©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Yeast Type II Topoisomerase Selected for Resistance to Quinolones
MUTATION OF HISTIDINE 1012 TO TYROSINE CONFERS RESISTANCE TO NONINTERCALATIVE DRUGS BUT HYPERSENSITIVITY TO ELLIPTICINE (*)

(Received for publication, July 27, 1994; and in revised form, October 12, 1994)

Sarah H. Elsea (1)(§) Yuchu Hsiung (3) John L. Nitiss (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 Biochemistry, University of Southern California and Division of Hematology/Oncology, Children's Hospital of Los Angeles, Los Angeles, California 90027

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A mutant yeast type II topoisomerase was generated by in vitro mutagenesis followed by selection in vivo for resistance to the quinolone CP-115,953. The resulting mutant enzyme had a single point mutation which converted His to Tyr (top2H1012Y). top2H1012Y was overexpressed in yeast, purified, and characterized in vitro. The mutant type II topoisomerase was slightly less active than the wild type enzyme, apparently due to a decreased affinity for DNA. The affinity of the mutant enzyme for ATP was similar to that of wild type topoisomerase II. As determined by DNA cleavage assays, top2H1012Y was resistant to CP-115,953 and etoposide both prior to and following the DNA strand-passage event. In marked contrast, the mutant enzyme displayed wild type sensitivity to amsacrine and was severalfold hypersensitive to ellipticine. A similar pattern of resistance was observed in yeast cells harboring the top2H1012Y allele. Thus, it appears that the mutant type II topoisomerase can distinguish between nonintercalative and intercalative agents. Finally, the His Tyr mutation defines a potential new drug resistance-conferring region on eukaryotic topoisomerase II.


INTRODUCTION

Topoisomerase II is required for a number of fundamental nuclear processes, including DNA replication, recombination, and chromosome structure/segregation(1, 2, 3) . In addition to its pivotal physiological roles, this enzyme is one of the most important targets for the treatment of human cancers(4, 5, 6, 7, 8) . Chemotherapeutic agents targeted to topoisomerase II are derived from several drug classes. However, despite the structural diversity of these drugs, they share a common basis for cytotoxic action. Their chemotherapeutic potential correlates with the ability to stabilize covalent topoisomerase II-cleaved DNA complexes that are fleeting intermediates in the catalytic cycle of the enzyme(4, 7, 8) . As a consequence of this action, cells that are treated with topoisomerase II-targeted agents contain high levels of transient protein-associated breaks in their genome(9, 10, 11, 12, 13, 14, 15) . These transient lesions, when traversed by DNA replication complexes, are converted to permanent breaks, which subsequently trigger a chain of events that ultimately leads to cell death(4, 16, 17, 18) .

Drugs appear to stabilize topoisomerase II-DNA cleavage complexes by two distinct mechanisms(8) . Etoposide and amsacrine have been shown to act primarily by impairing the ability of topoisomerase II to religate cleaved nucleic acids(19, 20, 21, 22) . In marked contrast, quinolones, nitroimidazoles, pyrimidobenzimidazoles, and genistein show little inhibition of enzyme-mediated DNA religation and appear to work by enhancing the forward rate of DNA cleavage(8, 22, 23, 24, 25) .

Even though topoisomerase II-targeted drugs play a critical role in cancer chemotherapy, their interactions with the enzyme-DNA complex are not well understood. Two approaches have been utilized in an effort to delineate interaction domains on topoisomerase II for these agents. First, genetic studies have identified a number of mutations in the enzyme that confer resistance to antineoplastic drugs(7, 8) . These mutations have defined two regions in topoisomerase II that appear to be important for drug-enzyme interactions. One is located in the gyrB homology domain near the consensus ATP binding sequence (at residues 461-466, based on the amino acid sequence of yeast topoisomerase II)(26, 27, 28, 29, 30) . The other, which is broader, is located in the gyrA homology domain and spans approximately 200 amino acids flanking the active site tyrosine residue (Tyr in yeast)(31, 32, 33, 34) . However, drug resistance profiles of individual mutants are often very different from one another. This finding makes it difficult to draw conclusions concerning the potential microenvironment of drug binding within these regions or even to conclude whether drugs share a common site on the enzyme. Therefore, to further characterize drug interactions with topoisomerase II, a second biochemical approach based on drug competition experiments has been employed(35, 36) . Results of this latter approach indicate that a number of antineoplastic agents, including etoposide, amsacrine, genistein, and the quinolone CP-115,953, share a common site of interaction on topoisomerase II. Taken together with the results of mutagenesis studies, this finding strongly suggests that the interaction domains for most (if not all) DNA cleavage-enhancing agents overlap one another, but that the specific points of contact on the enzyme probably differ between drug classes.

All drug resistance-conferring mutations previously described in topoisomerase II have been selected against agents that enhance DNA breakage primarily by inhibiting enzyme-mediated DNA religation. Little information exists concerning residues selected for resistance to drugs that act primarily by increasing the forward rate of DNA cleavage. Therefore, to broaden our understanding of drug-topoisomerase II interactions, the present study utilized random mutagenesis to select for type II enzymes that are resistant to the quinolone CP-115,953. A point mutation in yeast topoisomerase II was isolated that converts His to Tyr. top2H1012Y displays high catalytic activity in vitro and supports rapid rates of cell growth. Furthermore, this mutant shows high resistance, both in vitro and in vivo, to quinolones and to etoposide but displays wild type sensitivity to amsacrine. Finally, top2H1012Y is severalfold hypersensitive to ellipticine, and this mutation defines a new C-terminal region on the enzyme that may be involved in drug interactions.


