Doxorubicin-DNA Adducts Induce a Non-Topoisomerase II–Mediated Form of Cell Death Lonnie P

Research Article

Doxorubicin-DNA Adducts Induce a Non-Topoisomerase II–Mediated Form of Cell Death Lonnie P. Swift,1 Ada Rephaeli,2 Abraham Nudelman,3 Don R. Phillips,1 and Suzanne M. Cutts1

1Department of Biochemistry, La Trobe University, Victoria, Australia; 2Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Petach Tikva, Israel; and 3Chemistry Department, Bar Ilan University, Ramat Gan, Israel

Abstract catalyze the unwinding of DNA for transcription and replication, Doxorubicin (Adriamycin) is one of the most commonly used involving the process of cleavage of one strandof DNA duplexand chemotherapeutic drugs and exhibits a wide spectrum of passing a secondduplexthrough this transient cleavage. The activity against solid tumors, lymphomas, and leukemias. intermediate formed is termed the ‘‘cleavable complex.’’ Doxoru- Doxorubicin is classified as a topoisomerase II poison, bicin poisons the cleavable complex, inhibiting religation of the although other mechanisms of action have been character- cleaved duplex, a lesion that results in a DNA double-strand break ized. Here, we show that doxorubicin-DNA adducts (formed by (DSB; refs. 3, 4). Failure to repair DNA DSB results in an apoptotic the coadministration of doxorubicin with non-toxic doses of response. However, the mechanism by which doxorubicin induces formaldehyde-releasing prodrugs) induce a more cytotoxic topoisomerase II–mediated DNA DSB does not fully explain the wide spectrum of cytotoxicity that doxorubicin exhibits (5). Several response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic other mechanisms of action have been suggestedas modesof topoisomerase II–mediated cellular responses (as observed by doxorubicin-induced cell death (5, 6), including inhibition of DNA employing topoisomerase II catalytic inhibitors and HL-60/ and RNA synthesis (7, 8), production of free radicals (9), and the MX2 cells). Apoptosis induced by doxorubicin-DNA adducts formation of formaldehyde-mediated doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overex- (10, 11). None of these alternative modes of cell death have been pressed Bcl-2, suggesting that adducts induce a classic mode previously shown to be independent of topoisomerase II–mediated of apoptosis. A reduction in the level of topoisomerase II– cell effects. mediated double-strand-breaks was also observed with Doxorubicin-DNA adducts were initially characterized in a cell- increasing levels of doxorubicin-DNA adducts and increased free activation system, where doxorubicin-induced transcriptional blockages were observedat 5 V GpC sequences (12), indicating that levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic doxorubicin formed covalent adducts with DNA at these sites. topoisomerase II poison mode of cell death by doxorubicin Doxorubicin-DNA lesions were observedto function as interstrand alone. Collectively, these results indicate that the presence of cross-links in strandrenaturation assays (13) andhave also been formaldehyde transfers doxorubicin from topoisomerase II– observedin the DNA of tumor cells in culture (14). A thorough mediated cellular damage to the formation of doxorubicin- analysis of the cell-free activation system used to form adducts DNA adducts, and that these adducts are more cytotoxic than revealed that formaldehyde was a byproduct of the reaction conditions, and that formaldehyde was responsible for the topoisomerase II–mediated lesions. These results also show V that doxorubicin can induce apoptosis by a non-topoisomer- formation of an aminal linkage between the 3 amino group of ase II–dependent mechanism, and this provides exciting new doxorubicin and the N2 of guanine (15). Formaldehyde can also be prospects for enhancing the clinical use of this agent and for produced in cells under Fe-catalyzed oxidative stress conditions the development of new derivatives and new tumor-targeted induced by anthracyclines (16). Although the lesions are mono- therapies. (Cancer Res 2006; 66(9): 4863-71) adducts, they exhibit the characteristics of interstrand cross-links at GpC sequences, endowing strong duplex stabilization due to strong noncovalent interactions to the opposite strand(17, 18). Introduction Both nuclear magnetic resonance andX-ray crystal structures of The anthracyclines (including doxorubicin, daunomycin, epiru- the drug-DNA adduct have been solved (17, 19), and the formation bicin, andidarubicin)are one of the most clinically useful groups of of doxorubicin-DNA adducts has been studied in cells in culture anticancer chemotherapeutics. These drugs are routinely employed (20). It has been shown that doxorubicin