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J Biol Chem, Vol. 273, Issue 44, 29086-29092, October 30, 1998
From the Departments of To further characterize the mechanistic basis for
cellular resistance/hypersensitivity to anticancer drugs, a yeast
genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and
ellipticine. The mutant enzyme contained a single point mutation that
converted Gly437 Topoisomerase II is the primary cellular target for some of the
most effective drugs currently used for the chemotherapeutic treatment
of human cancers (1-9). These agents act in an unusual fashion. Rather
than killing cells by robbing them of the essential activities of
topoisomerase II, anticancer drugs "poison" the enzyme (10) and
convert it to a potent cellular toxin by increasing levels of covalent
topoisomerase II-cleaved DNA complexes that are normal but transient
intermediates in the catalytic cycle of the enzyme (1-8, 11-13). This
action leads to the generation of permanent double-stranded breaks in
the genetic material of treated cells and ultimately triggers cell
death pathways (2-8, 14-23).
There is a high degree of variability in the response of different
cells to topoisomerase II-targeted drugs (24-26). Despite the impact
of drug resistance and hypersensitivity on cancer chemotherapy, relatively few studies have shed light on the mechanistic basis that
underlies this physiological variability. In some cases, changes in the
cellular response to topoisomerase II poisons appear to result from
alterations in the activity or drug sensitivity of the enzyme. All
things being equal, increased expression of topoisomerase II tends to
produce drug hypersensitivity (27-29). In contrast, decreases in the
catalytic activity of the enzyme or in the level of nuclear
topoisomerase II (either by reduced protein expression/stability or by
improper localization) leads to drug resistance (1, 2, 30-43).
Finally, mutations in topoisomerase II have been identified that confer
drug resistance or hypersensitivity, depending on how they alter
interactions between the enzyme and anticancer agents (36, 44-56).
To characterize more fully the mechanistic basis for cellular
resistance/hypersensitivity to topoisomerase II poisons, a yeast genetic system was utilized to select mutant type II enzymes that confer resistance to anticancer agents in vivo (28, 49). The present study describes the characterization of one such yeast topoisomerase II in which Gly437 was mutated to Ser
(top2G437S). Although expression of this mutant enzyme conferred
cellular resistance to anticancer drugs, purified top2G437S was
hypersensitive to these agents in the absence of ATP. The enzyme lost
its hypersensitivity to anticancer agents and displayed wild-type
sensitivity in the presence of ATP, suggesting a conformational change
in the region of residue 437 upon binding of the high energy cofactor.
Further analysis indicated that in vivo resistance was
caused by decreased levels of active enzyme, while in vitro
hypersensitivity reflected (at least in part) an increased affinity for
drugs. Thus, top2G437S appears to have distinct physiological and
biochemical mechanisms for altered drug sensitivity.
Materials--
Negatively supercoiled bacterial plasmid pBR322
DNA was prepared as described previously (57). CP-115,953 was provided
by Drs. T. D. Gootz and P. R. McGuirk (Pfizer Central
Research); amsacrine was obtained from Bristol-Myers Squibb, and
etoposide and ellipticine were obtained from Sigma. All drugs were
prepared as 20 mM solutions in 100% Me2SO and
stored at 4 °C. Tris and ethidium bromide were obtained from Sigma;
SDS was from Merck; proteinase K was from U. S. Biochemical Corp.; ATP
was from Amersham Pharmacia Biotech; and restriction endonucleases and
T4 DNA ligase were from New England Biolabs. All other chemicals were
analytical reagent grade.
Yeast Strains and Plasmids--
Saccharomyces
cerevisiae strains employed were as described in Elsea et
al. (54). 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) and transformed as
described previously (58). The plasmid carrying the topoisomerase II
(TOP2) gene was YCpDED1TOP2 (28). Wild-type and mutant type II topoisomerases were purified using the inducible overexpression plasmid YEpGAL1TOP2 (59).