EXPERIMENTAL PROCEDURES

Materials

Negatively supercoiled bacterial plasmid pBR322 DNA was prepared as described previously(37) . Amsacrine, etoposide, ellipticine, and CP-115,953 were dissolved as 20 mM solutions in dimethyl sulfoxide and stored at 4 °C. CP-115,953 was kindly provided by Drs. T. D. Gootz and P. R. McGuirk (Pfizer Central Research), and amsacrine was obtained from Bristol Myers. Ellipticine, etoposide, Tris, App(NH)p, (^1)and ethidium bromide were obtained from Sigma; SDS was from E. Merck Biochemicals; proteinase K and Sequenase were from United States Biochemical Corp.; ATP was from Pharmacia Biotech; yeast nitrogen base, yeast extract, and Bacto-agar were from Difco; and restriction endonucleases and T4 DNA ligase were from New England BioLabs; Sequagel-6 was obtained from National Diagnostics; and [-P]ATP (3000 Ci/mmol) was from Amersham Corp. All other chemicals were analytical reagent grade.

Yeast Strains and Plasmids

The yeast strains employed were Saccharomyces cerevisiae JN394t2-4, whose genotype is ura3-52, leu2, trp1, his7, ade1-2, ISE2, rad52::LEU2 and carries the top2-4 mutant topoisomerase II allele in place of the wild type topoisomerase II gene (38) , and JEL1, whose genotype is leu2, trp1, ura3-52, prb1-1122, pep4-3, Deltahis3::PGAL10-GAL4(39) . The mutagenized plasmid carrying topoisomerase II was YCpDED1TOP2, which carries the yeast TOP2 gene under the control of the DED1 promoter(38) . Wild type and mutant type II topoisomerases were purified using the inducible overexpression plasmid YEpGAL1TOP2(40) .

In Vitro Mutagenesis

Mutations were introduced into the yeast TOP2 gene by hydroxylamine-induced in vitro mutagenesis(41) . Briefly, 10 µg of YCpDED1TOP2 was treated with 0.1 M hydroxylamine in 0.25 M potassium phosphate (pH 6.0), 5 mM EDTA for 1 h at 75 °C. Following treatment with hydroxylamine, the plasmid was dialyzed extensively against 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, precipitated with ethanol, and transformed into Escherichia coli strain XL-1 Blue. Transformants were pooled and grown for 10 h in Terrific Broth medium(41) . Plasmid DNA was purified by alkaline lysis, followed by banding in CsCl gradients containing ethidium bromide(37) .

Mutant Selection

Mutants were selected as described previously by Nitiss and co-workers(32) . The mutagenized pool of YCpDED1TOP2 was transformed into JN394t2-4. A total of about 20,000 individual yeast transformants were pooled, and a portion of the pool was suspended in SC-URA medium at a concentration of 2 times 10^6 cells/ml. CP-115,953 was added to a final concentration of 20 µM, and the cells were incubated for 48 h at 34 °C. Finally, cells were diluted and plated on SC-URA plates. Approximately 1000 colonies that grew on SC-URA were replica-plated on YPDA plates containing 5 µM CP-115,953, 20 µM CP-115,953, 170 µM etoposide, or 250 µM amsacrine to determine initial drug resistance phenotypes. Single colonies were selected, and cytotoxicity assays were performed as described below.

Yeast Cytotoxicity Assays

The sensitivity of yeast strain JN394t2-4 carrying wild type or mutagenized YCpDED1TOP2 to CP-115,953, etoposide, amsacrine, or ellipticine was determined as described previously(42) . Cells were cultured in YPDA or SC-URA selection medium at 34 °C. Following adjustment of logarithmically growing cultures to a titer of 2 times 10^6 cells/ml, drug (0-200 µM) was added to the medium, and cultures were incubated for 24 h. Initial phenotypes were established by following the absorbance of cultures at 600 nm. Transformants with phenotypes of interest subsequently were diluted with water and plated in duplicate on YPDA medium solidified with 1.5% Bacto-agar. Plates were incubated at 34 °C for 3-4 days, and drug sensitivity was quantitated by counting the number of surviving colonies.

Yeast Growth and Transformation

Yeast cells typically were grown in rich medium (YPDA) or, to select for plasmids carrying URA3 as a marker, in synthetic complete medium lacking uracil (SC-URA). Yeast transformation was carried out using the modified lithium acetate protocol of Schiestl and Gietz(43) .

Recovery of Plasmids Carrying Topoisomerase II Mutations

Plasmids carrying the mutant alleles were recovered as described previously(44) . In summary, cells from a 10-ml saturated culture growing in SC-URA were lysed using glass beads. After extraction with phenol, phenol/CHCl(3), and CHCl(3), total nucleic acids were precipitated with ethanol. Nucleic acids were resuspended in TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) containing ribonuclease A and precipitated again with ethanol. The DNA was transformed into E. coli strain XL-1 Blue. Plasmid DNA was purified using Qiagen plasmid kits (Qiagen).

