From the
Although a number of drugs currently in use for the treatment of
human cancers act by stimulating topoisomerase II-mediated DNA
breakage, little is known regarding interactions between these agents
and the enzyme. To further define the mechanism of drug action,
interactions between ellipticine (an intercalative drug with clinical
relevance) and yeast topoisomerase II were characterized. By utilizing
a yeast genetic system, topoisomerase II was identified as the primary
cellular target of the drug. Furthermore, ellipticine did not inhibit
enzyme-mediated DNA religation, suggesting that it stimulates DNA
breakage by enhancing the forward rate of cleavage. Finally,
ellipticine binding to DNA, topoisomerase II, and the enzyme
The ability to modulate the topological state of nucleic acids
is critical to the survival of eukaryotic and prokaryotic
cells(1, 2, 3, 4, 5) .
Topoisomerases, the enzymes that modulate DNA topology in
vivo, are involved in virtually every aspect of DNA metabolism
(1-5). In addition to their critical physiological functions,
topoisomerases are targets for a number of relevant chemotherapeutic
agents. For example, ciprofloxacin, which is targeted to the
prokaryotic type II topoisomerase, DNA gyrase, is the most active oral
antibiotic currently in clinical
use(6, 7, 8, 9, 10) .
Furthermore, camptothecin-based drugs, which target eukaryotic
topoisomerase I, and drugs such as etoposide, amsacrine, doxorubicin,
mitoxantrone, and ellipticine, which target eukaryotic topoisomerase
II, are effective agents for the treatment of several human
cancers(11, 12, 13, 14) . Although these
drugs are derived from diverse structural classes and act through three
different topoisomerases, they all exert their cytotoxic effects by
enhancing enzyme-mediated DNA breakage within the
genome(14, 15, 16, 17, 18, 19) .
Despite the clinical importance of topoisomerase poisons,
interactions between these agents and their enzyme targets are poorly
understood. It is likely that formation of a ternary complex between
topoisomerase, DNA, and drug is critical for nucleic acid breakage and
subsequent cell death(7, 9, 14, 19) .
However, the pathway by which this ternary complex is assembled has yet
to be determined. Three possible mechanisms for complex formation
exist. In the first, the drug binds only to the topoisomerase
To further define the
mechanism of action of antineoplastic drugs targeted to eukaryotic
topoisomerases, interactions between yeast topoisomerase II and
ellipticine were characterized. Ellipticine is an intercalative
alkaloid (26, 27) that stimulates topoisomerase
II-mediated DNA
breakage(28, 29, 30, 31) , and along
with several of its analogs, is currently in clinical
trials(32, 33, 34, 35, 36, 37, 38, 39, 40, 41) for the
treatment of human cancers. Results of the present study indicate that
topoisomerase II is the primary cellular target of the drug. In
addition, ellipticine shows little ability to inhibit the religation of
cleaved DNA by topoisomerase II, suggesting that this agent enhances
DNA breakage by increasing the forward rate of cleavage. Finally, as
determined by steady state and frequency domain fluorescence
spectroscopy, it appears that ellipticine forms a stable complex with
topoisomerase II in the absence of DNA, and that the enzyme dictates
the ionic state of the drug in the topoisomerase
II
If topoisomerase II is the
primary cellular target for ellipticine and if cytotoxicity is due to
the stimulation of topoisomerase II-mediated DNA cleavage, a reduction
in enzyme activity should greatly diminish drug-induced cell death.
Conversely, if topoisomerase II is the primary target, but cell death
results from the impairment of catalytic activity, cells with decreased
levels of enzyme activity should be hypersensitive to ellipticine.
Finally, if ellipticine targets other components in the cell, reduced
levels of topoisomerase II should not dramatically affect drug
toxicity.
At 25 °C, cell growth was abrogated by 20
µM ellipticine, and
To delineate the mechanism by which ellipticine enhances DNA
breakage, the effects of this drug on topoisomerase II-mediated DNA
religation were assessed. As determined by the conversion of
supercoiled to linear DNA, 10 µM ellipticine produced
maximal stimulation (
Ellipticine
can exist as a protonated or a deprotonated species
(pK
In
contrast to the emission increase at 420 nm, there was a decrease in
intensity at 520 nm upon the addition of topoisomerase II, and the
intensity of this peak was always less than that of free ellipticine.
