(Received for publication, September 6, 1995)
From the
Altered sensitivity of topoisomerase II to anticancer drugs
profoundly affects the response of eukaryotic cells to these agents.
Therefore, several approaches were employed to elucidate the mechanism
of drug hypersensitivity of the mutant yeast type II topoisomerase,
top2H1012Y. This mutant, which is 5-fold hypersensitive to
ellipticine, formed DNA cleavage complexes more rapidly than the
wild-type yeast enzyme in the presence of the drug. Conversely, no
change in the rate of DNA religation was observed. There was, however,
a correlation between increased cleavage rates and enhanced drug
binding affinity. The apparent dissociation constant for ellipticine in
the mutant topoisomerase II
drug
DNA ternary complex was
5-fold lower than in the wild-type ternary complex. Furthermore,
the apparent K
value for the mutant
binary (topoisomerase II
drug) complex was
2-fold lower than
the corresponding wild-type complex, indicating that drug
hypersensitivity is intrinsic to the enzyme. These findings strongly
suggest that the enhanced ellipticine binding affinity for
topoisomerase II is the mechanistic basis for drug hypersensitivity of
top2H1012Y.
Topoisomerase II is one of the most important targets currently available for the treatment of human cancers(1, 2, 3, 4, 5) . Drugs targeted to this essential enzyme act by increasing levels of covalent topoisomerase II-cleaved DNA complexes that are normal but fleeting intermediates in the catalytic cycle of the enzyme(1, 2, 3, 4, 5, 6, 7) . Treatment with these agents generates protein-associated breaks in the genome, which triggers a series of events that ultimately culminates in an apoptotic-like cell death(1, 3, 4, 8, 9) .
There is a high degree of variability in the response of different cancers and/or patients to topoisomerase II-targeted drugs(1, 10, 11, 12) . Although drug resistance or hypersensitivity greatly affects the success of cancer chemotherapy, mechanisms that alter drug sensitivity have yet to be fully defined. Several factors contribute to the sensitivity of cells toward agents targeted to the type II enzyme. First, altered rates of drug metabolism, cellular uptake, or efflux often result in resistance to a broad spectrum of agents(1, 8, 13, 14, 15, 16) . Second, mutations in enzymes that recognize or process topoisomerase II-induced lesions often lead to drug resistance, and diminished repair pathways render cells hypersensitive(1, 8, 14, 15, 16, 17, 18, 19, 20, 21) . Third, changes in cellular topoisomerase II content or activity dramatically affect the level of drug cytotoxicity (1, 2, 8, 14, 16, 20, 22-25). Finally, mutations within topoisomerase II that alter drug-induced DNA cleavage have produced a wide variety of phenotypes, ranging from high resistance(2, 3, 20, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) to severalfold hypersensitivity(35, 36) .
Although a
number of mutant type II topoisomerases have been characterized in
vitro(26, 27, 28, 29, 31, 32, 33, 34, 35, 36) ,
the mechanistic basis for altered drug sensitivity of these enzymes has
remained an enigma. It has been widely assumed that drug resistance of
mutants that maintain high rates of catalytic activity is due primarily
to decreased drug binding in the enzymedrug
DNA ternary
complex. While this has been demonstrated for one quinolone-resistant
mutant DNA gyrase (a prokaryotic type II topoisomerase) (gyrA S83W, which has a Ser
Trp mutation at position 83 of its A
subunit)(4, 37) , drug binding studies have never been
reported for any mutant eukaryotic enzyme. The strongest evidence for
decreased drug binding in the eukaryotic ternary complex comes from
dose-response relationships for quinolone-induced DNA cleavage mediated
by a yeast mutant (top2H1012Y, which has a His
Trp mutation at
position 1012) type II topoisomerase in which the potency of the drug
was diminished(35) .
The basis of drug hypersensitivity is even less well understood than resistance. To date, only two hypersensitive mutant type II topoisomerases have been described in eukaryotes, both of which are from yeast: top2H1012Y (hypersensitive to ellipticine) (35) and top2S741W (hypersensitive to etoposide)(36) . Although the mechanistic basis of altered drug sensitivity has not been defined for either enzyme, hypersensitivity of the latter mutant correlated with a decreased rate of DNA religation in the presence of etoposide(36) .