In Vitro Mutagenesis and Mutant Selection--
Mutations were
introduced into the yeast TOP2 gene by exposure to 0.1 M hydroxylamine in vitro (60). Following this
treatment, YCpDED1TOP2 was transformed into Escherichia coli
strain XL-1 Blue. Plasmid DNA was purified by the method of Sambrook
et al. (57). The mutagenized pool of YCpDED1TOP2 was
transformed into yeast strain JN394t2-4, and transformants were
selected for growth in the presence of 20 µM CP-115,953,
as described previously by Nitiss and co-workers (49). 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,
amsacrine, etoposide, or ellipticine was determined as described
previously (61). Cells were incubated in SC-URA selection media with
drug (0-200 µM) for 24 h, and initial phenotypes
were established by following the absorbance of cultures at 600 nm.
Transformants with phenotypes of interest were plated in duplicate on
YPDA medium solidified with 1.5% Bacto-agar and cultured for 3-4 days
at 34 °C. Drug sensitivity was quantitated by counting the number of
surviving colonies.
Recovery of Plasmids Carrying Topoisomerase II
Mutations--
Plasmids carrying the mutant alleles were recovered as
described (62). In summary, yeast cells were lysed, and total nucleic acids were extracted with phenol/CHCl3 and precipitated
with ethanol. RNA was degraded by RNase A, and the remaining DNA was
precipitated with ethanol and transformed into E. coli
strain XL-1 Blue. Plasmid DNA was purified using Qiagen plasmid kits
(Qiagen).
Construction of Plasmids for Sequencing and
Overexpression--
Plasmids were constructed according the procedure
of Elsea et al. (54). Briefly, mutagenized YCpDED1TOP2 was
digested with restriction endonucleases KpnI and
AvrII. DNA fragments were separated by electrophoresis, and
the 2.2-kb1 fragment (containing
the coding sequence for amino acids 317-1045 in yeast topoisomerase
II) was gel-purified. In addition, the wild-type plasmids YCpDED1TOP2
and YEpGAL1TOP2 were digested with KpnI and
AvrII, and the large fragment containing vector sequences was gel-purified. The mutagenized 2.2-kb fragment was then ligated into
YCpDED1TOP2 and YEpGAL1TOP2, replacing the wild-type fragments, and
subsequently transformed into E. coli strain XL-1 Blue.
YCpDED1TOP2 DNA was used for sequence analysis (63) and transformation
of the yeast strain JN394t2-4 for cytotoxicity assays. YEpGAL1TOP2 DNA
was used to transform the yeast strain JEL1 for overexpression and
purification of topoisomerase II.
Yeast Topoisomerase II Induction, Overexpression, and
Purification--
Yeast topoisomerase II was overexpressed in yeast
strain JEL1 (transformed with YEpGAL1TOP2) by the addition of galactose in glucose-free media (59). Wild-type and mutant enzymes were purified
to >95% homogeneity (as determined by visualization on silver-stained
polyacrylamide gels) by a modified procedure (54, 64) of Worland and
Wang (59). Although the mutant enzyme appeared to be stable for at
least 2 years in liquid nitrogen, it lost activity within a few days
following thawing and storage at Topoisomerase II-mediated DNA Relaxation--
DNA relaxation was
carried out as described by Osheroff et al. (65). Reaction
mixtures contained 0.1-3 nM 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
7.9), 175 mM KCl, 0.1 mM EDTA, 5 mM
MgCl2, and 2.5% glycerol). DNA relaxation was at 28 °C
for 15 min and stopped by the addition of 3 µl of 0.77% SDS and 77 mM EDTA. Samples were subjected to electrophoresis in 1%
agarose gels in TBE (100 mM Tris borate (pH 8.3), 2 mM EDTA). Gels were stained with 1 µg/ml ethidium
bromide, visualized by UV light, and photographed through Kodak 23A and
12 filters with Polaroid type 665 positive/negative film. Levels of DNA
relaxation were quantitated by scanning negatively supercoiled plasmid
bands in photographic negatives with an E-C apparatus model EC910
scanning densitometer in conjunction with Hoefer GS-370 Data System
software. The intensity of bands in the negative was proportional to
the amount of DNA present. The requirement of wild-type and mutant topoisomerase II for ATP was determined using relaxation assays in
which the concentration of ATP was varied from 0 to 0.5 mM.
Topoisomerase II-mediated DNA Cleavage--
DNA cleavage assays
were carried out as described by Osheroff and Zechiedrich (66).
