Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage (original) (raw)

Skip Nav Destination

Experimental Therapeutics, Molecular Targets, and Chemical Biology| October 17 2007

V. Ashutosh Rao;

1Laboratory of Molecular Pharmacology and

Search for other works by this author on:

Keli Agama;

1Laboratory of Molecular Pharmacology and

Search for other works by this author on:

Susan Holbeck;

2Developmental Therapeutics Program, National Cancer Institute, Department of Health and Human Services, Bethesda, Maryland

Search for other works by this author on:

Yves Pommier

1Laboratory of Molecular Pharmacology and

Search for other works by this author on:

Requests for reprints: Yves Pommier, National Cancer Institute, Department of Health and Human Services, NIH, 37 Convent Drive, 37-5068, Bethesda, MD 20892. Phone: 301-496-5944; Fax: 301-402-0752; E-mail: pommier@nih.gov.

Received: February 27 2007

Revision Received: July 09 2007

Accepted: August 08 2007

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2007

Cancer Res (2007) 67 (20): 9971–9979.

Split-Screen
Views Icon Views
Open the PDF for in another window
Tools Icon Tools
Search Site
Article Versions Icon Versions
- Version of Record October 17 2007

Abstract

Batracylin (8-aminoisoindolo [1,2-b_]quinazolin-10(12_H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI50) value for batracylin in HT29 colon carcinoma cells was 10 μmol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1- and Top2α/β-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (γ-H2AX) was tested to measure the extent of DNA damage. Kinetics of γ-H2AX “foci” showed early activation with low μmol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life of γ-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage–responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of γ-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and γ-H2AX could be considered a biomarker in the ongoing clinical trials. [Cancer Res 2007;67(20):9971–9]

Introduction

The DNA topoisomerase enzymes relax helical supercoiling generated during transcription, replication, and chromatin remodeling (1). Topoisomerase I (Top1) transiently cleaves a single strand of DNA, whereas topoisomerase II (Top2) cleaves double-stranded DNA (2). Interfacial inhibitors of topoisomerases reversibly trap these enzyme-DNA complexes, which become lethal lesions upon encountering a replication fork (3). Agents that target topoisomerases are successfully used as anticancer drugs (4, 5). However, drug resistance due to varying expressions of Top1 or Top2 and prolonged treatment schedules due to rapid reversibility of cleavage complexes impose serious therapeutic limitations (6, 7). Batracylin [8-aminoisoindolo [1,2-b_]quinazolin-10(12_H)-one; NSC320846] is a solid tumor-active compound developed by the Developmental Therapeutics Program of the National Cancer Institute (NCI; refs. 8, 9). Given i.p., batracylin was shown to inhibit tumor growth completely in 80% to 100% of mice with early-stage colon adenocarcinomas (9). Previous animal model studies have also shown s.c. and oral activity against solid tumors (implanted colon adenocarcinomas and pancreas ductal carcinomas) and Adriamycin-resistant P388 leukemia (9, 10). COMPARE analysis in the NCI-60 anticancer screen panel of cell lines suggested batracylin activity to be most closely related to the class of Top2 inhibitors (11–13).3

The overall dose-response curves revealed that the leukemia subpanel was most sensitive, with 50% growth inhibition of six leukemia cell lines ranging from 0.2 to 14.7 μmol/L. The colon cancer subpanel was ranked second most sensitive with GI50 between 4 and 15.8 μmol/L. Batracylin presents a promising agent because no correlation was found between drug sensitivity and activity of P-glycoprotein efflux pump [multidrug resistance (MDR) activity], as is seen for many MDR substrates (14).

Batracylin is undergoing evaluation as an anticancer agent at the NCI in patients. We therefore proposed to test the use of potential biomarkers of its DNA-directed actions. Members of the DNA damage response pathway are among the first responders to genomic alterations produced by topoisomerase-targeting agents. The ataxia telangiectasia mutated (ATM) kinase, a member of the PIKK family, is autophosphorylated on serine 1981 and acts on downstream substrates responsible for DNA repair, cell cycle arrest, and apoptosis. Histone H2AX phosphorylated on serine 139, termed γ-H2AX, is one of the earliest markers of camptothecin-induced replication-associated DNA double-strand breaks (DSB; refs. 15, 16) and of etoposide (VP-16; ref. 17). γ-H2AX is proposed to anchor broken chromosomes together and recruit DNA repair elements, including Mre11-Rad50-Nbs1 (18, 19). Another substrate of ATM is the cell cycle–associated kinase, Chk2 (2, 20). The Chk2 kinase undergoes a cascade of autophosphorylation, including on threonine 68, upon camptothecin-induced DNA damage (21).

In this report, we present results from our study on the topoisomerase-directed mechanism of actions of batracylin. We also present evidence for the use of phosphorylated histone γ-H2AX as a biomarker of its DNA damage.

Materials and Methods

Drugs, Enzymes, and Chemicals

Camptothecin was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). VP-16 was purchased from Sigma-Aldrich. MJ-III-65 (NSC 706744) was provided by Dr. Mark Cushman (Purdue University, Purdue, IN). Drug stocks were prepared at 1 mmol/L in DMSO, aliquoted, and stored at −20°C before use. Recombinant human Top1 was purchased from TopoGEN, Inc. Recombinant human Top2α was provided by Dr. John Nitiss (St. Jude Children's Research Hospital, Memphis, TN). Recombinant Top2β was a kind gift from Dr. Neil Osheroff. T4 polynucleotide kinase, DNA polymerase I (Klenow fragment), deoxynucleotide triphosphates, φX174 DNA, agarose, and polyacrylamide/bis were purchased from Invitrogen or New England Biolabs. DNA Quick Spin columns were purchased from Roche Diagnostics. [γ-32P]Deoxy-ATP and [α-32P]dGTP 5′-triphosphate were purchased from Perkin-Elmer Life and Analytical Science. Oligonucleotides were synthesized by MWG Biotech.

