Merbarone is a catalytic inhibitor of
topoisomerase II that is in clinical trials as an anticancer agent.
Despite the potential therapeutic value of this drug, the mechanism by
which it blocks topoisomerase II activity has not been delineated.
Therefore, to determine the mechanistic basis for the inhibitory action
of merbarone, the effects of this drug on individual steps of the catalytic cycle of human topoisomerase II
were assessed.
Concentrations of merbarone that inhibited catalytic activity
80%
had no effect on either enzyme·DNA binding or ATP hydrolysis. In
contrast, the drug was a potent inhibitor of enzyme-mediated DNA
scission (in the absence or presence of ATP), and the inhibitory
profiles of merbarone for DNA cleavage and relaxation were similar.
These data indicate that merbarone acts primarily by blocking
topoisomerase II-mediated DNA cleavage. Merbarone inhibited DNA
scission in a global (rather than site-specific) fashion but did not
appear to intercalate into DNA or bind in the minor groove. Since the drug competed with etoposide (a cleavage-enhancing agent that binds
directly to topoisomerase II), it is proposed that merbarone exerts its
inhibitory effects through interactions with the enzyme and that the
drug shares an interaction domain on topoisomerase II with
cleavage-enhancing agents.
 |
INTRODUCTION |
Topoisomerase II is the target for some of the most active
anticancer drugs used in the treatment of human malignancies (1-6). Among the topoisomerase II-targeted agents currently in clinical use
are etoposide, teniposide, doxorubicin, mitoxantrone, and amsacrine. These drugs kill cells in an unusual fashion. Rather than
inhibiting the overall catalytic activity of the type II enzyme, they
act by increasing levels of topoisomerase II-mediated DNA cleavage,
thus converting this essential enzyme into a potent cellular toxin (1,
3, 5, 7-10). Hence, to distinguish their unique mechanism of action,
they are referred to as topoisomerase II "poisons" (11).
A second class of drugs that affect the activity of topoisomerase II
also appears to have clinical potential (2, 5, 7). In contrast to
poisons, these agents act by inhibiting the catalytic activity of the
enzyme and display no ability to stimulate DNA cleavage. Originally,
topoisomerase II "catalytic inhibitors" were defined by
antibacterial compounds such as novobiocin and coumermycin (12). These
coumarin-based drugs block the DNA strand passage activity of the
prokaryotic type II enzyme, DNA gyrase, by interfering with the ability
of the enzyme to bind its ATP cofactor (13-17).
Recently, catalytic inhibitors that display high activity against
eukaryotic type II topoisomerases have been described. These are
typified by drugs such as merbarone (18), ICRF-193 (19), aclarubicin
(20), fostriecin (21), staurosporine (22), and mitindomide (23), which
reflect a variety of inhibitory mechanisms. Aclarubicin, for example,
blocks binding of the enzyme to its DNA substrate, the initial step of
the topoisomerase II catalytic cycle (24) (see Fig. 12). In contrast,
ICRF-193 blocks the final step of the catalytic cycle, ATP hydrolysis
(25). This action traps topoisomerase II on the DNA in its closed clamp
form and prevents both enzyme release and regeneration.
One of the catalytic inhibitors of topoisomerase II that has generated
the most interest is merbarone (18, 26-28) (see Fig. 1). This
thiobarbituric acid derivative was originally shown to inhibit the type
II enzyme by Drake et al. (18) and has been the subject of
phase II clinical cancer trials (29-32). In addition to its inhibitory
effects, merbarone has been shown in vitro and in cultured
cells to attenuate the DNA cleavage-enhancing properties of
topoisomerase II poisons such as teniposide and amsacrine (18, 33).
Although the cytotoxic actions of merbarone correlate with its ability
to block topoisomerase II function (18, 33, 62), the mechanism by which
this drug inhibits the enzyme has never been demonstrated. Therefore,
to determine the basis of merbarone inhibition, the effects of the drug
on individual steps of the catalytic cycle of human topoisomerase II
were characterized. Results indicate that merbarone is a specific
inhibitor of topoisomerase II-mediated DNA scission.
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EXPERIMENTAL PROCEDURES |
Human topoisomerase II
was expressed in Saccharomyces
cerevisiae (34) and purified by the protocol of Kingma et
al. (35). Yeast topoisomerase II was isolated from S. cerevisiae by the procedure of Elsea et al. (36) as
modified by Burden et al. (37). Drosophila
melanogaster topoisomerase II was purified from embryonic Kc cells
as described by Shelton et al. (38). Calf thymus
topoisomerase I was purchased from Life Technologies, Inc. Negatively
supercoiled pBR322 DNA was prepared as described (39). Hepes was
obtained from Boehringer Mannheim; proteinase K and SDS were from
Merck; bacteriophage T4 polynucleotide kinase, Klenow DNA
polymerase, and restriction endonucleases were from New England
Biolabs, Escherichia coli uracil DNA glycosylase, [
-32P]ATP (~6000 Ci/mmol), and
[
-32P]ATP (~3000 or ~6000 Ci/mmol) were from
Amersham Pharmacia Biotech; etoposide and ellipticine (stored at
4 °C as 20 mM stock solutions in 100%
Me2SO) as well as ethidium bromide were from Sigma; and fluorescein phosphoramidite was from Glen Research. Merbarone was the
generous gift of Dr. Randall K. Johnson (SmithKline Beecham) and was
stored at 4 °C as a 20 mM stock solution in 100%
Me2SO. All other chemicals were analytical reagent
grade.