can be preactivated by in combination regimes with other groups of drugs in which each formaldehyde to yield the compound doxoform, which consists of drug generally exhibits a different mechanism of action to increase two drug molecules bound together with three methylene groups tumor cell kill and to minimize induced resistance to these drugs. (21). This preactivated form of doxorubicin displays an accelerated Although doxorubicin is one of the most widely used anticancer uptake by cells, is retainedlonger in the nucleus, andis sub- agents, the mechanism of action for this drug is not fully stantially more cytotoxic than doxorubicin (22). Doxoform exhibits understood. Doxorubicin is normally described as a classic topoi- >200-foldgreater cytotoxicity than doxorubicinalone andis parti- somerase IIa poison (1, 2). The topoisomerase family of enzymes cularly cytotoxic to doxorubicin-resistant cell lines (21). Doxoform studies have highlighted the importance of formaldehyde-mediated doxorubicin-DNA adducts in the mechanism of action of Requests for reprints: Don R. Phillips, Department of Biochemistry, La Trobe doxorubicin. University, Victoria, 3086, Australia. Phone: 61-3-94792182; Fax: 61-3-94792467; E-mail: Formaldehyde-releasing prodrugs (such as AN-9) can be used to [email protected]. I2006 American Association for Cancer Research. enhance the intracellular levels of formaldehyde. The prodrug AN-9 doi:10.1158/0008-5472.CAN-05-3410 (pivaloyloxymethyl butyrate) is cleavedintracellularly by esterases, www.aacrjournals.org 4863 Cancer Res 2006; 66: (9). May 1, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research liberating formaldehyde, butyric acid, and pivalic acid. Formaldehyde varying concentrations of topoisomerase II inhibitors and/or formalde- 14 reacts with doxorubicin and induces doxorubicin-DNA adduct hyde-releasing prodrugs and [ C]doxorubicin (specific activity = 54 mCi/ formation anda synergistic cytotoxic response in tumor cells in mmol; GE Healthcare, Bucks, United Kingdom) for desired times and then culture (20, 23). A combination of daunomycin (an anthracycline that harvested(20). The genomic DNA was isolatedusing a QIAamp bloodkit (Qiagen, Hilden, Germany) and subjected to two phenol extractions and is structurally similar to doxorubicin) and AN-9 was shown to one chloroform extraction before being precipitatedin ammonium acetate increase the survival of mice inoculatedwith mouse monocytic andethanol. Pellets were resuspendedinTE buffer, andthe DNA leukemic cells (24). AN-9 was initially synthesizedas a butyric acid– concentration was calculatedat 260 nm. Samples were quantitatedto releasing prodrug to function as a histone deacetylase inhibitor (25). determine the incorporation of [14C]doxorubicin into DNA using a Wallac Given the logistic problem of treating cells with formaldehyde (a 1410 Liquid Scintillation Counter and expressed as doxorubicin adducts highly reactive molecule), the butyric acid–releasing prodrugs were per 10 kb. identified as a source of intracellular formaldehyde (20). The prodrug Comet assay. The comet assay used was based on methods developed by 6 AN-9 has been examinedas a single agent in phase I clinical trials Hartley andSalti (29, 30). Cell samples (1 Â 10 ) were treatedin 10-cm Petri dishes for 4 hours unless otherwise stated. To generate DNA strand breaks (26) andhas also been evaluatedin phase II clinical trials. During 137 phase I clinical trials, AN-9 doses given were limited by solubility of by ionizing radiation, cells were irradiated in media with a Cs Gammacell 1000 Elite (Nordion International, Inc., Ottawa, Canada). Standard the drug and not by drug-related toxic side effects. The maximal 2 microscope slides were precoated with low EEO agarose (Sigma) and feasible dose was determined to be 3.3 g/m /d, indicating that AN-9 allowed to dry. Treated cell samples in media (0.3-0.5 mL, depending on cell as a single agent is extremely well tolerated(26). numbers) were mixedwith 1 mL of type VII low gelling temperature agarose Here, we show that the cytotoxic potential of doxorubicin can be (molten andkept at 40 jC; Sigma), and1 mL was transferredto the increased dramatically, resulting in the induction of apoptosis under precoatedslidesandset with a 40 Â 22 mm coverslip over the agarose. conditions previously regarded as nontoxic. The cytotoxic lesion Once set, samples were subjectedto a 1-hour lysis in ice-coldlysis buffer responsible for the enhancement of doxorubicin-induced cell death [ref. 29; 100 mmol/L Na2EDTA, 2.5 mol/L NaCl, 10 mmol/L Tris-HCl (pH seems to be a formaldehyde-mediated doxorubicin-DNA adduct. We 10.5) plus 1% Triton X-100] then washedfour times for 15 minutes in ice- have