodies and tagged for 45 minutes with fluorescent secondary antibodies (Alexa-488 or Alexa-568, Molecular probes, Carlsbad, CA, 1/1000). All incubations were made in 1% bovine serum albumin, at room temperature. The primary antibodies were diluted 1/2,000 for γ-H2AX and 1/500 for 53BP1. Nuclei were stained with propidium iodide (0.05 mg/ml) and RNase A (0.5 mg/ml) for 10 minutes at 37°C.

For the simultaneous detection of γ-H2AX and replication foci, cells were treated with brostallicin for two hours and labeled with 20 µM EdU (5-ethenyl-2’-deoxyuridine; Invitrogen, Carlsbad, CA) for the last 80 minutes. Following treatment, the medium was aspirated out and cells were washed in phosphate-buffered saline (PBS). Cells were immediately fixed and permeabilized by a 20 minute incubation at room temperature with 2% paraformaldehyde and an overnight incubation in ice-cold 70% ethanol at 4ºC. We first performed the staining for γ-H2AX as described above. The staining for EdU was then performed with the Click-iT™ EdU flow cytometry assay kit from Invitrogen (Carlsbad, CA) following manufacturer’s instructions.

Slides were mounted using Vectashield mounting liquid (Vector Labs, Burlingame, CA) and visualized using either a Nikon Eclipse TE-300 confocal laser scanning microscope system or a Becton Dickinson Pathway confocal microscope (BD, Franklin Lakes, NJ). Images were captured and stored as TIF files. For each sample in each experiment, 50 to 200 cells were scored. The quantification of staining intensity was done with Adobe Photoshop 7.0 and normalized to the number of cells analyzed.

Cell viability assays

Cells were first seeded at a density of 1,000 per well in 96-well microtiter plates. One day later, brostallicin was added and incubations were continued for an additional 72 hours. Then 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well (0.5 mg/mL; Sigma Aldrich, St Louis, MO), and plates were maintained at 37°C for 4 hours. The medium was discarded and DMSO was added to each well to lyse the cells. Absorbance was measured at 450 nm using a multiwell spectrophotometer (Emax, Molecular Devices, Sunnyvale, CA).

BrdU Incorporation and Cell Cycle Analyses

Cells were incubated with brostallin for the indicated times and then pulse-labeled with 50 µM BrdU during the last 30 minutes. Cells were then harvested by trypsinization and washed twice with PBS. The pellet containing 2 to 4 × 106 cells was suspended in 50 µl PBS and fixed with 1.5 ml of ice-cold 70% ethanol. After overnight storage at −20ºC, cells were centrifuged and the pellet was suspended in 0.4 ml 2 N HCl. After 30 minute incubation at room temperature (RT), the medium was neutralized by the addition of 0.8 ml 0.1 M sodium borate pH 8.5. Cells were centrifuged and the pellet washed twice in PBS containing 0.5% Tween-20 and 0.5% BSA. Cells were incubated with 15 µl anti BrdU-FITC (BD Biosciences, San Jose, CA) for 1 hour at RT in the dark. To determine DNA content, 500 µl of staining solution containing 20 µg/ml propidium iodide and 50 units of RNase A in PBS was added to the pellet. Cells were analyzed with an FACScan flow cytometer (BD Biosciences, San Jose, CA) using the CellQuest software (BD Biosciences, San Jose, CA).

Results

Glutathione-dependent induction of histone γ-H2AX by brostallicin

To investigate whether brostallicin induced DNA damage, we treated HCT116 cells with various concentrations of brostallicin for 6 hours and analyzed the formation of γ-H2AX foci, which is among the most sensitive methods to detect DNA damage and particularly DNA double-strand breaks (DSBs) (29). Figure 2A shows that γ-H2AX foci appeared with a dose as low as 10 nM brostallicin. The quantification of several experiments (Figure 2B) shows that the γ-H2AX staining increased in a dose-dependent manner up to 1 µM. The H2AX response did not increase further at 10 µM, consistent with other alkylating DNA minor groove agents such as hedamycin that also show a plateau or even a decrease at high drug concentration (33). Comparable induction of γ-H2AX was observed in five other cell lines (see Figure 4B). We also found that brostallicin was able to induce γ-H2AX in normal prostate epithelial cells (Suppl. Fig. 1).