Reaction mixtures contained 150 nM topoisomerase II and 5 nM negatively supercoiled pBR322 DNA in a total volume of
20 µl of cleavage buffer (10 mM Tris-HCl (pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 5 mM
MgCl2, 2.5% glycerol, 10% Me2SO) and 0-200
µM drug. For dose-response studies, drug concentrations
ranged from 1 µM to 2 mM. Samples were
incubated at 28 °C for 6 min and cleavage products were trapped by
the addition of 2 µl of 5% SDS. To this mixture was added 1.5 µl
of 250 mM EDTA and 2 µl of 0.8 mg/ml Proteinase K,
followed by incubation at 45 °C for 30 min. Samples were subjected
to electrophoresis in 1% agarose gels in TAE (40 mM Tris
acetate (pH 8.3), 2 mM EDTA) containing 1 µg/ml ethidium bromide. When DNA cleavage was carried out in the presence of ATP (1 mM), reaction mixtures contained 40 nM
topoisomerase II and 10 nM negatively supercoiled pBR322
DNA. Samples were incubated at 28 °C for 3 min, and cleavage
products were trapped and analyzed as above.
Topoisomerase II-mediated DNA Religation--
DNA
cleavage/religation equilibria were established as in the preceding
section using 150 nM topoisomerase II, 5 nM
negatively supercoiled pBR322 DNA, and 100 µM drug in a
total volume of 200 µl of cleavage buffer. After 6 min at 28 °C,
religation was induced by the addition of 500 mM NaCl at
room temperature. At time points up to 75 s, 20-µl samples were
removed and added to 2 µl of 5% SDS to terminate the reaction.
Reaction products were analyzed as described above.
Thermal Stability of Topoisomerase II--
Purified
topoisomerase II (1 mg/ml) was diluted 1:250 and incubated at 34 °C.
At time points up to 30 min, 1-µl aliquots of topoisomerase II were
removed and added to assay buffer that contained 5 nM
negatively supercoiled pBR322 DNA and 1 mM ATP (total
volume of 20 µl). DNA relaxation assays were carried out as above at 28 °C for 6 min. Reaction products were analyzed as described above.
Steady-state Cellular Concentration of top2G437S--
The
S. cerevisiae strain JEL1 was transformed with YCpDED1TOP2,
carrying either the wild-type TOP2 or the mutant
top2G437S allele behind the DED1 promoter. Cells were grown
to confluency in SC-URA media at room temperature (25 °C). Whole
cell homogenates were prepared by adding glass beads to culture
aliquots and bead-beating for 2-3 1-min pulses. All homogenates were
diluted to 10 mg/ml, and serial dilutions of homogenates were subjected
to electrophoresis in a 7.5% homogeneous media PhastGel (PhastSystem,
Amersham Pharmacia Biotech). Proteins were transferred to Amersham
Pharmacia Biotech Hybond-P paper using the PhastSystem, and immunoblots
were prepared using an enhanced chemiluminescence kit (Amersham
Pharmacia Biotech). The primary antibody for yeast topoisomerase II was
from Topogen; the secondary antibody was supplied in the ECL kit.
Selection of a Mutant Yeast Type II Topoisomerase in Which
Gly437 Is Converted to Ser--
A yeast genetic system was
used to further analyze the mechanistic basis for cellular
resistance/hypersensitivity to topoisomerase II poisons (28, 49). In
this system, the chromosomal copy of the TOP2 gene was
replaced by a temperature-sensitive allele (top2-4) and the
resulting strain, JN394t2-4, contained a plasmid-based collection of
expressed mutated TOP2 genes. Cells were selected for
resistance to the quinolone CP-115,953 at the non-permissive temperature (34 °C), to ensure that viability was determined by the
phenotype of the expressed mutant type II enzymes. This initial procedure established a library highly enriched for type II
topoisomerases that confer altered physiological sensitivity to
anticancer drugs (54).