DNA Relaxation--
DNA relaxation assays were based on the
procedure of Osheroff et al. (40). Reactions contained 3 nM human topoisomerase II
, 5 nM negatively
supercoiled pBR322 DNA, and 1 mM ATP in a total of 20 µl
of reaction buffer (50 mM Tris-HCl (pH 7.9), 135 mM KCl, 10 mM MgCl2, 0.5 mM NaEDTA, and 2.5% glycerol). One microliter of merbarone
(or Me2SO for control reactions) was included, so that the
final [Me2SO] was 5% (v/v). Reactions were started by the addition of topoisomerase II, incubated 10 min at 37 °C, and stopped by the addition of 3 µl of 0.77% SDS, 77 mM
NaEDTA (pH 8.0). Alternatively, assays utilized either
Drosophila (4 nM) or yeast (6 nM)
topoisomerase II and were carried out as described previously (36, 40).
Samples were mixed with agarose gel loading buffer (30% sucrose, 0.5%
bromphenol blue, and 0.5% xylene cyanole FF in 10 mM
Tris-HCl (pH 7.9)) and subjected to electrophoresis in a 1% agarose
gel in TBE buffer (100 mM Tris borate (pH 8.3), 2 mM EDTA). DNA bands were stained with 1 µg/ml ethidium
bromide, visualized by UV light, photographed through Kodak 23A and 12 filters with Polaroid type 665 positive/negative film, and quantitated by scanning photographic negatives with an E-C apparatus model EC910
scanning densitometer in conjunction with Hoefer GS-370 Data System
software. The intensity of bands in the negative was proportional to
the amount of DNA present.
Topoisomerase II·DNA Binding--
The effect of merbarone on
topoisomerase II·DNA binding was characterized by two independent
techniques. In the first, an electrophoretic mobility shift assay was
employed (41). Reactions were carried out in 20 µl of reaction buffer
and contained 10 nM negatively supercoiled pBR322 DNA and
0-300 nM human topoisomerase II
in the presence or
absence of 100 µM merbarone (5% final
[Me2SO]). Reactions were incubated at 37 °C for 6 min,
loaded directly onto a 1% agarose gel, and subjected to
electrophoresis in TAE buffer (40 mM Tris acetate (pH 8.3),
2 mM EDTA) containing 0.5 µg/ml ethidium bromide. DNA
bands were visualized and photographed as described above for
relaxation assays.
Alternatively, the effect of merbarone on topoisomerase II·DNA
binding was monitored by fluorescence anisotropy. Assays were performed
at 25 °C on an ISS PC2 spectrofluorometer. A 40-mer double-stranded
oligonucleotide (same as utilized for AP site cleavage described below,
except that the uracil was replaced by guanine) with fluorescein
incorporated at the 5' terminus of the top strand was synthesized on an
Applied Biosystems DNA synthesizer using fluorescein phosphoramidite.
The fluoresceinated DNA substrate was excited at 430 nm with a xenon
arc lamp (18 A current) in a 1-cm silica quartz glass cuvette, and
emission was monitored through a 530-nm glass cut-off filter (Melles
Griot). Binding assays contained 5 nM fluoresceinated DNA
oligonucleotide and 0 or 100 µM merbarone (1% final
[Me2SO]) in 1.8 ml of fluorescence buffer (10 mM Hepes HCl (pH 7.9), 100 mM KCl, 5 mM MgCl2, 0.1 mM NaEDTA). Human
topoisomerase II
(0-50 nM) was titrated into the
sample, and anisotropies were determined for each topoisomerase II
concentration. Anisotropy binding curves were generated, and KD values were determined by nonlinear least squares regression analysis.
DNA Cleavage--
DNA cleavage reactions were carried out as
described previously (42), either in the absence or presence of a
nucleoside triphosphate. Assays contained 300 nM human
topoisomerase II
, negatively supercoiled pBR322 DNA (5 nM in reactions that lacked nucleotide triphosphate or
contained 1 mM
APP(NH)P1, or 10 nM in reactions that contained 1 mM ATP), and
0-200 µM merbarone (5% final [Me2SO]) in
a total of 20 µl of reaction buffer. Reactions were started by the
addition of topoisomerase II. Following a 6-min incubation at 37 °C,
cleavage intermediates were trapped by the addition of 2 µl of 4%
SDS and 2 µl of 250 mM NaEDTA (pH 8.0). Proteinase K was
added (2 µl of 0.8 mg/ml), and reactions were incubated 30 min at
45 °C to digest the topoisomerase II. Samples were mixed with
loading buffer, heated at 70 °C for 2 min, and subjected to
electrophoresis on a 1% TAE agarose gel containing 0.5 µg/ml
ethidium bromide. Photography of the gel and quantitation of cleavage
products were as described above.
