(Received for publication, March 14, 1995; and in revised form, June 5, 1995)
From the
In prokaryotic type II topoisomerases (DNA gyrases), mutations
that result in resistance to quinolones frequently occur at Ser or Ser
of the gyrA subunit. Mutations to
Trp, Ala, and Leu have been identified, all of which confer high levels
of quinolone resistance. Extensive segments of DNA gyrase are
homologous to eukaryotic topoisomerase II, and Ser
of
yeast TOP2 is homologous to Ser
of prokaryotic
DNA gyrA. Introduction of the Ser
Trp
mutation into yeast TOP2 confers resistance to
6,8-difluoro-7-(4`-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic
acid (CP-115,953), a fluoroquinolone with substantial activity against
eukaryotic topoisomerase II, whereas changing Ser
to
either Leu or Ala does not change sensitivity to quinolones.
Interestingly, Ser
Trp in the yeast TOP2 also confers hypersensitivity to etoposide. Sensitivity to
intercalating anti-topoisomerase II agents such as amsacrine is not
changed by any of the three mutations. The topoisomerase II protein
carrying the Ser
Trp mutation was overexpressed
and purified. The purified mutant enzyme had enhanced levels of
etoposide stabilized covalent complex as compared with the wild type
enzyme and reduced cleavage with CP-115,953. Unlike the wild type
enzyme, etoposide-stabilized cleavage is not readily reversible by
heat. We suggest that Ser
is near a binding site for both
quinolones and etoposide and that the Ser
Trp
mutation leads to a more stable ternary complex between etoposide, DNA,
and the mutant enzyme.
DNA topoisomerases play essential roles in a wide range of DNA metabolic processes(1) . Studies in both prokaryotic and eukaryotic cells have demonstrated their importance in transcription, DNA replication, and chromosome segregation(2) . The type II topoisomerases, which make transient double strand breaks and change the linking number of DNA in steps of two, play key roles in chromosome structure. In eukaryotic cells, these enzymes are essential for the chromosome condensation/decondensation process critical for the normal progression through mitosis(3, 4) . The ability of the enzyme to pass two DNA strands and change linking number in steps of two uniquely allows this class of enzyme to segregate fully replicated DNA molecules prior to cell division (reviewed in (5) and (6) ).
Type II topoisomerases have also been identified as a major target of chemotherapeutic agents that are specifically active against prokaryotic cells (7) or against rapidly proliferating cancer cells (8) . Fluoroquinolone antibiotics, such as norfloxacin and ciprofloxacin, along with their less active congeners nalidixic and oxolinic acid are antibacterial agents that target DNA gyrase(9) , whereas a variety of DNA-intercalating agents such as anilinoacridines, ellipticines, and mitoxantrone and nonintercalating agents such as epipodophyllotoxins are active against eukaryotic topoisomerase II and are clinically important anti-cancer agents(10, 11) .
Recently, it has been shown that
6,8-difluoro-7-(4`-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic
acid (CP-115,953), ()a fluoroquinolone closely related to
ciprofloxacin, is highly toxic to mammalian cells in culture and active
against topoisomerase II in vitro(12, 13, 14) . Genetic studies in yeast
demonstrated that topoisomerase II is the primary physiological target
for quinolone cytotoxicity(15) . Unlike etoposide, which
stabilizes cleavage mainly by inhibiting the religation reaction of
topoisomerase II, CP-115,953 stabilizes cleavage by enhancing the
forward rate of cleavage(12) . Therefore, fluoroquinolones are
nonintercalating anti-topoisomerase II agents with a different
biochemical mechanism of action than etoposide. Although the action of
quinolone-based drugs differs from agents such as etoposide, the
ultimate mechanism of cell-killing, enhanced levels of the covalent
complex is the same(15, 16) .
In Escherichia
coli, mutations that lead to quinolone resistance are most often
found in gyrA, the structural gene for the DNA gyrase A
subunit, although changes that lead to quinolone resistance have also
been found in the gyrB subunit. Ser of gyrA is the amino acid most frequently mutated in strains with high
levels of quinolone resistance(17) . Among the substitutions
noted in quinolone-resistant mutants are mutations of Ser
to Ala, Leu, or Trp, with higher levels of resistance in strains
carrying Ser
Trp or Ser
Leu
mutations. It has recently been reported that gyrase protein with the
Ser
Trp mutation in gyrA has greatly
reduced binding of ciprofloxacin compared with the wild type protein,
further demonstrating the importance of this region of the
protein(18) .