These data imply that deprotonated ellipticine is the major species
present in the ternary complex. This suggestion is confirmed by the
fact that the lifetimes observed for protonated ellipticine in the
presence of enzyme and oligonucleotide were the same as those of the
free or the DNA-bound drug.
Addition of
yeast topoisomerase II to ellipticine resulted in dramatic increases in
the fluorescence intensity and lifetime of the deprotonated form of the
drug (Fig. 6, left panel). These data are consistent
with formation of a binary complex between deprotonated ellipticine and
the enzyme. In contrast, no intensity increase at 520 nm was seen, and
the lifetime and anisotropy of the protonated drug were that of free
compound. Thus, it appears that there is no appreciable binding between
protonated ellipticine and topoisomerase II.
The dissociation
constant for the binary complex was determined by varying enzyme at a
fixed ellipticine concentration (Fig. 6, right panel).
The apparent K
It was not possible to utilize lifetime analysis for the direct
measurement of the fraction bound because of the large difference in
the lifetimes of bound and free ellipticine and the difficulty in
determining the lifetime of the free deprotonated form at the
frequencies available. In addition, anisotropy of the
topoisomerase
Although topoisomerase II is the primary cellular target for
a number of clinically relevant antineoplastic agents, virtually
nothing is known about enzyme
A summary scheme depicting ellipticine binding to DNA, topoisomerase
II, and the enzyme
It has been proposed
that intercalative drugs alter the cleavage/religation equilibrium of
topoisomerase II primarily through their effects on DNA
structure(72) . Two lines of evidence undercut this assertion.
First, there appear to be dominant interactions between ellipticine and
topoisomerase II in the ternary complex, indicating that there are
points of contact between this drug and a hydrophobic portion of the
enzyme. Second, the deprotonated form of ellipticine in the ternary
complex is not the preferred species for DNA intercalation. While the
deprotonation of ellipticine does not preclude intercalation, no
evidence for the interaction between the deprotonated form of
ellipticine and DNA was seen. This implies that interactions between
the drug and DNA in the ternary complex differ from that in the
ellipticine
Before the design and clinical efficacy of novel
topoisomerase II-targeted agents can be optimized, the mechanism of
drug action must be understood. The current study shows that
ellipticine interacts with the enzyme alone and raises the potential
for multiple pathways for ternary complex formation. This finding
presents a novel conceptual scaffold upon which to build new
theoretical models for topoisomerase II
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are grateful to the laboratory of Dr. Joseph
Lakowicz and the Center for Fluorescence Spectroscopy (University of
Maryland School of Medicine) for the use of the SLM 8000 steady state
fluorescence spectrometer, to Dr. John Nitiss for expert advice and the
yeast strains utilized, to Dr. Sarah H. Elsea and Michael Otto for
helpful discussion, to Erin Hannah for assistance with yeast and
preparation of pBR322 plasmid DNA, and to Paul Kingma, Dr. Kathy
Latham, and Dr. Andrew Burden for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
DNA
complex was assessed by steady-state and frequency domain fluorescence
spectroscopy. As determined by changes in fluorescence intensity and
emission maximum wavelength, and by lifetime analysis, only the
protonated species of ellipticine bound to a double-stranded 40-mer
oligonucleotide containing a topoisomerase II cleavage site (K
65 nM). In contrast,
predominantly deprotonated ellipticine bound to the enzyme
DNA
complex (K
1.5 µM) or
to the enzyme in the absence of nucleic acids (K
160 nM). These findings suggest that
ellipticine interacts directly with topoisomerase II and that the
enzyme dictates the ionic state of the drug in the ternary complex. A
model is presented in which the topoisomerase
II
ellipticine
DNA complex is formed via initial drug binding
to either the enzyme or DNA.