Further screening of the above library identified a yeast colony that
exhibited high resistance to CP-115,953, amsacrine, etoposide, and
ellipticine. A 2.2-kb fragment of its plasmid-based mutant
TOP2 gene (encoding amino acid residues 317-1045) was cut out of the construct and used to replace the corresponding fragment in
a wild-type construct, and cells containing the chimeric gene were
screened for drug resistance (Fig. 1). The
phenotype of the chimera was identical to that of the original isolate,
indicating that drug resistance resulted from a mutation(s) within the
coding region of the TOP2 gene.
To identify the nature of the resistance-conferring mutation(s), the
nucleic acid sequence of the 2.2-kb fragment was determined. A single
point mutation was identified that converted nucleotide position 1309 in the yeast TOP2 gene from a guanine to an adenine (consistent with the original hydroxylamine mutagenesis procedure). This mutation converts Gly437, a residue that is conserved
in many eukaryotic species (ranging from yeast to humans), to Ser in
the topoisomerase II protein (4). No wild-type species thus far
reported contains Ser at position 437.
Purification and Characterization of top2G437S--
Yeast
top2G437S was overexpressed in S. cerevisiae from a
multicopy plasmid that contained a gal1 promoter. The enzyme
was purified to >95% homogeneity, and its catalytic properties were compared with those of wild-type yeast topoisomerase II.
First, as determined by its ability to relax negatively supercoiled
plasmid DNA, the Gly
Second, since the mutation in top2G437S is contained within the GyrB
(ATPase) domain of topoisomerase II (4), the requirement of the mutant
enzyme for ATP was determined by analyzing its ability to relax DNA
over a 50-fold range of ATP concentrations. It is notable that two
previously described mutations in this region of human topoisomerase
II
Third, since drug resistance in vivo could result from a
decreased level of basal DNA scission activity, the ability of
top2G437S to cleave plasmid DNA in the absence of anticancer drugs was
compared with its parental enzyme. In both the absence (Fig.
4) and presence (not shown) of ATP, the basal
DNA cleavage activity of top2G437S appeared to be similar to that of
wild-type yeast topoisomerase II.
Taken together, the above data indicate that the native activity of
topoisomerase II is not significantly altered by the substitution of
Ser for Gly at position 437.
Hypersensitivity of Purified top2G437S to Anticancer Drugs in the
Absence of ATP--
The cytotoxic potential of topoisomerase II
poisons correlates with their ability to stimulate enzyme-mediated DNA
scission (1-8). Therefore, DNA cleavage assays were utilized to
characterize the sensitivity of purified top2G437S to the anticancer
drugs used in the original cytotoxicity screens. In the first set of experiments, the effects of drugs on the DNA scission event that precedes enzyme-catalyzed DNA strand passage were assessed. This was
accomplished by monitoring DNA cleavage in the absence of ATP.
In the absence of a high energy cofactor, top2G437S did not display the
expected resistance phenotype against CP-115,953, amsacrine, etoposide,
or ellipticine. Indeed, the mutant enzyme was hypersensitive to all of
these drugs (Fig. 5). Additional experiments
were carried out to explore the basis for this hypersensitivity. CP-115,953 and amsacrine were utilized for these studies because they
produced the greatest differential in sensitivity between the wild-type
and mutant enzymes.
As seen in Fig. 6, drug hypersensitivity was
not due to a fundamental change in the mechanistic basis for drug
action. Rates of DNA religation in the presence of CP-115,953 or
amsacrine were identical for wild-type topoisomerase II and top2G437S
(panels A and B). Furthermore, there did not
appear to be a change in the rate-determining step of DNA scission,
since the presteady-state profiles for drug-stimulated cleavage
(normalized so that maximal cleavage levels for both enzymes were set
to 100%) were the same for both type II enzymes (panels C
and D).
Dose-response studies for CP-115,953 and amsacrine were carried out
over a 3 log concentration range to further define their interactions
with top2G437S (Fig. 7). Maximal DNA cleavage
levels were attained at Sensitivity of Purified top2G437S to Anticancer Drugs in the
Presence of ATP--
Topoisomerase II requires ATP binding in order to
form a "closed protein clamp" on the double helix and promote the
catalytic DNA strand passage reaction (2, 11, 12, 65, 68, 69). Furthermore, the enzyme establishes two distinct DNA
cleavage/religation equilibria during its catalytic cycle (one prior to
and one following the double-stranded DNA passage event), and drugs
have been shown to affect both (2, 11, 12, 54, 70-72). Since the
pre-strand passage DNA cleavage activity of top2G437S was
hypersensitive to anticancer agents, the sensitivity of the enzyme to
drugs was also examined in the presence of ATP.