DNA cleavage assays that characterized the order of drug (merbarone and
etoposide) addition contained 75 nM human topoisomerase II
, 10 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in 20 µl of reaction buffer. Due to the presence
of both drugs, the final [Me2SO] in these experiments was
6%. As a prelude to DNA cleavage, mixtures were incubated with 20 µM etoposide, 0-400 µM merbarone, or a
combination of the two drugs for 2 min at room temperature or 37 °C.
As appropriate, the absent drug was added to reaction mixtures, and
samples were further incubated for 6 min at 37 °C. DNA cleavage
intermediates were trapped, resolved by electrophoresis, and
quantitated as above.
Site-specific DNA Cleavage--
A unique
32P-3'-end-labeled linear DNA substrate was prepared by
digesting pBR322 DNA with EcoRI, labeling with
[
-32P]ATP and Klenow DNA polymerase in the presence of
dGTP, dCTP, and dTTP, and digesting the labeled product with
PstI. The resulting 3609-bp EcoRI-PstI
fragment was purified by gel electrophoresis using DE81 ion exchange
paper (Whatman) (36).
DNA cleavage reactions contained 6 nM labeled 3609-bp DNA
substrate, 300 nM human topoisomerase II
, 1 mM ATP, and 0 or 100 µM merbarone (5% final
[Me2SO]) in 20 µl of reaction buffer. Reactions were
started by adding topoisomerase II and carried out as described in the
preceding section on DNA cleavage. Reaction products were resolved by
electrophoresis in a 1% agarose gel in TBE buffer. The gel was
dehydrated by blotting with paper towels and dried under partial vacuum
at 50 °C. Imaging and data analysis were performed using a Molecular
Dynamics PhosphorImager system.
Cleavage of a DNA Substrate Containing an Apurinic Site--
A
double-stranded 40-bp DNA oligonucleotide (residues 87-126 of plasmid
pBR322 (43)) that contained a position-specific apurinic site (44)
within the context of a preexisting topoisomerase II cleavage site was
employed (45, 46). To this end, complementary 40-base oligonucleotides
were synthesized on an Applied Biosystems DNA synthesizer. A uracil
(shown below in bold) was incorporated into the bottom strand. The
sequences of the top and bottom strands, respectively, were
5'-TGAAATCTAACAATG
CGCTCATCGTCATCCTCGGCACCGT-3' and
5'-ACGGTGCCGAGGATGACGATG
AUCGCATTGTTAGATTTCA-3'. Sites of
topoisomerase II-mediated DNA cleavage are denoted by the
arrows. The top strand was 32P-labeled on its 5'
terminus with polynucleotide kinase and [
-32P]ATP
(6000 Ci/mmol), and oligonucleotides were gel-purified (44) and
annealed (46) as described previously. An apurinic site was generated
in the oligonucleotide substrate by treatment with uracil-DNA
glycosylase (44).
Cleavage assays were carried out as described by Corbett et
al. (46), as modified by Kingma and Osheroff (44). Reactions contained 150 nM human topoisomerase II
, 100 nM apurinic site-containing 40-mer DNA, and 0-400
µM merbarone (5% final [Me2SO]) in a total of 20 µl of 10 mM Hepes-HCl (pH 7.9), 135 mM
KCl, 5 mM MgCl2, 0.1 mM NaEDTA, and
2.5% glycerol. Reactions were started by the addition of topoisomerase
II, incubated 10 min at 37 °C, and stopped by the addition of 2 µl
of 4% SDS and 2 µl of 250 mM NaEDTA. Proteinase K was
added (2 µl of 1.6 mg/ml), and reactions were incubated 20 min at
37 °C. Cleavage products were ethanol-precipitated twice and
resuspended in 5 µl of 95% formamide, 3% sucrose, 1 mM
Tris-HCl (pH 7.9), 0.05% bromphenol blue, 0.05% xylene cyanole FF.
Products were subjected to electrophoresis in a denaturing 7 M urea, 14% polyacrylamide sequencing gel in TBE buffer.
The gel was fixed in 10% methanol, 10% acetic acid for 30 s and
dried. DNA cleavage products were quantitated by analysis on a
PhosphorImager.
ATP Hydrolysis--
ATPase assays were performed as described by
Osheroff et al. (40). ATP hydrolysis reactions contained 50 nM human topoisomerase II
, 40 nM negatively
supercoiled pBR322 DNA, 1 mM [
-32P]ATP
(3000 Ci/mmol stock), and 0 or 200 µM merbarone (5%
final [Me2SO]) in a total of 60 µl of reaction buffer.