Construction of Plasmids for Sequencing and Overexpression

Mutagenized YCpDED1TOP2 was digested with restriction endonucleases KpnI and AvrII for 1 h at 37 °C. DNA fragments were subjected to electrophoresis on 1% agarose (MCB) gels in TBE (100 mM Tris borate, pH 8.3, 2 mM EDTA) containing 1 µg/ml ethidium bromide, and the 2.2-kb fragment (containing the coding sequence for amino acids 317-1045 in yeast topoisomerase II) was gel-purified using DE81 ion exchange paper. To ensure no cross-contamination, this mutagenized fragment was first subcloned into pSL1180 (Pharmacia Biotech), which had been cut with AvrII and KpnI and gel-purified. Following propagation in E. coli XL-1 Blue (Stratagene), this new plasmid was digested with KpnI and AvrII, and the resulting 2.2-kb fragment was gel-purified as above. In addition, the wild type plasmids YCpDED1TOP2 and YEpGAL1TOP2 were digested with KpnI and AvrII, and the large fragments were gel-purified. The mutagenized 2.2-kb fragment was then ligated into (wild type) YCpDED1TOP2 and YEpGAL1TOP2, replacing the wild type fragments, and subsequently transformed into E. coli XL-1 Blue. The YCpDED1TOP2 DNA was then used to transform the yeast strain JN394t2-4 for cytotoxicity studies, and the YEpGAL1TOP2 DNA was used to transform the yeast strain JEL1 for overexpression and purification of topoisomerase II.

DNA Sequencing

The DNA sequence of the mutant allele was determined by the dideoxynucleotide chain termination technique (45) with double-stranded YCpDED1TOP2 DNA templates using Sequenase. Ten 17-mer oligonucleotides (corresponding to the coding sequence) that spanned the 2.2-kb cassette described above were employed as primers. In all cases, the wild type gene was sequenced for comparison. The point mutation in top2H1012Y was confirmed by sequencing the noncoding strand.

Induction and Overexpression

A modification of the protocol of Worland and Wang (40) was used. Five-ml cultures (in SC-URA) of yeast strain JEL 1 (transformed with either wild type or mutant YEpGAL1TOP2) were grown overnight to saturation. Approximately 200 µl of saturated culture was added to 20 ml of induction media (synthetic complete medium without glucose, supplemented with 3% glycerol and 2% lactic acid) plus 2% glucose and grown overnight with vigorous shaking to an OD of 1.5-2.5. Cultures were diluted 1:100 in induction media and grown to an OD of 0.7. Galactose was added to 2% final concentration, and cells were grown for an additional 12-18 h to an OD of 0.8-1.2. Cells were harvested by centrifugation at 6000 rpm in a JA10 rotor for 15 min and washed with deionized water followed by harvest buffer (50 mM Tris-HCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 25 mM NaF, 1 mM Na(2)S(2)O(5), 1 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). Finally, cells were resuspended in 2 ml of harvest buffer per g of wet-packed cells, quick frozen in dry ice/ethanol, and stored at -80 °C until use.

Purification

Wild type and mutant enzymes were purified from 20-30 g of frozen wet-packed cells (obtained from 5-liter cultures). Prior to column chromatography, the purification scheme was based on the protocol of Worland and Wang(40) . All purification steps were carried out at 4 °C. Cells were disrupted on ice in Buffer I (50 mM Tris-HCl, pH 7.7, 1 mM EGTA, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM beta-mercaptoethanol, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin) using acid-washed glass beads in a Bead Beater (Biospec). Cell debris was removed by centrifugation for 15 min at 15,000 rpm in a JA20 rotor. Lysates were diluted to 5 mg/ml protein with Buffer I plus 25 mM KCl (typical lysates contained approximately 2 g of total protein). Nucleic acids and protein-nucleic acid complexes were precipitated by the slow addition of Polymin P (Life Technologies, Inc.) to a final concentration of 2%, followed by stirring for 30 min. Samples were subjected to centrifugation for 10 min at 9800 rpm in a JA20 rotor. Pellets were washed with 60 ml of Buffer I plus 150 mM KCl, stirred for 10 min, and centrifuged as above. The washed pellets were extracted twice by stirring for 15 min in 60 ml of Buffer I plus 750 mM KCl, followed by centrifugation as above. Supernatants were combined, brought to 35% saturation with the addition of powdered NH(4)(SO(4))(2), and stirred for 30 min. Following centrifugation for 25 min at 13,500 rpm in a JA14 rotor, the supernatant was brought to 65% saturation with NH(4)(SO(4))(2) and stirred for 30 min. Topoisomerase II was pelleted by centrifugation for 25 min at 13,500 rpm in a JA14 rotor. The pellet was resuspended in Buffer I to a conductivity that approximated that of the column buffer (10 mM Tris-HCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.5 mM dithiothreitol) containing 250 mM KCl.

Column chromatography was carried out by a modification of the protocol of Shelton et al.(12) . The sample was applied to a 10-ml phosphocellulose (P81, Whatman) column. The column was washed with 3 column volumes of column buffer containing 250 mM KCl and 0.1 mM phenylmethylsulfonyl fluoride. Topoisomerase II was eluted over a linear 10-column volume gradient with column buffer containing 250 mM KCl to 1 M KCl. Topoisomerase II eluted at 600 mM KCl, as monitored by PhastSystem (Pharmacia Biotech) protein gel electrophoresis on 7.5% homogenous media PhastGels stained with Coomassie Blue. Fractions containing topoisomerase II were pooled and diluted with Buffer I to a conductivity equal to that of the column buffer plus 250 mM KCl. Topoisomerase II was applied to a 2-ml phosphocellulose collection column, and the column was washed with 5 column volumes of column buffer plus 250 mM KCl. Topoisomerase II was eluted with 3 column volumes of column buffer containing 750 mM KCl and 30% glycerol. Fractions were assayed for protein concentration with the Bio-Rad reagent (Bio-Rad), using bovine serum albumin as a standard. Fractions containing topoisomerase II were pooled, aliquoted, and stored in liquid nitrogen. Typical yields were in excess of 0.3 mg/g of wet-packed cells.