To elucidate a mechanism of
drug hypersensitivity, the interactions of yeast top2H1012Y with
ellipticine were characterized. The mutant type II enzyme established
drug-induced enzyme-DNA cleavage complexes more rapidly than wild-type
topoisomerase II, but religated DNA at the same rate. As determined by
steady-state and frequency domain fluorescence spectroscopy, the
apparent affinity of ellipticine for topoisomerase II in the mutant
ternary complex was 5-fold higher (comparable to the level of
hypersensitivity; (35) ) than the wild-type complex. In
addition, the affinity of top2H1012Y for ellipticine in the absence of
DNA was
2-fold higher than that of the wild-type enzyme. These
results suggest a mechanistic basis for hypersensitivity of
topoisomerase II-targeted drugs and indicate that interactions between
ellipticine and the enzyme are representative of drug action in the
ternary complex.
The Saccharomyces
cerevisiae yeast strains employed were JN394T2-4, which possesses
the top2-4 temperature-sensitive mutant allele instead of the
wild-type topoisomerase II gene, and has the genotype: ura3-52, leu2, trp1, his7, ade1-2, ISE2, rad52::LEU2(24) ; and
JEL1, which has the genotype: leu2, trp1, ura3-52, prb1-1122, pep4-3, his3::PGAL10-GAL4(43) .
Figure 1: Time course for the formation of topoisomerase II-DNA cleavage complexes. DNA cleavage assays were carried out as described under ``Experimental Procedures.'' Assays that examined the wild-type (closed circles) or mutant top2H1012Y (open circles) enzymes contained 10 µM or 4 µM ellipticine, respectively. The inset shows assays that contained 10 µM ellipticine for both enzymes. All data shown are the averages of three independent experiments and standard deviations are represented by the error bars.
As seen in Fig. 1(inset), at equal concentrations of ellipticine (10 µM), top2H1012Y not only accumulated higher levels of DNA cleavage complexes but did so more rapidly than wild-type topoisomerase II. Even when cleavage levels for the two enzymes were normalized by decreasing the drug concentration to 4 µM in assays that employed the mutant enzyme, the rate of cleavage complex formation was severalfold faster for top2H1012Y than the wild-type enzyme (Fig. 1).
The enhanced rate of DNA cleavage complex formation observed with top2H1012Y may result from a number of possibilities. One possibility is that the mutant enzyme utilizes a broader spectrum of DNA cleavage sites than wild-type topoisomerase II. However, this does not appear to be the case. As determined by cleavage mapping experiments, the sites at which the two enzymes incised DNA were nearly identical (not shown). Two other possibilities to explain the enhanced rate of cleavage complex formation exist; 1) top2H1012Y may religate DNA more slowly, and/or 2) it may display a higher affinity for ellipticine than the wild-type enzyme. Experiments were performed to examine both of these alternatives.
Figure 2: DNA religation mediated by wild-type topoisomerase II or top2H1012Y. DNA religation assays were carried out as described under ``Experimental Procedures.'' Assays that examined the wild-type (closed circles) or mutant top2H1012Y (open circles) enzymes contained 10 µM or 4 µM ellipticine, respectively, in order to normalize initial levels of cleavage. At time zero, levels of cleavage were set to 100%. Data are the averages of three independent experiments.
Figure 3: Ellipticine binding to top2H1012Y or the wild-type enzyme in the ternary complex. Binding assays were carried out as outlined under ``Experimental Procedures.'' Interactions of ellipticine with the wild-type (closed circles) or mutant enzyme (open circles) were quantified by changes in fluorescence intensity of the deprotonated drug. Data are representative of three independent experiments and were analyzed by double-reciprocal plots (1/change in fluorescence intensity versus 1/enzyme concentration). Lines were fit by best-fit linear regression. Data for the wild-type enzyme are from Froelich-Ammon et al.(47) .