Reactions were started by addition of topoisomerase II and incubated at
37 °C. Samples (2 µl) were removed at intervals up to 16 min and
spotted on polyethyleneimine-impregnated thin layer cellulose
chromatography plates (J. T. Baker Inc.). Plates were developed by
chromatography in freshly made 400 mM
NH4HCO3 and analyzed by autoradiography. Radioactive areas corresponding to free inorganic phosphate released by
ATP hydrolysis were cut out and quantitated by scintillation counting.
Merbarone-DNA Interactions--
Interactions between merbarone
and DNA were assessed by two independent techniques. First, the ability
of the drug to intercalate into plasmid DNA was determined by a
topoisomerase I unwinding assay (47). Reactions contained 5 nM relaxed or negatively supercoiled pBR322 plasmid DNA and
10 units of topoisomerase I. Assays were carried out in the presence or
absence of 100 µM merbarone, etoposide, or ellipticine in
40 µl of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol,
0.1 mM EDTA, and 30 µg/ml bovine serum albumin. Following
a 15-min incubation at 37 °C, reaction mixtures were treated with 3 µl of 250 mM EDTA and extracted with phenol/chloroform.
Aqueous samples (20 µl) were treated with 2 µl of 2.5% SDS, mixed
with 2 µl of agarose gel loading buffer, and subjected to
electrophoresis in a 1% agarose gel in TAE buffer. DNA bands were
stained with 1 µg/ml ethidium bromide, visualized by UV light, and
quantitated as described above.
Second, an ethidium displacement fluorescence assay (48, 49) was
employed to determine whether merbarone binds in the minor groove of
DNA. Fluorescence emission spectra (
max = 605 nm,
excitation wavelength 510 nm) were obtained at 25 °C on an ISS PC2
spectrofluorometer. Assays contained 1 µM ethidium
bromide, 0-100 µM merbarone, and 5 nM
double-stranded 40-mer oligonucleotide in 2 ml of fluorescence buffer.
The oligonucleotide employed for this study was the same as utilized
for AP site cleavage (see above), except that the uracil was replaced
by guanine.
 |
RESULTS |
Inhibition of Topoisomerase II Catalytic Activity by
Merbarone--
Merbarone (Fig. 1) is a
catalytic inhibitor of topoisomerase II (18, 27, 28) that displays
curative activity against some murine cancer models (26, 50) and is
currently in human clinical trials (29-32). The effects of this drug
on the activity of human topoisomerase II
(which represents the
major topoisomerase II isoform present in rapidly proliferating human
cells) as well as the type II enzymes from Drosophila and
yeast are shown in Fig. 2. Merbarone was
considerably more effective against the mammalian type II enzyme than
it was against topoisomerase II from lower eukaryotes. While the drug
inhibited relaxation of negatively supercoiled pBR322 plasmid DNA by
human topoisomerase II
with an IC50 of ~40
µM, little inhibition was observed with either the
Drosophila or yeast enzyme up to 100 µM
merbarone. The IC50 values for these latter type II enzymes
were ~350 and ~700 µM merbarone, respectively (not
shown).

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Fig. 2.
Merbarone inhibits DNA relaxation catalyzed
by human topoisomerase II . The top panel shows the
effects of merbarone on DNA relaxation catalyzed by human topoisomerase
(Topo) II . Control reactions contained an equivalent
volume of Me2SO in the absence of enzyme ( Topo
II) or ATP ( ATP). The relative mobilities of
negatively supercoiled plasmid DNA (form I, FI) and nicked
circular plasmid DNA (form II, FII) are shown. The
bottom panel quantitates the effects of merbarone on DNA
relaxation catalyzed by human topoisomerase II ( ), D. melanogaster (D. mel.) topoisomerase II ( ), or yeast
topoisomerase II ( ). Error bars represent standard
deviations of 2 to 3 independent assays.
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Although the inhibition of topoisomerase II by merbarone is believed to
contribute to its efficacy against cancer cells, the mechanism by which
this drug blocks the function of the type II enzyme is not known.
Therefore, to determine the basis for merbarone action, the ability of
this drug to inhibit individual steps of the catalytic cycle (41, 51)
(see Fig. 12) of human topoisomerase II
was assessed.
Merbarone Does Not Impair Topoisomerase II·DNA Binding--
It
has been suggested that merbarone inhibits the catalytic activity of
topoisomerase II by blocking its ability to bind to its DNA substrate
(33). To test this hypothesis, two independent approaches were utilized
to characterize the effects of the drug on enzyme·DNA binding. One
hundred micromolar merbarone, a concentration that inhibited catalytic
activity ~80%, was employed for both.
In the first approach, interactions between human topoisomerase II
and negatively supercoiled pBR322 DNA were monitored by an
electrophoretic mobility shift assay. As determined by the upshift of
bound DNA to the gel origin, merbarone did not impair topoisomerase
II-DNA interactions (Fig. 3). If
anything, levels of DNA binding appeared to be slightly higher in the
presence of the drug.

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Fig. 3.