DNA
complex and has minimal interactions with either the enzyme or nucleic
acid independently. Support for this possibility is derived from
studies that characterized the binding of camptothecin with
topoisomerase I (20) and quinolones with DNA gyrase (21, 22). In
both cases, drugs were found to interact almost exclusively with the
enzyme
DNA complex. In the second mechanism, the drug becomes part
of the ternary complex primarily through interactions with DNA. Support
for this possibility stems from the fact that many
topoisomerase-targeted agents bind (in either an intercalative or
nonintercalative fashion) to DNA in the absence of
enzyme(23, 24) . It should be noted, however, that no
correlation has been observed between either the mode or strength of
DNA binding and the cytotoxicity or antineoplastic activity of these
drugs. In the third mechanism, the drug becomes part of the ternary
complex primarily through direct interaction with the enzyme in the
absence of DNA. Evidence for this last possibility comes from
surface-enhanced Raman scattering studies that suggest an interaction
between intoplicine and topoisomerase II (25).
ellipticine
DNA complex. A model is proposed in which
ellipticine enters the ternary complex through its prior association
with either DNA or the enzyme and does not require the presence of a
preformed topoisomerase II
DNA complex.
Materials and Yeast Strains
A 40-mer
double-stranded oligonucleotide containing a characterized cleavage
site for topoisomerase II (42, 43) was synthesized on an
Applied Biosystems DNA Synthesizer. The sequence of one of the two
complementary oligonucleotides is:
5`-TGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGT-3` where
the arrow denotes the site of topoisomerase II-mediated DNA cleavage.
Oligonucleotides were purified as described previously (44) and
diluted to the appropriate concentration in 5 mM Tris-HCl, pH
7.4, 0.5 mM EDTA. Negatively supercoiled bacterial plasmid
pBR322 DNA was prepared as described by Maniatis and
co-workers(45) . Ellipticine (Sigma) was solubilized as a 20
mM or 10 mM stock solution in dimethyl sulfoxide or
ethanol, respectively, and stored at -20 °C. The yeast
strains employed for the present study were Saccharomyces
cerevisiae JN394 with genotype ura3-52, leu2, trp1, his7, ade1-2, ISE2,
rad52:LEU2, and JN394t2-1, whose genotype is isogenic to JN394
except for the replacement of the wild type topoisomerase II gene (TOP2+) with the top2-1 mutant allele.
Dimethyl-POPOP
(
)and rose bengal were acquired
from Eastman; ultrapure HEPES was from VWR; Tris-HCl and ethidium
bromide were obtained from Sigma; SDS was purchased from E. Merck
Biochemicals; proteinase K was from United States Biochemical Corp.;
yeast nitrogen base, yeast extract, and Bacto-agar were from Difco. All
other chemicals were analytical reagent grade.
Yeast Topoisomerase II Overexpression and
Purification
Yeast topoisomerase II was overexpressed and
purified by the procedure of Worland and Wang (46) as modified
by Elsea et al.(47) . Briefly, overexpression was
achieved in yeast strain JEL 1 (transformed with YEpGAL1TOP2). Cells
were induced by the addition of galactose in glucose-free media, grown
for 16 h to an A of
1.0, and harvested by
centrifugation. Pellets were resuspended in buffer (50 mM
Tris-HCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 10% glycerol,
25 mM NaF, 1 mM
Na
S
O
, 1 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride),
quick-frozen, and stored at -80 °C until use. Cells were
lysed at 4 °C, and all purification procedures were carried out at
4 °C. Topoisomerase II was purified from cell extracts to apparent
homogeneity (as determined by visualization on silver-stained
polyacrylamide gels) by phosphocellulose column chromatography based on
the protocol of Shelton et al.(48) . Approximately 20
mg of the type II enzyme was isolated from 25 g of frozen wet-packed
yeast cells.
