©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Increased Drug Affinity as the Mechanistic Basis for Drug Hypersensitivity of a Mutant Type II Topoisomerase (*)

(Received for publication, September 6, 1995)

Stacie J. Froelich-Ammon (1) D. Andrew Burden (1) Marcia W. Patchan (3) Sarah H. Elsea (1)(§) Richard B. Thompson (3) Neil Osheroff (1) (2)(¶)

From the  (1)Departments of Biochemistry and (2)Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the (3)Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 IIbulletdrugbulletDNA ternary complex was 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent K value for the mutant binary (topoisomerase IIbulletdrug) 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.


INTRODUCTION

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 enzymebulletdrugbulletDNA 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.


EXPERIMENTAL PROCEDURES

Materials and Yeast Strains

A 40-mer oligonucleotide (corresponding to nucleotides 87-126 of pBR322; (38) ) containing a topoisomerase II cleavage site (39, 40) was synthesized and purified as outlined by Froelich-Ammon et al.(41) . Oligonucleotides were diluted to the appropriate concentration in 5 mM Tris-HCl, pH 7.4, 0.5 mM EDTA. Negatively supercoiled pBR322 DNA was prepared as formerly described(42) . Ellipticine was prepared as a 20 mM or 10 mM stock dissolved in dimethyl sulfoxide or ethanol, respectively, and stored at -20 °C. Ellipticine, ethidium bromide, and Tris-HCl were obtained from Sigma; UltraPure HEPES was from VWR; SDS was purchased from E. Merck Biochemicals; proteinase K was from U. S. Biochemical Corp.; yeast nitrogen base, yeast extract, and Bacto-agar were from Difco; Rose Bengal was from Aldrich; and dimethyl-POPOP was from Eastman. All other chemicals were analytical reagent grade.

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, Deltahis3::PGAL10-GAL4(43) .

Mutant Selection and Purification

The H1012Y mutation was introduced into the yeast TOP2 gene by hydroxylamine-induced in vitro mutagenesis (44) and selected for resistance to quinolones as described by Elsea et al.(35) . top2H1012Y displays resistance to CP-115,953 and etoposide, wild-type sensitivity to amsacrine, and 5-fold hypersensitivity to ellipticine(35) . Overexpression for purification of the wild-type and mutant type II enzymes was achieved using the plasmid YEpGAL1TOP2(45) . Mutant and wild-type enzymes were purified to apparent homogeneity (as determined by Coomassie or silver-stained polyacrylamide gels) using the protocol of Worland and Wang (45) as modified by Elsea et al.(35) .

Wild-type Topoisomerase II- and top2H1012Y-mediated DNA Cleavage

Assays were performed by a modification of the protocol of Robinson and Osheroff(46) . Cleavage reactions contained 100 nM wild-type yeast topoisomerase II or top2H1012Y, 5 nM negatively supercoiled pBR322 DNA, and the appropriate drug concentration in a total of 20 µl of reaction buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 5 mM MgCl(2), 0.1 mM EDTA, and 2.5% glycerol). Reaction mixtures were incubated at 28 °C for the designated times. Products were resolved by gel electrophoresis on ethidium bromide-containing 1% agarose gels (46) and DNA bands were visualized by transillumination with UV light (300 nm) and quantitated with an Alpha Innotech IS1000 imaging system. The density of the bands was proportional to the amount of DNA present. Control samples contained an equal amount of drug diluent. Drug-induced DNA cleavage was not seen in the absence of topoisomerase II.

Wild-type Topoisomerase II- and top2H1012Y-mediated DNA Religation

Assays were performed by a modification of the protocol of Robinson et al.(46) . Reactions contained 5 nM negatively supercoiled pBR322 DNA, 100 nM yeast topoisomerase II, and 10 µM ellipticine in a total of 20 µl of reaction buffer. DNA cleavage/religation equilibria were established by incubation at 28 °C for 6 min. 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 at various time points followed by 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 and frequency domain fluorescence spectroscopy were performed as outlined previously(47) . For all fluorescence experiments, samples were excited at 326 nm and emission light was monitored at 420 nm. The emission polarizer was fixed at the magic angle (54.7°), and a 420 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)(48) , were used as references. Data were acquired at 25 °C. Samples contained 10 µM ellipticine, the designated concentrations of enzyme, and 200 nM 40-mer oligonucleotide (when DNA was present) in a final volume of 500 µl of 20 mM HEPES, pH 7.9, 100 mM NaCl, 5 mM MgCl(2), and 0.1 mM EDTA and were incubated for 6 min prior to fluorescence measurements. All chemicals were ultrapure grade to minimize nonspecific fluorescence. The buffer background intensity was subtracted for binding calculations.


