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[CANCER RESEARCH 56, 1855-1862, April 15, 1996] Drug-specific Sites of Topoisomerase II DNA Cleavage in Drosophila Chromatin: Heterogeneous Localization and Reversibility1 Maria E. Borgnetto, Division of Experimental Franco Zunino, Stella Tinelli, Emmanuel ABSTRACT DNA cleavage stimulated by different topolsomerase H inhibitors shows in vitro a characteristic sequence specificity. Since chromatin struc tare and genome organization are expected to Influence drug-enzyme interactions and repair of drug-Induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxo rubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide,and amonafide Kits,2 and Giovanni in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencingby primer exten sion. VM-26 stimulated cleavage in the satellite Ill DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis In cloned DNA fragments with Drosophila topoisomerase II dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA deavage reverted faster in the satellite ifi than in the histone repeats While stimulating in a sequence-selective manner, resulting in drug-specific cleavage intensity patterns in sequencing gels (7—9). In past years, much attention has been focused on chromatin sites of topoisomerase II activity in living cells. This enzyme has been suggestedto regulate chromatin structure by specific interactions with SARs (1, 10—14)and scs regions (elements that determine domain boundaries; Ref. 15). Moreover, topoisomerase II cleavage sites often colocalize with DNase I-hypersensitive regions (16—19),which raises the possibility that the enzyme may function to establish or maintain the local topology at hypersensitive regions. In all of these studies, the topoisomerase II inhibitor VM-26 has been used as a tool to enhance enzyme-mediated DNA cleavage. This approach was based on the assumption that VM-26 is able to stimulate DNA cleavage at all in vivo showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major similar levels of DNA breakage in bulk genomic DNA, dh.EPI and VM-26 markedly differed for cleavage extent and reversibifity In specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells. INTRODUCTION sites of topoisomerase Received11/3/95;accepted2/14/96. The costsof publicationof this article weredefrayedin part by the paymentof page charges.This article mustthereforebe herebymarkedadvertisementin accordancewith 18 U.S.C.Section1734solely to indicatethis fact. work was supported partially by the Associazione Italians per Ia Ricerca sul II activity (14, 15, 17—19) and that chro matin structure is the primary determinant of cleavage site selectivity observed in vivo (16). Nevertheless, others groups have described differences in cleavage patterns in viral (20), episomal (21), and cellular chromatin (22) following cell treatments with structurally unrelated inhibitors, mAMSA and VM-26, suggesting that the in vivo site selectivity is determined, at least in part, by the inhibitor. Thus, to establish whether the drug sequence specificity is a deter minant of drug-enhanced DNA cleavage in the chromatin of living cells, we have examined at a sequence level the localization of drug-stimulated topoisomerase II DNA cleavage in Drosophila mela nogaster Kc cells. We selected two regions of the Kc cell genome (the satellite III DNA and the histone gene cluster) since their chromatin structures have been well characterized (see below); thus, the results may provide significant information on enzyme and drug activities also in human malignant cells. Our analysis has been focused on VM-26, dh-EPI (a potent doxorubicin derivative), mAMSA, and amonafide, since these four agents showed quite different sequence specificities in in vitro studies (23—26).The results demonstrate that each drug maintains in vivo a high degree of DNA sequence speci ficity, which correspondsto that establishedin vitro (9, 23, 27, 28), and suggest that the in vivo site selectivity of DNA cleavage is determined by several factors, including the chromatin structure as well as the inhibitor used. Moreover, a high degree of genomic heterogeneity was observed concerning cleavage sites, levels, and reversibility that might play a role in the drug antitumor activity. The chromatin of eukaryotic cells is organized into dynamic struc tural units, the DNA loops, the size and properties of which can vary among distinct genomic regions and during the cell cycle (1, 2). The complex topological problems that thus continuously arise are solved by DNA topoisomerases. These are widespread enzymes that regulate DNA topology through concerted DNA breakage and religation ac tivities (3). An important function of DNA topoisomerasesconsists in relieving the torsional stress generated during DNA replication and transcription (4). Type H DNA topoisomerase has an essential role in the decatenation ofdaughter DNA helices at the end of replication and in the segregation of intertwined chromosomes (5, 6). Moreover, topoisomerase II is the target of several antitumor agents, such as anthracyclines and the demethylepipodophyllotoxin VM-264 (7—9). These compounds stimulate enzyme-mediated DNA cleavage in vitro MATERIALS 1 This Capranico3 Oncology B. Istituto Nazionale per lo Studio e Ia Cura dei Tumori, 20133 Milan, Italy Drugs AND METHODS and Other Materials. VM-26 and dh-EPI were obtained from Bristol Italiana(Latina,Italy) andPharmacia-Farmitalia (Milan, Italy), respec tively. Amonafide Chemistry and mAMSA were obtained from the Drug Synthesis and Branch, National Cancer Institute (Bethesda, MD). Drugs were Cancro(Milan); by the Consiglio Nazionaledelle Ricerche,ProgettoFinalizzatoAppli cazioni Cliniche della RicercaOncologica(Rome); and by the Ministero della Sanità freshly prepared in DMSO and then diluted in deionized water. Drosophila (Rome).E. K. receivedgrantsupportfromthe AssociationpourIa Recherchesurle otide kinase and polyacrylamide were purchased from Life Technologies, Inc. Cancer. 