shown that doxorubicin-induced topoisomerase II–mediated coldMilli-Q water (lysis andwash step doneon ice). Samples were DNA damage does not contribute to adduct induced cell death, and transferredto a Horizon 20.25 (Invitrogen, San Diego, CA) electrophoresis apparatus andallowedto sit in ice coldalkali electrophoresis buffer (ref. 30; that with increasing levels of adducts, there was a decrease of 300 mmol/L NaOH, 1 mmol/L EDTA) for 1 hour then subjectedto topoisomerase II–mediated DNA damage, indicating a preference electrophoresis (30 V) for 30 minutes at 4jC. The remaining cell sample was for the formation of the more cytotoxic adduct lesion. To study typically usedto analyze sub-G 1 events for quantitation of apoptosis as doxorubicin-DNA adducts as separate lesions (compared with described below. topoisomerase II–mediated DNA damage), we inhibited topoiso- Following electrophoresis, each slide was flooded with 1 mL neutraliza- merase II–mediated DNA damage by employing various top- tion buffer [0.5 mol/L Tris-HCl (pH 7.5)] for 10 minutes andrinsedtwice for oisomerase II catalytic inhibitors, andthis didnotseem to affect 10 minutes with PBS. Milli-Q water was usedto rinse andrehydrate slides adduct formation and adduct-induced apoptosis. We have also (30 minutes) before staining twice with 1 mL of 2.5 Ag/mL propidium iodide shown that adduct formation and apoptosis can be efficiently for 5 minutes. The stain was rinsedoff with Milli-Q water, andcomet tails induced in topoisomerase II–defective cells. The cell cycle effects of were analyzedusing a fluorescence microscope andKomet software (Kinetic Imaging, Nottingham, UnitedKingdom).For each sample, 50 comet doxorubicin-DNA adducts were also different from the character- tails were countedto yieldthe average olive tail moment (OTM; ref. 31). All istic G2 block observedwith doxorubicin as a single treatment, comet results are representative of the average OTM of two or more with a predominant S-phase cell cycle arrest. These data also show individual experiments with the error being the SE. that doxorubicin can induce apoptosis by a non-topoisomerase II– Analysis of apoptosis and cell cycle distribution by flow dependent mechanism, and this provides exciting new prospects for cytometry. Analysis of cell cycle distribution and cellular events in enhancing the clinical use of this agent andfor the developmentof the sub-G1 phase was from a methodusedfor cell cycle analysis as new derivatives and new tumor-targeted therapies. The formation of previously described (32). After drug treatment, cells (1 Â 106 in 10 mL) doxorubicin-DNA adducts, therefore, provides potential advantages were pelletedandfixedby resuspension in 70% ethanol andincubated for reducing dose-limiting side effects and enhancing tumor cell kill. at room temperature for 30 minutes. After fixing, samples were pelleted at 2,000 rpm for 5 minutes, andpellets were washedonce with ice-cold PBS andcentrifugedfor a further 5 minutes. Pellets were resuspendedin Materials and Methods 0.5 mL DNA staining solution (25 Ag/mL propidium iodide, 100 Ag/mL Cell lines. The promyelocytic leukemic cell line HL-60 andthe RNase A in PBS) andincubatedat 37 jC for 30 minutes in the dark. mitoxantrone resistant variant HL-60/MX2 (which exhibits decreased Samples were transferredto 5-mL Falcon tubes andstoredon ice until expression of topoisomerase IIa andno detectablelevels of topoisomerase assayed. IIh; ref. 27) were obtainedfrom the American Type Culture Collection Samples were analyzedon a FACSCalibur employing CellQuest software (Rockville, MD). HL-60 cells overexpressing Bcl-2 (HL-60/Bcl2) andthe (BD Biosciences, San Jose, CA). The FL2-H filter was usedto measure event parental control HL-60/Puro were generous gifts from Dr. Gino Vairo (CSL size (being a measure of nuclei size andtherefore indicatingcells with Limited, Melbourne, Australia; 28). HL-60/Puro and HL-60/Bcl2 cell lines normal DNA content), or cells with fragmentedDNA (sub-G 1 or apoptotic). contain a stably insertedplasmidexpressing puromycin resistance and,in Cells with normal DNA content were analyzedfor G 1, S, andG 2 basedon the case of the HL-60/Bcl2 cells, overexpress the Bcl-2 gene. These cells were DNA content of these events. Samples (30,000 events) were gatedto routinely maintainedby passaging in the presence of 2 Ag/mL puromycin distinguish doublets and small debris by employing a FL2-A versus FL2-W (Sigma, St. Louis, MO; ref. 28). All HL-60 cell lines were maintainedin RPMI dot plot and applying an appropriate gate. The gated events were then 1640 (JRH Biosciences, Lenexa, KS) supplementedwith 10% FCS (Trace plottedas a FL-2H histogram, andregions were set up to acquire quan-