Primary Cellular Target Assay
The primary cellular
target of ellipticine in yeast was determined by the protocol of Nitiss
and co-workers(49, 50, 51) . Briefly, yeast
strain JN394t2-1 was cultured in YPDA media at 25 °C. Following the
adjustment of logarithmically growing cells to a titer of 2
10
cells/ml, ellipticine (10-200 µM) was
added to the medium. Cultures were incubated with the drug for 18 h at
25 °C or 30 °C. Cells were diluted into sterile water and
plated in duplicate onto YPDA medium solidified with 1.5% Bacto-agar
(Difco). Plates were incubated at 25 °C or 30 °C, and surviving
colonies were counted.
Topoisomerase II-mediated DNA Cleavage
Assays were
performed using a modification of the protocol described by Robinson
and Osheroff(52) . Experiments were carried out in the absence
of ATP, thus monitoring topoisomerase II-mediated cleavage that occurs
prior to the strand passage event(53) . DNA cleavage reactions
contained 100 nM yeast topoisomerase II and 5 nM negatively supercoiled pBR322 in reaction buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 5 mM
MgCl, 0.1 mM EDTA, and 2.5% glycerol) with a total
volume of 20 µl. The DNA cleavage/religation equilibrium was
established by incubating reaction mixtures at 28 °C for 6 min.
Cleavage products were trapped by the addition of 2 µl of 10% SDS,
followed by the addition of 1.5 µl of 250 mM EDTA and 2
µl of a 0.8 mg/ml solution of proteinase K. Samples were incubated
at 45 °C for 20 min to digest the topoisomerase II, mixed with 2
µl of 10 mM Tris-HCl, pH 7.9, 0.05% bromphenol blue, 0.05%
xylene cyanol, and 60% sucrose, and heated at 70 °C for 2 min.
Reaction products were resolved by electrophoresis in 1% agarose gels
in 40 mM Tris acetate, pH 8.0, 2 mM EDTA, and gels
were stained with 1 µg/ml ethidium bromide. DNA bands were
visualized by transillumination with UV light (300 nm) and were
photographed through Kodak 23A and 12 filters with Polaroid type 665
positive-negative film. The amount of DNA present was quantitated by
scanning photographic negatives with an E-C Apparatus model EC910
scanning densitometer using Hoefer GS-370 Data System software.
Alternatively, DNA cleavage bands were quantitated with an Alpha
Innotech IS1000 Imaging System. In both cases, the densities of the
bands were proportional to the amount of DNA present. The effects of
ellipticine were studied over a range of 250 nM to 100
µM. All control samples contained an equal amount of drug
diluent, either dimethyl sulfoxide or EtOH. No drug-induced DNA
cleavage was seen in the absence of topoisomerase II.
Topoisomerase II-mediated DNA Religation
Assays
were performed by a modification of the protocol of Robinson et
al.(54) . Reactions contained 100 nM yeast
topoisomerase II and 5 nM negatively supercoiled pBR322. DNA
cleavage/religation equilibria were established as described above.
Topoisomerase II-mediated religation of cleaved DNA was induced by
rapidly shifting samples from 28 °C to 65 °C. Religation was
terminated by the addition of SDS (2 µl of 10%) at various time
points. Following the cessation of religation, samples were treated
with EDTA and proteinase K. Reaction products were analyzed by agarose
gel electrophoresis and quantitated as described above.
Steady-state and Frequency Domain Fluorescence
Spectroscopy
Steady-state fluorescence experiments were
performed utilizing an SLM 8000C fluorometer with SLM software Version
4.0. Spectral analysis was carried out at 25 °C. Fluorescence
excited-state lifetimes were determined using an ISS K2 multifrequency
phase-modulation fluorometer, and data were analyzed using the ISS
software package Version 2.0. A Liconix 4214NB helium-cadmium laser
emitting 3 milliwatts at 326 nm or 12 milliwatts at 442 nm was the
excitation light source. For both steady-state and frequency domain
experiments, emitted light was monitored while the emission polarizer
was fixed at the magic angle (54.7°), and either a 420 nm or 520 nm
interference band pass filter was employed to separate fluorescence
from scattered light. Dimethyl-POPOP, with a lifetime value of 1.45 ns,
or rose bengal, with a lifetime of 732 ps (in EtOH)(55) , were
used as references. Data were acquired at 25 °C between 1 and 250
MHz. Samples contained the designated concentrations of yeast
topoisomerase II and/or 40-mer oligonucleotide and ellipticine in a
final volume of 500 µl of 20 mM HEPES, pH 7.9 (unless
otherwise noted), 100 mM NaCl, 5 mM MgCl,
and 0.1 mM EDTA and were incubated for 6 min prior to
fluorescence measurements. All chemicals were ultrapure grade to
eliminate scatter and nonspecific fluorescence. The buffer background,
shown as a reference in Fig. 6, was subtracted from intensities
for binding calculations.
