Download Drug-specific Sites of Topoisomerase II DNA

[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 SanitÃ
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
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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.
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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
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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
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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
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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:
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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. DNA topoisomerase 11 mutant of
Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter
molecules at the termination of DNA replication. Proc. Natl. Acad. Sci. USA, 81:
2616—2620,
1984.
6. HoIm, C., Goto,T., Wang,J. C., andBotstein,D. DNA topoisomeraseII is required
at the time of mitosis in yeast. Cell, 41: 553—563,1985.
7. Pommier, Y., and Kohn, K. W. Topoisomerase II inhibition by antitumor intercalators
and demethylepipodophyllotoxins.
In: R. I. Glazer (ed). Developmentsin Cancer
Chemotherapy, pp. 175—195.
Boca Raton, FL: CRC Press, 1989.
8. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem.. 58:
351—375.1989.
9. Capranico,G., andZunino, F. DNA topoisomerase-trapping
antitumourdrugs.Eur. J.
Cancer,28A: 2055—2060,
1992.
10. Gasser,S. M., and Laemmli,U. K. The organisationof chromatinloops:character
ization of a scaffold attachment site. EMBO J., 5: 511—518,1986.
11. Earnshaw, W. C., and Heck, M. M. Localization of topoisomerase II in mitotic
chromosomes.J. Cell Biol., JIYk 1716—1725,1985.
12. Pommier, Y., Cockerill, P. N., Kohn, K. W., and Garrard, W. T. Identification within
the simian virus 40 genomeof a chromosomalloop attachmentsite that contains
topoisomeraseII cleavagesites.J. Virol., 64: 419—423,
1990.
13. Adachi, Y., Kits. E., and Laemmli, U. K. Preferential, cooperative binding of DNA
topoisomerase II to scaffold-associated
regions. EMBO
J.. 8: 3997—4006. 1989.
14. Gromova,I. I., Thomsen,B., andRazin,S. V. Different topoisomeraseII antitumor
drugsdirect similar specific long-rangefragmentationof an amplified c-MYC gene
locus in living cells and in high-salt-extracted
nuclei. Proc. NatI. Acad. Sci. USA, 92:
102—106,1995.
15. Udvardy,A., andSchedl,P.The dynamicsof chromatincondensation:redistribution
oftopoisomerase II in the 87A7 heat shock locus during induction and recovery. Mol.
Cell. Biol., 13: 7522—7530,
1993.
16. Udvardy,A.. and Schedl,P. Chromatinstructure,not DNA sequencespecificity. is
the primary determinantof topoisomeraseII sitesof actionin vivo. Mol. Cell. Biol.,
11: 4973—4984, 1991.
17. Muller, M. T., and Mehta, V. B. DNase I hypersensitivity is independent of endog
enoustopoisomerase
II activity duringchickenerythrocytedifferentiation.Mol. Cell.
Biol., 8: 3661—3669,1988.
18. Reitman,M., andFelsenfeld,0. Developmentalregulationof topoisomeraseII sites
andDNaseI-hypersensitivesitesin thechickenfJ-globinlocus.Mol. Cell. Biol., 10:
2774—2786,1990.
19. Käs,E., and Laemmli, U. K. In vivo topoisomeraseII cleavageof the Drosophila
histone and satellite III repeats: DNA sequenceand structural characteristics. EMBO
J., Ii:
S Unpublished
U. K., KSs, E., Poljak.
cis-acting determinants of chromatin structural loops and functional domains. Curr.
Opin. Gen. Dev., 2: 275—285,1992.
705-716,
1992.
20. Yang. L., Rowe, T. C., and Liu, L. F. Identification of DNA topoisomerase II as an
1861
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1996 American Association for Cancer Research.
TOPOISOMERASE
II DNACLEAVAGEIN CHROMATIN
intracellular target of antitumor epipodophyllotoxins
32. Kohn, K. W. Principles and practice of DNA filter elution. Pharmacol. & Ther., 49:
55—77,1991.
in simian virus 40-infected
monkeycells. CancerRes.,45: 5872-5876, 1985.
21. Cullinan, E. B., and Beerman,T. A. A study of drug-inducedtopoisomeraseH-
33. Binaschi,M., Capranico,G., De Isabella,P.,Mariani, M., Supino,R.. Tinelli, S.,and
mediated DNA lesions on episomal chromatin. J. Biol. Chem., 264: 16268—16275,
1989.
22. Pommier,Y., Orr, A., Kohn, K. W., andRiots,1.F. Differentialeffectsof amsacrine
andepipodophyllotoxins
ontopoisomerase
II cleavage
in thehumanc-mycprotoon
Zunino, F. Comparison of DNA cleavage induced by etoposide and doxorubicin in
two humansmall-celllung cancerlines with different sensitivitiesto topoisomerase
II inhibitors. tnt. J. Cancer,45: 347—352,
1990.