Scientific, Melbourne, Australia) at 37jC, 5% CO2. Cells were countedusing titative data of either cell cycle distribution or of sub-G1 events compared a Sysmex CDA-500 particle analyzer. Cell viability was assessedby trypan with the events that fell into the normal G1, S andG 2 regions. blue staining. Analysis of DNA fragmentation. Treatedcells (1 Â 106) were harvested, Detection of doxorubicin-DNA adducts. HL-60 cells (2 Â 106) were andDNA was extractedusing a QIAamp bloodkit (Qiagen) accordingto seeded in six-well plates and left overnight. The cells were incubated with the manufacturer’s protocol. ExtractedDNA was quantitated,andequal

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Doxorubicin-DNA Adducts amounts were loaded into a 1% agarose gel. Gels were electrophoresed for induced was too high for accurate detection by the apoptosis 2 to 3 hours at 70 V, andthe resulting gel was stainedwith 2 Ag/mL (measuredas sub-G 1 events) assay. The level of AN-9 chosen was, ethidium bromide and destained with Milli-Q water before visualization. therefore, 50 Amol/L, and under these conditions the dose- dependent increase in adduct formation was maintained (data Results not shown). The comet assay was usedto measure drugDNA Doxorubicin-DNA adduct formation compared with DNA damage and results obtained as an OTM (34–36). An increase in strand breaks. We established conditions where formaldehyde- DNA strandbreakage was observedwith doxorubicin as a single mediated doxorubicin-DNA adducts were observed by using the agent, but this was not observedwhen increasing concentrations of formaldehyde releasing prodrug AN-9 in the human promyelocytic doxorubicin were combined with the formaldehyde-releasing leukemic cell line HL-60 andthe topoisomerase II defectivesubline prodrug AN-9. The background level of DNA damage observed HL-60/MX2 (Fig. 1A). Only low levels of adducts were observed is thought to reflect normal levels of damage associated with with doxorubicin as a single agent (Fig. 1A) andare thought to be DNA repair and perhaps also to breaks induced during the experi- due to endogenous levels of formaldehyde (33) and to formalde- mental processes. Under identical treatment conditions, apoptosis C hyde released by redox cycling of doxorubicin (16). We observed a was analyzedby sub-G 1 DNA content analysis (Fig. 1 ) andDNA doxorubicin concentration-dependent increase in formation of fragmentation (Fig. 1D). The adduct-forming treatments of doxo- adducts in the presence of formaldehyde from f2 adducts per 10 rubicin (1, 2, and4 Amol/L) andAN-9 (50 Amol/L) induced a kb DNA for 1 Amol/L doxorubicin and 100 Amol/L AN-9 to f8.5 striking doxorubicin concentration-dependent increase in detect- adducts per 10 kb for 4 Amol/L doxorubicin and 100 Amol/L AN-9. able apoptosis in HL-60 (30-50%), HL-60/MX2 (20-50%), andHL-60/ The z4-fold levels of doxorubicin-DNA adducts with doxorubicin Puro (25-50%) cell lines following 4 hours of treatment (Fig. 1C andAN-9 combinedtreatments have also been detectedinMCF-7 and D), but no apoptosis was observedfor doxorubicinor AN-9 as andIMR-32 cells (20). Doxorubicin-DNA adductswere also single agents. Caspase-3/7 activity was also measuredto verify observedin the topoisomerase II a–defective HL-60/MX2 cell line apoptosis where the pattern of active caspase-3/7 (data not shown) (27), where a 3.5-fold increase in adducts was detected with doxo- matchedthe pattern of inductionof apoptosis as measuredby sub- rubicin andAN-9 comparedwith doxorubicin as a single agent. G1 DNA content analysis andby DNA fragmentation. To assess We then investigatedthe resulting DNA strandbreakage induced whether apoptotic DNA fragmentation contributedto the OTM by similar concentrations of doxorubicin alone or in combination detected in HL-60 cells under adduct-inducing treatments, the cells with the formaldehyde releasing prodrug AN-9 (i.e., adduct forming were cotreatedwith 20 Amol/L of the apoptosis inhibitor Z-VAD- treatments; Fig. 1B). The concentration of AN-9 was reduced in fmk. There was no detectable decrease of the OTM, whereas a these studies compared with Fig. 1A, as the level of apoptosis substantial decrease of apoptosis was observed (data not shown).

Figure 1. Formation of formaldehyde-mediated doxorubicin- DNA adducts is independent of DNA strand breaks. HL-60 and topoisomerase-defective HL-60/MX2 cells were treated for 4 hours with doxorubicin (Dox) and AN-9, both as single agents and in combination at concentrations as indicated. A, adduct levels were detected in HL-60 cells following treatment with doxorubicin (1-4 Amol/L) in the absence and presence of 100 Amol/L AN-9 and in HL-60/MX2 cells following treatment with 4 Amol/L doxorubicin in the absence and presence of 100 Amol/L AN-9 (n = 4). B, the comet assay OTM was assessed in HL-60 (solid columns), HL-60/MX2 (horizontal lines), HL-60/Puro (diagonal lines), and HL-60/Bcl2 (vertical lines) cell lines following a 4-hour treatment with doxorubicin (1-4 Amol/L) in the absence and presence of AN-9 (50 Amol/L). Columns, average of three or more independent experiments where 50 cells per experiment were analyzed; bars, SE. The extent of apoptosis was determined as the sub-G1 cell population (C) and also by electrophoretic DNA fragmentation analysis (D) for HL-60, HL-60/MX2, HL-60/Puro, and HL-60/Bcl2 cell lines (column designation as in B) following 4-hour treatments with doxorubicin (0-4 Amol/L) alone or in combination with AN-9 (50 Amol/L), or a representative selection of treatments as indicated. Sub-G1 analysis represents the average of at least three individual experiments, while DNA fragmentation gels are representative of patterns that were reproduced at least three times. For HL-60, HL-60/MX2, and HL-60/Puro, the left hand lane is a 1-kb DNA molecular weight marker.

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Furthermore, no apoptosis was detected in the antiapoptotic Bcl-2 overexpressing HL-60/Bcl2 cells (Fig. 1C and D), although formaldehyde-mediated doxorubicin-DNA adducts were induced in these cells (data not shown). To further investigate the relationship between doxorubicin-DNA adducts and DNA strand breakage, we examined adduct formation at a constant doxorubicin concentration with increasing levels of formaldehyde (by increasing AN-9 concentrations from 0 to 30 Amol/L over 6 hours). As anticipated, increasing adducts were formed(Fig. 2 A). Under these conditions, we noted a decrease in OTM (Fig. 2B) that was accompaniedby an increase in apoptosis (Fig. 2C). This relationship suggests that by controlling the formaldehyde levels within a cell, doxorubicin can preferentially form drug-DNA adducts compared with mechanisms that induce DNA strandbreaks. To examine the DNA strand breaks induced by doxorubicin as a single agent in further detail (and to yield some insight into how they were formed), a time course analysis was undertaken (Fig. 3A). The topoisomerase II inhibitor etoposide was also examined under the same conditions for comparison. There was a time-dependent increase in the OTM values obtainedwith a plateau not being Figure 3. DNA damage detected by the comet assay. HL-60 cells were obtained within 4 hours; however, etoposide-induced OTM values subjected to drug and/or ionizing radiation treatment before determination of remainedessentially constant over the time periodof the study. DNA strand breakage by the comet assay. A, comparative analysis of OTM induced by doxorubicin and etoposide (both at 1 Amol/L) for 0.5, 1, 2, and As formaldehyde is a reactive species that can form protein-DNA 4 hours. Columns, average of two independent experiments, where 50 cells cross-links, and doxorubicin-DNA adducts are DNA stabilizing were counted per experiment; bars, SE. B, cells were treated for 4 hours with either no drug as a control (x), 4 Amol/L doxorubicin (n), 50 Amol/L AN-9 (E), or a combination of doxorubicin and AN-9 (.). The cells were then exposed to 2.5 or 5 Gy of radiation before determination of the OTM (n = 3).