Cell Culture and Drug or Ionizing Radiation Treatment

Human colon carcinoma HT29, HCT116, and MCF-7 cells were obtained from the Developmental Therapeutics Program, NCI. HCT116-p53–deficient cells were obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). MCF-7/VP cells were maintained in 4 μmol/L VP-16 during culture. Cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C before experimental use. Cells grown in T75 flasks were exposed to indicated doses of ionizing radiation from a 137Cs source in a Mark I irradiator (J.L. Shepherd and Associates).

Colony Formation Assay

Cell survival was determined using a colony formation assay after indicated treatments. HT-29 cells were trypsinized, counted, and plated (in triplicate, 50/500/5,000 cells) on six-well, 60-mm sterile polystyrene culture plates. The cells were maintained in 3 mL of culture medium and incubated unperturbed for 10 days. Before colony counting, culture medium was aspirated, and colonies were treated with 2 mL of fixation solution (50% methanol, 5% acetic acid) for 1 h. After removal of fixation solution, colonies were stained with 3 mL of Wright's Giemsa stain (Sigma Diagnostics) for 1 h. Colonies were counted manually.

Assessment of Cell Survival

Cytotoxicity of batracylin was assessed by the sulforhodamine B (SRB; Sigma-Aldrich) assay as described previously (22). IC50 was calculated using the software Prism 4 (GraphPad Software, Inc.). Data were obtained from at least three independent experiments.

Alkaline Elution

DNA damage was detected using alkaline elution assays as described previously (22, 23). HT29 cells were prelabeled with [3H]thymidine (1 μCi/mL) for 72 h. Cells were chased overnight (16 h) with radioisotope-free medium before receiving drug treatments. The cells were harvested after specified incubations by scraping them into ice-cold HBSS.

Detection of DNA-protein cross-links and protein-associated strand breaks. DNA-protein cross-links (DPC) and protein-associated strand breaks were analyzed using DNA-denaturing (pH 12.1) alkaline elution carried out under nondeproteinizing conditions as described previously (24).

Detection of DNA single-strand breaks. DNA single-strand breaks (SSB) were analyzed using DNA-denaturing (pH 12.1) alkaline elution carried out under deproteinizing conditions as reported previously (24).

Detection of DSBs. DSBs were analyzed using non–DNA-denaturing (pH 9.6) elution carried out under deproteinizing conditions as described previously (23, 25).

Top1- and Top2-Induced DNA Cleavage Assays

Top-mediated reactions have been described previously (22). The 161-bp fragment from pBluescript SK(−) phagemid DNA (Stratagene) was 3′-end labeled with [α-32P]dGTP. For Top1 cleavage assays, labeled DNA (50 fmol/reaction) was incubated with 5 ng of recombinant Top1 with or without drug at 25°C in 10 μL reaction buffer [10 mmol/L Tris-Cl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 15 μg/mL bovine serum albumin (BSA), final concentrations]. Maxam Gilbert loading buffer [3.3 volumes of 80% formamide, 10 mmol/L sodium hydroxide, 1 mmol/L sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue (pH 8.0)] was added to the reaction mixtures. Aliquots were separated in 16% denaturing polyacrylamide gels (7 mol/L urea) in 1× Tris-borate EDTA (45 mmol/L Tris, 45 mmol/L boric acid, and 1 mmol/L EDTA) for 2 h at 40 V/cm at 50°C.

For Top2 assays, the same pSK fragment used for Top1 assays or single-stranded oligonucleotides were 5′-end–labeled with [32P]ATP and T4 polynucleotide kinase. Labeling mixtures were subsequently centrifuged through Mini Quick Spin DNA columns (for pSK fragment) or Oligo columns (for oligonucleotides; Roche Diagnostics) to remove the unincorporated label. Annealing to the complementary strand of the oligonucleotides was done by heating the reaction mixture to 95°C and overnight cooling to room temperature in 10 mmol/L Tris-HCl (pH 7.8), 100 mmol/L NaCl, and 1 mmol/L EDTA. DNA substrates (10 pmol/reaction) were incubated with 500 ng of Top2α or Top2β (kind gift from Dr. Neil Osheroff) in the presence or absence of drugs for the indicated times at 25°C in 10 μL of reaction buffer [10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L ATP, 0.2 mmol/L DTT, 0.1 mmol/L EDTA, and 15 μg/mL BSA]. Reactions were stopped by adding SDS (final concentration 0.5%). Samples were separated on 16% (for pSK DNA) or 20% (for the oligonucleotides) denaturing polyacrylamide gels (7 mol/L urea). Imaging and quantitation were done using a PhosphorImager (Molecular Dynamics).

