(Received for publication, July 27, 1994; and in revised form, October 12, 1994)
From the
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.
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.
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.
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.
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.
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 II
from mouse (M
), Chinese hamster ovary (CHO
), and human (H
), and of topoisomerase II
from human (H
) 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.
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.
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).
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.
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. ()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 enzyme
DNA 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 enzyme
DNA 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.
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
enzymeDNA 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 enzymeDNA 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.