ICANCKR RKSHARCH 5.1. 3.WI-3596. August 1. 1993]
Use of Yeast in the Study of Anticancer Drugs Targeting DNA Topoisomerases:
Expression of a Functional Recombinant Human DNA Topoisomerase
Ila in Yeast1
Robin A. Wasserman, Caroline A. Austin,2 L. Mark Fisher, and James C. Wang3
Department of Biochemistry {¡nelMolecular Bioloitv. Han-ard University. Camhrid^e. Massachusetts 021.W ¡R.A, \V., J. C. W.I, ami Division of Biochemistry.
Cellular and Molecular Sciences. St. George's Hospital Medical School, Cratimer Terrace, London SWI7 ORE. Enfianti ¡C.A. A.. L. M. F.]
ABSTRACT
A plasmid was constructed for the expression of human DNA topo
isomerase Ila in yeast from a galactose-inducible promoter of the yeast
GALI gene. Expression of a recombinant human enzyme, in which the
first 28 of the 1531 codons of human DNA topoisomerase Ha were re
placed by the first five codons of yeast DNA topoisomerase II, was shown
to rescue the lethal phenotype of thermal sensitive yeast DNA topo
isomerase II mutants at 35°C.Purification of the human enzyme overexpresscd in yeast yielded a single polypeptide with an apparent mass of 170
kDa, and the properties of the purified recombinant enzyme were found to
be the same as those reported for human DNA topoisomerase Ha purified
from HeLa cells. Studies with the anticancer drug amsacrine indicated
that the human enzyme, either inside yeast cells or in its purified form, is
a target of the drug; inhibition of the purified enzyme by teniposide
(VM-26) and merbarone was also demonstrated.
These studies demon
strate that yeast strains expressing human DNA topoisomerase Ila pro
vide a convenient system for studying drugs targeting the enzyme: unlike
mammalian systems, potential complications due to the presence of hu
man DNA topoisomerase ll/i can be eliminated in this system. Overex-
Department
of
expressed in yeast from cloned human TOPI cDNA. and appropriate
yeast strains expressing human instead of yeast DNA topoisomerase I
have also been constructed to provide a convenient system for mutational studies, drug screening, and analysis of the mechanisms of cell
killing by drugs acting on the human enzyme (23. 24). In the present
work, we describe the expression of a functional recombinant human
DNA topoisomerase Ila in yeast and demonstrate that, similar to the
case of human DNA topoisomerase I. yeast expressing human DNA
topoisomerase Ha is suitable for studies of drugs targeting the en
zyme. Overexpression of human DNA topoisomerase Ila in yeast and
purification of the enzyme to near homogeneity have also been
achieved. The catalytic properties of the purified recombinant human
enzyme appear to be indistinguishable from those of DNA topo
isomerase Ha purified from human cells (25).
MATERIALS
AND METHODS
Chemicals and Drugs. Merhurone. amsacrine. and oAMSA were obtained
from the Drug Synthesis and Chemistry Branch. Developmental Therapeutics
Program. Division of Cancer Treatment. National Cancer Institute. Teniposide
was kindly provided by Dr. Leroy F. Liu. Robert Wood Johnson Medical
School. All other reagents viere obtained from commercial sources.
Plasmids and Yeast Strains. The construction of pBS-hTOP2. which con
pression of human DNA topoisomerase Ha in yeast also provides a con
venient source of the enzyme for in vitro studies.
INTRODUCTION
With the identification of DNA topoisomerases as the targets of a
variety of therapeutics including anticancer drugs, structural and
mechanistic studies of these enzymes, especially human DNA topo
isomerases, have assumed added significance (1-5). Three human
DNA topoisomerases are presently known. Human DNA topo
isomerase I, the target of an antitumor plant alkaloid camptothecin
(6-9), is a type I DNA topoisomerase characterized by its transient
breakage of DNA strands one at a time. Human DNA topoisomerases
Ila and Ilßare two closely related members of the type II DNA
topoisomerase family (10-12). a class of structurally and evolutionarily related enzymes characterized by its transient breakage of pairs
of strands in duplex DNA ( 13-15). Studies with mammalian cell lines
and purified enzymes indicate that a large number of anticancer drugs,
including mAMSA.4 VM-26. VP-16. merbarone. and bis(2,6-dioxopiperazine) derivatives, act on one or both of the a and ßvariants of
human DNA topoisomerase II (1-5. 16-1X).