Immunocomplex of Top1-DNA Detection Assay

Top1-DNA adducts were detected as described previously (16, 26). Briefly, 106 treated or untreated cells were pelleted and immediately lysed in 1% sarkosyl. Following homogenization with a Dounce homogenizer and pestle B, cell lysates were gently layered on cesium chloride step gradients and centrifuged at 165,000 × g for 20 h at 20°C. Half-milliliter fractions were collected and the fractions 6 to 10 were pooled together. The pooled fractions were then diluted with 25 mmol/L sodium phosphate buffer (pH 6.5) to make a 1×, 2×, 4×, or 8× scaled dilution for better resolution of differences and applied to Immobilon-P membranes (Millipore) in a slot-blot vacuum manifold. Top1-DNA complexes were detected using the C21 Top1 monoclonal antibody (gift from Yung-Chi Cheng, Yale University, New Haven, CT) using standard Western blotting procedures. Top2α and top2β antibodies were obtained from Abcam.

Confocal Microscopy of Nuclear Protein Localization, Antibodies Used

Cells grown in Nunc chamber slides (Nalgene) using 0.5 mL of tissue culture medium were fixed and permeabilized as described previously using 4% paraformaldehyde and ice-cold 70% ethanol (16). Nonspecific binding was blocked using 8% bovine serum albumin in PBS. Fixed cells were stained overnight with primary antibodies (in 1% BSA at 4°C) and tagged with fluorescent secondary antibodies (Molecular Probes) for 2 h at room temperature. Slides were mounted using Vectashield mounting liquid (Vector Labs) and sealed. Immunofluorescence microscopy was done in a Nikon Eclipse TE-300 confocal laser scanning microscope system. The mouse anti–γ-H2AX antibody was purchased from Upstate USA, Inc., and the rabbit anti–γ-H2AX antibody was obtained from Dr. William Bonner (NCI). The phosphorylated S1981-ATM and phosphorylated T68-Chk2 antibodies were purchased from Cell Signaling Technologies. Images were collected as tif files and processed with Adobe Photoshop (Adobe Systems, Inc.). Images were converted to black and white and ellipses were drawn over the nuclear boundaries for clarity.

Results

Cytotoxicity profile of batracylin. Batracylin is a synthetic heterocyclic amine (Fig. 1A, structure) that has advanced through the NCI drug development program based on its anticancer activity in mouse colon adenocarcinoma 38 and other tumor models. Using a colony formation assay, we determined the IC50 of batracylin to be 10 μmol/L after a 6-h pulse exposure in the human colon carcinoma cell line, HT29 (Fig. 1B). Figure 1C shows the activity profile of batracylin in the NCI-60 panel of human cancer cell lines using a SRB dye–based cell proliferation assay. The colon cancer subset of cell lines was ranked second most sensitive, after leukemic subset, with GI50 between 4.07 and 15.85 μmol/L. The mean value for concentration corresponding to GI50 across all cell lines (MG_MID) was 4.4 μmol/L. COMPARE analysis of batracylin has shown that its pattern of activity is most closely related to that of the Top2-targeting family of drugs (Supplementary Table S1). The sensitivity to batracylin did not seem to be predictable based on the p53 status of these cells from the NCI-60 panel. Thus, we confirmed the cytotoxicity of batracylin in human cancer models and chose the colon carcinoma cell line, HT29, to further analyze its topoisomerase-directed molecular mechanism.

Figure 1.

Cytotoxicity profile of batracylin (NSC320846). A, chemical structure of batracylin (NSC 320846). B, cell killing of HT-29 cells by batracylin for 6 h using colony formation assay. The IC50 value in HT-29 cells was 10.02 μmol/L. C, mean graph of the growth inhibition profile for batracylin as tested by SRB assay in the NCI-60 anticancer drug screen cell line panel. GI50 values for each cell line are plotted relative to the mean GI50 across all cell lines (MG_MID, mean graph midpoint). The maximum divergence from the mean (Delta) and the difference between the greatest and smallest values (Range) are indicated below the profile. CNS, central nervous system; NSCLC, non–small cell lung carcinoma.

Figure 1.

Close modal

Batracylin induces DPCs. As a consequence of the proposed Top2-mediated actions of batracylin (11), we proposed that exposure of HT29 cells would produce protein-associated DNA breaks formed as a result of the Top2-DNA cleavage complexes trapped by batracylin. To quantify the protein-associated DNA breaks, we did alkaline elutions (23). Figure 2A shows that treatment with 100 μmol/L of batracylin for 3 h produced DNA SSB under deproteinizing conditions. In contrast, under nondeproteinizing alkaline elution conditions, no such breaks were observed (Fig. 2A,, right), which is consistent with induction of topoisomerase cleavage complexes (23). A time course of 100 μmol/L batracylin revealed an increasing level of DPC formation (Fig. 2B). The formation of DPC was concentration dependent when measured at 3 h (Fig. 2C). As expected for topoisomerase cleavage complexes (23), very low levels of protein-free breaks were observed, and the formation of SSB was nearly equivalent with the extent of DPC (Fig. 2C).

Figure 2.