Figure 4: Ellipticine binding to top2H1012Y and the wild-type enzyme in the binary complex. Binding assays were carried out as outlined under ``Experimental Procedures.'' Interactions of ellipticine with the wild-type (closed circles) or mutant enzyme (open circles) were quantified by changes in fluorescence intensity of the deprotonated drug. Data are representative of three independent experiments and were analyzed by double-reciprocal plots (1/change in fluorescence intensity versus 1/enzyme concentration). Lines were fit by best-fit linear regression. Data for the wild-type enzyme are from Froelich-Ammon et al.(47) .
To examine interactions within the binary and ternary
complex in greater detail, drugenzyme binding was analyzed by
frequency domain fluorescence spectroscopy (Table 1). The
fluorescence lifetime of free deprotonated ellipticine increased
dramatically from
60 ps to
24 ns upon binding to the
wild-type enzyme in the absence of DNA(47) . A similar increase
was observed following the formation of the wild-type ternary
complex(47) . In contrast, the lifetime of deprotonated
ellipticine rose to
15 ns in the presence of top2H1012Y or upon
formation of the mutant ternary complex. The fact that the lifetimes
for ellipticine bound to the wild-type or mutant enzymes paralleled
those for the respective ternary complexes supports the conclusion that
similar enzyme
drug interactions occur in the binary and ternary
complexes, and further argues for direct interactions between
topoisomerase II and ellipticine.
Little is understood concerning the factors that govern the sensitivity of topoisomerase II to anticancer drugs. Changes in the cellular levels of topoisomerase II often correlate with either drug resistance or hypersensitivity(1, 2, 8, 14, 22, 23, 24, 25) ; however, the mechanism(s) by which mutations within the enzyme contribute to drug sensitivity has not been defined. In an effort to delineate potential mechanisms underlying altered drug sensitivity, the hypersensitivity of top2H1012Y to ellipticine was characterized.
As
determined by steady-state and frequency-based time domain fluorescence
spectroscopy, the affinity of topoisomerase IIdrug binding within
the mutant ternary complex was higher than that of the wild-type
complex. The increase in binding was comparable to the enhanced drug
cytotoxicity of yeast harboring the top2H1012Y gene and the
increased DNA cleavage mediated by the mutant enzyme in
vitro(35) . Thus, it appears that enhanced drug binding in
the top2H1012Y ternary complex is the primary mechanistic basis of
hypersensitivity to ellipticine.
Furthermore, the binding affinity
of ellipticine for top2H1012Y in the absence of DNA was 2-fold
higher than that of the wild-type binary complex. This indicates that
drug hypersensitivity is intrinsic to topoisomerase II and provides
compelling evidence for direct interactions between anticancer drugs
and the enzyme. Moreover, these findings suggest that the
enzyme
ellipticine interactions in the binary complex are
indicative of those in the ternary complex. However, it is likely that
DNA also modulates topoisomerase II
drug interactions as shown by
the greater increase in binding affinity in the ternary complex.
A
difference in fluorescence lifetimes of ellipticine in the wild-type
and mutant binary and ternary complexes was observed, indicating that
the His Tyr mutation alters the environment of bound drug.
Although a three-dimensional structure is not yet available for
topoisomerase II, these findings suggests that aminoacyl residue 1012
is in the vicinity of the ellipticine interaction domain.
Ellipticine does not impair topoisomerase II-mediated DNA religation and presumably increases levels of DNA cleavage complexes primarily by stimulating the forward rate of scission(47) . It is notable that top2H1012Y displayed an increased rate of enzyme-DNA cleavage complex formation as compared to the wild-type enzyme, but had the same rate of DNA religation. Thus, the effects of ellipticine were exacerbated with the hypersensitive mutant, but the drug mechanism remained unchanged. Recently, the mutant type II topoisomerase top2S741W was found to be hypersensitive to etoposide (a drug that acts primarily by inhibiting the rate of religation; (2) )(36) . Consistent with the mechanism of etoposide, a slower rate of DNA religation was observed with top2S741W. Therefore, a recurring theme among hypersensitive mutant type II topoisomerases appears to be the exaggeration of normal drug action. Clearly enhanced drug binding may be the underlying cause for this effect.
In
summary, results of the present study indicate that increased drug
binding affinity may be a common mechanistic basis for the enhanced
activity of anticancer agents toward the type II enzyme and suggest a
correlation between drugenzyme binding and the sensitivity of
cells to topoisomerase II-targeted drugs.