34. Käs,
E., Poljak, L., Adachi, Y., and Laemmli, U. K. A model for chromatin opening:
cogene.CancerRes.,52: 3125—3130,
1992.
23. Pommier, Y., Capranico, 0., Orr. A., and Kohn. K. W. Local base sequence prefer
stimulation of topoisomerase II and restriction enzyme cleavage of chromatin by
distamycin.EMBO J., 12: 115—126,
1993.
ences
forDNAcleavage
bymammalian
topoisomerase
II inthepresence
ofamsacrine 35.Worcel,A.,Gargiulo,
0., Jesee,
B.,Udvardy,
A.,Louis,C.,andSchedl,
P.Chromatin
or teniposide [erratum in Nucleic Acids Res.. 19: 7003, 1991). Nucleic Acids Res.,
19: 5973—5980,1991.
fine structure of the histone gene complex of Drosophila
melanogaster.
Nucleic
Acids Res.,11: 421—439,
1983.
24. Bigioni, M., Zunino,F., andCapranico,0. Basemutationalanalysisof topoisomerase 36. Capranico,
0., Jaxel,C.,Roberge,
M., Kohn,K. W.,andPommier,
Y. Nucleosome
ll-idarubicin-DNA
ternary complex formation. Evidence for enzyme subunit coop
erativity in DNA cleavage.Nucleic Acids Res.,22: 2274—2281,
1994.
25. Capranico,G., Supino,R., Binaschi,M., Capolongo,L., Grandi,M., Suarato,A., and
Zumno, F. Influence of structural modifications at the 3' and 4' positions of doxo
rubicin on the drug ability to trap topoisomeraseII and to overcomemultidrug
resistance.Mol. Pharmacol.,45: 908—915,
1994.
26. Dc Isabella, P., Zunino, F.. and Capranico, G. Base sequencedeterminants of
amonafide stimulation of topoisomerase II DNA cleavage. Nucleic Acids Res., 23:
positioning as a critical determinant for the DNA cleavage sites of mammalian DNA
topoisomeraseII in reconstitutedsimianvirus 40 chromatin.Nucleic Acids Res.,18:
4553—4559,1990.
37. Clark,J. M. Novel non-templatednucleotideadditionreactionscatalyzedby procary
otic and eucaryotic DNA polymerases. Nucleic Acids Res., 16: 9677—9686,1988.
38. Hu, G. DNA polymerase-catalyzed
additionof nontemplatedextranucleotidesto the
3' end of a DNA fragment. DNA Cell Biol., 12: 763—770,1993.
39. Brou,C.,Kuhn,A.,Staub,A.,Chaudhary,
S.,Grummt,I., Davidson,
I., andTora,L.
223—229,
1995.
Sequence-specific
transactivatorscounteracttopoisomerase11-mediated
inhibition of
in vitro transcriptionby RNA polymerasesI and II. Nucleic Acids Res.,21: 4011—
27. Pommier, Y., Kohn, K. W., Capranico, 0., and Jaxel, C. Base sequenceselectivity of
topoisomerase inhibitors suggestsa common model for drug action. In: T. Andoh, H.
Ikeda, and M. Oguro (eds.). Molecular Biology of DNA Topoisomerases and Its
4018, 1993.
40. Roca, J., Berger, J. M., and Wang, J. C. On the simultaneous binding of eukaryotic
Application to Chemotherapy.pp. 215—227.
BocaRaton,FL: CRC Press.1993.
28. Freudenreich,C. H., and Kreuzer, K. N. Localization of an aminoaciidineantitumor
DNA topoisomerase
H to a pairof double-stranded
DNA helices.J. Biol. Chem.,268:
agentin a type H topoisomerase-DNAcomplex. Proc. Nail. Aced. Sci. USA, 91:
11007—11011,
1994.
14250—14255,
1993.
41. Zunino, F., and Capranico, G. DNA topoisomeraseII as the primary target of
anti-tumor anthracyclines. Anti-Cancer Drug Design, 5: 307—317,1990.
42. Hentschel,C. C., andBirnstiel, M. L. The organizationandexpressionof histonegene
29. Sambrook,
J.,Fritsch,
E.F.,andManiatis,
T.Molecular
Cloning:A Laboratory
Manual.
pp. 9.31—9.57.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 1989.
families.Cell, 25: 301—313,
1981.
30. Hsieh,T-S.,andBrutlag,D. L. Sequence
andsequence
variationwithinthe 1.688
g/cm3satelliteDNA ofDrosophila melanogaster.J. Mol. Biol.. 135:465—481,
1979.
43. Bunch, R. T., Povirk, L. F., Orr, M. S., Randolph.J. K.. Fomari, F. A., and Oewirtz,
D. A. Influenceof amsacrine(m-AMSA) on bulk andgene-specificDNA damageand
31. Saluz, H., and lost, J-P. 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.
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