Figure 6:
Ellipticinetopoisomerase II binding.
The emission spectra for 1 µM ellipticine in the presence
of topoisomerase II concentrations ranging from 0-150 nM are shown in the left panel. Samples were excited at 326
nm. A double reciprocal binding analysis is shown in the right
panel. Data are plotted as 1/
fluorescence intensity at 420
nm versus 1/[topoisomerase II] and represent the
averages of two independent experiments.
Primary Cellular Target of
Ellipticine
Ellipticine and several of its analogs are currently
being evaluated for their clinical efficacy against human
cancers(32, 33, 34, 35, 36, 37, 38, 39, 40, 41) .
Since these drugs stimulate topoisomerase II-mediated DNA breakage, and
the stabilization of enzyme-DNA cleavage complexes induces cell death,
it has been assumed that topoisomerase II is the primary cytotoxic
target of this drug class. However, there is no direct evidence to
support this assumption. Therefore, to determine the primary cellular
target and cytotoxic mechanism of ellipticine, a yeast genetic system
that exploits a temperature-sensitive chromosomal copy of the
topoisomerase II allele (top2-1) was employed. At 25 °C,
enzyme activity in the top2-1 strain is 100% (i.e. wild type), but at 30 °C, activity is diminished to
<10%(56) . Since the enzyme is
essential(57, 58, 59, 60) , it is not
possible to fully delete the activity.
90% of the initial culture was
killed by 200 µM drug (Fig. 1). However, at 30
°C, no cell death was observed at any ellipticine concentration
employed. Even at 200 µM ellipticine,
30% cell growth
occurred. These results identify topoisomerase II as the primary
cellular target of ellipticine (at least in yeast) and demonstrate that
the drug acts by converting the type II enzyme to a cellular poison.
Figure 1:
Cytotoxicity of ellipticine toward
yeast cells (JN394t2-1) that express a temperature-sensitive mutant
topoisomerase II (top2-1). Ellipticine cytotoxicity at 25
°C () and at 30 °C (
) is shown for increasing
concentrations of ellipticine at 18 h. Relative cell survival at time
zero was set arbitrarily to 100%. Results are representative of four
independent experiments.
As a control, the cytotoxicity of ellipticine toward yeast cells
that contain wild type topoisomerase II (JN394 cells) was determined at
25 °C and 30 °C (data not shown). Similar drug cytotoxicity was
observed at either temperature. Furthermore, the relative survival
curve resembled that of the top2-1 strain at 25 °C. Thus,
resistance of the JN394t2-1 cells to ellipticine at 30 °C does not
result from a difference in cellular efflux or metabolism of the drug
at the elevated temperature.
Effects of Ellipticine on Topoisomerase II-mediated DNA
Religation
The ability to cleave and religate DNA is not only
central to the physiological functions of topoisomerase II, but also
provides the basis for the action of antineoplastic agents targeted to
the enzyme. As discussed above, these agents act by increasing the
amount of topoisomerase-mediated DNA
breakage(11, 12, 13, 14) . In this
regard, two drug mechanisms have been reported based on religation
assays. Drugs that function by the first mechanism (including several
quinolones and genistein) have little effect on religation and are
presumed to act by enhancing the forward rate of
cleavage(14, 54, 61, 62) . Agents that
function by the second mechanism (including amsacrine and etoposide)
strongly inhibit religation of the severed DNA and appear to act
primarily at this step of the cleavage/religation event (52, 61, 63).