2 Present address: Laboratoire de Biologic Moléculaire Eucaryote, CNRS, Toulouse, 31062France. 3 To whom requests for reprints should be addressed, at Division of Experimental OncologyB. Istituto NazionaleTumori, 20133Milan, Italy. Phone:39-2-2390-203;Fax: 39-2-2390-764;E-mail: [email protected]. 4 The abbreviations used are: VM-26, teniposide; SAR, scaffold-attachment region; mAMSA, 4'-(9-acridinylamino)-methanesulfon-m-anisidide; dh-EPI, 4-demethoxy-3'deamino-3'-hydroxy-4'-epi-doxorubicin;DSB, double-strandbreak. topoisomerase II was purchased from USB (Cleveland, OH). T4 polynucle (Basel,Switzerland).Restrictionenzymeswerefrom BioLabs (Taunus,Ger many).[y-32P]ATPwasobtainedfrom Amersham(Milan, Italy). In Vivo Topoisomerase II Cleavage Assays. Exponentially growing Kc cells (3—4x 106cells/ml at 25°C)were treated with VM-26, dh-EPI, mAMSA, or amonatidefor 30 mm at theindicatedconcentrations.Whenindicated,cells were first treated with 25 p.Mdistamycin for 30 mm before the addition of topoisomerase inhibitors. Heat shock was performed at 37°Cfor 30 mm before 1855 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPO1SOMERASE 11DNA CLEAVAGE IN CHROMATIN the addition of drugs and continued cell incubation at 37°C.Aliquots of 2 X l0@cells were pelleted for 5 mm at 800 X g and lysed in 5 ml of 20 mai cycle (18—20 h) before drug treatments (30 mm at 25°C).Untreated cells were Tris-HC1 (pH 8)-l% SDS, followed by the addition of EDTA to 15 mM and proteinase K to 400 @g/ml.The lysate was incubated at 37°Cfor 16 h with and drug-treated cells (5 X l05/sample) were layered on polycarbonate mem irradiated on ice with ‘y-rays and kept at 0°Cuntil lysis on the filter. Control branes of 25-mm diameter and 2-sm pore size (Nucleopore, Pleasanton, CA) andlysedwith 2% SDS,0.1Mglycine,25 mIstEDTA (pH 9.6),and0.5 mg/ml proteinase K (Merck, Darmstadt, Germany). DNA on the filter was eluted with mediumafter drug treatmentsand incubatedat 25°Cfor the indicatedtime. 0. 1% SDS, 20 nmiEDTA (free-acid form), and tetrapropylammonium hydrox Cellswerethencollectedandlysedasdescribedabove.PurifiedDNA samples ide (Eastman Kodak, Rochester, NY), pH 9.6, for 15h. A calibration curve was were digested with Hindlll and electrophoresed through 1.2% agarose gels derived from elution values of untreated control cells, 2000-md, 5000-md, and 8000-md irradiated cells. DNA DSBs in drug-treated cells were then calculated overnight at 65 V in ThE buffer (89 mM Iris-borate and 2 mM EDTA, pH 8). as described (32) and expressed in md-equivalents. Southern blots were then performed by standard protocols (29). Filters were prehybridized in 10 ml of 6X SSC (1X SSC = 150 miiiNaCI, 50 mi@i sodium gentleshaking,and DNA wasthen purified by standardprocedures(29). To measure cleavage reversibility, cells were resuspended in fresh drug-free citrate), 5X Denhardt's solution, 0.5% SDS, and 0.1 mg/mI denatured salmon sperm DNA for 4 h at 65°C.Cleavage products in the histone gene cluster were detected with indirect end-labeling by using either a 300-bp HindIII-PstI RESULTS (HP Cleavage of Satellite HI DNA Is Stimulated by VM-26 Only. Satellite III DNA (1.688 g/cm3) is a highly repetitive heterochromatic coding regions of the Hi gene, respectively. Hybridization probes generated by DNA (about 5% of the whole genome) of the centromeric region of D. the random primer labeling procedure (29) were added to the filters, together melanogaster X chromosomes. The repeat unit is a SAR-like, AT-rich with fresh salmon sperm DNA, and hybridizations were performed overnight sequence of 359 bp (30). Two nucleosomes are accommodated by at 65°C. To detectcleavagein the satelliteIII repeat,genomicDNA samples were electrophoresed onto 1.2% agarose gels without prior digestion with each repeat, and a strong VM-26-stimulated topoisomerase II cleav age site has been mapped to one of the two nucleosomal linkers (19). restriction enzymes. Hybridization to a cloned satellite III 359-bp repeat was Exponentially growing Kc cells were exposed to VM-26, dh-EPI, as described above. Dried gels were routinely exposed to phosphorimager screens to measure cleavage levels at the different sites with a Molecular mAMSA, or amonafide for 30 mm at the indicated concentrations, and Dynamics phosphorimager 425 model and then were exposed to Amersham DNA cleavage stimulation was assayed by Southern blotting. Since MP films. topoisomeraseII cleavesthissatelliteonceper repeat,hybridizationof In Vitro Topoisomerase II Cleavage Assay. A 5744-bp I DNA fragment DNA extracted from VM-26-treated Kc cells to a satellite III probe containing 16repeatsof satelliteIII DNA from D. melanogasterwascloned results in a DNA ladder of 359-bp unit length (Ref. 19; Fig. lA). In into the AccI site of pUC18. The 5744-bpfragmentwas uniquely 5'-end striking contrast to VM-26, dh-EPI, arnonafide, and mAMSA did not labeledasdescribedpreviouslyandpurifiedby agarosegelelectrophoresisand stimulate detectable cleavage products in this genomic locus, either in electroelution (25, 26). DNA cleavage reactions were performed in 20 @l of 10 probe) or a 530-bp HindIII-BglII (HB probe) fragments spanning the 5' or 3' mM Tris-HC1 (pH 7.9), 50 mM NaC1, 50 mM KC1, 5 mM MgCl2, 0. 