$Figure 2. Batracylin induces protein-linked DNA breaks consistent with topoisomerase targeting. A, SSBs formed by 100 μmol/L batracylin for 3 h as measured by alkaline elution. Ionizing radiation (3 Gy) was used as positive control. HT29 cells were prelabeled with [3H]thymidine and exposed to either ionizing radiation (*) or batracylin (▪), or were left untreated (○). To measure protein-associated SSB, alkaline elutions were done in the presence (left) or absence (right) of proteinase K. The extent of SSB is expressed as the fraction of DNA remaining on the filter after elution. B, time course for the formation of DPCs induced by 100 μmol/L batracylin. Results show four independent experiments with the shaded area corresponding to the range of the DPC calculated as Rad equivalents. C, concentration-response graph for batracylin-induced DPC, total SSBs, and protein-free breaks (deproteinizing conditions, PFB). Batracylin treatments were for 3 h. DNA lesion frequencies are expressed in Rad equivalents (23). Each unit of 1,000 Rad equivalents corresponds to about one lesion per 106 nucleotides. D, representative alkaline elution experiments show the extent of DSB (left) and SSB (right). Following batracylin treatments, the same cell culture was divided into two fractions, one for DSB and one for SSB. DSB and SSB experiments were done under non-DNA denaturing (pH 9.6) and DNA-denaturing conditions (pH 12.1), respectively. Cells were exposed to 100 μmol/L (⧫) or 300 μmol/L (▴) of batracylin for 6 h followed by alkaline elution. VP-16 (10 μmol/L, ▪) and 50 Gy ionizing radiation (*) were used for comparison. The weak induction of DSB at 3 h (□) by batracylin is included for comparison. For SSB analysis, the cells were the same as for the DSB except for the irradiated cells that were exposed to 3 Gy (*). The extent of DNA lesions is expressed as the fraction of DNA remaining on the filter after elution.$

Batracylin induces protein-linked DNA breaks consistent with topoisomerase targeting. A, SSBs formed by 100 μmol/L batracylin for 3 h as measured by alkaline elution. Ionizing radiation (3 Gy) was used as positive control. HT29 cells were prelabeled with [3H]thymidine and exposed to either ionizing radiation (*) or batracylin (▪), or were left untreated (○). To measure protein-associated SSB, alkaline elutions were done in the presence (left) or absence (right) of proteinase K. The extent of SSB is expressed as the fraction of DNA remaining on the filter after elution. B, time course for the formation of DPCs induced by 100 μmol/L batracylin. Results show four independent experiments with the shaded area corresponding to the range of the DPC calculated as Rad equivalents. C, concentration-response graph for batracylin-induced DPC, total SSBs, and protein-free breaks (deproteinizing conditions, PFB). Batracylin treatments were for 3 h. DNA lesion frequencies are expressed in Rad equivalents (23). Each unit of 1,000 Rad equivalents corresponds to about one lesion per 106 nucleotides. D, representative alkaline elution experiments show the extent of DSB (left) and SSB (right). Following batracylin treatments, the same cell culture was divided into two fractions, one for DSB and one for SSB. DSB and SSB experiments were done under non-DNA denaturing (pH 9.6) and DNA-denaturing conditions (pH 12.1), respectively. Cells were exposed to 100 μmol/L (⧫) or 300 μmol/L (▴) of batracylin for 6 h followed by alkaline elution. VP-16 (10 μmol/L, ▪) and 50 Gy ionizing radiation (*) were used for comparison. The weak induction of DSB at 3 h (□) by batracylin is included for comparison. For SSB analysis, the cells were the same as for the DSB except for the irradiated cells that were exposed to 3 Gy (*). The extent of DNA lesions is expressed as the fraction of DNA remaining on the filter after elution.

Figure 2.

Close modal

Formation of SSB or DSB by batracylin. Because batracylin was known to induce Top2 cleavage complexes (11), we expected batracylin to produce both SSBs and DSBs (23). We used pH-based alkaline elution assays to quantitate the extent of SSB or DSB formation. Contrary to our expectation, batracylin was a weak inducer of DSBs at 3 or 6 h even at concentrations up to 300 μmol/L (Fig. 2D,, left). The extent of clear DSB formation by known Top2-targeting VP-16 was included in the analysis for comparison. Under the same conditions, batracylin efficiently produced SSB within 3 h of exposure (Fig. 2D , right). Thus, batracylin produced SSBs to a greater extent than DSBs, which is unusual for a Top2 inhibitor (23, 25).

Topoisomerase-directed actions of batracylin. The favored production of SSBs over DSBs by batracylin led us to hypothesize that batracylin could be targeting Top1. Induction of DNA cleavage in the presence of Top1 was tested using recombinant Top1 on a purified DNA fragment (Fig. 3A; ref. 22). The cleavage sites observed with camptothecin are indicated by arrows for comparison. Batracylin showed a DNA cleavage pattern distinct from the known Top1-targeting drugs, camptothecin, and the indenoisoquinoline, MJ-III-65 (NSC 706744) (2). We also tested the actions of batracylin on Top2α and Top2β. VP-16 (100 μmol/L) was used as a positive control in these experiments. Shown in Fig. 3B, batracylin exhibits extremely low Top2α inhibition compared with VP-16. We found mainly Top2β-mediated DNA cleavage in the presence of increasing concentrations of batracylin (Fig. 3C). Thus, batracylin is a dual inhibitor of both Top1 and Top2β enzymes.

Figure 3.

Batracylin is a dual Top1 and Top2 poison. A, DNA corresponding to the 3′ end-labeled _Pvu_II/_Hin_dIII fragment of pBluescript SK(−) phagemid DNA (pSK) was incubated with recombinant Top1 in the absence of drug (Top1) or presence of camptothecin, MJ-III-65, or increasing concentrations of batracylin (10–300 μmol/L). Reactions were done at 30°C for 20 min and stopped by addition of 0.5% SDS. Arrows, migration positions of DNA fragments cleaved at the indicated sites (sequence indicated to the right). B, to probe Top2α-mediated cleavage activity, the _Pvu_II/_Hin_dIII fragment of pBluescript SK(−) phagemid DNA (pSK) was labeled at the 5′ ends, and reactions were carried out with recombinant Top2α. VP-16 (100 μmol/L for 30 min) was included as a positive control. C, for testing Top2β-mediated activity, recombinant Top2β was used in the reactions as above with increasing concentrations of batracylin or with VP-16. Arrows, the batracylin-induced cleavage sites. D, cellular topoisomerase cleavage complexes were determined using the ICE bioassay at 3 h. Positive controls were 1 μmol/L camptothecin for Top1 or 100 μmol/L VP-16 for Top2α and Top2β.