These findings have led us to examine the
effects of mutations in the yeast TOP2 gene that change
Ser, the amino acid that is homologous to Ser
in gyrA of E. coli, on the yeast type II enzyme
for sensitivity to quinolones as well as other topoisomerase II
inhibitors. We have found that the Ser
Trp
mutation results in resistance to quinolones that act against
eukaryotic topoisomerase II. In addition, this mutation causes
hypersensitivity to etoposide. Overexpression, purification, and
characterization of yeast topoisomerase II carrying the Ser
Trp demonstrated that the protein is relatively
insensitive to fluoroquinolones and is hypersensitive to etoposide. Our
results demonstrate that Ser
of yeast TOP2 plays
a critical role in the action of some anti-topoisomerase II agents and
may be directly involved in the interactions of the eukaryotic enzyme
with etoposide.
In order to construct a
plasmid for overexpressing the Ser
Trp mutant
topoisomerase II, plasmid pMJ2-S*W and YEpTOP2-PGAL1 (25) were
digested with KpnI and AvrII. The 2.2-kilobase
fragment from pMJ2-S*W and the 11.5-kilobase fragment from
YEpTOP2-PGAL1 were gel purified, ligated, and transformed into E. coli DH5
competent cells. Plasmids that had an
identical restriction pattern to YEpTOP2-PGAL1 were identified, and the
presence of the Ser
Trp mutation in YEpTOP2-PGAL1
was confirmed by DNA sequencing. The plasmid carrying the mutation was
designated YEptop2-S*W-PGAL1.
We determined the IC of cells carrying either wild type
or the Ser
Trp top2 mutation. The
IC
of wild type cells is about 1 µM, whereas
the IC
of cells carrying the Ser
Trp top2 mutation is about 10 µM. The results in Fig. 1show that strains carrying the Ser
Trp mutation grow in medium containing 20 µM CP-115,953,
although the growth is considerably less than in drug-free medium.
However, 10 µM CP-115,953 is very cytotoxic for
cell-carrying wild type TOP2 (Fig. 1). At a drug
concentration of 20 µM, the viability of TOP2
cells is reduced to less than 0.1% after
24 h of exposure to CP-115,953, whereas the Ser
Trp mutant shows no reduction in viability compared with the time of
drug addition. The presence of the Ser
Leu or the
Ser
Ala mutation did not have significant effects
on sensitivity to CP-115,953, compared with TOP2
(Table 1).
Figure 1:
Mutation of Ser
Trp of yeast TOP2 results in resistance to the fluoroquinolone
CP-115,953. Strain JN394 or an isogenic derivative carrying the
Ser
Trp (Ser741Trp) mutation in TOP2 was treated with CP-115,953 for the indicated times. Samples were
removed, diluted, and plated to YPDA medium to determine the number of
viable colony-forming units. Results are expressed relative to T
= 0.
Because the Ser
Trp
mutation conferred resistance to CP-115,953, we examined whether the
sensitivity to other anti-topoisomerase II agents was affected. Cells
carrying either the Ser
Trp mutation or wild type TOP2 have the same minimum lethal concentration as the
intercalating topoisomerase II inhibitors amsacrine and mitoxantrone (Table 1).
A very different result was obtained with the
nonintercalating topoisomerase II inhibitor etoposide. Strains carrying
the Ser
Trp mutation are hypersensitive to
etoposide. The IC
for etoposide is approximately 10
µg/ml. By contrast the IC
for etoposide for cells
carrying the Ser
Trp mutation is less than 1
µg/ml. Mutant cells are efficiently killed by 20 µg/ml
etoposide, whereas the isogenic wild type strain is able to grow at
this drug concentration (Fig. 2). At 100 µg/ml of etoposide,
the viability of cells carrying the Ser
Trp
mutation is approximately 0.1% after 24 h drug exposure compared with
about 3% for cells carrying wild type TOP2. The minimum lethal
concentration for etoposide for cells carrying the Ser
Trp mutant TOP2 is 5 µg/ml, versus 50 µg/ml for cells carrying wild type TOP2 (Table 1). The mutant strain also shows hypersensitivity to
another epipodophyllotoxin teniposide; the minimum lethal concentration
for the mutant strain is about 10 µg/ml teniposide, compared with
50 µg/ml teniposide for strains carrying wild type topoisomerase II (Table 1).