1 mu EDTA, 1mr@i ATP, 15 @xg/ml BSA with 10unitsofDrosophila topoisomerase II (USB, Cleveland, OH), and 50 ,.@M of VM-26 or 10 @M ofdh-EPI at 30°Cfor 20 mm. Reactionswere stoppedwith SDS and proteinaseK (1% and 0.1 mg/ml, respectively) and further incubated at 42°Cfor 45 mm. DNA cleavage was examined by agarose gel electrophoresis (25, 26). Dried gels were exposed to A B CVDAN CTVD -1 Amersham MP films. Genomic Sequencing of Cleavage Sites. Histone gene sequence informa tion usedfor theselectionof primerswasobtainedfrom theEMBL Nucleotide Sequence Database (accession number X 14215; D. melanogaster histone gene cluster). Primers DlO-l (5'-CACTI1@GCCACC1TrTCCACG-3'), from 160 to 180 of the upper strand, and Dl0-2 (5'-GTlTFCGGAGGCATFGUCAC 3'), from 429 to 409 of the lower strand, were used for extension through the H2A-H2B spacer region to sequence site 10. DNA sequence information used for analyses of the 359-bp satellite III repeat was derived from Fig. 4 in the report of Hsieh and Brutlag (30). The sequence coordinates of satellite for primers Dl0-l and Dl0-2 and with Hindill or DdeI for primers V-l and V-2, respectively. Primer extensions were carried out as described (31) using 1 i.@g ofdigested DNA and 1—5 X 106cpm of labeled primer per sample. Taq DNA polymerase (1.2 unit; Perkin-Elmer-Cetus or Promega) or Ultma DNA polymerase (0.8 unit; Perkin-Elmer-Cetus) were added to 50-pi reactions in the buffer recommended by the manufacturer supplemented with 2.5 ms@ MgC12and 10 g.LM (final concentrations) of each deoxynucleotide (Boehringer Mannheim). Samples were subjected to 30 cycles of 1 mm denaturation at 94°C, 2 mm hybridizationat 58°C, and 3 mm extensionat 72°C in a Perkin Elmer-Cetus similarly DNA thermal cycler. Sequence digested, appropriately diluted, ladders were generated cloned DNA templates; (Boehringer Mannheim). Reaction products were elution method (32, 33). Briefly, Kc cells separated were :@ . from on a 6 or 8% sequencing gel (29). Dried gels were exposed to Amersham MP films. Filter Elution Technique. DNA DSBs in intact cells were determined by the filter @.. reaction mixtureswerepreparedexactlyasabove,exceptthat extensionswerecarried out in thepresenceof400 @M ddATP,300 @LM ddCTP,100p.MddGTP,or 600 LM ddTFP U, ifi primers were 273—299 (primer V-I, upper strand) and 299—273(primer V-2, lower strand). Primers were 5'-end-labeled with [y-32P]ATPand T4 polynu cleotide kinase as described (24). Genomic DNA samples were digested with MboII *-@ labeled with Fig. I. Topoisomerase11DNA cleavage in the centromericsatellite III DNA in vi@'o (A) and in vitro (B). A, Kc cells were treated with 25 gui distamycin for 30 mm and then incubated with or without topoisomerase II inhibitors for an additional 30 mm at 25C. Lane C, no inhibitor; Lane V. 50 @M VM-26; Lane D, 10 @iMdh-EPI; Lane A. 25 @ssi mAMSA; lime N, 50 ,@Mamonafide. DNA samples were electrophoresed on a 1.2% agarosegel without prior restrictionenzymedigestionandhybridizedto a clonedsatellite m repeat.B, a cloned5744-bpDNAfragmentcontaining16repeatsof the satelliteIII DNA was uniquely 5' end-labeled and then incubated with 10 units of Drosophila [2-'4Clthymidine (0.025 mCi/mI; Amersham International, Milan, Italy) for 20 topoisomeraseII without or with drugs.Lane C, no inhibitor; Lane T, topoisomeraseII h. Cells were then chased in fresh radioactive-free medium for at least one cell alone;Lane V. 50 @LM of VM-26; Lane D, 10 @M of dh-EPI. @, a contaminatingfragment. 1856 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPOISOMERASEII DNA CLEAVAGE IN CHROMATIN 60 age stimulation were lower than those of VM-26 (Fig. 1B). The present findings strongly indicate that the in vivo VM-26 site is also the target for VM-26 stimulation in vitro. Therefore, although satellite 40 5 ifi repeats were accessible to topoisomerase II in the chromatin of a) living cells as shown by VM-26-stimulated cleavage, the other studied drugs were unable to trap the covalent DNA-enzyme complex in the 20 a) heterochromatic satellite ifi DNA. 34 -@10. Drug-specific Patterns of Topoisomerase H DNA Cleavage in 2 ii ;; ; I the Drosophila Histone Repeat. To determine whether differences H3YH4 @‘ H2A H2B H1J might be observed in another locus, we extended our analysis to the a [email protected] gene cluster, a transcriptionally active region whose chromatin 0 ,.. A AAAAAAAA AA A A A _________ structure has been well characterized (35). The major histone repeat U 3b4•@• •• U I C) 2 5 6b7b7a 8b 8a 9b 9a spans a 5-kb DNA region that contains a SAR localized in the (13 10 6a intergenic spacer between the Hi and H3 genes (see map in Fig. 2) and is repeated about 110 times in the Drosophila genome (35). DNA samples from the same experiments of Fig. 1A were digested o_ with Hindffl and analyzed by Southern blotting using the indirect end-labeling technique with probes from the 5' or 3' portions of the Hi gene (HP and HB probes, respectively, in Fig. 3). Drug-stimulated cleavage sites were numbered according to the convention used pre Fig. 2. Relative levels of in vivo topoisomeraseII DNA stimulatedby VM-26 or viously for VM-26-stimulated sites (19). Similarly to VM-26, dh-EPI dh-EPIcleavagein the histonegenecluster.The histonegeneclusteris representedasa stimulated DNA cleavage at several