Remarkably, the drug hypersensitivity observed in the absence of a
nucleoside triphosphate was lost in the presence of ATP (Fig.
8). In fact, the sensitivity of top2G437S
toward CP-115,953, amsacrine, etoposide, and ellipticine was similar to
that displayed by wild-type topoisomerase II. The drug concentrations
utilized for this study represented those that produced the highest
levels of scission without generating multiple DNA breaks per plasmid. As seen in the inset, a wild-type DNA cleavage stimulation
also was observed at sub-saturating concentrations of amsacrine (the drug that produced the highest level of hypersensitivity in the absence
of ATP). Thus, top2G437S appears to be the first reported mutant type
II topoisomerase that displays an ATP-dependent drug phenotype.
Basis for the in Vivo Drug Resistance Conferred by
top2G437S--
Although the drug hypersensitivity described in the
absence of ATP is lost in the presence of this nucleoside triphosphate, the wild-type profile observed with ATP in vitro is
insufficient to explain the resistant phenotype conferred to yeast
cells carrying top2G437S. Therefore, it is unlikely that drug
resistance in vivo results from altered interactions between
the mutant enzyme and topoisomerase II poisons.
Decreased levels of topoisomerase II activity have been linked to
cellular drug resistance (1, 2, 30, 31, 33, 34, 36, 39, 43, 52). As
described above, the catalytic activity of purified top2G437S was
indistinguishable from that of the wild-type enzyme. However, since the
in vitro activity assays were carried out at 28 °C and
the mutant selection, screening, and cytotoxicity protocols were
performed at 34 °C (necessitated by the requirement for a
temperature-sensitive chromosomal TOP2 allele), it is
possible that the Gly
In this experiment, the mutant and wild-type enzymes were incubated at
34 °C in the absence of ATP and DNA. At various times, ATP and
negatively supercoiled plasmid were added, and DNA relaxation was
monitored at 28 °C. As seen in Fig. 9, the
thermal stability of top2G437S at 34 °C was markedly decreased
compared with that of wild-type topoisomerase II. While the
t1/2 of the wild-type enzyme was estimated to be 60 min, that of the mutant enzyme was ~2.5 min. A similar
t1/2 was observed when top2G437S was incubated at 34 °C in the presence of ATP (not shown). Thus, under the
temperature conditions utilized for characterization of physiological
drug sensitivity, the cellular activity of plasmid-encoded top2G437S would be expected to be significantly lower than that of the wild-type control enzyme.
The decreased thermal stability of top2G437S raises the possibility
that the Gly
Two conclusions can be drawn from the above experiments. First, beyond
its effects on drug-enzyme interactions, the Gly Alterations in the sensitivity of topoisomerase II to anticancer
agents can profoundly affect the efficacy of chemotherapeutic regimens
(4, 36, 44-55). In an effort to further our understanding of the
mechanistic basis for altered drug sensitivity, a mutant yeast type II
enzyme was selected that conferred cellular resistance to several
structurally distinct topoisomerase II poisons. The resistance
conferring mutation was identified as a single base change that
converted Gly top2G437S exhibited two properties that were not predicted by the
cellular resistance phenotype. First, in the absence of ATP, the enzyme
was hypersensitive to all of the topoisomerase II poisons examined.
This hypersensitivity correlated, at least for amsacrine and
CP-115,953, with an increased affinity for drug. Second, in the
presence of ATP, top2G437S lost its drug hypersensitivity and displayed
wild-type sensitivity toward the same topoisomerase II poisons.