Topoisomerase II-mediated DNA Relaxation

DNA relaxation assays were carried out as described by Osheroff et al. (46) . Reaction mixtures contained 0.65-22 nM yeast topoisomerase II, 5 nM supercoiled pBR322, and 0.5 mM ATP in a total volume of 20 µl of assay buffer (10 mM Tris-HCl, pH 7.7, 10 mM MgCl(2), 100 µM EDTA, and 2.5% glycerol) that contained 175 mM KCl. DNA relaxation was carried out at 30 °C for 15 min. Reactions were stopped by the addition of 3 µl of 0.77% SDS, 77 mM EDTA. Samples were mixed with 2 µl of loading buffer (60% sucrose, 0.05% bromphenol blue, 0.05% xylene cyanol FF, 10 mM Tris-HCl (pH 7.9)), heated for 2 min at 70 °C, and subjected to electrophoresis on 1% agarose gels in TBE. 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 was quantitated by scanning negatives with an E-C Apparatus model EC910 scanning densitometer using Hoefer GS-370 Data System software. Under the conditions employed, the intensity of bands in the negative was proportional to the amount of DNA present.

Binding of Topoisomerase II to DNA

Topoisomerase IIbulletDNA binding was monitored by electrophoretic mobility shift assays(47) . Reaction mixtures contained 25-250 nM wild type or mutant topoisomerase II and 5 nM negatively supercoiled pBR322 DNA in a total of 20 µl of assay buffer that contained 175 mM KCl. Reactions were incubated at 30 °C for 6 min and were terminated by the addition of 2 µl of loading buffer. Samples were subjected to electrophoresis on 1% agarose gels in TBE containing 1 µg/ml ethidium bromide. DNA in gels was visualized as described above.

Topoisomerase II-mediated DNA Cleavage

DNA cleavage assays were carried out as described by Osheroff and Zechiedrich(48) . Reaction mixtures contained 110 nM (wild type) or 140 nM (mutant) topoisomerase II and 5 nM negatively supercoiled pBR322 DNA in a total of 20 µl of assay buffer containing 50 mM NaCl. Reactions that monitored the DNA cleavage/religation equilibria established prior to the strand-passage event of the enzyme contained no ATP analog, while reactions that monitored the DNA cleavage/religation equilibrium established after strand-passage contained 0.5 mM App(NH)p. DNA cleavage/religation equilibria were established by incubating samples at 30 °C for 6 min. Cleavage products were trapped by the addition of 2 µl of 5% SDS, followed by the addition of 1 µl of 250 mM EDTA and 2 µl of an 0.8 mg/ml solution of proteinase K. Samples were incubated at 45 °C for 30 min to digest topoisomerase II. Final products were mixed with 2 µl of loading buffer, heated at 70 °C for 2 min, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris acetate (pH 8.3), 2 mM EDTA containing 1 µg/ml ethidium bromide. The effects of drugs were examined over a concentration range of 0-1000 µM. An amount of diluent equal to that in drug-containing samples was added to all control samples. No drug-induced DNA cleavage was observed in the absence of topoisomerase II. DNA in the gels was visualized and quantitated as described above.

Hydrolysis of ATP by Topoisomerase II

ATPase assays were carried out as described by Osheroff et al.(46) . Reactions included 1 or 10 units of topoisomerase II (1 unit is equivalent to the amount of enzyme required to relax 5 nM supercoiled pBR322 DNA in 15 min at 30 °C) and 75 nM negatively supercoiled pBR322 plasmid DNA in 20 µl of assay buffer containing 175 mM KCl and 0.5 mM [-P]ATP (3 µCi/reaction). Mixtures were incubated at 30 °C. Samples were removed at various time points up to 20 min, spotted onto thin layer cellulose plates impregnated with polyethyleneimine (Polygram CEL 300 PEI, Brinkmann), and resolved by chromatography in freshly made 400 mM NH(4)HCO(3). Reaction products were visualized by autoradiography with Reflection film (DuPont NEN). Radioactive areas corresponding to inorganic phosphate released by ATP hydrolysis were cut out of the chromatograms and quantitated by liquid scintillation counting. Ten ml of Ecolume aqueous counting scintillant were added, and radioactivity was determined using a Beckman LS-7500 liquid scintillation counter.


RESULTS

Little is understood concerning the residues of topoisomerase II that interact with antineoplastic drugs in the ternary complex. The only available evidence comes from genetic studies that mapped drug resistance-conferring mutations(7, 8) . However, to date, only a limited number of mutant type II topoisomerases have been purified and characterized in vitro(31, 49, 50) . Thus, in most cases, drug resistance with the purified enzyme has not been confirmed. Moreover, in virtually every case, mutants were selected against demethylepipodophyllotoxins (etoposide or teniposide) or amsacrine(26, 30, 31, 32, 50, 51, 52, 53) , both of which enhance DNA breakage primarily by inhibiting DNA religation(19, 20, 21, 22) . As yet, no drug resistance-conferring mutations that were selected against agents that act by stimulating the forward rate of DNA cleavage have been identified. Therefore, to extend genetic studies to this latter mechanistic class and to further define the potential interaction domain for antineoplastic drugs on topoisomerase II, mutants were selected for resistance to the quinolone CP-115,953.