sites in the Drosophila histone 5-kbHindlll DNA fragment:thin lines,noncoding regions;thickhorizontalarrows, repeat (Fig. 3); however, cleavage intensity patterns were clearly coding sequencesand direction of transcription; 0 betweenthe HI and H3 genes representphasednucleosomes;a SAR is indicatedby a line below the nucleosomes.in different between the two compounds. vivo topoisomeraseII DNA cleavagesites,determinedby experimentssimilar to those A striking cleavage of dh-EPI at site 10 was mapped at about 100 shown in Fig. 3, are indicated by arrowheads and columns above (VM-26) and below bp from the ATG codon of the H2A gene (Fig. 3, middle panel). (dh-EPI) the histone repeat. The percentage of cleaved DNA at each site relative to total cleavage in the repeat is reported on the Y-axis; the heights of columns correspond to the VM-26 could stimulate cleavage in this region only after heat shock cleavage levelateachsite.Radioactive determinations werecarriedoutwithaMolecular of Kc cells (Ref. 19 and see below). Site 10 is located in one of the Dynamicsphosphorimager425 model. three DNase I-hypersensitive regions that were previously mapped in the promoter regions between the H2A and H2B genes,the H3 and H4 the presence or absenceof distamycin (Fig. 1A and data not shown). genes,and at the 5' end of the Hi gene (35). We detected no cleavage The addition of distamycin (25 @i)to Kc cells has been shown to stimulation by dh-EPI at the 5' end of the Hi gene and only weak stimulation between the H3 and H4 genes (sites 8a and 8b in Fig. 3). markedly increase VM-26-stimulated cleavage levels in SAR regions Weaker sites stimulated by dh-EPI were observed in the H4-H2A (19), probably by displacing Hi histone from chromatin, thus increas intergenic region (Fig. 3, site 9). In the Hl-H3 spacer, dh-EPI sites ing DNA accessibility to topoisomerase H (34). were located within the SAR and at the 3' end of the H3 gene (Figs. Cleavage intensity patterns were drug specific in a cloned satellite 2 and 3). The Hl-H3 spacercontains a number of phasednucleosomes HI fragment of 16 repeats in the presence of purified Drosophila topoisomerase II (Fig. 1B). Interestingly, VM-26 stimulated a very (35), and several dh-EPI sites were localized in nucleosomal DNA strong cleavage site once per repeat that appearsto be the sameas that linkers (Fig. 2), in agreementwith publisheddata(19, 36). Neverthe observed in vivo. The dh-Epi-stimulated cleavage intensity pattern less, dh-EPI-stimulated sites 3a and 6a mapped in nucleosomal DNA (Fig. 2). DNA cleavage levels were measured at each site with a was markedly distinct from that of VM-26, although levels of cleav 50@ @ VM-26 z @ @ :@dh-EPI 1@1 CVD Fig.3. Topoisomerase II DNAcleavage listen sity patterns stimulated by VM-26, dh-EPI, mAMSA, and amonafide in the histone gene clus tel. Kc cells were treated with no dnig(Lane C), 50 @LMVM-26 (Lane V), 10 (Lane D), 25 @LM mAMSA @si4dm-3'OH H2B H2A (Lane A), or 50 @as@ I * , * @l:9t HI I H4 epirubicin amonafide(Lane N) for 30 mm at 25°C.DNAS were purified and analyzedby indirect end-label tag. , minor histonerepeatvariants.Left andright CDAN CVD Hi H3 8— H3 *1 F 7— 6- panth, cleavage was detected with a 300.bp Hin dIll-PstI fragment (HP probe) spanning the 5' cod tag region of the HI gene. Middle panel. cleavage bands were detected with a 530-bp HindflI-Bglll 5— 4— fragment(HB probe)spanningthe3' endofthe HI gene.Left andmiddlepanels,major “VM-26― and “@-@rsites are indicated by numbers to the left andtheright of gels,respectively.Thehistonegene repeatis schematizedon the sidesof gels; black ..-@ 4— 3:: .4 3- @H2B arrows. coding sequences and relative direction of transcription; hatched boxes, the positions of the HP and MBprobes.Rightpanel,numbersindicate 2- drug-stimulatedcleavagesites. 2- HP@ @HB 1857 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPOISOMERASEII DNA CLEAVAGE IN CHROMATIN phosphorimager and were expressed as percentages of cleaved DNA Taq Tma at that site over the total cleaved DNA in the histone repeat (Fig. 2). VM-26 stimulated 60—65% of DNA cleavage at sites 5 and 9, whereas 50—55%of dh-EPI-stimulated cleavage occurred at site 10 C V CV C AT (Fig. 2). Site specificity of drug-stimulated DNA cleavage was not restricted to VM-26 and dh-EPI. Amonafide and mAMSA also stimulated topoisomerase II DNA cleavage in the histone repeat, although to a lower extent than the other two drugs (Fig. 3, right panel). In this case, strong cleavage stimulation was detected in the intergenic region between the H4 and H2A genes at sites 9a and 9b (Fig. 3). Cleavage stimulated by amonafide and mAMSA in the histone SAR region was low, even in the presence of 25 ,.@Mdistamycin (data not shown), which enhanced VM-26-stimulated cleavage in SAR linker sites (19). In summary, all four drugs yielded specific cleavage intensity patterns in the Drosophila histone gene repeat. Together with the results shown above for the satellite III DNA, the present data dem onstrate that topoisomerase II inhibitors maintain a high degree of site selectivity in the chromatin of Kc cells. tu Drug-specific Topoisomerase II DNA Cleavage in the H2A II Histone Gene Promoter. We next analyzed at a nucleotide level the sites of cleavage by dh-EPI and VM-26 in the intergenic H2A-H2B 5'-AGGAGGGGGGTCAT region, where the major cleavage site stimulated by dh-EPI was mapped (Figs. 2 and 3). Sequencing of topoisomerase II cleavage sites 3-N C C C C C C A 0 1 A .