The genes TOPI. TOP2a. and TOP2ßencoding the human DNA
topoisomerases have been cloned and mapped to chromosomal loca
tions 20ql2. l7q2l-22, and 3p24. respectively (19-22). We have
previously shown that functional human DNA topoisomerase 1can be
Received 3/29/9.?: accepted 5/20/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore he hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by grants from the NIH (CA47958 to J. C. W.I and the Cancer Research
Campaign (to L. M. F.l.
: Present address: Department of Biochemistry and Genetics. Medical School. Cathe
rine Cookson Building. University of Newcastle. Newcastle Upon Tyne. NE2 4HH En
gland.
1To whom requests for reprints should be addressed.
4 The abbreviations used are: mAMSA. amsacrine: VM-26. tcniposide: VP-16. ctoposide; cDNA. complementary DNA: oAMSA. ortho analogue of amsacrine: PCR. polymerasc chain reaction.
tains the entire coding sequences of human TOP2a. has been reponed previ
ously (24); YEpTOP2-PGAL I. a YEp24 derivative for the expression of yeast
TOP2 gene from a galactose-inducible promoter of the yeast GALI gene, and
the plasmid ScTOP2-myc, derived from YEpTOP2-PGALI.
have been de
scribed (26. 27). The splicing of the first five codons of yeast TOP2 to the 29th
codon of human TOP2a was accomplished by the application of the PCR. A
pair of primers was used to synthesize a human TOP2a cDNA segment from
the 29th codon to the Hin<\\\\ site after nucleotide 1520 (Refs. 21 and 28;
Footnote 5). The 5' primer had the sequence 5' CGGGTACCGGTA [TCT GTT
GAA AGA ATC TAT].V. in which the underlined sequence is an Agel restric
tion site and the sequence in brackets corresponds to that of human TOP2a
starting at codon 29; the 3' primer was complementary to nucleotides 1517 to
1536 of the human TOP2« cDNA sequence containing a WmdIII restriction
site. The Age\ site in the primer was used to facilitate the joining of the
PCR-svnthesi/ed fragment to the A<>e\site at the beginning of the yeast TOP2
coding region. Multiple rounds of routine cloning were then carried out to
construct YEpWOB6 from this segment and other segments derived from
YEpTOP2-PGALl. ScTOP2-myc. and pBS-hTOP2 (see Fig. 1 legend in -Resuits"). YCpDEDWOBlO was derived from YCpPDEDlT2 (29) by the dele
tion of an SpeI site between the TAG stop of the yeast TOP2 gene and the
unique Sfil site downstream of it.
Yeast strains CHI 106 MATa acìi-2-IOIIi'ti2\l. l\s2-8(H irpIM nra3-52
top2-4. and CHI585 MATa U-u2\l iiyj/AiW ura3-52 his3\200 were kindly
provided by Dr. C. Holm. Harvard University: strains JN394t2-4 MATa ISE2
imi3-52 H>p2-4 nu/52::LEU2 (29). SDÌ
17 MATa ude2 Ieu2 his? top2-I rr/>l
uni.i-52. and SDÌ
19 MATa to/il-l top2-l tipi uni3-52 (30) have been de
scribed previously. BCY123 MATa ¡H'p4::HIS3 prhi::l.EU2
barl::HISG
lys2::GALI/l()-GAL4
nini utlc2 rr/,1 um3 /i/.v.i Ieu2-3.ll2. a protease-defis We have confirmed the findings in Ret. 28 that the human TOP2 cDNA nucleotide
sequence reported in Ref. 2 I missed two Ts (after nueleotides 362 and 376) and an A (after
nucleotide 372) and that nucleotide 3097 is a T rather than C (M. Tsai-Pflugfclder and J.
C. Wang, unpublished results); nucleotide numbers in this work were those in Ref. 21
corrected for the missini: nueleolidcs.
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USF. OF YF.AST IN THE
dent strain with a GALI promoter-linked
STUDY
OF ANTICANCF.R
DRUGS
GAL4 gene, was provided by Dr.