lesions, we were interestedto establish if either of these lesions inhibitedthe electrophoretic mobility of the DNA duringthe comet assay, hence resulting in artificially low comet OTM values. To investigate this possibility, we induced ionizing radiation-mediated DNA damage to HL-60 cells and compared the OTM obtained with untreatedcells to the OTM obtainedwith doxorubicin andAN-9 as single agents andin combination. The increase in OTM with increasing ionizing radiation in the control samples (from f0.3 with 0 Gy to f0.9 with 5 Gy) were mimickedwith all drugtreatments (Fig. 3B). Both AN-9 (50 Amol/L) andAN-9 in combination with doxorubicin (4 Amol/L) resultedin similar increases of the OTM from f0.3 to 0.8. Doxorubicin (4 Amol/L) as a single agent increasedthe OTM from f0.95 with 0 Gy radiation to 1.6 at 5 Gy. It shouldbe notedthat there was no detectableincrease of DNA fragmentation (assessedby sub-G 1 analysis) accompanying increasing levels of irradiation for either doxorubicin treated cells, or for doxorubicin plus AN-9 combination treatments (data not shown). Catalytic inhibition of topoisomerase II damage does not affect adduct induced apoptosis. To further characterize the DNA strand breaks induced by doxorubicin, and test whether inhibition of this damage also affected the magnitude of doxorubicin-DNA adducts, topoisomerase II catalytic inhibitors were employed(staurosporine, suramin, andmaleimide).All three inhibitors reduced doxorubicin-induced DNA strand breaks in single agent– andcombination-treatedsamples, as evidentby a reduction of OTM (Fig. 4A, C, and E). Significantly, under adduct- forming conditions, the level of adduct induced apoptosis was Figure 2. Increasing adduct levels results in reduced DNA strand breaks and increased apoptosis. HL-60 cells were treated with doxorubicin (1 Amol/L) not reduced when topoisomerase catalytic inhibitors were present and increasing concentrations of AN-9 (0, 5, 10, 20, and 30 Amol/L) for 6 hours, (Fig. 4B, D, and F). and the level of doxorubicin-DNA adducts (A), OTM (B), and sub-G1 apoptosis Cell growth and viability. To assess the effect of different drug (C) was determined for each treatment. A and C, columns, average of two independent experiments; bars, SD. B, columns, average of two independent treatments on cell growth, cell numbers were determined at the experiments; bars, SE (analysis two sets of 50 cells). endof the 4 hours of treatment following drugremoval andagain

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Doxorubicin-DNA Adducts at 24 and48 hours after treatment in HL-60 (Fig. 5 A) andHL-60/ only f20% apoptosis was detected at the 48-hour post-treatment MX2 cells (Fig. 5B). It was notedthat the control andAN-9-treated time point. Trypan-positive cells indicate a measure of nonviable cell populations exhibitedlinear increasing cell growth, whereas cells. The mode of death displayed by a cell that is measured as populations treatedwith doxorubicin as a single agent or in trypan blue positive is not indicated by this assay; however, it does combination with AN-9 showedinhibition of cell growth. Given reveal viability based on a number of modes of cell death (e.g., late that doxorubicin-DNA adducts were shown to induce significant apoptosis andnecrosis). Trypan staining of these samples indicated levels of apoptosis following the 4 hours of incubation, the cell that all cells assayedat the 4-hour time point were in fact viable viability was investigatedto ensure that datawere not obtainedfor cells (or consistedof a small proportion of early apoptotic cells as a predominantly necrotic cell population. All samples were assayed evidenced by lack of trypan blue staining of combination treated for levels of apoptosis induced following 4 hours of treatment as HL-60 cells at 4 hours). Following 24 hours after treatment, a high well as 24 and48 hours after treatment (Fig. 5 C and D). The level of trypan-positive cells existedin the HL-60 line ( f80%), resulting trypan-positive cells were also measuredto assess cell whereas a lesser number were measuredin HL-60/MX2 cells viability (Fig. 5E and F). Although apoptosis was detected at 4 (f50%; Fig. 5E and F). These results correlate with the apoptosis hours with combination treatments in both HL-60 andHL-60/MX2 data in Fig. 5C and D, showing a delay in progression through cells, no apoptosis was detected with doxorubicin as a single agent apoptosis before a cell becomes trypan positive. until 24 hours after treatment in HL-60 cells and48 hours after Alterations to cell cycle distribution by doxorubicin-DNA treatment in HL-60/MX2 cells. Very high levels of apoptosis were adducts. The delayed cell growth data (Fig. 5A and B) suggest the detected with doxorubicin (4 Amol/L) andAN-9 (50 Amol/L) at possible involvement of cell cycle checkpoints in response to drug 24 and48 hours after treatment, andthese levels (>60%) are treatments. No alterations to the cell cycle profile were observed approaching the high enddetectionlimit of this assay. However, with AN-9 single agent treatments in either HL-60 cells or HL-60/ apoptosis induced by doxorubicin as a single agent was much less MX2 cells (Fig. 6C and G, respectively). As expected, doxorubicin as pronouncedat all time points, especially in HL-60/MX2 cells, where a single agent induced a G2-phase cell cycle accumulation in HL-60

Figure 4. Doxorubicin-DNA adducts are independent of topoisomerase II–mediated cell effects. Effect of 30 minutes of pretreatment with topoisomerase II catalytic inhibitors staurosporine (A and B), suramin (C and D), and maleimide (E and F) on comet OTM (A, C,andE) and apoptosis (B, D, and F) induced by 1 Amol/L doxorubicin as a single agent and in combination with 50 Amol/L AN-9 following a 4-hour exposure to drug(s) (n = 2).