Topoisomerase II·DNA binding is not impeded
by merbarone. Binding reactions contained 0-300 nM
human topoisomerase II (Topo II), 10 nM
pBR322 plasmid DNA, and either 0 µM (No Drug)
or 100 µM merbarone. DNA products were analyzed by gel
electrophoresis, and the presence of enzyme·DNA complexes was
indicated by a shift in the mobility of negatively supercoiled DNA
(form I, FI). Topoisomerase II-bound DNA exhibited a slower
electrophoretic mobility or remained at the gel origin
(Ori). The position of nicked circular DNA (form II,
FII) is shown for reference.
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In the second approach, interactions between human topoisomerase II
and a double-stranded 40-mer oligonucleotide that contained a single
cleavage site were monitored by fluorescence anisotropy. As seen in
Fig. 4, merbarone had no effect on
enzyme·oligonucleotide binding. The apparent KD
values calculated in the absence or presence of the drug were 43 ± 9 or 45 ± 10 nM topoisomerase II
,
respectively.

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Fig. 4.
Merbarone has no effect on topoisomerase
II·DNA binding in a fluorescence anisotropy assay. Samples
contained 5 nM fluorescein end-labeled double-stranded
40-mer DNA oligonucleotide and either 0 µM ( ) or 100 ( ) µM merbarone. The fluorophore was excited at 430 nm, and emission was monitored through a 530-nm cut-off filter.
Anisotropies were determined over a concentration range of 0-50
nM human topoisomerase II (Topo II).
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Merbarone Blocks Topoisomerase II-mediated DNA Cleavage--
Since
merbarone does not interfere with enzyme·DNA binding, its effects on
other steps of the topoisomerase II catalytic cycle were assessed in
order to delineate the mechanism by which it exerts its inhibition.
The step that immediately follows DNA binding is pre-strand passage DNA
cleavage (i.e. scission monitored in the absence of a
nucleoside triphosphate (41, 51)). As seen in Fig.
5 (bottom panel), merbarone
strongly inhibited this reaction step (IC50
50
µM). A similar inhibition was observed for post-strand
passage DNA cleavage (i.e. scission monitored in the
presence of the nonhydrolyzable ATP analog, APP(NH)P (41, 51)) (not
shown). Finally, the effects of merbarone on DNA scission were
determined in the presence of ATP, conditions that support the overall
catalytic activity of the enzyme. The IC50 of the drug in
ATP-containing reactions was identical to that determined in the
absence of a nucleoside triphosphate (Fig. 5, top and
bottom panels).

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Fig. 5.
Topoisomerase II-mediated DNA cleavage is
blocked by merbarone. Cleavage reactions contained 300 nM human topoisomerase II (Topo II), pBR322
plasmid DNA (5 nM for ATP cleavage, 10 nM for
+ATP cleavage), and 0-200 µM merbarone. The gel shown in
the top panel depicts the effects of merbarone on
enzyme-mediated DNA cleavage in the presence of 1 mM ATP.
Double-stranded DNA cleavage converts negatively supercoiled plasmid
(form I, FI) to linear molecules (form III,
FIII). The position of nicked circular DNA (form II,
FII) is shown for reference. The bottom panel
quantitates the effects of merbarone on DNA cleavage as described above
in the absence ( ) or presence ( ) of ATP. Standard deviations of 2 to 3 independent assays under each condition are shown as error
bars.
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The concentration of merbarone required to inhibit topoisomerase
II-mediated DNA cleavage by 50% was similar to the
IC50 value (~40 µM) for its
inhibition of enzyme-catalyzed DNA relaxation. This finding strongly
suggests that the primary mechanism by which merbarone inhibits the
overall catalytic activity of human topoisomerase II
is by blocking
DNA cleavage.
Merbarone Does Not Inhibit Topoisomerase II-catalyzed ATP
Hydrolysis--
Coumarin-based topoisomerase II inhibitors as well as
a number of DNA cleavage-enhancing topoisomerase II poisons impair
interactions between the enzyme and ATP (40, 52-55). However, the fact
that the IC50 for merbarone inhibition of DNA cleavage is
unaffected by ATP implies that this drug does not interfere with ATP
utilization. To address this issue directly, the effects of merbarone
on the ATPase activity of human topoisomerase II
were determined
(Fig. 6). Even at a drug concentration of
200 µM, no inhibition of ATP hydrolysis was observed.
This result is similar to that reported recently by Hammonds and
Maxwell (55) and supports the conclusion that merbarone is specific for
topoisomerase II-mediated DNA scission.

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Fig. 6.
Merbarone does not affect topoisomerase
II-mediated ATP hydrolysis. ATPase reactions contained 50 nM human topoisomerase II , 40 nM pBR322
plasmid DNA, and 1 mM [ -32P]ATP. A time
course of ATP hydrolysis was performed at 0 µM ( ) or
200 µM ( ) merbarone. Three independent assays were
carried out for each drug concentration. Error bars depict
standard deviations.