Figure 3.

Close modal

Batracylin induced Top1 and Top2 cleavage complexes in vivo. Using the in vivo complex of enzyme (ICE) bioassay, we measured in vivo trapping of Top1, Top2α, and Top2β after batracylin treatment at 3 h in HT-29 cells. Camptothecin (1 μmol/L) or VP-16 (100 μmol/L) were used as positive controls. The pooled fractions were diluted as described in Materials and Methods and probed for Top 1, Top2α, or Top2β. Batracylin at 100 and 300 μmol/L showed increased enzyme complexes at 3 h (Fig. 3D). The complex formation was most apparent for Top1. Thus, batracylin exhibits in vivo trapping of both Top1 and Top2 enzymes.

Batracylin cell killing in cells deficient for p53 and Top1, or resistant to VP-16. Loss in cellular viability by batracylin was assayed using a SRB dye–based test (22). A dose-dependent reduction in cell survival at 72 h was observed in HCT116 colon carcinoma cells deficient or proficient for p53 (Fig. 4A), suggesting a p53-independent mode of cell killing. MCF-7 breast cancer cells stably transfected with Top1 siRNA (27) were partially resistant to batracylin (∼2.9-fold reduction in IC50 value). MCF-7 cells that are resistant to VP-16 (MCF-7/VP; ref. 28) were 5.4-fold resistant to batracylin compared with their sensitive parental cells. Thus, batracylin displays a p53-independent but partially Top1- and Top2-mediated cell killing.

Figure 4.

Antiproliferative activity of batracylin in p53-deficient, Top1-deficient, and VP-16–resistant cancer cell lines. SRB dye–based assays were used to measure growth inhibition. Pairs of HCT116 (p53+/+ or p53−/−; A), siRNA-transfected MCF-7 (Top1 siRNA or control siRNA; B), and MCF-7 (sensitive and VP-16 resistant; C) were treated with increasing concentrations of batracylin and cell viability was measured after 72 h.

Figure 4.

Close modal

Phosphorylated histone, γ-H2AX, as a biomarker of batracylin-induced DNA damage. We tested the phosphorylation of a variant of histone H2A, termed γ-H2AX, as a viable biomarker for the early detection of DNA damage by batracylin. We analyzed whether we would be able to detect nuclear aggregates (“foci”) of γ-H2AX before the earliest detection of DNA breaks that would be indicative of DNA damage. In Fig. 5A, we used confocal microscopy to detect the formation of γ-H2AX foci with increasing concentrations of batracylin for 3 h. Doses as low as 1 μmol/L were able to increase the number of cells that were positive for γ-H2AX. VP-16 was included as a positive control and the time course of γ-H2AX formation in response to batracylin was determined. Figure 5B shows distinct foci formation after 1 h of treatment. The signal for γ-H2AX seemed to increase with time up to 15 h.

Figure 5.

γ-H2AX foci as a cellular biomarker of DNA damage by batracylin. A, batracylin-treated HT29 cells were permeabilized and fixed for visualization of microscopic foci by histone γ-H2AX. Images were converted to gray scale and nuclear boundaries are indicated by white ellipses for clarity. Kinetics of concentration response are shown in representative images. B, HT-29 cells were exposed for various times to batracylin at 10 μmol/L. VP-16 at 20 μmol/L for 3 h was used as a reference. Representative images. C, phosphorylation of ATM kinase (pS1981-ATM) and colocalization with γ-H2AX. HT-29 cells exposed to 1 μmol/L batracylin for 6 h were co-stained with antibodies against γ-H2AX and pATM-S1981. Inset, single enlarged nuclei representative of colocalization. D, batracylin also induces T68 phosphorylated Chk2 foci at 6 h. The weak signal for pChk2-T68 was found to colocalize with foci by γ-H2AX. Inset, single enlarged nuclei representative of colocalization.

Figure 5.

Close modal

The formation of γ-H2AX has been associated with the kinase activity of ATM, a large protein mutated in the disorder ataxia telangiectasia (20, 29). ATM itself is autophosphorylated at S1981 (pS1981-ATM) and is emerging as yet another responsive element to DNA damage (20, 29). We tested the formation of pS1981-ATM along with γ-H2AX and found an equivalent increase in nuclear foci for pS1981-ATM with 3-h treatment of batracylin in HT29 cells (Fig. 5C). The signal for γ-H2AX seemed to colocalize with that of pS1981-ATM. Additionally, we tested the phosphorylation of the cell cycle checkpoint kinase, Chk2 (pT68-Chk2), a substrate of ATM and other phosphatidylinositol 3-kinases (30, 31). In Fig. 5D, we observe weak induction of pT68-Chk2 compared with γ-H2AX. The pT68-Chk2 signal was also colocalized with γ-H2AX.

Thus, γ-H2AX could serve as a biomarker for early detection of DNA damage by batracylin.