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Figure 5. Post-treatment cell viability. Cell growth was measured in HL-60 (A) and HL-60/MX2 (B) cells as an increase in cell number following treatments with either control (no drug, x), doxorubicin (1 Amol/L, n), AN-9 (50 Amol/L, E), or a combination of doxorubicin and AN-9 (.) for 4 hours. These cells were also assessed for levels of apoptosis in HL-60 (C) and HL-60/MX2 cells (D) and for the percentage of trypan blue–positive HL-60 (E) and HL-60/MX2 cells (F). The four treatments for each time point in C to F (left to right) were a control (no drug treatment), 1 Amol/L doxorubicin, 50 Amol/L AN-9, and doxorubicin in combination with AN-9. Cell growth plots are representative of the trend observed in three independent experiments. Columns, average of two independent experiments; bars, SE. Trypan viability was reproduced twice, counting at least 100 cells per sample. Columns, average; bars, SE.

cells (Fig. 6B) but did not induce an altered cell cycle profile in the DNA adducts (10–13), we sought to investigate the role of topoisomerase II–deficient HL-60/MX2 cells (Fig. 6F) as has been topoisomerase II–mediated cell effects compared with drug-DNA previously described(37).In HL-60 andHL-60/MX2 cells, adducts. Although it is well known that topoisomerase II is not the doxorubicin in combination with AN-9 induced a dramatically sole cytotoxic target of doxorubicin, numerous reports have alteredS-phase cell cycle accumulation. Apoptosis levels associated highlightedthe important role of topoisomerase II, predominantly with these treatments were low in all single-agent treatments in forming protein-associatedDNA DSBs. Previous studieshave (percentages in Fig. 6A-H) but elevatedfor doxorubicin plus AN-9 assayedthe formation of these topoisomerase-associateddoxoru- treatments in both cell lines (Fig. 6D and H). bicin-induced DSB through alkaline elution and K-SDS precipita- tion (38, 39). Due to the experimentally demanding nature of the former assay andthe potential nonspecific effects pickedup by Discussion the latter assay, we sought to identify an alternative assay for the Following the discovery that the topoisomerase II poison measurement of doxorubicin-mediated topoisomerase II damage. doxorubicin also induced formaldehyde-mediated doxorubicin- Initially, we employedthe in vivo complex of enzyme bioassay

Figure 6. Effect of doxorubicin and doxorubicin/ AN-9 treatments on cell cycling. Cell cycle distributions following 24 hours of treatment of HL-60 cells (A-D) and topoisomerase II–deficient HL-60/MX2 cells (E-H) in the absence of drug (A and E) or in the presence of 100 nmol/L doxorubicin (B and F), 25 Amol/L AN-9 (C and G), and the combination of 100 nmol/L doxorubicin and 25 Amol/L AN-9 (D and H). The level of apoptosis (sub-G1 fraction) accompanying each treatment was measured separately and is shown for each cell treatment. Representative of a reproducible cell cycle distribution seen under these conditions (n = 3).