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Global Inhibition of Topoisomerase II-mediated Cleavage by
Merbarone--
Topoisomerase II poisons act in a site-specific fashion
and dramatically alter the spectrum of DNA sites cleaved by the enzyme (2, 4, 56, 57). However, it is not known whether catalytic inhibitors
of the enzyme act in a DNA site-specific manner. To this end, the
effects of merbarone on site-specific DNA scission mediated by human
topoisomerase II
were characterized (Fig.
7).

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Fig. 7.
Merbarone inhibits topoisomerase II-mediated
DNA cleavage in a global manner. DNA cleavage reactions contained
an end-labeled 3609-bp fragment derived from pBR322 (6 nM),
300 nM human topoisomerase II , 1 mM ATP, and
either 0 µM ( ) or 100 µM ( )
merbarone. Ten topoisomerase II-mediated DNA cleavage sites were
observed. Relative levels of DNA cleavage were normalized to site
8 at 100 µM merbarone (relative cleavage = 1).
The standard deviations of two independent experiments are represented
by error bars.
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A uniquely end-labeled 3609-bp fragment of pBR322 DNA was employed for
this experiment. Under the conditions utilized, 10 sites of
topoisomerase II-mediated scission were observed, with cleavage levels
varying by ~7-fold. In the presence of 100 µM merbarone, scission at all sites was inhibited by ~60-80%. The average level of inhibition at these sites, ~70%, correlated well with values determined for the cleavage of circular plasmid DNA at this
drug concentration (see Fig. 5). These data indicate that merbarone
does not block cleavage in a DNA site-specific manner but rather
inhibits this reaction globally.
Interactions between Merbarone and DNA--
Since chemical
compounds that alter the gross structure of DNA either by intercalation
or by minor groove binding can have dramatic effects on the activity of
type II topoisomerases (1, 2, 58-61), it is possible that merbarone
induces its global inhibition of enzyme-mediated DNA cleavage by one of
these two mechanisms. Therefore, two approaches were utilized to
determine whether this was the case.
First, the ability of merbarone to intercalate into DNA was determined
by a topoisomerase I-catalyzed unwinding assay (Fig. 8). In the presence of a strongly
intercalative drug such as ellipticine, a net negative supercoiling of
relaxed DNA substrate was induced following treatment with the type I
enzyme. Conversely, no unwinding was observed in the presence of the
nonintercalative drug etoposide. As seen in Fig. 8, 100 µM merbarone also had no effect on the topological state
of the plasmid. To ensure that this latter result reflected a lack of
DNA intercalation rather than an inhibition of topoisomerase I by the
drug, negatively supercoiled DNA was utilized as the initial substrate
for assays (not shown). Once again, relaxed plasmid resulted after
treatment with topoisomerase I in the presence of 100 µM
merbarone. These findings strongly suggest that merbarone is not
intercalative in nature.

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Fig. 8.
Merbarone does not intercalate into DNA.
The ability of merbarone to intercalate into DNA was investigated using
a topoisomerase I-catalyzed DNA unwinding assay. Relaxed pBR322 DNA
(generated by treatment of negatively supercoiled pBR322 DNA (form I,
FI) with topoisomerase I) was incubated with 100 µM merbarone, etoposide, or ellipticine, or with
Me2SO as a control (Topo I control), in the
presence of topoisomerase I. A negatively supercoiled plasmid control
(DNA Control) is shown. The position of nicked circular DNA
molecules (form II, FII) is indicated.
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Second, the ability of merbarone to displace ethidium bromide from DNA
was determined by a fluorescence emission assay. The DNA-bound form of
ethidium has a significantly stronger fluorescence emission than does
free ethidium; thus, displacement of ethidium from DNA can be monitored
by a decrease in fluorescence signal (48, 49). Moreover, since ethidium
bromide intercalates into DNA through interactions in the minor groove,
this assay is capable of detecting drugs that either intercalate or
bind in the minor groove of DNA.
As seen in Fig. 9, 100 µM
merbarone was incapable of displacing 1 µM ethidium
bromide. Less than 6% displacement was observed at 200 µM merbarone (not shown). In contrast, the intercalative drug amsacrine readily dislodged the bound fluorophore
(IC50
50 µM). Taken together, these
results indicate that if merbarone does in fact bind to DNA, it neither
intercalates nor interacts with the minor groove.

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Fig. 9.
Merbarone does not displace ethidium from the
minor groove of DNA. The ability of merbarone to interact with the
minor groove of DNA was determined by a fluorescence-based ethidium
displacement assay. Samples contained 1 µM ethidium
bromide and 5 nM double-stranded 40-mer DNA
oligonucleotide. Increasing concentrations of merbarone ( ) or
amsacrine ( ) were added, and ethidium fluorescence at 605 nm
( max) was monitored (510 nm excitation
wavelength).
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Attenuation of Topoisomerase II Poisons by Merbarone--
Previous
studies demonstrated that merbarone could block the actions of
topoisomerase II-targeted DNA cleavage-enhancing drugs both in
vitro and in cultured cells (18, 33). Moreover, the attenuation of
teniposide action in culture was observed only if cells were exposed to
merbarone prior to the addition of the topoisomerase II poison (33).