Gly437, which is conserved in a number of eukaryotic
species (4), is located in the GyrB domain of topoisomerase II in a
tight loop that connects Clearly, the resistance phenotype of yeast cells harboring top2G437S
cannot be explained by alterations in enzyme-drug interactions. However, since the "wild-type" and "mutant" yeast strains are isogenic except for the replacement of TOP2 with
top2G437S, it is reasonable to assume that the Gly Topoisomerase II is the primary cytotoxic target for a number of widely
prescribed anticancer drugs (1-9, 56). Understanding the mechanistic
basis for resistance/hypersensitivity to these agents is critical to
the continued development of successful chemotherapeutic regimens. This
study highlights the need to analyze both the biochemistry and
the physiology of topoisomerase II mutants with altered drug
sensitivity in order to define the mechanistic bridge that links enzyme
function to cellular phenotype. Assumptions made on the basis of
either one alone may lead to erroneous conclusions and obscure the
actual mechanism that underlies drug resistance or
hypersensitivity.
We are grateful to Dr. Sarah H. Elsea for
preliminary work on this project; to Drs. Paul S. Kingma and D. Andrew
Burden for helpful discussions; and to Dr. Paul S. Kingma and John M. Fortune for critical reading of the manuscript.
*
This work was supported by Grants GM33944 (to N. O.),
CA21765 (to J. L. N.), and CA52814 (to J. L. N.) from the National
Institutes of Health, Grant NP-812 from the American Cancer Society (to
N. O.), and support from the American Lebanese Syrian Associated
Charities (to J. L. N.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Trainee under National Institutes of Health Grant 5 T32 CA09582.
**
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; E-mail: osheron{at}ctrvax.vanderbilt.edu.
The abbreviation used is:
kb, kilobase
pair.
A Mutant Yeast Topoisomerase II (top2G437S) with Differential
Sensitivity to Anticancer Drugs in the Presence and Absence of ATP*
§,,,
**
Biochemistry and
Medicine (Oncology), Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146 and the ¶ Department of
Molecular Pharmacology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38101
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Ser (top2G437S). Purified
top2G437S displayed wild-type enzymatic activity in the absence of
drugs but exhibited two properties that were not predicted by the
cellular resistance phenotype. First, in the absence of ATP, it was
hypersensitive to all of the drugs examined and hypersensitivity
correlated with increased drug affinity. Second, in the presence of
ATP, top2G437S lost its hypersensitivity and displayed wild-type drug
sensitivity. Since the resistance of yeast harboring top2G437S could
not be explained by alterations in enzyme-drug interactions,
physiological levels of topoisomerase II were determined. The
Gly437 Ser mutation reduced the stability of
topoisomerase II and decreased the cellular concentration of the
enzyme. These findings suggest that the physiological drug resistance
phenotype conferred by top2G437S results primarily from its decreased
stability. This study highlights the need to analyze both the
biochemistry and the physiology of topoisomerase II mutants with
altered drug sensitivity in order to define the mechanistic bridge that
links enzyme function to cellular phenotype.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
20 °C. Therefore, enzymes were
aliquoted and stored at 80 °C and utilized for assays within 2 days of their transfer from 80 to 20 °C.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Drug resistance profile of yeast cells
carrying the mutant top2G437S or wild-type TOP2
allele. The effects of CP-115,953, amsacrine, etoposide, or
ellipticine on the survival of cells expressing either wild-type
(WT, closed circles) or mutant (G437S,
open circles) topoisomerase II are shown. Data are plotted
as relative percent 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 to 2000% in the absence of drug.
Results are representative of two independent experiments, each carried
out in triplicate.
Ser mutation had no effect on the overall
catalytic activity of top2G437S. Indeed, over a wide range of enzyme
concentrations, rates of DNA relaxation catalyzed by top2G437S were
indistinguishable from those of the wild-type enzyme (Fig.
2).
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Fig. 2.
DNA relaxation activity of yeast wild-type
topoisomerase II and top2G437S. Results of DNA relaxation assays
utilizing purified enzymes are shown. Closed circles,
wild-type topoisomerase II (WT); open circles,
top2G437S (G437S). Data represent the averages of four
independent experiments. An ethidium bromide-stained agarose gel
showing a representative titration of top2G437S is shown at top.
Lanes 1-8, the concentration of topoisomerase II was 0.12, 0.16, 0.20, 0.26, 0.40, 0.80, 1.6, and 3.1 nM, respectively;
lane 9, DNA standard (std). The positions of
negatively supercoiled (FI) and nicked (FII)
plasmid molecules are indicated; relaxed plasmid products migrate
between these two forms.