Figure 2. Brostallicin induces γ-H2AX foci in a glutathione-dependent manner.

Figure 2

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A–B: Dose-dependent induction of γ-H2AX foci by brostallicin in human colon cancer HCT116 cells. Cells were exposed to the indicated concentrations of brostallicin for 6 hours and analyzed for γ-H2AX by immunofluorescence confocal microscopy. A: Representative images; B: Quantification of the intensity of γ-H2AX staining normalized to the number of cells analyzed (mean ± SD of 3 independent experiments). The intensity of γ-H2AX staining in cells treated with 1 µM brostallicin was set as 1. C–D: Depletion of intracellular glutathione by L-buthionine sulfoximine (BSO) decreases brostallicin-induced γ-H2AX. HCT116 cells were pretreated with BSO (100 µM) for 16 hours and then treated with 1 µM brostallicin for 6 hours. D: Quantification of the intensity of γ-H2AX staining normalized to the number of cells analyzed (mean ± SD of at least 3 independent experiments). The intensity of γ-H2AX staining in cells treated with 1 µM brostallicin was set as 1.

Figure 4. Brostallicin and trabectedin both induce γ-H2AX foci but neither the induction of γ-H2AX foci nor the antiproliferative activity of brostallicin depend on NER, and brostallicin-induced γ-H2AX foci are Mre11-dependent.

Figure 4

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A: Comparison between brostallicin and trabectedin for the induction of γ-H2AX foci. HCT116 cells were treated for 6 hours with either brostallicin or trabectedin, as indicated. γ-H2AX induction was measured by immunofluorescence confocal microscopy. Left panels: Representative images; Right panels: Quantification of γ-H2AX staining intensity normalized to the number of cells analyzed (mean ± SD of three independent experiments). B: Brostallicin-induced γ-H2AX foci are independent of the NER status of the cells. NER-deficient cells (XPD, HCT116-ER5 and XPF) and their NER-proficient counterparts (XPD-c, HCT116 and GM00637, respectively) were incubated with the indicated concentrations of brostallicin for 6 hours. The induction of the γ-H2AX foci was then measured by immunofluorescence. Quantification of the γ-H2AX staining intensities normalized to the number of cells analyzed (mean ± SD of three independent experiments). For each panel the intensity in the NER-proficient cell line incubated with 10 µM brostallicin was set as 1. C: The antiproliferative activity of brostallicin is independent of NER. The same pairs used in panel B (NER-deficient and proficient cells) were incubated with the indicated concentrations of brostallicin for 72 hours and cell survival was analyzed by MTT assays (mean ± SD of three independent experiments). D: HCT116 cells and their Mre11-complemented clone (38) were treated with the indicated brostallicin concentrations for 6 hours. Quantitation is shown at left and representative images at right.

As mentioned in the Introduction, Geroni et al. (18) have shown that glutathione is necessary for the activation of brostallicin and its covalent binding to DNA (see Figure 1). To determine whether the γ-H2AX induction by brostallicin was dependent on glutathione, we used BSO (L-buthionine sulfoximine), which is an inhibitor of the gamma glutamylcysteine synthetase (one of the enzymes implicated in glutathione synthesis) to deplete intracellular glutathione (21, 34). As shown on the representative immunofluorescence confocal microscopy images (Figure 2C) and on the quantification of several independent experiments (Figure 2D), BSO decreased the intensity of γ-H2AX staining. This result is consistent with the fact that brostallicin-induced DNA damage is dependent on intracellular glutathione levels. The partial effect of BSO could be attributed to residual GSH or H2AX activation by the non-covalent binding of brostallicin in the DNA minor groove.

Brostallicin-induced γ-H2AX foci persist for several hours after drug removal and co-localize with 53BP1, another marker of DSB

To further characterize brostallicin-induced DNA damage, we determined the kinetic of the appearance of γ-H2AX foci. As shown on the representative pictures (Figure 3A) and on the quantification of the intensity of γ-H2AX staining (Figure 3B), γ-H2AX foci were detectible after short treatments (1 hour). The intensity of the γ-H2AX staining then increased with the time of exposure in HCT116 cells. Comparable kinetics were observed in different cell lines, the NER-deficient XPD and their counterpart complemented for XPD, XPD-c (data not shown).