These results have been interpreted as an indication that merbarone
blocks a topoisomerase II reaction step prior to cleavage, namely DNA
binding (33). In light of the finding that merbarone acts by inhibiting
the DNA cleavage step, the effects of merbarone on the actions of
topoisomerase II poisons were reexamined.
Consistent with previous reports, merbarone diminished the stimulation
of topoisomerase II-mediated DNA cleavage by etoposide (Fig.
10). Furthermore, merbarone curtailed
the cleavage-enhancing effects of an apurinic site located within a
topoisomerase II recognition sequence (Fig.
11). Thus, merbarone is capable of
attenuating cleavage enhancement induced both by anticancer drugs and
by lesions that are an intrinsic part of the genetic material.

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Fig. 10.
Etoposide-enhanced topoisomerase II-mediated
DNA cleavage is attenuated by merbarone. DNA cleavage reactions
contained 75 nM human topoisomerase II , 10 nM pBR322 plasmid, and 1 mM ATP. Each reaction
included etoposide (20 µM) and merbarone (100-400
µM). The order of drug addition was varied such that the
enzyme·DNA complex was incubated with Etoposide First,
Merbarone First, or the two drugs simultaneously
(Etoposide/Merbarone). Cleavage levels are reported relative
to cleavage (set at 100%) in control reactions which contained
etoposide in the absence of merbarone. Error bars represent
standard deviations of 2 to 6 data sets.
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Fig. 11.
Merbarone decreases topoisomerase
II-mediated cleavage at a DNA site that contains an apurinic
lesion. A double-stranded 40-mer DNA oligonucleotide that
contained an apurinic site located within a topoisomerase II cleavage
site was utilized as the cleavage substrate. Reactions contained 100 nM oligonucleotide, 150 nM human topoisomerase
II , and 0-400 µM merbarone. The Control
reaction (assigned a relative cleavage value = 1) contained no
merbarone and employed an oligonucleotide lacking an apurinic site.
Standard deviations of 8 independent data sets are presented as
error bars.
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Although the above results are compatible with the fact that merbarone
blocks the DNA cleavage step of the topoisomerase II catalytic cycle,
they shed little light on potential relationships between this
catalytic inhibitor and topoisomerase II poisons. However, by comparing
Fig. 5 with Figs. 10 and 11, it is clear that higher concentrations of
merbarone were required to diminish cleavage in the presence of
etoposide or a DNA lesion. This finding at least suggests that beyond
its inhibition of DNA scission, merbarone may compete with DNA
cleavage-enhancing drugs for a binding site within the topoisomerase
II·DNA complex.
Therefore, to dissect further the mechanism by which merbarone
attenuates the actions of etoposide, an order of addition experiment was performed (Fig. 10). The diminution of cleavage was greatest when
the enzyme·DNA complex was incubated with merbarone prior to the
addition of etoposide and was largely overcome when the poison was
added first. An intermediate degree of cleavage attenuation was
observed when etoposide and merbarone were added simultaneously. These
data suggest that the actions of the poison and the catalytic inhibitor
on human topoisomerase II
are mutually exclusive and imply that they
may act within an overlapping interaction domain on the enzyme.
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DISCUSSION |
Merbarone is a catalytic inhibitor of human topoisomerase II
that exhibits clinical potential as an anticancer agent (18, 26-32,
50). This drug disrupts chromosome separation during mitosis (33,
62-64) in a manner that is reminiscent of temperature-sensitive mutations in topoisomerase II (65-67) or treatment of cells with ICRF-193 (68, 69), a specific catalytic inhibitor of the enzyme (70,
71). Furthermore, merbarone blocks the stimulation of DNA cleavage by
topoisomerase II poisons in cultured cells (33). Taken together, these
findings provide strong (albeit circumstantial) evidence that
topoisomerase II is an important in vivo target for
merbarone.
Despite the potential therapeutic value of merbarone, the mechanism by
which it inhibits the catalytic activity of topoisomerase II has never
been delineated. Based on the fact that merbarone blocks the actions of
topoisomerase II poisons, it has been suggested that this compound acts
by obstructing enzyme·DNA binding (33). On the contrary, results of
the present study indicate that this is not the case. Rather, it
appears that merbarone inhibits topoisomerase II specifically by
blocking enzyme-mediated DNA cleavage.
This conclusion was derived by dissecting the actions of merbarone on
the individual steps of the topoisomerase II catalytic cycle.
Concentrations of merbarone that inhibited catalytic activity
80%
had no effect on either enzyme·DNA binding or ATP hydrolysis. In
contrast, the drug was a potent inhibitor of DNA scission mediated by
human topoisomerase II
(in the absence or presence of ATP), and the
IC50 values of merbarone for DNA cleavage and relaxation were similar.