(CEM/VM-1-5, Arg450 Gln (46, 67); HL60/AMSA,
Arg487 Lys (44)) display either an altered affinity for
ATP or an altered response to anticancer drugs in the presence of the
nucleoside triphosphate. (The positions of these mutated residues
correspond to residues 439 and 476 in the sequence of S. cerevisiae topoisomerase II (4).) Contrary to the mutant human
enzymes, top2G437S displayed a requirement for ATP that was similar to
that of wild-type topoisomerase II (Fig.
3).
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Fig. 3.
Requirement of wild-type topoisomerase II and
top2G437S for ATP. The effects of ATP concentration on DNA
relaxation activity are shown. Results with the wild-type
(WT) and mutant (G437S) enzymes are denoted by
the closed and open circles, respectively. Data
represent the averages of two independent experiments.
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Fig. 4.
DNA cleavage activity of wild-type
topoisomerase II and top2G437S. DNA cleavage activity was
determined over a range of topoisomerase II concentrations. Results
with the wild-type (WT) and mutant (G437S)
enzymes are denoted by the closed and open
circles, respectively. Data represent the averages of two
independent experiments.
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Fig. 5.
Effects of topoisomerase II poisons on the
pre-strand passage DNA cleavage activity of top2G437S. Assays were
carried out in the absence of an ATP cofactor. The effects of
CP-115,953, amsacrine, etoposide, or ellipticine on the pre-strand
passage DNA cleavage/religation equilibrium of wild-type
(WT, closed circles) or mutant (G437S,
open circles) topoisomerase II are shown. The relative level
of DNA cleavage in the absence of drug was set to 1.0. Data represent
the averages of two to five independent experiments.
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Fig. 6.
Effects of CP-115,953 and amsacrine on DNA
religation and presteady-state DNA cleavage mediated by wild-type
topoisomerase II and top2G437S. Assays were carried out in the
absence of an ATP cofactor. Panels A and B show
the effects of 100 µM CP-115,953 and amsacrine,
respectively, on DNA religation (squares) mediated by
wild-type topoisomerase II (WT, closed symbols) and
top2G437S (G437S, open symbols). The level of DNA cleavage
prior to induction of religation was set to 100%. Panels C
and D show the effects of 100 µM CP-115,953
and amsacrine, respectively, on presteady-state DNA cleavage
(circles) mediated by wild-type topoisomerase II (WT,
closed symbols) and top2G437S (G437S, open symbols).
Data were plotted relative to the maximal level of DNA cleavage (which
was normalized to 100% in all cases).
1 mM drug in all cases; higher
concentrations were inhibitory. As determined from the drug
concentration required to stimulate one-half maximal DNA scission, the
potency of both drugs against top2G437S was 2-3-fold higher than that
for wild-type topoisomerase II. Thus, it appears that the conversion of
Gly437 to a Ser yields an enzyme that has a higher kinetic
affinity for these topoisomerase II poisons. In addition, the mutant
displayed an efficacy (determined by the maximal level of cleavage
induced) for amsacrine that was ~2-fold greater than wild type. This
suggests that once complexed with the mutant enzyme, amsacrine
stimulates higher levels of DNA scission than normally induced in the
wild-type topoisomerase II·DNA·drug complex.
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Fig. 7.
Dose-response curves for the enhancement of
pre-strand passage DNA cleavage by CP-115,953 and amsacrine. The
left panel shows dose-response curves for CP-115,953, and
the right panel shows dose-response curves for amsacrine.
Data for wild-type topoisomerase II (WT, closed
circles) and top2G437S (G437S, open circles)
represent the averages of two independent experiments. Results are
plotted as the percent maximal DNA cleavage achieved by the wild-type
enzyme to allow direct comparison between the wild-type and mutant
enzymes.
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Fig. 8.