@primer-5' was carried out by primer extension techniques with thermostable DNA polymerases (31). 3-C C C C C C A G T A -primer-5' To map a break site correctly, it must be noted that thermostable Fig. 4. Genomicsequencingof theVM-26-stimulatedtopoisomeraseII DNA cleavage DNA polymerases have been shown to add an extra, template-inde site in satellite Ill repeats with Taq and Ultma (Tim) polymerases. Genomic DNA from pendent nucleotide at 3' ends of extended DNA fragments (28, 37, cells treatedwith no drug (Lane c) or with 50 @sM VM-26 (Lane s') weredigestedwith Hindu! and sequencedwith primer V-l. The sequencesof the two major extended 38). Such an activity should be counteracted by the proofreading cleavageproducts(t andu) are shownat the bottom.The t productis one nucleotidelonger activity of DNA polymerases (38). Thus, to test DNA polymerases than the u product.*, pausesitesfor both polymerases.See “Materials and Methods― for further details. under our conditions, we sequenced the Hinfl restriction site and the VM-26-stimulated topoisomerase II DNA cleavage site in satellite III DNA using DNA polymerases without (Taq) or with (Ultma) 3' to 5' gesting that topoisomerase II might be bound to this sequencemotif in proofreading activity (Fig. 4). In the case of Hinfi-digested DNA, Taq vivo. polymerase added an extra nucleotide in 93% of the extended mole Since VM-26 strongly stimulated cleavage in the H2A-H2B spacer cules, whereas Ultma polymerase did so only in 25% of molecules, as only in heat-shocked cells (19), we compared VM-26 and dh-EPI sites determined by phosphorimager analyses of gels (data not shown). following heat shock of the cells. Kc cells were cultured at either 25°C Consistently, at the VM-26-stimulated cleavage site, a one-nucleotide or 37°Cfor 30 mm and then exposed to VM-26 or dh-EPI for 30 mm. difference was observed between DNA fragments extended by Taq The cleavage site stimulated by dh-EPI was not significantly influ and Ultma polymerases (Fig. 4). These findings have to be taken into enced by heat shock (compare d lanes at 25°Cand 37°Cin Fig. SB). account when interpreting data based on primer extension techniques; In contrast, VM-26 stimulated DNA cleavage only after heat shock (at using Taq polymerase, a strand break would be assigned to the 37°C)but at a distinct site that was mapped to the A at the —109 phosphodiester bond immediately 5' to the actual cleaved bond, position, exactly 29-bp apart from the dh-EPI site (compare v lanes at resulting in an apparent stagger of 6 basesinstead of 4 for topoisomer 25°Cand 37°Cin Fig. SB). These results thus demonstrate that the ase II DNA cleavage, as reported previously (19). two drugs stimulate site-selective cleavage also at nucleotide levels. The strong dh-EPI-stimulated cleavage site 10 was then sequenced Base Sequences at the in Vivo Sites of Drug-stimulated Topo using Taq DNA polymerase since it was the most efficient enzyme isomerase II DNA Cleavage. The above sequence data provide (Fig. 5). Two bands could be observed for the extension products of evidence that topoisomerase H double-stranded DNA cleavage occurs the upper strand that comigrated with an A and a T corresponding to with a 4-base stagger also in vivo, in contrast to a previous report, positions —80and —81relative to the first ATG codon of the H2A which did not take into account quantitative nontemplated nucleotide gene (Fig. 5, A and B). By analogy to the results described above, the addition by Taq polymerase (19). This prompted us to re-evaluate the DNA cut was assigned to the 5' side of the T at —80on the lower basesequencesat 10 sites of VM-26-stimulated DNA cleavage (Table strand (Fig. SC). Only one band was detected in the case of extension 1), including those of nine sites published previously (19), as well as of the lower strand (Fig. SA), corresponding to the T at —76;thus, the new site in the H2A gene promoter region (Fig. SC). A cytosine cleavage in the upper strand was mapped to the 5' side of the T at —77 appears to be preferred at the —1 position (Table 1), in agreement with (Fig. SC). dh-EPI-stimulated DNA cleavage was also investigated in in vitro sequence preferences of VM-26 (23). The dinucleotide 5'a cloned histone DNA fragment with purified topoisomerase II, and TA-3' was instead present at both the 3' termini of the major in vivo the results showed that the in vivo site 10 was also a target for dh-EPI dh-EPI-stimulated site of the histone repeat (Fig. SC). Again, this stimulation in vitro (data not shown). Interestingly, a close examina observation was in complete agreement with the in vitro sequence tion of the DNA sequence showed that the “dh-EPI― site mapped specificity of doxorubicin and dh-EPI (24, 25). These findings, to gether with the cleavage analysis after heat shock, showed that topoi exactly to the TATA box of the H2A gene promoter (Fig. 5), sug 1858 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPOISOMERASE 11 DNA CLEAVAGE IN CHROMATIN A B C dATCG — C --@ 25°C 37°CU @vc@@v d CATG dCATG -@ •, @ : p C -77 @I,I 5, GCGAAAT CGCT 3' -106 I A'T T T AT ACT T T AT AAATAAT IA -80 T T T CCGT T T GCCT GAAAAGGCAAA GCGCAT CGGACGCGT 3' T CAGT AAGT dh-EPI T AGGGGT CAA5T IA -109 GG CCCCACC 5' VM-26 Fig. 5. Sequencing of in visa topoisomerase II DNA cleavage sites stimulated by dh-EPI and VM-26 in the H2A-H2B intergenic region. A. DNAs were from control cells (Lane c) or from cells treated with 10 ,.LMdh-EPI (Lane d). Extension was performed with Taq polymerase from primer DlO-l (left panel. upper strand) or primer DlO-2 (right panel, lower strand)labeledat 5' ends.Sequencingladderswereobtainedfrom extensionwith thesameprimerandtheappropriatedideoxynucleotide.