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(a cesium chloride gradient isolation of DNA-protein complexes doxorubicin, but are not cross-resistant to a range of other detected with an anti-topoisomerase II antibody); however, the drugs with non-topoisomerase II–mediated mechanisms of action complexes were not stable enough to survive this treatment (41). HL-60/MX2 cells exhibit reduced expression of the topoiso- (although complexes couldbe detectedusingthe topoisomerase merase IIh isoform andexpress a truncated a isoform that results inhibitor etoposide). We, therefore, employed the comet or single in an altered subcellular distribution and decreased topoisomer- cell gel electrophoresis assay to measure DNA strandbreak damage ase II activity anddonot exhibit up-regulation of P-glycoprotein induced by doxorubicin. This assay has previously been shown to (27). No doxorubicin-mediated OTM was observed in the HL-60/ detect topoisomerase II–mediated effects induced by a range of MX2 cells, whereas comet tails were observedin HL-60, HL-60/ drugs, including doxorubicin and mitoxantrone (30, 40). However, Puro, and HL-60/Bcl2 cell lines (41). Moreover, three independent because it is known that doxorubicin can induce DNA strand topoisomerase II catalytic inhibitors inhibitedthe doxorubicin breakage through other mechanisms of action (5), we validated the OTM in a dose-dependent fashion. Although we cannot com- use of the comet assay to detect topoisomerase II–mediated pletely rule out some contribution from free radical–mediated damage by the use of topoisomerase II–deficient sublines and and other forms of DNA damage, the major form of doxorubicin- topoisomerase II catalytic inhibitors. induced DNA damage detected in these assays seems to be Doxorubicin-mediated DNA damage detected using the topoisomerase II mediated, fully consistent with the main docu- comet assay. The comet assay has been widely used to measure mented mode of action of doxorubicin as a topoisomerase II DNA damage in response to a variety of DNA-damaging agents inhibitor (2–5). (40). DNA damage in the form of single-strand breaks, DSB, and The exact nature of the topoisomerase-mediated damage protein-associatedstrandbreaks can all be measuredby the comet detected by the comet assay is less clear. Although the primary assay (40). However, it is not possible to easily distinguish among lesion mediated by doxorubicin is a topoisomerase II–mediated these various forms of DNA damage. Because the comet assay is DSB, due to other dynamic cellular processes, this may be usedto measure both single strandandDSB, it can potentially quickly translated to secondary topoisomerase II–mediated pick up a range of doxorubicin-mediated effects. For doxorubicin, lesions by stalledreplication forks andtranscriptional com- the main forms of DNA damage that are likely to occur are plexes with trappedtopoisomerase II complexes, although these topoisomerase II–mediated DSB and other secondary damage, types of lesions have been better documented for topoisomerase free-radical-inducedsingle-strandbreaks, andapoptotic DNA I cleavage complexes (42). Most of the data generated were at a fragmentation. The molecular basis for the DNA damage was, time point of 4 hours. To attempt to gain some insight into the therefore, examinedin further detail.We examinedOTM values in nature of this topoisomerase II damage, a time course study was HL-60 andtopoisomerase II–deficientHL-60/MX2 cells andfound undertaken where the amount of doxorubicin-induced damage high levels of strand breakage induced by doxorubicin in HL-60 at 30 minutes was limitedcomparedwith more extensive cells but no strandbreakage at any of the dosesemployedin the damage (roughly 3-fold increase) at 4 hours. In contrast, etopo- MX2 cells. Because the basis of the resistance in MX2 cells is side exhibited a similar degree of damage at all time points topoisomerase II mediated (see below), this cell line should still be with the exception of the 4-hour time point where repair pro- susceptible to free radical–mediated effects. Although we cannot cesses may be commencing, thus resulting in a decreased OTM. rule out some contribution of free radical mediated effects, it Because early time points are appropriate for the measurement seems that there is little free radical mediated strand breakage at of strandbreaks that derivefrom the inhibition of DNA the treatment conditions employed in our study. religation in trappedcleavable complexes, it is likely that much Another potential contributor to DNA strandbreakage is of the damage detected at 4 hours reflects secondary forms of apoptotic DNA fragmentation. The conventional comet assay is topoisomerase II-DNA lesions. This is also supportedby reports not used to detect apoptosis, as these severely damaged cells that DNA lesions induced by doxorubicin persist and even normally harbor small pieces of DNA that disappear during lysis or increase following drug removal (43). electrophoresis, sometimes leaving ghost tails behind(40). Topoisomerase IIa expression profile is foundto correlate with a However, we cannot completely rule out very early stages of G2-phase doxorubicin cell cycle block, with an increase in expres- apoptosis leading to long-range DNA strand breakages that may sion predominantly in late S and early G2 phases of the cell cycle contribute to the OTM in comet assays. To inhibit apoptotic DNA (44, 45). To confirm that HL-60 cells in this study displayed a typical fragmentation, the apoptosis inhibitor Z-VAD-fmk was employed. topoisomerase II–mediated cell cycle response to doxorubicin, cell Although Z-VAD-fmk inhibitedthe apoptosis that was inducedby cycle analysis was undertaken. Doxorubicin treatment induced both doxorubicin and combination treatments, there was no effect aG2-phase cell cycle arrest in HL-60, presumably due to DNA on the comet tails, indicating that the OTM measurements damage recognition at the G2 check point as has been previously obtainedare unlikely to includea significant contribution from described (45), whereas this check point was absent in HL-60/MX2 apoptotic DNA fragmentation. This conclusion was further cells, consistent with absent or reduced topoisomerase II–mediated supportedby the fact that 4 hours of drugtreatments of HL-60/ DNA damage in response to doxorubicin. Bcl2 cells did not induce any apoptosis (assessed by sub-G1 analysis Effect of formation of DNA adducts on topoisomerase II– andDNA fragmentation gels) but yieldedsimilar OTM values as in mediated damage. After validating the comet assay as a measure all other HL-60 cell lines. of topoisomerase II–mediated damage induced by doxorubicin as a It is likely that the majority of DNA damage detected by single agent (where DNA adduct formation was negligible), we used the comet assay in our experiments reflects topoisomerase-II– AN-9 to provide the formaldehyde required for adduct formation mediated DNA damage. As alluded to above, the most convincing andassess the effect of the combination on OTM values. The level evidence is from experiments using the HL-60/MX2 cells. These of AN-9 chosen did not induce apoptosis, did not inhibit cell cells exhibit varying degrees of resistance to a range of topoi- growth, and did not produce any OTM. However, a potential somerase II inhibitors, including mitoxantrone, etoposide, and complication of the comet assay is that DNA cross-linking agents www.aacrjournals.org 4869 Cancer Res 2006; 66: (9). May 1, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research