Figure 3. Kinetics of brostallicin-induced γ-H2AX foci and focal co-localization of γ-H2AX with 53BP1.

Figure 3

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A–B: Kinetics of γ-H2AX induction. HCT116 cells were treated with 1 µM brostallicin for the indicated times and γ-H2AX foci were visualized by immunofluorescence confocal microscopy. A: Representative images; B: Quantification of the intensity of γ-H2AX staining normalized to the number of cells analyzed. The intensity of γ-H2AX staining in cells treated with brostallicin for 6 hours was set as 1. C: Colocalization of brostallicin-induced γ-H2AX foci with 53BP1. HCT116 cells were treated with 1 µM brostallicin for 6 hours, and analyzed by immunofluorescence confocal microscopy for both γ-H2AX and 53BP1. D: Persistence of the brostallicin-induced γ-H2AX foci. HCT116 cells were treated for 2 or 4 hours with brostallicin and then allowed to recover in a drug-free medium for 4 and 2 hours, respectively. γ-H2AX foci were then analyzed and compared to the γ-H2AX signal measured in cells continuously incubated with brostallicin for 6 hours, which was set as 1 (mean ± SD).

To further characterize the γ-H2AX foci induced by brostallicin, we performed co-staining experiments with both γ-H2AX and another landmark marker for DNA double-strand breaks, 53BP1 (35). As shown on Figure 3C, brostallicin induced the formation of 53BP1 foci that co-localized with the γ-H2AX foci. Similar induction of 53BP1 colocalizing with γ-H2AX foci was confirmed in the XPD and XPD-c cell lines (data not shown). The co-staining γ-H2AX and 53BP1 is consistent with the induction of DNA double-strand breaks by brostallicin.

Next we investigated whether γ-H2AX foci induced by brostallicin would disappear when the drug was washed out. We treated HCT116 cells with 1 µM brostallicin for 2 hours or 4 hours. After which we let the cells recover in a drug free medium for 4 hours or 2 hours, respectively. As shown in Figure 3D, the intensity of γ-H2AX staining was the same whether the cells were treated continuously for 6 hours with brostallicin or treated for 2 or 4 hours and then allowed to recover in a drug free medium. These result shows that 2 hour exposure to brostallicin is sufficient to induce the formation of γ-H2AX foci that persist for at least 4 hours after the drug is washed out from the culture medium.

Brostallicin-induced γ-H2AX foci and antiproliferative activity are independent of nucleotide excision repair, which sets brostallicin apart from trabectedin

Like brostallicin, trabectedin (Et743), another DNA minor groove alkylating agent, is very efficient at inducing γ-H2AX foci (26–28). Figure 4 (panels A-B) shows that brostallicin induced comparable levels of γ-H2AX foci as trabectedin in HCT116 cells.

Because in the case of trabectedin, the induction of γ-H2AX is dependent on transcription-coupled nucleotide excision repair (TC-NER) (26, 28), we tested whether brostallicin would also induce NER-dependent γ-H2AX. To that effect, we treated three NER-deficient cell lines (XPD-deficient fibroblasts, XPG-deficient and trabectedin-resistant HCT116-ER5 cells and XPF-deficient fibroblasts) with brostallicin, and compared the induction of γ-H2AX in those cells to the induction of γ-H2AX in their complemented or wild-type counterparts (the XPD-complemented XPD-c cells, HCT116 or GM00637 wild type fibroblasts, respectively). Those cell pairs are the same we recently used to demonstrate the TC-NER-dependent induction of γ-H2AX by trabectedin (28). As shown on Figure 4B, the induction of γ-H2AX after brostallicin treatment was similar in NER-deficient or NER-proficient cell lines, showing that, by contrast to trabectedin, brostallicin-induced γ-H2AX foci are NER-independent.