Effects of topoisomerase II poisons on the
DNA cleavage activity of top2G437S in the presence of ATP. The
effects of drugs on DNA cleavage mediated by wild-type (closed
bars) or mutant (open bars) topoisomerase II in the
presence of ATP (1 mM) are shown. Assays were carried out
in the presence of 35 µM CP-115,953, etoposide, or
ellipticine, or 10 µM amsacrine. Inset,
relative DNA cleavage mediated by wild-type topoisomerase II
(closed circles) or top2G437S (open circles) in
the presence of 1 mM ATP and 1-10 µM
amsacrine (AMSA). For all data, the relative level of DNA
cleavage in the absence of drug and ATP was set to 1.0. Data represent
the averages of two to four independent experiments. Standard
deviations are indicated.
Ser mutation at position 437 generated an
enzyme that was temperature-sensitive in nature. Therefore, the thermal
stability of top2G437S was characterized at 34 °C.
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Fig. 9.
Thermal stability of wild-type topoisomerase
II and top2G437S at 34 °C. The effects of incubation at
34 °C on the catalytic activity of wild-type (WT, closed
circles) and mutant (G437S, open circles) topoisomerase
II are shown. The relative level of DNA relaxation at incubation
time = 0 was set to 100%. Data represent the averages of two to
four independent experiments.
Ser mutation may reduce the general stability of this
enzyme. This is supported by the finding that 1) top2G437S displayed a
lifetime at 20 °C that was considerably shorter (on the order of
days, not shown) than that of the wild-type enzyme; and 2) the yield of
mutant enzyme was ~2-fold lower than that of wild-type topoisomerase
II when purified from an equivalent weight of yeast cells. Since a
destabilizing mutation might decrease cellular concentrations of
top2G437S even at the permissive temperature, physiological levels of
the mutant polypeptide were determined at 25 °C. As characterized by
immunoblot analysis, the steady-state concentration of plasmid-encoded
top2G437S polypeptide was ~10% that of wild-type topoisomerase II
expressed from the same plasmid system (Fig.
10).
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Fig. 10.
Physiological levels of plasmid-encoded
wild-type topoisomerase II and top2G437S in yeast cells.
Steady-state levels of wild-type topoisomerase II (WT, left)
and top2G437S (G437S, right) polypeptide were determined by
immunoblot analysis of whole cell homogenates. The relative level of
wild-type topoisomerase II in 10 mg/ml whole cell homogenate was set to
100%. Immunoblots (shown at top) were quantitated by
scanning densitometry (shown at bottom).
Ser mutation at
position 437 leads to temperature-sensitivity and an apparent
destabilization of topoisomerase II. This is despite the fact that the
mutation does not affect the catalytic activity of the enzyme at the
permissive temperature. Second, the basis for the drug resistance of
yeast cells harboring top2G437S appears to be related to the
instability of the mutant enzyme (and the consequent loss of active
enzyme from the cell), rather than its altered interaction with
topoisomerase II poisons.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Ser at amino acid position 437 (top2G437S). This
point mutation did not affect the intrinsic ability of the purified
enzyme to relax or cleave its DNA substrate (at 28 °C) in the
absence of anticancer drugs.
sheet B'1 to helix B'1 in the
crystal structure of the yeast enzyme (73). The residue is located
~23 Å from the hydroxyl group of the active site Tyr (residue 783), diagonally across the cleft that is proposed to bind the DNA cleavage helix (73, 74). Given the distance between the mutated residue and the
point of DNA cleavage, it is unlikely that either Gly or Ser at
position 437 makes intimate contact with a bound drug molecule.
However, the fact that position 437 lies along the proposed path of the
DNA cleavage helix strongly suggests that alterations at this residue
could have ramifications for drug action (73). Finally,
Gly437 is located in the region of the GyrB domain that is
proposed to undergo a substantial conformational change upon ATP
binding and the subsequent closing of the N-terminal protein gate
(73-76). It is this structural rearrangement that may mask the effects of the Gly Ser mutation and ultimately restore wild-type drug sensitivity to top2G437S in the presence of ATP.
Ser
mutation in topoisomerase II is the root cause of the cellular
phenotype. As determined by in vitro and in vivo
experiments, the substitution of Ser for Gly at amino acid 437 reduces
the stability of topoisomerase II and decreases the cellular
concentration of the enzyme. Therefore, it is proposed that the
physiological drug resistance phenotype associated with top2G437S
results primarily from its decreased stability.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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