may retardthe movement of comet tails andthus can complicate in doxorubicin/AN-9 combination treatments. Three different interpretation of these results because the OTM wouldyieldan topoisomerase II catalytic inhibitors were used to independently underestimate of DNA strand breaks. Unlike conventional DNA inhibit topoisomerase II–mediated damage. These treatments cross-linking agents, such as cisplatin, it is unlikely that inhibited the damage induced by doxorubicin as a single agent doxorubicin-DNA adducts have the ability to retard movement andalso combination treatments (although this was less dramatic of comet tails because of the limited stability of these adducts than seen with doxorubicin alone as the initial OTM value was under the alkaline conditions that were employed in the comet substantially less). However, the levels of apoptosis in combination assay (46). This was confirmedby subjecting drug-treatedHL-60 treatments remainedunaffectedin the presence of each inhibitor. cells (with extensive comet tails) to increasing times (1 to 3 It shouldbe notedthat results obtainedby the three catalytic hours) in alkaline conditions (pH 12.5), and this treatment did not inhibitors couldbe complicatedby other mechanisms of action. alter the observed comet OTM. Although formaldehyde-mediated For example, staurosporine is an inhibitor of the pre-strand DNA protein adducts can be detected using the alkali comet passage step of the catalytic decatenation of topoisomerase II but assay, higher formaldehyde concentrations than used in our is also a known inhibitor of protein kinases (48). Suramin inhibits experiments are usually required(47). Nevertheless, to verify binding of topoisomerase II to DNA but also inhibits the binding of whether DNA adduct formation by doxorubicin or DNA protein certain growth factors to their receptors (49). Maleimide is thought cross-links (a potential consequence of the formaldehyde released to covalently modify topoisomerase II cysteine residues, thereby from AN-9) affectedany of the comet tails formed,radiation reducing the amount of catalytically active enzyme, but could also experiments were done. Increasing doses of ionizing radiation modify multiple cysteine-containing proteins (50). The similar resultedin similarly increasedOTM values for control, AN-9, results obtained with the three independent inhibitors provide doxorubicin, and combination drug treatments, confirming that goodsupport for the view that the early apoptosis induced DNA adducts and DNA-protein cross-links formed in these assays by adduct-forming treatments is independent of topoisomerase do not lead to any significant under estimation of the observed II–mediated effects. OTM values. We have shown that the apoptotic potential of doxorubicin can OTM values for doxorubicin in the presence of AN-9 were be increased dramatically, resulting in the induction of apoptosis reduced in magnitude compared with doxorubicin as a single under conditions previously regarded as non-toxic. The mechanism agent, implying that topoisomerase-mediated damage is less underlying the enhancement of doxorubicin-induced cell death significant in these drug combination treatments. Despite loss of seems to be the formation of doxorubicin-DNA adducts. We have the dose-dependent topoisomerase II–mediated damage, a dose- shown that doxorubicin-induced topoisomerase II–mediated dependent increase in apoptosis was observed. A titration effect DNA damage does not contribute to adduct-induced cell death, from predominantly topoisomerase II–mediated damage for and that with increasing levels of adducts, there was a decrease of doxorubicin alone to predominantly adduct formation for the topoisomerase II–mediated DNA damage, indicating a preference AN-9 combination was illustratedin Fig. 2. It is apparent that the for the formation of adducts when sufficient formaldehyde is adduct-forming conditions are much more damaging in terms of available. induction of apoptosis than doxorubicin alone. However, this was In clinical trials of advanced breast cancer patients, increased observedat the relatively early time points of 4 to 6 hours. To topoisomerase II expression resultedin a greater response to assess longer-term effects of this damage, cells were monitored at doxorubicin treatments (51), strongly linking single-agent various times after treatment. It is apparent that adduct-forming doxorubicin to topoisomerase-mediated cell death. However, treatments induce apoptosis at earlier time points than with treatments that preferentially induce doxorubicin-DNA adducts doxorubicin as a single agent. However, doxorubicin eventually may prove to be useful where molecular profiling of topoi- causes apoptosis at later time points, suggesting that apoptosis is somerase II status reveals patients who wouldnot be good delayed compared with adduct-forming treatments, where apopto- candidates for therapy with doxorubicin as a single agent (11). sis occurs rapidly. However, a large population of viable cells still There is now considerable potential to enhance the cytotoxic remainedfor cells treatedwith doxorubicinas a single agent, even response of doxorubicin, particularly in doxorubicin-resistant after 48 hours. The HL-60/MX2 cells displayed a similar short-term tumors or tumors that express low levels of topoisomerase II, induction of apoptosis in response to DNA adducts but did not thereby providing a specific strategy for the treatment of these display delayed apoptosis in response to doxorubicin as a single tumors. The activation process also provides the potential to agent, consistent with the lack of topoisomerase II–mediated develop new targeting strategies to localize doxorubicin-induced damage in these cells. cytotoxicity to tumors by localization of the formaldehyde- Treatment with doxorubicin and AN-9 resulted in an S-phase releasing prodrug. cell cycle accumulation. As this was observedin both HL-60 and HL-60/MX2 cells, the observedS-phase accumulation seems to be independent of topoisomerase II. Presumably, S-phase accumula- Acknowledgments tion following 24 hours of drug exposure indicates a cell cycle Received9/23/2005; revised1/30/2006; accepted3/3/2006. checkpoint block in response to doxorubicin-DNA adducts. The Grant support: Australian Research Council (S.M. Cutts andD.R. Phillips), the Marcus Center for Pharmaceutical andMedicinalChemistry, the Bronia andSamuel observation of two distinctly different cell cycle responses provides Hacker Fundfor Scientific Instrumentation at Bar Ilan University, LaTrobe University further support for the view that there are different mechanisms of postgraduate scholarship (L.P. Swift), and Australian Research Council’s Australian action displayed by doxorubicin as a single agent, compared with Research Fellowship (S.M. Cutts). The costs of publication of this article were defrayed in part by the payment of page doxorubicin-DNA adducts that form following combination treat- charges. This article must therefore be hereby marked advertisement in accordance ments. with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Prof. John Hartley (Department of Oncology, University College London) To further test any possible interdependence between the two for help with the comet assay andProf. Geoff Pietersz andDr. DodiePouniotis for types of lesions, topoisomerase II–mediated damage was inhibited assistance with irradiation experiments.

Cancer Res 2006; 66: (9). May 1, 2006 4870 www.aacrjournals.org

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