RESULTS

Rate of Topoisomerase II-DNA Cleavage Complex Formation

The mutant type II topoisomerase, top2H1012Y, is hypersensitive to ellipticine as determined by the enhancement of DNA cleavage (Fig. 1, inset)(35) . Yeast cells that harbor top2H1012Y (and lack wild-type enzyme activity) display a similar degree of hypersensitivity toward this drug(35) , underscoring the relationship between drugbulletenzyme interactions in vitro and cellular phenotype. Therefore, as a first step toward defining the mechanistic basis of enhanced ellipticine sensitivity of top2H1012Y, the rate of formation of drug-induced topoisomerase II-DNA cleavage complexes was determined for the wild-type and mutant enzymes.


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.

Topoisomerase II-mediated DNA Religation

Ellipticine does not affect the rate of DNA religation mediated by wild-type topoisomerase II(47) . Therefore, the drug apparently increases the level of DNA scission primarily by enhancing the forward rate of cleavage. It may be that the mechanism of ellipticine action on the mutant enzyme differs and that the hypersensitivity of top2H1012Y is due (at least in part) to an additional effect on DNA religation. As determined by DNA religation assays, this is not the case. The apparent first order rate of religation mediated by top2H1012Y was identical to that of the wild-type enzyme when ellipticine concentrations were equal (not shown) or corrected to normalize cleavage levels (Fig. 2).


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.



Ellipticine Binding to top2H1012Y in the Ternary Enzymebullet DNAbulletDrug Complex

Recently, the binding affinities of ellipticine for DNA, yeast wild-type topoisomerase II, and the ternary complex were characterized by steady-state and frequency domain fluorescence spectroscopy(47) . Therefore, these techniques were utilized to ascertain whether the drug hypersensitivity of top2H1012Y correlates with an increased binding affinity of ellipticine for topoisomerase II in the ternary complex. The apparent K(D) value was calculated from changes in fluorescence intensity of ellipticine in the presence of DNA with increasing concentrations of mutant topoisomerase II. As determined by double-reciprocal analysis (Fig. 3), the apparent dissociation constant of ellipticine for top2H1012Y was 310 nM (Table 1). This K(D) value is 5-fold lower than that previously reported (47) for the wild-type ternary complex (1.5 µM) (Table 1, Fig. 3). The increase in binding affinity of ellipticine for topoisomerase II in the ternary complex is comparable to the level of drug hypersensitivity of top2H1012Y observed in vivo and in vitro(35) . It should be emphasized that the lower dissociation constant of top2H1012Y in the ternary complex is not due to enhanced DNA binding by the mutant enzyme; indeed, the affinity of top2H1012Y for DNA is lower than that of the wild-type enzyme(35) .


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) .





Ellipticine Binding to top2H1012Y

While protonated ellipticine binds to DNA, it is the deprotonated form of the drug that binds to topoisomerase II and is present in the ternary complex(47) . On this basis, it has been suggested that ellipticine has contacts with topoisomerase II in the ternary complex and that the interactions between the enzyme and the drug in the absence of DNA (i.e. binary complex) may reflect those occurring within the topoisomerase IIbulletdrugbulletDNA complex. To investigate whether alterations that lead to hypersensitivity are intrinsic to the enzyme (in the absence of DNA), the binding of ellipticine to top2H1012Y was characterized (Fig. 4). The apparent K(D) value for the mutant enzyme (90 nM) was 2-fold lower than that of wild-type topoisomerase II (160 nM) (Table 1)(47) . The enhanced binding of ellipticine to top2H1012Y provides strong evidence that interactions within the binary complex mimic those observed for the ternary complex and contribute to the hypersensitivity of this mutant enzyme.


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, drugbulletenzyme 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 enzymebulletdrug interactions occur in the binary and ternary complexes, and further argues for direct interactions between topoisomerase II and ellipticine.


DISCUSSION

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 IIbulletdrug 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 enzymebulletellipticine interactions in the binary complex are indicative of those in the ternary complex. However, it is likely that DNA also modulates topoisomerase IIbulletdrug 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 drugbulletenzyme binding and the sensitivity of cells to topoisomerase II-targeted drugs.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM33944 (to N. O.), by American Cancer Society Research Grant NP-812 (to N. O.) and Faculty Research Award FRA-370 (to N. O.), by Office of Naval Research Contract N00014-91-J-1572 (to R. B. T.), and by a traineeship (to D. A. B.) under National Institutes of Health Grant 5 T32 CA 09582. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Neurology, Baylor College of Medicine, Houston, TX 77030.

To whom reprint requests should be addressed: Dept. of Biochemistry, 654 Medical Research Bldg. I, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-4338; Fax: 615-343-1166; osheron@ctrvax.vanderbilt.edu.


ACKNOWLEDGEMENTS

We are grateful to Jo Ann Byl, Paul S. Kingma, and John M. Fortune for critical reading of the manuscript.


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