Selection of a Mutant Yeast Type II Topoisomerase with Resistance to the Quinolone CP-115,953

Quinolone-resistant type II topoisomerases were selected using the yeast genetic system of Nitiss and co-workers(32, 38) . This system takes advantage of a yeast strain (JN394t2-4) that carries the ISE2 permeability mutation (that greatly enhances drug uptake), the rad52 double-stranded DNA repair mutation (that renders cells hypersensitive to DNA damaging agents), and the top2-4 temperature-sensitive topoisomerase II allele (that is inactive at the nonpermissive temperature of 34 °C)(32, 38, 42, 54, 55, 56) .

Briefly, a plasmid-encoded copy of the yeast topoisomerase II gene under control of the pDED1 constitutive promoter was mutagenized in vitro with hydroxylamine(57) . Following amplification in E. coli, the mutagenized plasmid was transformed into JN394t2-4 and selected in 20 µM CP-115,953 at 34 °C. Since the chromosomal copy of the gene is inactive at 34 °C, growth at this temperature requires the presence of an active plasmid-encoded copy of topoisomerase II. Individual transformants were picked, and resistance toward a variety of antineoplastic agents was determined by cytotoxicity assays. A single colony that displayed high resistance to CP-115,953 but differential sensitivity to other drug classes was chosen for further study. The mutagenized plasmid was isolated and retransformed into JN394t2-4. The drug resistance profile of the retransformed yeast was similar to that of the original colony, indicating that the phenotype was plasmid-based.

In an attempt to localize the drug resistance-conferring mutation(s) to a specific region of the topoisomerase II gene, a 2.2-kb cassette (which encompassed the nucleotides encoding amino acids 317 through 1045) was excised and subcloned. This cassette was used to replace a corresponding cassette in the wild type gene either under the control of the DED1 promoter (for subsequent cytotoxicity studies) or under the control of the GAL1 promoter (for subsequent overexpression and purification of the mutant enzyme). The KpnI-AvrII cassette that was chosen includes the active site tyrosine and spans all previously described resistance-conferring mutations in topoisomerase II(8) . The resulting chimeric TOP2 constructs exhibited the same phenotype as did the original isolate (see Fig. 3) and were used for all further experiments.


Figure 3: Drug resistance profile of yeast cells carrying the mutant top2H1012Y or wild type TOP2 allele. The effects of CP-115,953 (circles), etoposide (squares), amsacrine (diamonds), or ellipticine (triangles) on the survival of cells carrying either wild type (WT, open symbols) or mutant (H1012Y, closed symbols) topoisomerase II are shown. Data are plotted as percent relative cell survival after 24-h exposure to drug versus drug concentration. The number of cells at time = 0 was set to 100%. Over the course of a 24-h experiment, cell populations routinely increased from 100% to 2000% in the absence of drug. Results are the averages of 2-5 independent experiments.



DNA Sequence of Chimeric TOP2 Constructs

To localize the base change(s) that led to the resistance phenotype of the mutant type II topoisomerase, the DNA sequence of the 2.2-kb cassette under the control of the DED1 promoter was determined. A single point mutation was found that converted C to T at base position 3034 (Fig. 1). This is consistent with hydroxylamine mutagenesis, which produces G to A or C to T transitions(57) . The resulting mutation in the TOP2 gene converts His to Tyr. This mutant enzyme henceforth will be referred to as top2H1012Y. As seen in Fig. 2, residue 1012 is located in a highly conserved region of yeast topoisomerase II that is 200 amino acid residues C-terminal to the active site tyrosine (Tyr in yeast topoisomerase II). Although this position is not conserved through the species sequenced to date, it is aromatic in all multicellular eukaryotes but is never a tyrosine.


Figure 1: DNA sequence of top2H1012Y cDNA. A polyacrylamide gel is shown. The sequence of the wild type yeast topoisomerase II cDNA is shown for comparison. The arrow indicates the single point mutation of C T at position 3034. The DNA sequence of the indicated region of wild type cDNA is shown at left. The asterisk denotes the base that is mutated in top2H1012Y. Lanes 1-4, wild type topoisomerase II (WT); lanes 5-8, mutant topoisomerase II (H1012Y).




Figure 2: Predicted amino acid sequence of yeast top2H1012Y. The single base change at position 3034 in the cDNA of top2H1012Y resulted in a conversion of His Tyr at position 1012 in the mutant polypeptide. The primary structures of wild type topoisomerase II from S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Drosophila melanogaster (Dm), of topoisomerase IIalpha from mouse (Malpha), Chinese hamster ovary (CHOalpha), and human (Halpha), and of topoisomerase IIbeta from human (Hbeta) are shown for comparison. Identical or highly conserved amino acids are indicated by the shaded regions. The amino acid at position 1012 is denoted by the asterisk and the open box.



Drug Resistance Phenotype of top2H1012Y

pDED1top2H1012Y was used to transform JN394t2-4, and the phenotype of the resulting transformant was determined. top2H1012Y supported rates of cell growth in the absence of drug at 34 °C that were comparable to that of the plasmid-encoded wild type TOP2 allele. The drug resistance profile of yeast containing the mutant type II allele is shown in Fig. 3. Cells carrying top2H1012Y were highly resistant to the quinolone CP-115,953. While 5 µM quinolone killed over 90% of the initial wild type culture, 200 µM drug had no effect on the growth rate of mutant cells. In addition, mutant cultures also displayed high resistance toward etoposide.