12.07/0.00
Neal Lue, Harvard University. Strain JCW2 is a ura3 derivative of strain
TG205 \topl top2-4 (31) isolated by Dr. M-A. Bjornsti, Thomas Jefferson
University Medical School, and strains JCW26 wp2-4 and JCW28 \wpi
tor>2-4 were constructed from their TOP' parent CHI585 by targeted gene
replacement or deletion.11
pBR322
Methods. Assays of decatenation of kinetoplast DNA and relaxation of
supercoiled DNA were carried out as described (25), with minor modifications
as specified in "Results" and in the appropriate figure legends. Protein con
centrations
were measured
spectrophotometrically
using
Coomassie-Plus
(Pierce); bovine serum albumin was used as a calibration standard. The DNA
concentration was calculated from its optical absorbance at 260 nm. Viability
of yeast strains harboring various plasmids was tested by imprinting serially
diluted cultures in microtiter plate wells onto agar plates of different growth
media, using a sterili/.ed aluminum block with an array of cylindrically shaped
prongs that could be dipped into the wells. The agar plates were incubated at
25°Cor 35°C.as specified.
2U
RESULTS
TAA (stop)
4.52
Expression of a Functional Human DNA Topoisomerase Ha in
the Yeast Saccharomyces cerevisiae. Fig. 1 depicts YEpWOBó, a
multicopy yeast plasmid, for the expression of human DNA topoisomerase Ila. This plasmid is closely related to YEpTOP2-PGALl,
which was constructed previously for the expression of yeast TOP2
from a galactose-inducible and glucose-repressible promoter of the
yeast GAL! gene. Inspection of amino acid sequence alignments of
various type II DNA topoisomerases (32) suggested that the human
coding sequences might be functionally expressed in yeast by fusing
the first five codons of yeast TOP2. which contains an Age\ restriction
site, to the 29th codon of human TOP2a: YEpWOB6 was constructed
to express such a recombinant enzyme.
Results illustrated in Fig. 2 show that the recombinant human
enzyme expressed from YEpWOBó is functional inside yeast cells. In
these experiments, several S. cerevisiae ura3 strains with top2 ts or
both topi and top2 is mutations were transformed to URA * with
YEpWOBó or YCpDEDWOBlO, a single-copy control plasmid con
structed for the expression of yeast TOP2 gene from a constitutive
yeast DED1 gene promoter (29). Untransformed or transformed
top2-I or top2-4 strains grew normally at 25°C,a permissive temper
Eco R1
6.70
Eco R1
5.47
Fig. I. Construction of YEpWOB6 for the expression of human DNA topoisomerase
lia in yeast. The 0.9-kilobase Sail to Agel fragment containing the first 5 codons of yeast
TOP2 downstream of the CALI promoter was derived from YEpTOP2-PGALl.
previ
ously constructed for the overexpression of yeast TOP2 in yeast (32). The filth codon of
yeast TOP2 was spliced to the 29th codon of human TOP2a at the Agel site through the
use of a PCR-generated human TOP2a cDNA fragment (see "Materials and Methods").
The human TOP2a coding sequences ending at a TAA translation stop and approximately
0.95-kilobases of a 3' untranslated region ending at an LcttRl linker were derived from the
full-length human TOP2a cDNA clone pBS-hTOP2 (24). The short segment from this
EcoRl site to the Xlio\ site was derived from the polylinker region of pBluescript (Stralagene): the short segment from the Xhol to the Sintil site, with a nucleotide sequence of
S'-CTCGAGGCATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATTAATTCCTGCAGCCCGGG-3'.
is a remnant of the plasmid ScTOP2-Myc (27). The 7.09-kilobase
segment from Snuil to Still, which contains the URA3 marker gene, the yeast 2-jim
plasmid replication origin, and the ß-lactamaseand replication origin of Escht'richiu ctiii
pBR322. was derived from YEp24 (39). Restriction sites in boldface are unique in the
plasmid.
25°C
35°C
ature for these temperature-sensitive alÃ-eles(Fig. 2, left). As illustrated
in Fig. 2, right, no growth was observed in untransformed strains or
strains transformed with the URA3 + top2~ control plasmid YCpSO at
35°C,a nonpermissible temperature for the top2 alÃ-elesused; trans
formants of the control yeast URAJ ' TOP2 * single-copy plasmid
YCpDEDWOBlO, on the other hand, grew well under all conditions.