6-fold) of topoisomerase II-mediated DNA
breakage (Fig. 2, inset). This concentration of
ellipticine had virtually no effect on the apparent first order rate of
religation (Fig. 2). In contrast, 25 µM etoposide
(which produced equivalent cleavage levels to that of ellipticine)
decreased the apparent first order rate of religation
10-fold.
This finding suggests that ellipticine stimulates enzyme-mediated DNA
breakage primarily by increasing the forward rate of cleavage. This is
the first strongly intercalative drug found to function by this
mechanism(14) .
Figure 2:
Effects of ellipticine on topoisomerase
II-mediated DNA cleavage and religation. Results are shown for
religation reactions performed in the absence of drug () or in the
presence of 10 µM ellipticine (
). Data for
reactions performed in the presence of 25 µM etoposide
(
) are shown for comparison. Data are plotted as the loss of
linear DNA versus time. The proportion of linear DNA was set
to 100% at time zero. The inset shows the effect of 0-10
µM ellipticine (x axis) on relative DNA cleavage (y axis). Levels of DNA cleavage in the presence of drug
diluent were set to 1.0. Results of DNA cleavage and religation
represent the averages of three independent
experiments.
Fluorescence Properties of
Ellipticine
Fluorescence spectroscopy is a sensitive technique
that can be used to characterize ligandmacromolecule binding and
to describe the molecular environment of the bound ligand. However, the
presence of 18 tryptophan residues per subunit of yeast topoisomerase
II (64) makes it difficult to utilize the intrinsic fluorescence
of the enzyme. Therefore, the interactions of ellipticine with DNA,
topoisomerase II, and the enzyme
DNA complex were elucidated by
monitoring the fluorescence properties of ellipticine.
= 7.4), and the fluorescence
emission of the drug is highly pH-dependent (Fig. 3). At high pH,
where the deprotonated form predominates, the drug fluoresces weakly
with peak excitation at 360 nm (data not shown) and emission at 420 nm.
In addition, deprotonated ellipticine has a short lifetime (
60 ps, , see Fig. 7) and a low quantum yield, suggesting
that the fluorescence of this drug species is substantially quenched in
water. This suggestion is supported by the fact that the fluorescence
lifetime and apparent efficiency of ellipticine fluorescence increase
in ethanol () or glycerol (not shown) at low temperatures.
Figure 3:
pH-dependent emission of ellipticine. The
fluorescence emission spectra for 10 µM ellipticine at pH
values ranging from 2.0 to 10.3 are shown. Samples were excited at 326
nm.
Figure 7:
Frequency domain fluorescence lifetime
measurements of ellipticine. Phase shifts, in degrees for free ()
and complexed (
) ellipticine, and demodulation ratios, in percent
for free (
) and complexed (
) ellipticine in the ternary
complex are shown. The residual values (top panel) represent
the differences between the measured and best fit calculated values.
Dimethyl-POPOP was utilized as a reference.
At low pH, where the protonated form of ellipticine is most
prevalent, the drug displays maximal absorption at 440 nm and emission
at 520 nm. Both the lifetime (3.5 ns) and the apparent quantum yield of
this species are considerably larger than that of the deprotonated
drug. Since fluorescence assays were carried out at pH 7.9 (the optimal
pH for topoisomerase II activity), both deprotonated and protonated
ellipticine were present in an 60:40 ratio. The dramatic
differences in emission maxima and lifetimes of these two ionic species
allow both forms to be monitored simultaneously.
Ellipticine
The binding of
ellipticine to a double-stranded 40-mer oligonucleotide was
characterized. This 40-mer contains a cleavage/recognition site for
topoisomerase II(42, 43) , and enzyme-mediated cleavage
at this site is stimulated by the drug (not shown). In the presence of
the oligonucleotide, the fluorescence intensity, anisotropy, and the
lifetime of the protonated form of ellipticine increased (Fig. 4, left panel, ). No changes in the fluorescence
properties of the deprotonated species were observed in the presence of
DNA. These findings are consistent with the anionic nature of the
genetic material and suggest that protonated ellipticine binds to DNA.