CP-115,953 and etoposide are nonintercalative with respect to DNA(23, 58) . In marked contrast to the phenotype displayed toward these drugs, cells carrying top2H1012Y showed no resistance toward the intercalative agents (59, 60) amsacrine and ellipticine (Fig. 3). Yeast displayed wild type sensitivity toward amsacrine over a wide range of drug concentrations. Furthermore, cells harboring top2H1012Y appeared to be severalfold hypersensitive toward ellipticine. In fact, levels of ellipticine that allowed high growth rates of wild type cultures killed over 90% of mutant cultures.

As discussed above, the phenotype of cultures carrying the chimeric plasmid-encoded top2 allele was similar to that observed in the original transformant. Thus, the His Tyr mutation at position 1012 appears to be solely responsible for the resistance profile observed in the initial isolate.

Purification of Wild Type and Mutant Yeast Type II Topoisomerases

Prior to purification, the presence of the C to T mutation at base position 3034 in pGAL1top2H1012Y was confirmed by sequence analysis. Both the wild type and mutant enzymes were purified by a modification of the protocol of Worland and Wang(40) . Details are given under ``Experimental Procedures.'' Briefly, the present scheme departed from that of Worland and Wang following NH(4)SO(4) precipitation of polymin P extracts. In place of the Celite column, the extract was applied to a phosphocellulose column as described by Shelton et al. (12) . Following chromatography with a linear KCl gradient (both proteins eluted at 600 mM KCl), samples were concentrated on a mini phosphocellulose collection column and eluted with high salt. As seen in Fig. 4, this protocol yields highly purified yeast topoisomerase II with little or no degradation. Typically, the concentration of purified preparations was 5-10 mg/ml protein, and the yield of topoisomerase II exceeded 0.3 mg/g of wet-packed yeast cells.


Figure 4: Purification of wild type and top2H1012Y yeast type II topoisomerases. A Coomassie-stained PhastGel 7.5% homogenous media polyacrylamide gel is shown. Lane 1, molecular weight markers; lane 2, wild type cell homogenate; lane 3, purified wild type topoisomerase II; lane 4, top2H1012Y cell homogenate; lane 5, purified top2H1012Y topoisomerase II.



The mutant enzyme was stable in liquid nitrogen for at least 12 months (the longest period monitored to date) and showed no loss of activity following storage at -20 °C for several weeks. Finally, the thermal stability of top2H1012Y was similar to that of the wild type enzyme (not shown).

Characterization of top2H1012Y

The catalytic activity of purified top2H1012Y was monitored by DNA relaxation assays (Fig. 5). The mutant enzyme displayed high rates of DNA relaxation but was somewhat less active than wild type topoisomerase II. In addition, top2H1012Y appeared to be slightly less processive than the wild type yeast enzyme (not shown). Both characteristics are consistent with a decrease in the affinity of top2H1012Y for its DNA substrate. To determine if this was the case, topoisomerase IIbulletDNA binding was monitored using an electrophoretic mobility shift assay (Fig. 6)(47) . Based on the relative retardation of the negatively supercoiled DNA band, the affinity of top2H1012Y for pBR322 DNA appeared to be less than that of wild type topoisomerase II. Similar results were obtained in the presence or absence of magnesium (not shown). Finally, the His Tyr mutation at position 1012 did not affect the ability of topoisomerase II to recognize the topological state of DNA. Like the wild type enzyme, top2H1012Y bound negatively supercoiled molecules preferentially over nicked plasmids (Fig. 6).


Figure 5: Catalytic activity of yeast wild type and top2H1012Y topoisomerase II. Results of DNA relaxation assays utilizing purified topoisomerase II are shown. Open circles, wild type topoisomerase II; closed circles, top2H1012Y topoisomerase II. Data represent the averages of 2-3 independent experiments.




Figure 6: The binding of yeast wild type and top2H1012Y topoisomerase II to DNA. An ethidium bromide stained agarose gel is shown. An electrophoretic mobility shift assay was employed. Assays contained 5 nM negatively supercoiled pBR322 DNA and 0 (lane 1), 25 nM (lanes 2 and 9), 50 nM (lanes 3 and 10), 75 nM (lanes 4 and 11), 100 nM (lanes 5 and 12), 150 nM (lanes 6 and 13), 200 nM (lanes 7 and 14), or 250 nM (lanes 8 and 15) topoisomerase II. Results with the wild type and mutant enzymes are shown in lanes 2-8 and lanes 9-15, respectively. The positions of negatively supercoiled (Form I, FI) and nicked (Form II, FII) plasmid molecules are indicated.



At least one mutant topoisomerase II, CEM/VM-1-5, has been found to have a decreased affinity for ATP(61) . Two experiments were carried out to characterize the interaction of top2H1012Y with this high energy cofactor (Fig. 7). In both cases, mutant and wild type enzyme concentrations were adjusted so that the same DNA relaxation units were employed. First, the effect of ATP concentration on enzyme-catalyzed DNA relaxation was determined (panel A). Comparable titration curves were observed for both type II topoisomerases. In both cases, 50% relaxation was observed at approximately 0.1 mM ATP. Second, the rate of ATP hydrolysis was determined for the mutant and wild type enzymes (panel B). Under the conditions employed, top2H1012Y hydrolyzed ATP slightly faster than did wild type topoisomerase II. Therefore, the His Tyr mutation at position 1012 appears to have no significant effect on the interaction of topoisomerase II with its ATP cofactor.