@. a pausesite;arrowheads.extendedcleavage products.B, cleavagesitesstimulatedby dh-EPIandVM-26 undernormalor heat-shocked conditions.DNAs werefrom controlcells(Lanec) andfrom cellstreatedwith 10,.LMdh-EPI (Lane d) or 10 @LM VM-26 (Lane s') at the indicated temperatures. Sequencing was performed with Taq polymerase from primer DlO- 1. Arrowheads. the extended cleavage products. C. basesequenceof the regionanalyzed.Cleavagesitesstimulatedby drugsare shown.Numbersindicatethe nucleotidescovalentlylinked to topoisomeraseII subunitsat the cleavage site. Bases are numbered from the start codon of the H2A gene. Arrowheads. the observed cleaved bonds. The ‘@VM-26― cleavage in the upper strand ( —106 nucleotide) was assigned based on the cleaved bond observed in the lower strand and a 4-bp stagger. A line indicates the TATA box of the H2A gene promoter. somerase II was already present in the H2A promoter region at 25°C but that VM-26 was unable to reveal its activity, presumably because the site recognized by the enzyme in the —77/—80region did not fulfill the local base preference of this inhibitor (Fig. 5). Heterogeneity of Cleavage Levels at Different Genomic Loci. The observation that dh-EPI, mAMSA, and amonafide in vivo did not stimulate cleavage in the satellite III DNA, whereas VM-26 did (Fig. 1A), suggested that drug-stimulated cleavage levels might be mark edly heterogeneousin different genomic loci. However, since the drug potency in stimulating DNA cleavage might be different among the cells―PositionBase —6 studied inhibitors, we therefore measured the DNA breakage levels in the whole genome of Kc cells with the filter elution technique (32). The results showed that similar concentrations of VM-26 and dh-EPI produced nearly the same amounts of DNA DSBs (Fig. 6A ). At the same time, overall cleavage levels in the histone gene locus stimulated by VM-26 and dh-EPI displayeddifferent dose-responsecurves;the doxorubicin analogue was able to stimulate 2—3-foldhigher levels of cleavage than VM-26, as determined by a phosphorimager analysis of Southern blots (Fig. 6B). Thus, unrelated inhibitors may promote about the same amount of DNA breakage in the genome as a whole, Table INucleotide frequencies atJO sites of VM.26.stimulated topoisoinerase II DNA cleatage ii, thechroinat:is of Kc —5—4—3—2—1I2345678 9 10 A 40 0 40 20 60 10 20 10 30 20 20 10 20 20 10 30 34 C G 30 10 60 10 30 20 0 0 10 0 60 10 30 30 30 30 60 0 60 0 10 30 40 20 30 10 20 20 20 40 20 10 14.5 13 _i._ 20 30 10 80 30 20 20 30 10 20 40 30 40 40 30 40 38.5 aThe10topoisomerase IIDNA cleavage sites analyzed were sites I,2.3.4a,5a, 5b,6,and 7ofthehistone repeat. thesatellite IIIsitedescribedalready (Ref. 19:Figs. I.2.and 4B), and site 10 stimulated by VM-26 after heat shock (Fig. SC). Position coordinates (—6 to + 10) are positive and negative for the bases 3' and 5' of the strand cut, respectively. Only the upperstrandis shown,correspondingto thepolarity of the mapin Fig. 3 for thehistonerepeat.andfor thesatellitesite,to thepublishedsequence(30). Numbersto theright (shownin italics) indicatesinglenucleotidefrequenciesin the upperstrandof the 1400-bphistoneHl—H3intergenicspacersequence(19). The topoisomeraseII cuts for the sites reportedpreviously(seeTable I in Ref. 19)werereassignedonenucleotidefurther3' to takeinto accountthequantitativenontemplatednucleotideadditionby Taq polymerase(Fig. 4 andseetext) andto reflect a staggerof 4 basesinsteadof 6 for in rho DNA cleavageby topoisomeraseII. No statisticalanalysiswasattempteddueto the very small numberof sites. 1859 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPOISOMERASEII DNA CLEAVAGE IN CHROMATIN @ @ @ yet the extent of DNA cleavage can substantially diverge in specific chromatin regions. Heterogeneous Reversibility of Topoisomerase II DNA Cleav age in the Drosophila Genome. We then asked whether DNA cleav age reversion was also heterogeneous among genomic loci or struc turally unrelated drugs. Cleavage reversibility was first investigated in the histone gene and sateffite HI repeats after cell treatments with VM-26 (Fig. 7A). These two regions displayed different behaviors; in the satellite ifi DNA, cleavage was completely reversed within 30 mm from drug removal, whereas in the histone gene region, some cleavage still persisted at 30 mm, and overall cleavage was as low as 10% of initial levels after 1 or 2 h (Fig. 7A). Reversion kinetics of VM-26stimulated DNA cleavage were instead similar in cloned histone and satellite ifi DNA repeats with purified Drosophila topoisomerase II (data not shown). It is interesting to observe that not all cleavage sites in the histone gene cluster reversed with the samerate (Fig. 7A). Only 40—50%of DNA cleavage at sites 4 and S was reversed after 30 mm, whereas cleavage reversal was almost complete at the other sites. We then analyzed the reversibility of DNA cleavage stimulated by dh-EPI in the histone gene repeat (Fig. 7B). Forty % of DNA cleavage persisted for up to 2 h after removal of dh-EPI at sites 6a and 10, whereas some dh-EPI-stimulated sites reversed completely after only 30 mm (Fig. 7B, site 7b, •).Interestingly, the three closely positioned sites 6a, 6b, and lb (Figs. 2 and 3) reversed with distinct rates (Fig. 7B). Overall, DNA cleavage stimulated by dh-EPI appeared to persist for longer periods of time than