When human DNA topoisomerase Ha instead of the yeast enzyme
was expressed in the top2 ts strains JCW26 top2-4 and SDÌ
17 top2-l.
the lethal phenotype of the parent strains at 35°Cwas again rescued.
It was noted, however, that in medium containing galactose instead of
glucose or raffinose. the YEpWOBó transformants of these strains
reproducibly gave smaller colonies than the corresponding YCpDED
WOBlO transformants (data not shown); this was probably due to a
high cellular concentration of the human enzyme in the galactose
medium. Previously, we observed that galactose induction of yeast
DNA topoisomerase II from a GALI promoter-linked yeast TOP2
gene on the multicopy plasmid YEpTOP2-PGALl was toxic to cells
harboring the plasmid (33).
Fig. 2 also shows that transformation of yeast mutants defective in
both DNA topoisomerases I and II by the control yeast TOP2+ plas
mid YCpDEDWOBlO rescued their lethality at 35°C,which is ex
pected from the published results that topi null mutants of yeast are
viable (31, 34-36). When the strain JCW28 \topl top2-4 was transh R. A. Kim, J. Roca, and J. C. Wang, unpublished results.
Fig. 2. Viability of various yeast strains untransformed or transformed with plasmids in
a glucose medium at 25°C(left) or 35°C(right}. The strains and the plasmids they
harbored were as follows: first row, from left to right: JCW26 ti>p2-4. none: JCW26.
YCp5(); JCW26. YCpDEDWOBlO; JCW26. YEpWOBô: JCW28 AM/J/ Mp2-4. none;
JCW28, YCp5(); JCW28. YCpDEDWOBlO; JCW28. YEpWOB6. Fnurlh HIM-,from left
to right: SDII? top2-l. none; SDÌ
17, YCpSO: SDÌ
17. YCpDEDWOBlO; SDÌ
17. YEpWOB6; SDÌ
19 t,,pl-l tnp2-l. none; SDÌ
19, YCpSO: SDÌ
19. YCpDEDWOBlO: SDÌ
19.
YEpWOBò. Each sample well of a microtiter plate contained 200 /il of water, and the first
and fourth row wells were inoculated with yeast cells from single colonies: the suspen
sions were then serially diluted twice, hy 10-fold each time, into the two rows of sample
wells below the inoculated wells; imprints of the 4K cultures were made on two YPDA
plates (40). The plates were incubated separately at 25°Cand 35°Cand then photo
graphed. See Table I for results from this and similar experiments.
formed with the human DNA topoisomerase II expression plasmid
YEpWOBó. poor growth was observed at 35°Cin rich media con
taining glucose, raffinose. or galactose, and no growth was evident in
minimal medium containing glucose: similar observations were made
with YEpWOBó transformants of SDÌ
19 lopl-ìtop2-ì(Fig. 2, right.
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USE OF YEAST IN THE STUDY
OF ANTICANCER
Table I Cnm-th of top2 is and topi Iop2 is yeast strains untransformed
DRUGS
or transformed with various pltismids at 35°C
See the legend to Fig. 2 for experimental details. No growth is indicated by a minus sign, and poor to proliferous growth by +/-. +. ++, and +++ signs. In the example shown
in Fig. 2, right. +++ was assigned to growth of all YCpDEDWOBK) transformants and the YEpWOB6 transformant of strain JCW26. ++ to SDÌ
17 bearing YEpWOBfi. + to strains
JCW28 hearing YEpWOBn. and +/- to SDÌ
19 bearing YEpWOB6. Agar plates containing various growth media were prepared according to Ret'. 40; -uracil + dextrose was minimal
medium lacking uracil and containing dextrose as the carbon source. YPA + dextrose was the same as YPDA. and the other media were YPDA with dextrose substituted by the sugar
specified.