Figure 7: Affinity of wild type and top2H1012Y topoisomerase II for ATP. Panel A shows the effect of ATP concentration on DNA relaxation activity. Panel B shows the results of ATP hydrolysis assays. Results with the wild type (WT) and mutant (H1012Y) enzymes are denoted by the open and closed circles, respectively. Data represent the averages of 2-3 independent experiments.



In Vitro Drug Resistance of top2H1012Y

Since the cytotoxicity of topoisomerase II-targeted antineoplastic drugs correlates with their ability to enhance enzyme-mediated nucleic acid breakage, DNA cleavage assays were utilized to assess the drug resistance of top2H1012Y in vitro. Topoisomerase II carries out two cleavage reactions, one prior to and one following the strand passage event(3, 8) . Pre-strand passage DNA cleavage was monitored in the absence of a high energy cofactor, whereas post-strand passage DNA cleavage was monitored in the presence of a nonhydrolyzable ATP analog, App(NH)p (20) . Both reactions were used to characterize the mutant enzyme.

Results from pre-strand passage DNA cleavage studies are shown in Fig. 8. Data are plotted as relative DNA cleavage in which the amount of cleavage in the absence of drug was set to 1.0. (^2)In the presence of the quinolone CP-115,953, large differences between the mutant and wild type enzymes were observed. At quinolone concentrations less than 100 µM, 5-fold less cleavage was observed with the mutant enzyme. Similar results were observed with the quinolones CP-115,955 and CP-67,804, which are related to but are less potent than CP-115,953 (not shown)(23, 24, 62) . The mutant enzyme also displayed resistance to etoposide in cleavage assays (Fig. 8). Levels of resistance were similar to those obtained with CP-115,953.


Figure 8: Pre-strand passage DNA cleavage. Assays were carried out in the absence of an ATP cofactor. The effects of CP-115,953 (circles), etoposide (squares), amsacrine (diamonds), or ellipticine (triangles) on the pre-strand passage DNA cleavage/religation equilibrium of wild type (WT, open symbols) or mutant (H1012Y, closed symbols) type II topoisomerases are shown. The relative level of DNA cleavage in the absence of drug was set to 1.0. Data represent the averages of 2-5 independent experiments.



Despite the resistance of top2H1012Y toward nonintercalative drugs, the mutant enzyme was highly sensitive to two intercalative drugs. At all drug concentrations tested, top2H1012Y displayed wild type sensitivity to amsacrine. In addition, the mutant enzyme was severalfold hypersensitive to ellipticine. While the wild type topoisomerase II was only moderately affected by ellipticine, DNA cleavage with the mutant enzyme was enhanced more than 10-fold. Thus, top2H1012Y is the first eukaryotic type II topoisomerase found to be hypersensitive toward antineoplastic agents.

A similar pattern of resistance was observed for each drug in post-strand passage DNA cleavage assays (Fig. 9). top2H1012Y displayed resistance toward CP-115,953 and etoposide, wild type sensitivity toward amsacrine, and hypersensitivity toward ellipticine. In all cases, the in vitro resistance profile of top2H1012Y paralleled the phenotype of yeast cells carrying the mutant allele. These results strongly suggest that the drug resistance profile observed for top2H1012Y in vivo is due solely to the characteristics of the mutant enzyme.


Figure 9: Post-strand passage DNA cleavage. Assays were carried out in the presence of 1 mM App(NH)p. The effects of CP-115,953 (circles), etoposide (squares), amsacrine (diamonds), or ellipticine (triangles) on the post-strand passage DNA cleavage/religation equilibrium of wild type (WT, open symbols) or top2H1012Y (H1012Y, closed symbols) type II topoisomerases are shown. The relative level of DNA cleavage in the absence of drug was set to 1.0. Data represent the averages of 2-5 independent experiments.



As a first step toward determining the mechanistic basis for the resistance of top2H1012Y toward quinolones, a dose-response curve was generated for the enhancement of DNA cleavage by CP-115,953 (Fig. 10). In this experiment, the effect of the quinolone on pre-strand passage DNA cleavage of both the wild type and mutant enzymes was examined over a concentration range that spanned 2 orders of magnitude. Saturating drug levels were reached at 300 µM and 1000 µM for wild type topoisomerase II and top2H1012Y, respectively. At higher drug concentrations, levels of cleavage begin to decrease (not shown). As seen in Fig. 10, the maximal cleavage enhancement observed with top2H1012Y was 50% that observed with wild type enzyme. This decrease in drug efficacy indicates that the mutation partially abrogates the ability of CP-115,953 in the enzymebulletDNA complex to enhance DNA cleavage. In addition, the concentration of the quinolone required to reach 50% saturation with top2H1012Y (230 µM) was nearly 5-fold higher than that required for wild type topoisomerase II (50 µM). This decrease in drug potency strongly suggests that the H1012Y mutation decreases the affinity of CP-115,953 for the enzymebulletDNA complex.


Figure 10: Dose-response curve for the enhancement of pre-strand passage DNA cleavage by CP-115,953. Data for wild type topoisomerase II (WT, open circles) and top2H1012Y (H1012Y, closed circles) represent the averages of two independent experiments. Results are plotted as the percent maximal DNA cleavage to allow direct comparison between the wild type and mutant enzymes.