Also, we recently reported that the increased induction of γ-H2AX foci in NER-proficient cells was correlated to an increased antiproliferative activity of trabectedin (26, 28). In order to further investigate the lack of implication of NER in brostallicin activity, we performed MTT assays in the 3 pairs of cell lines mentioned above (XPD vs. XPD-c, HCT116-ER5 vs. HCT116 and XPF vs. GM00637). Figure 4C shows similar antiproliferative activity whether the cells were NER-proficient or NER-deficient. Altogether, the γ-H2AX and the cell proliferation assays show that brostallicin acts independently of the NER and thus demonstrate that brostallicin is different than trabectedin. Additionally, brostallicin still retained activity in the HCT116-ER5 cell line generated in our laboratory for trabectedin resistance (31). These findings also suggest that cross-resistance between trabectedin and brostallicin may not occur.

Mre11 increases the brostallicin-induced γ-H2AX response

Mre11 is a DNA repair endonuclease, which has been associated with DSB repair (36). To determine whether Mre11 was involved in brostallicin-induced DSBs, we compared the induction of γ-H2AX in Mre11-deficient HCT116 cells (37, 38) and in their Mre11-complemented clone (38). Figure 4D shows enhanced induction of γ-H2AX in Mre11-complemented cells, which indicates the γ-H2AX response to brostallicin is partially dependent on Mre11 activity.

Replication-dependence of the brostallicin-induced γ-H2AX foci

Examination of the γ-H2AX responses of different cells simultaneously treated in a given experiment with brostallicin showed that not all cells responded similarly. Some cells exhibited intense response with multiple foci while some had only a few foci and some others showed minimal γ-H2AX foci (see Fig. 2–Fig. 4). Our prior studies revealed that such differential staining could be attributed to replication-dependent γ-H2AX induction in response to replication-associated DNA damage (39, 40). To investigate whether brostallicin-induced DNA damage was related to replication, we treated cells with aphidicolin, a DNA polymerase inhibitor, which we previously used to prevent γ-H2AX induction by replication-associated DNA damage (39, 40). Figure 5A shows that aphidicolin reduced brostallicin-induced γ-H2AX, which is consistent with replication-induced DNA damage by brostallicin.

Figure 5. Brostallicin induced γ-H2AX foci are mostly replication-dependent.

Figure 5

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A: γ-H2AX inhibition by the DNA polymerase inhibitor aphidicolin (APD). HCT116 cells were treated with brostallicin in the absence or presence of aphidicolin, as indicated. left: Representative pictures; right: Quantification of γ-H2AX staining intensity normalized to the number of cells analyzed (mean ± SD of 3 independent experiments). The intensity of the γ-H2AX staining in HCT116 cells treated with brostallicin alone was set as 1. B: Brostallicin induced γ-H2AX foci co-localize with replication foci visualized by EdU incorporation. HCT116 cells were treated with 1 µM brostallicin for 2 hours and pulse-labeled with 20 µM EdU. The γ-H2AX staining is shown on top and the EdU staining at the bottom. Insets: arrowheads indicate cells with γ-H2AX and EdU colocalization; asterisks indicate γ-H2AX positive cells without significant EdU staining. C: Brostallicin induced γ-H2AX foci in human lymphocytes. D: Quantification of γ-H2AX staining intensity normalized to the number of cells analyzed.

To gain further insight in the relationship between brostallicin-induced γ-H2AX formation and DNA replication, we performed experiments to determine whether the brostallicin-induced γ-H2AX were colocalized with replication foci (41). To that effect, we took advantage of the fact that replication foci can be labeled by incorporation of thymidine precursors and detected by indirect fluorescence (42, 43). We used (EdU) (5-ethenyl-2’-deoxyuridine; Invitrogen, Carlsbad, CA), which is readily incorporated into DNA in actively replicating cells and detected its incorporation by immunofluorescence confocal microscopy. Figure 5B[NN1] shows that the cells with most intense γ-H2AX were also positive for EdU, and that the cells negative for EdU tended to have the lowest γ-H2AX levels, suggesting preferential γ-H2AX induction in cells that were actively replicating at the time of the brostallicin treatment. Moreover, in the EdU positive cells, nuclear distribution of γ-H2AX foci generally coincided with the replication (EdU) foci. Together these results are consistent with the fact that brostallicin induces replication-associated DNA damage.