Figure 2:
The Ser
Trp mutation
confers hypersensitivity to etoposide. Strain JN394 or an isogenic
derivative carrying the Ser
Trp mutation in TOP2 was treated with etoposide for the indicated times.
Samples were removed, diluted, and plated to YPDA medium to determine
the number of viable colony-forming units. Results are expressed
relative to T = 0.
Figure 3:
Purification and catalytic activity of
yeast topoisomerase II protein carrying Ser
Trp
mutation. A, yeast topoisomerase II (TopoII) was
expressed from the yeast GAL1 promoter in strain JEL1. Protein
samples were analyzed by SDS-polyacrylamide gel electrophoresis. The
samples were: lanes 1 and 6, molecular mass markers (Std); lane 2, the JEL1 homogenate (Homo)
from cells expressing wild type (WT) yeast topoisomerase II; lane 3, purified wild type yeast topoisomerase II; lane
4, the JEL1 homogenate from cells expressing Ser
Trp (S741W) topoisomerase II; and lane
5, purified topoisomerase II carrying the Ser
Trp mutation. B, catalytic activity of wild type (WT)
topoisomerase II or the Ser
Trp (Ser741Trp) mutant was determined by monitoring ATP-dependent
relaxation of pBR322. The reaction mixtures were analyzed by agarose
gel electrophoresis, and a negative of the gel was scanned by
densitometry to determine the percentage of the input substrate DNA
that was relaxed by the enzyme.
Figure 4:
K/SDS assay of cleavage
stabilized by the fluoroquinolone CP-115,953. Covalent complex
formation stabilized by CP-115,953, and purified yeast topoisomerase II
was measured using the K
/SDS assay as described in the
text. Drug concentrations are indicated on the figure. WT,
wild type; Ser741Trp, Ser
Trp
mutation.
We then
examined the sensitivity of the Ser
Trp protein to
etoposide using the K
/SDS assay. An etoposide
concentration of 1 µg/ml is required to effect a 2-fold increase in
precipitated counts with wild type TOP2 protein, whereas the same level
of precipitated counts occurs at <0.1 µg/ml for the Ser
Trp protein (Fig. 5). At 1 µg/ml etoposide, the
Ser
Trp protein has greater than a 5-fold increase
in precipitated counts. The etoposide hypersensitivity is less
pronounced at higher drug concentrations; at 5 µg/ml etoposide
there is a 2-fold difference in the level of cleavage between
Ser
Trp and wild type TOP2 protein (Fig. 5).
Figure 5:
K/SDS assay of cleavage
stabilized by etoposide. Covalent complex formation stabilized by
etoposide and purified yeast topoisomerase II was measured using the
K
/SDS assay as described in the text. Drug
concentrations are indicated on the figure. WT, wild type; Ser741Trp, Ser
Trp
mutation.
The K+/SDS cleavage reaction was carried out with wild
type TOP2 or the Ser
Trp protein as above except
that the reaction was incubated at 65 °C for various times prior to
the addition of SDS. In the absence of the drug, the 65 °C
treatment reduces the amount of precipitated counts to about 25% of the
level observed without heat for both the wild type and Ser
Trp proteins (Fig. 6A). Because the
cleavage complex formed in the absence of drugs is heat reversible, it
is likely that the Ser
Trp protein does not form a
complex that is qualitatively different than the wild type protein.
Similarly, for both the wild type and Ser
Trp
proteins, the complexes formed in the presence of 10 µM
CP-115,953 were completely reversed by 1 min of incubation at 65 °C
prior to the addition of SDS.
Figure 6:
Heat reversibility of covalent complexes
formed by the Ser
Trp mutant protein. A,
wild type and Ser
Trp (Ser741Trp)
proteins (8 units) were used in a K
/SDS assay under
the same conditions as in Fig. 4. The samples treated with heat
were incubated at 65° for 1 min prior to the addition of SDS. B, wild type (WT) and Ser
Trp
proteins (8 units) were used in a K
/SDS assay in the
presence of 10 µg/ml etoposide. Samples were placed at 65 °C,
and at the indicated times, SDS was added to an aliquot of the
reaction. The percentage of precipitable counts relative to unheated
samples is shown.