Slow reversibility of the DPCs and γ-H2AX foci induced by batracylin. To ascertain the reversibility of DNA damage induced by batracylin, we measured the reversal of γ-H2AX after a 3-h exposure to 10 μmol/L of drug. In Fig. 6A, we show persistence of γ-H2AX foci after 3-h incubation in drug-free medium. By 15 h, most cells had returned to their untreated state for γ-H2AX signal. We then measured the stability of Top1- and Top2-DNA cleavage complexes by alkaline elution. The reversal of DPC after batracylin removal was found to be in agreement with γ-H2AX disappearance (Fig. 6B). The DPC reversal was compared with that of VP-16 and observed to be significantly slower. The half-life of VP-16–induced DPCs was within minutes (<30 min), whereas that of batracylin was between 1 and 2 h. Thus, the reversibility of DNA damage by batracylin was slower than for VP-16.

Figure 6.

Reversal of DNA damage by batracylin. A, reversal of γ-H2AX foci in HT-29 cells after 3-h treatment with 10 μmol/L batracylin. Cells were analyzed at 1, 3, or 6 h after treatment. Images were converted to gray scale and nuclear boundaries were indicated by white ellipses for clarity. B, reversal of DPC by batracylin 1, 2, or 3 h after a 3-h exposure. Reversal from 10 μmol/L VP-16 (circles) is included as comparison. Data from three independent experiments. Shaded area, range of DPC. Dashed line, half-life of DPC reversal after drug removal.

Figure 6.

Close modal

Discussion

Batracylin has advanced through the NCI drug development pipeline based on its activity against colon carcinomas as well as cisplatin- and doxorubicin-resistant tumors. Initial studies had suggested its mechanism to be Top2 targeted (Supplementary Table S1; refs. 11–13). We aimed to perform preclinical studies to test the activity of batracylin on topoisomerases and the DNA damage it caused, as well as provide evidence for a viable biomarker for its DNA-damaging activity. Current anticancer therapy with topoisomerase-directed agents is limited by drug resistance as well as long treatment schedules to overcome reversibility of cleavage complexes. Therefore, we also questioned whether batracylin would present an advantageous option based on its mechanism of action. Previous studies have already indicated that batracylin sensitivity did not correlate with MDR activity (14). Thus, batracylin could present alternative therapy against doxorubicin- or cisplatin-resistant tumors.

The topoisomerase-directed actions of batracylin. We confirmed the toxicity of batracylin in colon carcinoma cell line HT29 by colony formation assay and provided evidence for a dose-dependent cell killing (Fig. 1). The COMPARE algorithm was used to analyze sensitivity patterns in the NCI-60 human tumor cell line panel, and activity of batracylin was most comparable with the Top2 class of drugs. The Top2 inhibitory actions of batracylin were hypothesized to reveal predominantly DSB in cells. However, when alkaline elutions were done to measure DSB and SSB, we observed almost none at 3 h under conditions where SSB were clearly detected. Only a few DSB were detectable at longer times (6 h; Fig. 2D). This led us to hypothesize that batracylin might be targeting Top1 in addition to Top2. In the present study, we show batracylin activity on both Top1- and Top2-mediated DNA cleavage (Fig. 3A–C). Batracylin exhibited a unique cleavage pattern when compared with VP-16 or camptothecin or VP-16, suggesting that it might also target distinct genomic sites. These DNA breaks observed were protein associated because no significant breaks could be measured in the presence of proteinase K (Fig. 2A). Also, we present evidence for increasing DPCs after batracylin exposure with time and increasing concentration (Fig. 2B). Thus, batracylin seems to possess the properties of a dual topoisomerase inhibitor. We confirmed the cellular activity of batracylin on Top1 and Top2 using the immunocomplex of enzyme assay (ref. 26; Fig. 3D). The activity of batracylin was only partially diminished in Top1-deficient or VP-16–resistant cells, confirming the redundancy of the topoisomerase targeting (Fig. 4). The varying expression level of Top1 and Top2 in cancer cells treated with a selective inhibitor can potentially be overcome by use of a dual inhibitor such as batracylin. Thus, batracylin seems to be a nonclassic topoisomerase inhibitor. It belongs to the group of dual topoisomerase inhibitors. A limited number of dual topoisomerase inhibitors have been identified and some are being developed as anticancer agents. These include the anthracycline-like anthraquinone saintopin, indenoquinolones (TAS-103), quaternary alkaloids (fagaronine), the indolocarbazole NB-506, GA3P polysaccharide, the acridine DACA and pyrazoloacridine, the pyridoindole intoplicin, XR5944, and the homocamptothecin derivative BN-80297 (32–40). The DPCs produced by batracylin had a significantly longer half-life than those induced by VP-16 (Fig. 6) and camptothecin (data not shown; ref. 41). Hence, similar to the bisindenoisoquinolines (22), batracylin has an advantage of producing cleavage complexes with slower reversibility than conventional topoisomerase-targeting drugs that require longer treatment schedules. Only a few dual topoisomerase inhibitors have been identified and are being developed as anticancer agents (42, 43). Further preclinical studies on batracylin are therefore warranted to evaluate its clinical potential.