Because the suppressive effect of aphidicolin on brostallicin-induced γ-H2AX was only partial (Fig. 5A) and some cells without significant EdU labeling still showed γ-H2AX foci (Fig. 5B inset, asterisks), we investigated whether brostallicin could also induce γ-H2AX in non-replicating cells. Figure 5 (panels C and D) shows that brostallicin could induce γH2AX foci in normal human lymphocytes, albeit with higher doses of brostallicin. Those results demonstrate that, at low dose, the γH2AX induction by brostallicin was mostly dependent on replication whereas micromolar concentrations of brostallicin were able to induce γ-H2AX independently of replication.

Inhibition of DNA replication and S-phase synchronization by brostallicin

As we found that brostallicin induced replication-associated DNA damage, we tested the effect of brostallicin on cell cycle progression and DNA synthesis. Flow cytometry analyses (Fig. 6A) showed that brostallicin induced a marked accumulation of the cells in the S-phase of the cell cycle after 24 hour drug exposure. The G1- and G2/M-phases were almost completely depleted. The percentage of cells in G1 dropped from 44.0 ± 2.5% in untreated cells to 4.9 ± 2.0% in brostallicin-treated cells and the percentage of cells in G2/M dropped from 14.3 ± 2.1% in untreated cells to 2.0 ± 2.0% in brostallicin-treated cells. Almost all the cells were distributed in S-phase (from 41.8 ± 1.5% in control cells to 93.1 ± 0.1% in brostallicin-treated cells) (Fig. 6A).

Figure 6. S-phase arrest and inhibition of DNA synthesis by brostallicin.

Figure 6

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A: HCT116 cells were treated with 1 µM brostallicin for the indicated times and DNA was stained with propidium iodide. Left panels: Measurement of the cell cycle distribution using Modfit software (mean ± SD of 3 independent experiments). Right panel: Representative cell cycle histogram; B: Short exposures to brostallicin inhibit BrdU incorporation. HCT116 cells were incubated with 1 µM brostallicin for the indicated times, and were pulse-labeled with 50 µM BrdU during the last 30 minutes. Left panel: Representative plots of the FACS analysis. Right panel: Measurement of the percentage of BrdU positive cells using CellQuest software (mean ± SD of 3 independent experiments).

In parallel experiments we measured DNA synthesis in brostallicin-treated cells by BrdU pulse incorporation. As shown on the representative FACS plots and by quantifying independent experiments (Fig. 6B), brostallicin reduced BrdU incorporation, with BrdU-positive cells dropping from 34.8 ± 3.2% BrdU positive cells in control cells to 16.8 ± 2.3% and 13.0 ± 2.7% in cells treated with brostallicin for 4 and 6 hours, respectively. These results are consistent with inhibition of DNA synthesis by brostallicin.

Discussion

In this study we have shown that brostallicin induces DNA damage, in particular DNA double-strand breaks, as revealed by the induction of γ-H2AX and 53BP1 foci (Fig. 2 and Fig. 3). Consistently with the alkylation of DNA by brostallicin, we show that washing out the drug did not decrease the induction of γ-H2AX and that the induction of DNA damage upon brostallicin treatment was persistent (Fig. 2). Also in accordance with the proposed glutathione-dependent DNA alkylation by brostallicin (7, 18) (Fig. 1), we found that the induction of γ-H2AX was reduced when the intracellular levels of glutathione were depleted by BSO. Together the above results further justify the rationale for using brostallicin in tumors with high glutathione levels, which is a relatively common feature of cancer cells especially after prior chemotherapy (7, 18–20, 23–25, 44).

The inhibition of brostallicin-induced γ-H2AX in cells treated with the DNA polymerase inhibitor, aphidicolin (45) and the co-localization of those γ-H2AX foci with replication factories detected by the incorporation of an analog of thymidine, EdU (Fig. 5 and Fig. 6), demonstrate replication-dependent induction of γ-H2AX by brostallicin. The fact that brostallicin inhibited DNA synthesis and led to an accumulation of brostallicin-treated cells in S-phase (Fig. 6) is also consistent with a prominent effect of brostallicin on DNA replication. Replication-coupled DSB (RC-DSB) are well-known for other anticancer agents like the camptothecins (38, 39). In that case, DNA polymerase run-off converts the DNA single-strand breaks generated by the trapping of topoisomerase I-DNA complexes into DSB (46). We have recently reported an alternate mechanism with another novel anticancer agent, aminoflavone derivative (NSC 686288), which produces DNA-protein complexes that arrest replication fork progression (40) and may involve “replication fork collapse” (47). Abnormal DNA structures and helicase deficiencies have also been shown to produce RC-DSB that can be detected as γ-H2AX foci (48). RC-DSB have also been reported for DNA alkylating agents, which like brostallicin alkylate guanine N2 in the DNA minor groove. Indeed, both Et743 and S23906 (9, 27, 28) generate γ-H2AX foci that were proposed to occur when an advancing DNA replication fork runs into a DNA adduct. Both models (collision of the replication fork either with a DNA-protein-drug complex or a DNA adduct) are consistent with the decrease of γ-H2AX induction produced by aphidicolin and the co-localization of γ-H2AX foci with replication factories (see Fig. 5) (41).