Media
Plasmid
Strain
>p2/.v
JCW26(Jo/>2-/l
SDÌ
17 (lop2-D
topi top2 ts
JCW2X iStopl. top2^t)
SDÌ
19 (tiipl-l. tnp2-l)
-Uracil + dextrose
YPA + dextrose
YPA + raffinose
YPA + galactose
None
YCpSO
YCpDEDWOBIO
YEpWOBo
None
YCpSO
YCpDEDWOBIO
YEpWOBo
None
YCpSO
YCpDEDWOBIO
YEpWOBo
None
YCpSO
YCpDEDWOBIO
YEpWOB6
and results not shown). The results illustrated in Fig. 2 and those from
similar experiments are summarized in Table 1.
In agreement with the results described above, it was observed
previously that expression of the human TOP2 gene directed by the
yeast TOPI gene promoter also complemented the top2 defect in yeast
strains CHI 106 top2-4 or SDÌ
17 top2-ìbut failed to rescue the
lethality of the double topoisomerase mutant JCW2 \topl iop2-4 at
35°C.7
was achieved by replacing the growth medium by one containing
galactose, following the procedure described in Ref. 33. Overexpres
sion of the human enzyme was also enhanced by the use of strain
BCY123. which contains a GALI promoter-linked GAL4 gene; when
inactive yeast top2-4 mutant enzyme is not responsible for cell killing
by amsacrine, and therefore the amsacrine sensitivity of YEpWOBo
transformants of JN394t2-4 at 35°Cis due to the presence of the
revealed that this band was recognized by the rabbit antibodies (Fig.
3/j). No band was detected when rabbit antibodies raised against yeast
DNA topoisomerase II were used, indicating that the purified enzyme
was essentially free of host DNA topoisomerase II (result not shown).
From 2 liters of yeast culture, approximately 0.7 mg of purified human
enzyme were obtained.
Properties of Purified Recombinant Human DNA Topoi
somerase Ha. Relaxation of supercoiled DNA by the purified recombinant human DNA topoisomerase Ha showed that the activity of
the purified fraction was strictly dependent on the presence of Mg(II)
and ATP; thus, the purified enzyme was free of contaminating yeast
DNA topoisomerase I, which is capable of relaxing supercoiled DNA
in media without Mg(II) and ATP. By varying one component at a time
of an assay buffer containing 50 mw Tris-HCl, pH 7.4. 100 nui KC1.
10 nui MgCl2, 5 mm 2-mercaptoethanol. 0.5 mM EDTA. and 300
fig/ml of bovine serum albumin, the optimal concentrations of MgCI-,
and KCI at 37°Cwere observed to fall in rather broad ranges of 2 to
BCY123 cells harboring YEpWOBo were placed in a galactose me
dium, the expression of GAL4 protein, which has a key role in the
induction of the GALI promoter, was also elevated, and this increase
in turn maximized the induction of the GALI promoter-linked human
Drug Sensitivity of Yeast Cells Expressing Human DNA Topoi
DNA topoisomerase Ila.
somerase Ha. To test whether the recombinant human DNA topo
Purification of the recombinant human enzyme was carried out as
isomerase Ha inside yeast can serve as a target of the anticancer drugs,
described (33) with modifications as described in the legend to Fig. 3.
strain JN394t2-4 ura3-52 ISE2 top2-4 rad52::LEU2 was transformed
Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel identi
with YEpWOBo and tested for sensitivity to amsacrine. At 25°C.
fied a Coomassie blue-stained 170-kDa protein band in extracts of
JN394t2-4 was found to be sensitive to 10 p.g/ml of amsacrine. indi
galactose-induced BCY123 cells harboring YEpWOBo (Fig. 3a, Lane
cating that the endogenous yeast top2-4 mutant enzyme is sensitive to
c). but not in extracts of uninduced cells (Fig. 3o, Lane b). The
the drug at the permissive temperature. At 35°C.JN394t2-4 cells
molecular mass of this galactose-inducible protein is that expected for
harboring YEpWOBo were viable in the absence but not in the pres
the recombinant human enzyme. Purification by polymin-P fractionence of 10 p.g/ml of the drug. This result by itself cannot distinguish
ation (Fig. 3«,Lane d). ammonium sulfate precipitation, and finally
whether the yeast top2-4 mutant enzyme, the recombinant human
phosphocellulo.se chromatography yielded fractions that eluted at a
enzyme, or both were sensitive to the drug. It was found, however, that
KCI concentration around 0.5 M and exhibited a single CoomassieJN39412-4 cells harboring YCpDEDWOBIO derivatives expressing
stained 170-kDa band (Fig. 3«,Lane e). Immunostaining of gel blots
amsacrine-resistant yeast DNA topoisomerase II were viable in the
with human DNA topoisomerase Ha-specific rabbit antibodies and
presence of up to 100 ;u.g/ml of amsacrine at 35°C.I<Because of the
phosphatase-coupled anti-rabbit immunoglobulin G goat antibodies
dominance of amsacrine sensitivity, we conclude that, at 35°C.the
recombinant human enzyme.