Similar results have been found for other intercalators such as
ethidium bromide and acridine yellow(65, 66) .
DNA Binding
Figure 4:
EllipticineDNA binding. The emission
spectra for 1 µM ellipticine in the presence of
oligonucleotide concentrations ranging from 25-200 nM are shown in the left panel. Samples were excited at 442
nm. A double reciprocal binding analysis is shown in the right
panel. Data are plotted as 1/
fluorescence intensity at 520
nm versus 1/[oligonucleotide] and represent the
averages of two independent experiments. The change in anisotropy of
ellipticine with increasing concentrations of 40-mer is shown (inset).
The
affinity of ellipticine for DNA binding was monitored by quantitating
the change in intensity at 520 nm. The apparent K value was calculated (67) from the slope of the line (K
/
I
) in
a plot of the inverse of the intensity increase (
I) versus the inverse of the DNA concentration (Fig. 4, right panel). The y-intercept (i.e. 1/
I
) is defined as the inverse of
the intensity enhancement expected when all the ellipticine is bound.
The apparent K
(
65 nM) for
DNA binding determined by this method is comparable to values reported
previously for ellipticine (68, 69, 70, 71) and other cationic
aromatic intercalators(65, 66) . The ratio of the
apparent maximal intensities (
I
) of
DNA-bound ellipticine and the free protonated drug is comparable to the
ratio of the lifetimes of these two species. The increased steady-state
anisotropy of the protonated drug in the presence of DNA (Fig. 4, inset in right panel), together with the concomitant increase
in lifetime, provides strong evidence that the rotational diffusion of
the drug is less rapid and/or its motion is restricted.
Formation of a Ternary Topoisomerase
II
Formation of the
topoisomerase IIEllipticine
DNA Complex
ellipticine
DNA complex was monitored by
adding increasing amounts of topoisomerase II to a solution of
ellipticine and the oligonucleotide or by adding DNA to a solution
containing enzyme and the drug. In either case, an increase in
fluorescence intensity at 420 nm was observed, indicating that the
deprotonated form of the drug is present in the ternary complex (Fig. 5, left panel). In addition, the
of the 420 nm peak was blue-shifted, suggesting that the drug in
the ternary complex is located in a hydrophobic environment. Binding
affinity was evaluated, as described above, by plotting the inverse of
the apparent intensity change (
I) as a function of the
inverse of the enzyme concentration at fixed drug and DNA
concentrations (Fig. 5, right panel). The calculated
apparent K
is
1.5 µM,
which is in the range of clinical efficacy for
ellipticine(32, 33, 34, 35, 36, 37, 38, 39, 40, 41) .
Figure 5:
Topoisomerase IIellipticine
DNA
binding. The emission spectra for 1 µM ellipticine and 200
nM oligonucleotide in the presence of topoisomerase II
concentrations ranging from 0-150 nM are shown in the left panel. Samples were excited at 326 nm. A double
reciprocal binding analysis is shown in the right panel. Data
are plotted as 1/
fluorescence intensity at 420 nm versus 1/[topoisomerase II] and represent the averages of two
independent experiments.
To further define the fluorescence properties of deprotonated
ellipticine in the ternary complex, the frequency domain lifetimes were
determined. A dramatic increase in lifetime from 60 ps for free
ellipticine to
23 ns for the bound drug was observed (, see Fig. 7). This finding suggests that the
quantum yield of bound ellipticine is much higher than the uncomplexed
drug. The fact that ellipticine exhibits a stronger fluorescence (not
shown) and a longer lifetime in a less polar solvent such as ethanol () supports the suggestion that the ellipticine binding
site in the ternary complex is hydrophobic in nature. The relatively
high X
value suggests that the emission may be
more complex than the two-component model used to fit the data.