It was not possible to assess the effects of merbarone on the DNA
strand passage step of the catalytic cycle due to drug inhibition of
DNA cleavage (the step that immediately precedes strand passage (see
Fig. 12)). If merbarone is affecting
the strand passage step, it is not doing so by interfering with
topoisomerase II-ATP interactions, the primary mechanism that is
employed by several other inhibitory drugs (15, 40, 52-55).

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Fig. 12.
Effects of inhibitors on the catalytic cycle
of topoisomerase II. The topoisomerase II homodimer (shown in
green) is modeled after the crystal structure reported by
Berger et al. (72). The catalytic cycle of the enzyme has
been described previously as a series of six individual reaction steps
(1, 51). Step 1, topoisomerase II binds its DNA substrate
(41, 73, 74). Step 2, a transient enzyme-linked
double-stranded break is formed in the "cleavage" helix (shown in
yellow) (75, 76). Step 3, ATP binding induces a
conformational change in the enzyme (77) that converts topoisomerase II
into a "protein clamp" on the DNA (41, 78). Concomitant with this
structural reorientation, the "passage" helix (shown in
purple) is translocated through the break in the cleavage
helix (40). Step 4, the enzyme religates the break in the
cleavage helix (41). Step 5, upon ATP hydrolysis, the
protein clamp opens (41, 78), allowing Step 6, release of
the DNA and the initiation of a new round of catalysis (41). Drugs that
inhibit topoisomerase II catalytic function act at different steps of
the catalytic cycle. Aclarubicin is an anthracycline that blocks
enzyme·DNA binding, the initial step of the cycle (24).
Staurosporine, a tyrosine kinase inhibitor that also acts on
topoisomerase II, inhibits DNA cleavage and ATP interactions (22).
Novobiocin and coumermycin, coumarin-based drugs that are primarily
active against prokaryotic type II topoisomerases (15), block binding
of ATP to the enzyme (40, 52). ICRF-193 is a bis(2,6-dioxopiperazine)
derivative that blocks ATP hydrolysis, an action that traps the enzyme
on DNA in its closed clamp form (25). Finally, as described in the
present study, merbarone specifically blocks topoisomerase II-mediated
cleavage of its DNA substrate.
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A number of chemical compounds that inhibit the catalytic activity of
type II topoisomerases do so by distorting the gross structure of DNA.
This is most often seen with drugs that intercalate or bind in the
minor groove of the genetic material (58-61). As assessed by two
independent DNA binding assays, merbarone does not appear to act in
this manner. Instead, it seems likely that this drug exerts its effects
on topoisomerase II catalytic activity by a specific interaction with
either the enzyme or the enzyme·DNA complex. This suggestion is
supported by two lines of evidence. First, merbarone competes for
action with etoposide, a DNA cleavage-enhancing drug that enters the
topoisomerase II·DNA complex primarily through its interactions with
the enzyme (37). Second, merbarone displays a dramatic species
dependence, inhibiting human topoisomerase II
with a potency that is
vastly greater than that for either the yeast or Drosophila
type II enzymes.
A number of drugs that inhibit the catalytic activity of topoisomerase
II have been identified. Although detailed studies have not been
carried out for all of these agents, the available data indicate that
catalytic inhibitors act by a variety of mechanisms (depicted in Fig.
12). Depending upon which step(s) of the topoisomerase II catalytic
cycle is targeted by a specific agent, the cellular effects of that
drug could be significantly different. For example, a compound such as
aclarubicin, which disrupts topoisomerase II·DNA binding (24), would
undermine both the catalytic and structural roles of the enzyme. In
contrast, drugs such as merbarone, which act by blocking DNA cleavage,
or coumarins, which act by blocking enzyme-ATP interactions (40, 52),
would only impact the catalytic functions of topoisomerase II. Since
ICRF-193 acts by blocking ATP hydrolysis, it not only inhibits
catalytic activity but also traps the enzyme on the DNA in its
"closed clamp" form (25). This in turn might generate a physical
barrier on the chromosome that impedes the actions of other DNA
enzymes. Finally, in addition to their differential cellular effects,
drugs that act at or before the DNA cleavage step are likely to
attenuate the actions of topoisomerase II poisons. Clearly, the
"mechanistic fingerprint" of a catalytic inhibitor could seriously
impact its therapeutic potential.
Several compounds that inhibit the activity of topoisomerase II are
under consideration as anticancer agents. Although these drugs are
categorized under the general "umbrella" of catalytic inhibitors,
they exert their effects by a variety of mechanisms. On the basis of
the present study, merbarone acts by blocking the DNA cleavage reaction
of topoisomerase II. This is the first drug found to specifically
inhibit this reaction step and defines a new mechanistic class of
catalytic inhibitors.
We are grateful to Dr. Randall K. Johnson
(SmithKline Beecham) for the gift of the merbarone; to Drs. Paul S. Kingma, D. Andrew Burden, and David E. Graves (University of
Mississippi) for helpful discussions; to Susan D. Cline for critical
reading of the manuscript; and to Dr. Paul S. Kingma for expert
assistance in the preparation of Fig. 12.