DISCUSSION

A mutant type II topoisomerase initially selected for resistance to the quinolone CP-115,953 was generated using a yeast genetic system coupled with in vitro mutagenesis. Drug resistance was conferred by a single point mutation that converts the histidine at position 1012 to a tyrosine. This conversion represents the first resistance-conferring mutation identified for a type II topoisomerase specifically selected against a drug that stabilizes enzymebulletDNA cleavage complexes without inhibiting religation. Residue 1012 is located in the C-terminal portion of the gyrA homology domain, 20 amino acids from the putative leucine zipper(63) . As determined by deletion studies of the C terminus of topoisomerase II, this residue is located in a region that is essential for catalytic activity(64, 65, 66) .

top2H1012Y displays resistance to quinolones and etoposide both in vitro and in vivo. In contrast, the mutant enzyme is sensitive to amsacrine and hypersensitive to ellipticine. Previous biochemical studies indicate that DNA cleavage-enhancing drugs share an overlapping interaction domain on topoisomerase II(35, 36) . Together with previous mutagenesis studies(23, 24, 49, 50) , the present results strongly suggest that, within this common interaction domain, different drugs may interact with different residues on topoisomerase II.

Quinolones targeted to the prokaryotic type II topoisomerase, DNA gyrase, are in wide clinical use and represent the most potent class of oral antibiotics currently available(67, 68) . The vast majority of mutations in DNA gyrase that confer resistance to quinolones are localized to the A subunit of the enzyme in the vicinity of Ser(69, 70) . (The homologous residue in yeast topoisomerase II is Ser.) No quinolone resistance-conferring mutations in DNA gyrase have been found in the region of the present mutation. Furthermore, position 1012 is not conserved in the prokaryotic type II enzyme(71, 72) . Thus, the H1012Y mutation in yeast topoisomerase II suggests that there are unique aspects to the interactions of quinolones with the eukaryotic type II enzyme.

Point mutations in DNA gyrase and T4 topoisomerase II that result in hypersensitivity to quinolones also have been described(73, 74) . However, top2H1012Y is the first mutant eukaryotic type II enzyme reported to display drug hypersensitivity. Unlike the hypersensitivity-conferring mutations reported for the prokaryotic type II topoisomerases (which are located in the gyrB homology domain), the point mutation in top2H1012Y is in the gyrA domain of the eukaryotic enzyme.

Most mutant eukaryotic type II enzymes selected for drug resistance display at least some level of resistance to a broad spectrum of topoisomerase II-targeted antineoplastic agents(7, 8) . The notable exception is the HL-60/AMSA enzyme which is resistant to intercalative drugs but is highly sensitive to nonintercalative agents(50, 75) . Although the scope of the present study was limited to four drugs, top2H1012Y appears to display the opposite phenotype (i.e. resistant to nonintercalative drugs but sensitive to intercalative agents). The basis for the differential drug resistance of top2H1012Y is not known. However, at least for CP-115,953, it appears as though the mutation decreases both the affinity of the drug for the enzymebulletDNA complex and the ability of the bound drug to enhance DNA cleavage. Finally, the differential drug resistance observed for top2H1012Y suggests that at least some malignancies that are resistant to one class of topoisomerase II-targeted drugs may respond to other antineoplastic agents targeted to the enyzme.

Previous genetic and biochemical studies indicate that topoisomerase II is the primary cellular target for quinolones, etoposide, and amsacrine (7, 8, 32, 54, 62) . Comparable studies have not been carried out for ellipticine. However, since the hypersensitivity of top2H1012Y toward ellipticine seen in vitro also is observed in cells carrying the mutant enzyme, it is likely that topoisomerase II is an important cytotoxic target for ellipticine in yeast and that ellipticine acts by converting topoisomerase II into a cellular toxin.

Thus far, mutagenesis studies have defined two regions in topoisomerase II that are important for interactions with antineoplastic drugs(7, 8) . One region is located in the gyrA homology domain surrounding the active site tyrosine, and the other is located in the gyrB homology domain near the consensus ATP binding sequence. The present mutation at position 1012 potentially defines a new drug resistance-conferring region on topoisomerase II and suggests that amino acid residues toward the C terminus of the enzyme may play an important role in drug-enzyme interactions.


FOOTNOTES

*
This work was supported in part by Grants GM33944 (to N. O.) and CA52814 (to J. L. N.) from the National Institutes of Health, Grant NP-812 from the American Cancer Society (to N. O.), and by the Neil Bogart Laboratory of the Martell Foundation for Leukemia, Cancer, and AIDS (to J. L. N.). 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.

§
Trainee under Grant 5 T32 CA09582 from the National Institutes of Health.

Supported by a special fellowship from the Leukemia Society of America. Present address: St. Jude Children's Research Hospital, Dept. of Pharmacology, 332 N. Lauderdale, Memphis, TN 38101.

**
Supported by Faculty Research Award FRA-370 from the American Cancer Society. To whom correspondence and 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.

(^1)
The abbreviations used are: App(NH)p, adenyl-5`-yl beta,-imidodiphosphate; kb, kilobase(s).

(^2)
When adjusted for catalytic activity, absolute levels of DNA cleavage observed for the mutant enzyme in the absence of drug were 20% less than those observed for wild type topoisomerase II (not shown).


ACKNOWLEDGEMENTS

We are grateful to M. S. Grotewiel for helpful discussions, to E. Hannah for assistance with yeast and bacteria preparations, to T. D. Gootz and P. R. McGuirk for generously providing CP-115,953, and to P. S. Kingma, S. J. Froelich-Ammon, and D. A. Burden for critical reading of the manuscript.


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