Formation of the Topoisomerase II
Although the protonated form of ellipticine binds to
DNA, it is the deprotonated form that is present in the ternary
complex. This finding implies that the enzyme rather than the DNA
dictates the protonation state of the bound drug and suggests that
topoisomerase II may play a role in recruiting the drug to the ternary
complex. Therefore, the ability of ellipticine to bind directly to
topoisomerase II in the absence of DNA was assessed.
Ellipticine
Complex
value (
160
nM) was
10-fold lower than that calculated for the
ternary complex. Under conditions approaching stoichiometric binding of
the drug to topoisomerase II, a lifetime component of
24 ns was
observed (, Fig. 7). Thus, binding of the drug to the
enzyme greatly increases the quantum yield of the deprotonated
ellipticine. As expected, the large increase in lifetime was
proportional to the increase in intensity
(
I
) of the bound over the free form.
ellipticine complex could not be measured because
excitation near the peak of the absorption band at 360 nm, where the
limiting anisotropy is maximal (r
= 0.4,
data not shown), resulted in overlap between the water Raman -OH
stretching band emission and the sample emission.
drug interactions. In general,
binding studies have been hampered by the limited number of techniques
available and the relatively large amount of enzyme required for these
methods. However, by taking advantage of the optical properties of
ellipticine and the ability to overexpress topoisomerase II in yeast,
it was possible to characterize the interactions of this DNA cleavage
enhancing drug with topoisomerase II and the enzyme
DNA complex.
DNA complex is shown in Fig. 8. At
neutrality, two species of ellipticine, a protonated and a deprotonated
form, exist in equilibrium. While protonated ellipticine intercalates
in free DNA, it is predominantly deprotonated ellipticine that binds to
topoisomerase II in the absence of DNA and is present in the ternary
complex. This finding implies an interaction between the drug and
topoisomerase II in the ternary complex and further suggests that the
enzyme dictates the ionic state of ellipticine. Furthermore, it appears
that the binding of ellipticine to topoisomerase II is indicative of
the enzyme
drug interactions in the ternary complex, since a
mutant type II topoisomerase that is hypersensitive to ellipticine has
a higher intrinsic binding affinity for the drug.
(
)
Figure 8:
Model of ellipticine binding to DNA,
topoisomerase II, and the topoisomerase IIDNA complex. Protonated
and deprotonated forms of ellipticine are shown. Topoisomerase II is
depicted by the handlebar-shaped structure, and DNA is
represented by the cylinders. The enzyme is shown binding to
DNA at a point of helix-helix crossover as proposed by Zechiedrich and
Osheroff (73) and Roca et al. (74). In this model, the
topoisomerase II
ellipticine
DNA ternary complex (complex
3) is formed via the prior association of the drug with either the
DNA (complex 1) or the enzyme (complex 2). Formation
of the ternary complex results in increased levels of covalent
topoisomerase II
DNA cleavage complexes (complex
4).
Based on previous studies that examined drug
binding to topoisomerase I (20) or DNA gyrase(21, 22) ,
it has been suggested that topoisomerase II-targeted agents may bind
specifically to the enzymeDNA complex (14, 19). The generality of
this theory is challenged by the finding that ellipticine and
intoplicine (25) bind directly to the enzyme. Moreover, since
the apparent dissociation constants for the topoisomerase
II
ellipticine and ellipticine
DNA complexes are an order of
magnitude lower than that of the ternary complex, it is likely that the
formation of the topoisomerase II
ellipticine
DNA complex
occurs via initial drug binding to either the enzyme or the DNA (Fig. 8). In the latter case, the ionic state of ellipticine
bound to DNA must be converted by the enzyme to the deprotonated form
during the formation of the ternary complex.
DNA complex. Because the enzyme and its nucleic acid
substrates prefer different ionic states of the drug, the juxtaposition
of ellipticine, the enzyme, and the DNA may produce an inherent stress
in the ternary complex. If so, the resulting strain could contribute to
the increased dissociation constant of the ternary complex and may be
the underlying mechanistic basis for the enhancement of enzyme-mediated
DNA cleavage.
drug
DNA
interactions.
Table: Ellipticine lifetime data
/EMBL Data Bank with accession number(s) L41827.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.