that stimulated by VM-26 (Fig. 7). No relation could be observed between in vivo cleavage levels and re versibility. In brief, DNA cleavage tended to be reversible at the majority of sites following drug removal but with different kinetics that were clearly dependent upon the drug, the genomic locus, and the particular site studied. A ioci :: 0 40 z 20 0 0 a a w _____ 0@_io 4:Ø@0801Ô0120 B w -J 0 TNE(nin) 100 1 8O@ 60 40 \‘@ 20 \‘ Th20w40 801Ô0 TIME (mm) Fig. 7. Reversibility of drug-stimulated DNA cleavage in the studied Drosophila genome regions. Cellswereexposed to50 VM-26or 10 @&xi dh-EPIfor30rainat25°C andthenwashedandresuspended in drug-freemediumandfurtherincubatedat 25°C for the indicated time. DNA was then purified and analyzed by Southern blotting. Cleavage levelswere then determinedby countingthe radioactivity on Southernblottings with a MolecularDynamicsphosphorimager425model.A. timecoursesofcleavagereversibility at specific VM-26-stimulated sites. In the histone locus: A and V. sites 5 and 4, respectively; @, site9; S, site 6; • , site 3. inset, satelliteifi DNA site.B, time courses .,sites 6a and 10, respectively; 0and •, sites 6b and 2,respectively; Vand V, sites 5 of cleavage reversibility atspecificdh-EPI-stimulated sitesin thehistonecluster.0 and DISCUSSION and 3, respectively; 0 and • , sites 9 and 7b. respectively; & site 7a. Our results demonstrate a high degree of heterogeneity in the genomic localization and reversibility of drug-stimulated topoisomer ase H DNA cleavage in the chromatin of living cells. Among dh-EPI, VM-26, mAMSA, and amonafide, only VM-26 could stimulate cleav age in the centromeric satellite ifi DNA, whereas all four drugs did at the histone gene repeat. In this locus, however, cleavage intensity patterns markedly differed among the four drugs. Using mAMSA and VM-26, some differences in cleavage patterns have been reported previously in viral (20), episomal (21), and cellular chromatin (22). A B genome !:: Moreover, our study now shows that, in the case of VM-26 and dh-EPI, base sequences at specific drug-stimulated sites conform to the known drug sequence specificities established in vitro with pun fled topoisomerase II (9, 23, 24). Thus, the DNA sequencespecificity of the studied topoisomerase II inhibitors plays an important role in determining the sites of cleavage in the chromatin of living cells. This is best seenin the caseof dh-EPI and VM-26, which stimulate cleavage at a different subset of sites in the histone gene repeat (Figs. 2 and 3) and exert all-or-none effects in the satellite ifi DNA (Fig. 1). The observed high degree of in vivo cleavage site selectivity is likely due to a combination of several factors, including: (a) the sequencespecificity imparted by the inhib itor; (b) the local chromatin structure; (c) the loose sequence selec tivity of the enzyme itself; and (d) possibly other protein factors. A likely hypothesis is that histones and other nuclear proteins modulate 4000 DNA accessibility, and drugs then select among the accessible sites z Q8000 where topoisomerase H can bind to and cleave DNA. For instance, Cl since dh-EPI requires a relatively AT-rich sequence at the cleavage site (9, 24), this is likely to preclude stimulation by dh-EPI and related @0:4@0:60:81:0 @.Whole anthracyclines at sites stimulated by VM-26 (such as the satellite ifi site), which appear to be (IC-rich instead (Table 1). Sequencing of DRUG CONCENTRATION(@tM) additional in vivo drug-enhanced cleavage sites will establish this Fig. 6. Levelsof DNA DSBsin the whole genome(A) andin the histonegenelocus point more definitively. (B) of Drosophila Kc cells. Cells were exposed to VM-26 or dh-EPI for 30 mm at 25°C The identification of genomic sites of topoisomerase II activity is at the indicated drug concentrations. 0 and •,dh-EPI and VM-26, respectively. A, DNA DSBs were determinedwith the filter elution method and expressedas rad-equivalents. under intense investigation in several laboratories (14—19).Although Datapointsare means(bars, SE)of at leastthreeindependentdeterminations.B. DNA DSBsweremeasuredwith a phosphorimagerfrom indirectend-labelingexperiments,such sites of in vivo drug-enhanced cleavage and sites of enzyme activity and/or DNA binding are certainly related, nevertheless our data as those in Fig. 3, and expressed as percentages of cleavage products over total DNA. 1860 @, : /,$f C,) Cl 4: Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. TOPO1SOMERASE II DNA CLEAVAGE IN CHROMATIN clearly demonstrate that cleavage site analysis can miss certain sites of enzyme activity, and this may lead to inaccurate conclusions regard to topoisomerase binding sites in nuclear chromatin. with The common use of VM-26 to detect sites of enzyme activity is based on the following key assumptions: (a) the inhibitor increases cleavage uniformly at physiological sites; and (b) sites of enzyme binding to DNA are always sites of DNA cleavage (14—19). However, the present results demonstrate that this is not always the case. In in vitro investigations, VM-26 (or any other inhibitor) did not uniformly stimulate cleavage sites in any given DNA fragment, even if, to our knowledge, this drug was the least sequence-specific inhibitor (9, 23). Since in vivo cleavage sites are not detectable without drugs, cleavage intensity in the chromatin of intact cells is likely determined in large part by the particular drug used. Thus, it is not possible to derive even semiquantitative information on chromatin