Histone γ-H2AX as a biomarker of the DNA damage by batracylin. We have previously presented γ-H2AX as an experimental biomarker of replication-mediated DNA damage by anticancer agents (16, 22). Phosphorylation of histone H2AX occurs rapidly after replication stress and can be readily detected by microscopic analysis of the nuclear foci that appears after fluorescent antibody staining (44). The phosphorylation of H2AX is mediated, among other kinases, by the ATM kinase. We show clear increase in γ-H2AX foci formation along with an increased colocalized signal for phosphorylated ATM and Chk2 (Fig. 5). The γ-H2AX signals are within similar dose range as the antiproliferative activity of batracylin, as indicated in Fig. 4. Lower doses of batracylin used for these microscopic analyses of γ-H2AX are indicative of the high sensitivity of this assay (15, 45, 46). Pilot studies, including those from our group, with surgical tissue specimens and cell lines have shown that the γ-H2AX foci are present at higher levels in cancerous samples when compared with normal tissue of the same origin (15, 44, 47). These foci are suggested to be indicative of aggressive replication stress–mediated genomic instability in these cells. The early detection of such foci is an early hallmark of DNA damage caused by batracylin and similar to other replication stress models likely signals for the concentration of DNA repair factors at the lesion sites (45, 48). Efforts are under way to characterize the low basal level of γ-H2AX foci in various cancer models (44). An increase in γ-H2AX foci signal is being tested in the clinic as a biomarker of increased DNA damage by ionizing radiation (49) and computed tomography examination (50). The slower rate of reversal of DPCs by batracylin was in agreement with the rate of reversal of γ-H2AX foci after drug removal (Fig. 6). Consequently, batracylin caused DNA damage after topoisomerase inhibition that was measured by alkaline elution as well as γ-H2AX foci formation. We have also previously presented evidence for the use of γ-H2AX in the measurement of DNA damage by camptothecin, 4-nitroquinoline 1-oxide, and aminoflavone in cancer cell lines (14, 24, 25). Thus, γ-H2AX presents a sensitive biomarker of therapeutic efficacy of anticancer drugs that could be readily measured in tissue biopsies or blood cells. It would be most beneficial to use it in combination with other biomarkers of disease progression during the upcoming clinical trials of batracylin.

Acknowledgments

Grant support: Intramural Research Program, Center for Cancer Research, National Cancer Institute.

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

We thank Dr. Jerry Collins for encouraging this work; Drs. Kurt Kohn, Bill Bonner, Andy Jobson, and Melanie Simpson for helpful discussions; and Dr. Neil Osheroff and Jo Ann Byl (Department of Biochemistry, Vanderbilt University, TN) for generously providingTop2α and Top2β enzymes.

References

Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective.

Nat Rev

2002

;

430

–40.

Pommier Y. Topoisomerase I inhibitors: camptothecins and beyond.

Nat Rev Cancer

2006

;

789

–802.

Pommier Y, Redon C, Rao VA, et al. Repair of and checkpoint response to topoisomerase I-mediated DNA damage.

Mutat Res

2003

;

532

173

–203.

Pizzolato JF, Saltz LB. The camptothecins.

Lancet

2003

;

361

2235

–42.

Baldwin EL, Osheroff N. Etoposide, topoisomerase II and cancer.

Curr Med Chem Anti-Canc Agents

2005

;

363

–72.

Rasheed ZA, Rubin EH. Mechanisms of resistance to topoisomerase I-targeting drugs.

Oncogene

2003

;

7296

–304.

Pommier Y, Leteurtre F, Fesen MR, et al. Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors.

Cancer Invest

1994

;

530

–42.

Mucci-LoRusso P, Polin L, Bissery MC, et al. Activity of batracylin (NSC-320846) against solid tumors of mice.

Invest New Drugs

1989

;

295

–306.

Plowman J, Paull KD, Atassi G, et al. Preclinical antitumor activity of batracylin (NSC 320846).

Invest New Drugs

1988

;

147

–53.

Atassi G, Dumont P, Kabbe HJ, Yoder O. A new antitumour agent, batracylin, selected by a preclinical solid tumour model.

Drugs Exp Clin Res

1988

;

571

–4.

Luo Y, Ren YF, Chou TC, et al. A structure-activity relationship study of batracylin analogues.

Pharm Res

1993

;

918

–23.

Paull KD, Shoemaker RH, Hodes L, et al. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm.

J Natl Cancer Inst

1989

;

1088

–92.

Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines.

J Natl Cancer Inst

1991

;

757

–66.

Moreau P, Holbeck S, Prudhomme M, Sausville EA. Cytotoxicities of three rebeccamycin derivatives in the National Cancer Institute screening of 60 human tumor cell lines.

Anticancer Drugs

2005

;

145

–50.

Sedelnikova OA, Bonner WM. γH2AX in Cancer Cells: A Potential Biomarker for Cancer Diagnostics, Prediction and Recurrence.

Cell Cycle

2006

;

2909

–13.

Rao VA, Fan AM, Meng L, et al. Phosphorylation of BLM, dissociation from topoisomerase IIIα, and colocalization with γ-H2AX after topoisomerase I-induced replication damage.

Mol Cell Biol

2005

;

8925

–37.

Banath JP, Olive PL. Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks.

Cancer Res

2003

;

4347

–50.

Bassing CH, Alt FW. H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity.

Cell Cycle

2004

;

149

–53.

Furuta T, Takemura H, Liao ZY, et al. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes.

J Biol Chem

2003

;

278

20303

–12.

Shiloh Y. ATM and related protein kinases: safeguarding genome integrity.

Nat Rev

2003

;

155

–68.

Takemura H, Rao VA, Sordet O, et al. Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks.

J Biol Chem

2006

;

281

30814

–23.

Antony S, Agama KK, Miao ZH, et al. Bisindenoisoquinoline bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-_c_]isoquinoline)-6-propylamino}propane bis(trifluoroacetate) (NSC 727357), a DNA intercalator and topoisomerase inhibitor with antitumor activity.