In addition to replication mediated-γ-H2AX foci, we show here that brostallicin can induce γ-H2AX independently of replication based on the finding that aphidicolin only partially reduces the H2AX response (see Fig. 4A), based on the presence of γ-H2AX foci in cells outside of S-phase, and based on the H2AX response of post mitotic peripheral lymphocytes at micromolar concentrations of brostallicin (see Fig. 4B). Replication-independent DSB have also been reported for trabectedin (28) and the pluramycin derivative, hedamycin (33). Whether such DSB are related to transcription, as in the case of trabectedin (28) remains to be elucidated.

Because brostallicin and trabectedin are both DNA minor groove alkylators and are clinically active in the same types of tumors (soft tissue sarcomas) (4, 16), we compared the molecular and cellular effects of the two drugs. We find that trabectedin and brostallicin do not have the same dependency on NER, neither in terms of antiproliferative activity, nor in terms of induction of γ-H2AX foci. γ-H2AX foci induction upon brostallicin treatment was the same in cells proficient or deficient for NER (Fig. 4B–C). Accordingly, NER-deficient cells did not exhibit differential sensitivity to brostallicin (Fig. 4C). We also show that brostallicin retains full activity in the HCT116-ER5 cells (31) (Fig. 4C), which further differentiates brostallicin from trabectedin. Thus, our study demonstrates key mechanistic differences between brostallicin and trabectedin with respect to the molecular and mechanistic determinants of γ-H2AX induction (primarily replicative in the case of brostallicin and NER-dependent in the case of trabectedin) and nucleotide excision repair-dependency. By contrast, although the detailed molecular mechanisms leading to the formation of DSBs remain to be fully determined, we found that the DSB-associated nuclease Mre11 increased the γ-H2AX-induction by brostallicin was partially dependent on the DSB-associated nuclease Mre11 (see Fig. 4D). We also recently reported that Mre11 was involved in the induction of γ-H2AX by trabectedin (28), which indicates that both alkylating agents are able to induce DSBs in connection with Mre11.

In conclusion, our study establishes some unique characteristics that set brostallicin apart from presently used anticancer agents. Indeed, the DNA damaging activity of brostallicin is enhanced by high levels of glutathione, which is a hallmark of drug-resistant tumor cells (7, 18, 21–25). We also show for the first time that brostallicin selectively damages and blocks replicating DNA within replication factories. These results, with the detection of γ-H2AX foci in peripheral lymphocytes upon brostallicin treatment, suggest γ-H2AX detection as a useful pharmacodynamic biomarker (28, 29, 49) in the upcoming clinical trials with brostallicin. The rapid appearance and persistence of γ-H2AX for several hours following brostallicin removal make γ-H2AX a potentially useful pharmacodynamic marker for the ongoing brostallicin clinical trials.

Supplementary Material

1

Acknowledgements

We wish to thank Dr. Elizabeth Bruckheimer and the drug development team of Systems Medicine, LLC, Scottsdale, AZ, for kindly providing brostallicin. We also wish to thank Dr. Daniel Von Hoff for bringing brostallicin to our attention and for his sustained interest in the present study.

Funding information: This study was solely supported by the intramural program of the National Cancer Institute, Center for Cancer Research, Bethesda, Maryland.

Abbreviations

DSB

DNA double-strand breaks

Et743

Ecteinascidin 743

γ-H2AX

phosphorylated histone H2AX at serine 139

NER

Nucleotide Excision Repair

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Supplementary Materials

1
NIHMS120798-supplement-1.doc (72.5KB, doc)

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