sites of enzyme binding/ activity from cleavage patterns stimulated by any single inhibitor. In the extreme case, an enzyme binding site can be missed because the drug usedcannotstabilizethecleavablecomplexat that particularsite (see the opposite effects of VM-26 and dh-EPI in satellite III repeats derivatives, this relation is lost when topoisomerase II inhibitors of different chemical classes are compared (7, 8, 41). Therefore, we suggest that the genomic site and reversion kinetics of in vivo DNA cleavage might be important factors influencing drug cytotoxic po tency. Interestingly, VM-26 has been shown to stimulate higher cleavage levels in regions surrounding the c-myc and /3-globin genes than in the a-satellite and Ll repeat DNAs in human cells, indicating that certain DNA regions may be preferentially damaged by VM-26 (43). In conclusion, the present findings demonstrate that the studied drugs maintain, in the chromatin of living Kc cells, a high degree of sequence specificity that may correspond to that established in vitro (9, 27). In addition, the present data provide strong evidence that drug-stimulated topoisomerase II DNA cleavage is highly heteroge neous in the chromatin of intact cells, in terms of the extent (at specific genomic loci), localization, and reversibility. The present information should be taken into account in investigations on the genomic sites of enzyme activity, the chromatin structure, and the antitumor activity of topoisomerase II inhibitors. and at site 10 of the histone gene locus in Figs. 1, 2, and 5). Moreover, it is possible that important functions of DNA topoisomerase ACKNOWLEDGMENTS II might be independent from enzyme catalysis (39, 40). The high degree of heterogeneity we observed in the localization of topoisomerase II cleavagesitesstimulatedby unrelateddrugsis accom panied by an equally heterogeneous reversibility of cleavage, which varies as a function of the drug, the genomic locus, and the particular cleavage site studied. In contrast, DNA cleavagereversibility in cloned DNA fragments in the presence of purified Drosophila topoisomerase II was similar between the histone and satellite ifi repeats.5 The heteroge neity in the reversibility of dh-EPI- and VM-26-enhanced cleavagemay be due to different drug interactions with DNA or different cellular pharmacokinetics (33, 41). However, it is more difficult to explain the heterogeneityamong sitesstimulated by the samedrug. It is possiblethat either particular aspects of chromatin structure or ongoing metabolic processes may influence the persistence of DNA cuts. In this respect, it is interesting to note that the reversibility of topoisomeraseII DNA cleav age was much faster in the silent satellite ifi repeats than in the transcrip tionally active histone gene repeats(Fig. 6). It remains to be established whether a relation between transcription and lack of complete cleavage reversion is a general event. An unexpected finding of this work is that the striking topoi somerase II DNA cleavage stimulated by dh-EPI at site 10 of the histone cluster coincides precisely with the TATA box of the H2A gene promoter (42). This indicates that topoisomerase II may bind to this sequence motif in the chromatin of living cells, suggesting that TATA boxes of promoter regions might be in vivo sites of topoisomerase II activity. Although the functional significance of this result remains to be fully established, one possibility is that topoisomerase II binding to the TATA box may be related to the proposed activity of this enzyme as a general repressor of tran scription (39). However, we do not know at present whether all of the histone gene repeats are equally cleaved by the enzyme or if cleavage occurs only in a subpopulation of the repeats; a functional assay will then be needed to determine whether topoisomerase II may play a direct role in gene expression. The present findings raise the question as to whether the extensive genomic heterogeneity in DNA cleavage localization, extent, and reversibility has any role in the biological activity of topoisomerase II inhibitors. Although drug cytotoxic potency correlates well with the extent of drug-stimulated DSBs when considering closely related We thank M. Binaschi for help in the sequencing experiments and L. Poljak and M. Binaschi for critical comments on the manuscript. REFERENCES 1. Laemmli, data. L, and Adachi, Y. Scaffold-associated regions: 2. Roberge, M., and Gasser, S. M. DNA loops: strucwral and functional properties of scaffold-attached regions. Mol. Microbiol., 6: 419—423.1992. 3. Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem., 54: 665—697,1985. 4. Liu, L. F., and Wang, J. C. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA. 84: 7024—7027,1987. 5. DiNardo, S., Voelkel, K., and Sternglanz, R. 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A simple high-resolutionprocedureto study DNA methyl ation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes. Proc. Natl. Acad. Sci. USA, 86: 2602—2606,1989. c-myc expression in MCF-7 breast tumor cells. Biochem. Pharmacol., 47: 3 17—329, 1994. 1862 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research. Drug-specific Sites of Topoisomerase II DNA Cleavage in Drosophila Chromatin: Heterogeneous Localization and Reversibility Maria E. Borgnetto, Franco Zunino, Stella Tinelli, et al. Cancer Res 1996;56:1855-1862. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/8/1855 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.