Mol Pharmacol

2006

;

1109

–20.

Kohn KW. DNA filter elution: a window on DNA damage in mammalian cells.

Bioessays

1996

;

505

–13.

Kohn KW. Principles and practice of DNA filter elution.

Pharmacol Ther

1991

;

–77.

Pommier Y, Schwartz RE, Kohn KW, Zwelling LA. Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents.

Biochemistry

1984

;

3194

–201.

Pourquier P, Pommier Y. Topoisomerase I-mediated DNA damage.

Adv Cancer Res

2001

;

189

–216.

Miao ZH, Rao VA, Agama K, Antony S, Kohn KW, Pommier Y. 4-nitroquinoline-1-oxide induces the formation of cellular topoisomerase I-DNA cleavage complexes.

Cancer Res

2006

;

6540

–5.

Sinha BK, Haim N, Dusre L, Kerrigan D, Pommier Y. DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action.

Cancer Res

1988

;

5096

–100.

Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.

Nature

2003

;

421

499

–506.

Pommier Y, Sordet O, Rao VA, Zhang H, Kohn KW. Targeting chk2 kinase: molecular interaction maps and therapeutic rationale.

Curr Pharm Des

2005

;

2855

–72.

Melchionna R, Chen XB, Blasina A, McGowan CH. Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1.

Nat Cell Biol

2000

;

762

–5.

Etievant C, Kruczynski A, Barret JM, et al. F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103.

Cancer Chemother Pharmacol

2000

;

101

–13.

Leteurtre F, Fujimori A, Tanizawa A, et al. Saintopin, a dual inhibitor of DNA topoisomerases I and II, as a probe for drug-enzyme interactions.

J Biol Chem

1994

;

269

28702

–7.

Denny WA. Dual topoisomerase I/II poisons as anticancer drugs.

Expert Opin Investig Drugs

1997

;

1845

–51.

Finlay GJ, Riou JF, Baguley BC. From amsacrine to DACA (_N_-[2-(dimethylamino)ethyl]acridine-4-carboxamide): selectivity for topoisomerases I and II among acridine derivatives.

Eur J Cancer

1996

;

32A

708

–14.

Ishida K, Asao T. Self-association and unique DNA binding properties of the anti-cancer agent TAS-103, a dual inhibitor of topoisomerases I and II.

Biochim Biophys Acta

2002

;

1587

155

–63.

Larsen AK, Grondard L, Couprie J, et al. The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II.

Biochem Pharmacol

1993

;

1403

–12.

Adjei AA, Charron M, Rowinsky EK, et al. Effect of pyrazoloacridine (NSC 366140) on DNA topoisomerases I and II.

Clin Cancer Res

1998

;

683

–91.

Demarquay D, Coulomb H, Huchet M, et al. The homocamptothecin, BN 80927, is a potent topoisomerase I poison and topoisomerase II catalytic inhibitor.

Ann N Y Acad Sci

2000

;

922

301

–2.

Spicer JA, Gamage SA, Rewcastle GW, et al. Bis(phenazine-1-carboxamides): structure-activity relationships for a new class of dual topoisomerase I/II-directed anticancer drugs.

J Med Chem

2000

;

1350

–8.

Covey JM, Jaxel C, Kohn KW, Pommier Y. Protein-linked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I.

Cancer Res

1989

;

5016

–22.

Vicker N, Burgess L, Chuckowree IS, et al. Novel angular benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents.

J Med Chem

2002

;

721

–39.

Denny WA. Emerging DNA topisomerase inhibitors as anticancer drugs.

Expert Opin Emerg Drugs

2004

;

105

–33.

Nakamura A, Sedelnikova OA, Redon C, et al. Techniques for γ-H2AX detection.

Methods Enzymol

2006

;

409

236

–50.

Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM. Characteristics of γ-H2AX foci at DNA double-strand breaks sites.

Biochem Cell Biol

2003

;

123

–9.

Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM. Quantitative detection of (125)IdU-induced DNA double-strand breaks with γ-H2AX antibody.

Radiat Res

2002

;

158

486

–92.

Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.

Nature

2005

;

434

907

–13.

Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone guardian of the genome.

DNA Repair

2004

;

959

–67.

Kao J, Milano MT, Javaheri A, et al. γ-H2AX as a therapeutic target for improving the efficacy of radiation therapy.

Curr Cancer Drug Targets

2006

;

197

–205.

Lobrich M, Rief N, Kuhne M, et al. In vivo formation and repair of DNA double-strand breaks after computed tomography examinations.

Proc Natl Acad Sci U S A

2005

;

102

8984

–9.

2007

Supplementary data

550 Views

71 Web of Science

69 Crossref

Batracylin (NSC 320846), a Dual Inhibitor of DNA Topoisomerases I and II Induces Histone γ-H2AX as a Biomarker of DNA Damage (original) (raw)

Abstract

Introduction

Materials and Methods

Drugs, Enzymes, and Chemicals

Cell Culture and Drug or Ionizing Radiation Treatment

Colony Formation Assay

Assessment of Cell Survival

Alkaline Elution

Top1- and Top2-Induced DNA Cleavage Assays

Immunocomplex of Top1-DNA Detection Assay

Confocal Microscopy of Nuclear Protein Localization, Antibodies Used

Results

Discussion

Acknowledgments

References

Supplementary data

Citing articles via

Email alerts