In the presence of etoposide, a
different result is seen. At 10 µg/ml etoposide, the cleavage
complexes formed with the wild type protein are rapidly reversed by
heat treatment, as indicated by a 50% reduction in precipitable counts
after 1 min at 65 °C. However, little reversal is seen when the
Ser
Trp protein treated with etoposide is exposed
to 65 °C prior to SDS addition. An incubation of 20 min is required
to reduce the precipitated counts by 50% under these conditions (Fig. 6B). These results taken together strongly
suggest that the etoposide-DNA-Ser
Trp protein
ternary complex has enhanced stability compared with the wild type
protein, which is consistent with a more stable interaction between
etoposide and the Ser
Trp mutant protein.
Acquired drug resistance by mutation of the drug target
likely represents a contributor to the failure of drug treatment for
both antibacterial and anti-cancer drugs. This has been clearly
demonstrated with quinolone antibiotics, where mutations in the
structural genes of bacterial DNA gyrase are frequently observed in
quinolone-resistant clinical isolates(7) . In this report, we
have demonstrated that mutations in yeast TOP2 at a position
homologous to Ser of gyrA gives rise to
fluoroquinolone resistance. The same mutation also generates
hypersensitivity to another class of drugs, epipodophyllotoxins. Our
results suggest that in some contexts, it may be possible to overcome
acquired drug resistance due to a mutation in the drug target by use of
a different class of drugs that act against the same target.
We
constructed several changes at Ser in the yeast TOP2 gene of Saccharomyces cerevisiae and have demonstrated
that mutation of Ser
Trp of yeast topoisomerase II
confers hypersensitivity to etoposide and resistance to quinolone
CP-115,953 in vivo. In agreement with the in vivo results, the Ser
Trp protein has increased
drug-stabilized cleavage in response to etoposide and decreased drug
stabilized cleavage when treated with the fluoroquinolone CP-115,953.
The results of Maxwell and Willmott strongly suggest that Ser
of gyrA is critical for the binding of quinolones to
gyrase(18) . We reasoned that the homology between gyrase and
eukaryotic topoisomerases (32) suggested that the equivalent
amino acid would be important, at least for the action of
fluoroquinolones that have activity against eukaryotic topoisomerase
II. However, the homology between yeast TOP2 and gyrA does not
result in a perfect correspondence in sensitivity to fluoroquinolones.
Most notably, the Ser
Leu mutant is not resistant
to CP-115,953, nor is it etoposide-hypersensitive. By contrast, a
Ser
Leu mutation in gyrA results in almost
the same level of quinolone resistance as Ser
Trp.
Although there are likely to be several ways that alterations in TOP2 lead to a drug-resistant protein, it would seem that
there are only a limited number of ways where mutations could generate
drug hypersensitivity. Because the Ser
Trp protein
has essentially wild type activity and because the drug
hypersensitivity is specific for epipodophyllotoxins (i.e. sensitivity to intercalating drugs is not affected by the mutant
protein), the simplest explanation is that the protein's affinity
for epipodophyllotoxins is affected. The increased stability of the
etoposide-stabilized covalent complex at 65 °C also supports the
hypothesis that the drug hypersensitivity is due to a more stable
interaction between the mutant TOP2 protein and etoposide. The nature
of the covalent interaction is unlikely to be qualitatively different
with the Ser
Trp protein because the covalent
complex formed in the absence of drug or in the presence of CP-115,953
is fully heat-reversible. Because gyrA protein carrying a
Ser
Trp mutation has reduced fluoroquinolone
binding(17, 18) , Ser
either is part of a
domain of gyrase that binds to quinolones or is fairly close to the
quinolone binding site. Our results would suggest that the domain
including Ser
in yeast TOP2 interacts with both
fluoroquinolones and epipodophyllotoxins. This result is consistent
with recently reported results that suggest that fluoroquinolones and
etoposide can compete for binding to topoisomerase II(33) . A
simple interpretation is that the two agents share (at least in part)
the domain required for drug/protein interactions. Our results suggest
that Ser
may be a key part of that domain. Drug binding
assays with eukaryotic topoisomerase II have yet to be reported,
however, so we have not been able to directly demonstrate the roles of
specific domains in interaction with etoposide or other topoisomerase
II agents.
It should be possible to design etoposide derivatives
that generate covalent complexes that behave like the TOP2 Ser
Trp-etoposide-DNA complexes with the wild type protein.
Because other agents such as clerocidin have been described that can
generate heat stable complexes(34) , we suggest that such
agents have a high enough affinity for topoisomerase II such that at 65
°C the covalent complex is still favored. It will be of
considerable interest to determine whether agents that form such stable
cleavage complexes might result in more effective antibacterial or
anti-cancer agents.