Overexpression of Human DNA Topoisomerase Ila in Yea.st and
the Purification of the Human Enzyme. The complementation re
sults described above provide strong evidence that the recombinant
human enzyme is functional in yeast. To study further the properties
of the recombinant human DNA topoisomerase Ila. the enzyme was
overexpressed in yeast and purified to near homogeneity.
A protease-deficient and ura3 yeast strain BCYI23 was trans
formed with YEpWOBo and grown in minimal medium lacking uracil
and containing 2% (w/v) glucose, 3% (v/v) glycerol. and 2% (w/v)
lactic acid. In this medium, expression from the GALI gene promoter
is repressed, and induction of the human enzyme from this promoter
7 C. Kanik-Ennulat and J. C. Wang, unpublished observation.
* R. A. Wasserman and J. C. Wang, unpublished results.
18 triMand 75 to 170 niM. respectively; tests of the effect of pH at the
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USE OF YEAST IN THE STUDY
(a)
a
b
e
d
OF ANTICANCER
DKI'C.S
mixture without the enzyme, and each of these mixtures was then
diluted 10-fold with the same buffer containing 1 HIMATP instead of
500 niM KCI to provide ATP and to reduce at the same time the KCI
concentration to 50 mst. Reactions were incubated at 37°Cfor 20 min.
and each 20-ju.l reaction was terminated by the addition of an equal
volume of 7.5 M ammonium acetate and 10 p,g of tRNA. Following
phenol extraction and ethanol precipitation, the samples were redissolved and analyzed by agarose gel electrophoresis in a Tris-borate-
e
EDTA buffer containing 0.6 jug/ml of ethidium bromide.
As can be seen in Fig. 4. in the 50 RIMKCI assay medium used the
reaction appeared to be processive: both the starting negatively su
percoiled pBR322 DNA and the same DNA after relaxation to com
pletion by the human enzyme migrated as sharp bands in the electro
phoresis buffer containing ethidium. Assuming strict processivity and
random distribution of enzyme molecules on DNA, in an equal molar
enzyme-DNA mixture 64% of the DNA molecules would be relaxed
if all enzyme molecules are active. In the experiment shown in Fig. 4,
more than 649f of the DNA molecules were relaxed at an estimated
enzyme/DNA molar ratio of I (Lane 2). suggesting that within the
experimental errors of the molar concentration estimates the enzyme
was fully active. Relaxation of supercoiled DNA by the human en
zyme is not strictly processive even in the 50 niM KCI low salt
medium used, however, and therefore this experiment probably over
estimated the fraction of active enzyme molecules in the purified
preparation. In one experiment. 1.5 pmol of the dimeric human en
zyme and 0.35 pmol of pBR322 were mixed in the high salt buffer and
diluted to 50 nui KC1, as described above except that ATP was omitted
in the dilution buffer: 0.35 pmol of pUCI9 DNA was then mixed in.
and ATP was added to a final concentration of I HIMto initiate the
relaxation reaction. Both DNAs were found to be completely relaxed
after 20 min at 37°C(result not shown), whereas strict processivity
(b)
Fig. 3. a, patterns of Coomassie blue-stained proteins upon 1'ractionation by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Lane a. molecular mass standards:
ÃŒMne
b. cleared cell lysate from uninduced cells: ÕMHC
c. cleared cell lysate from induced
cells; IMHI'd. fraction alter pulymin-P fraetionation; IMIICe, the peak fraction eluted troni
a phosphocellulose column at a KCI concentration of about 0.5 M. The polymin-P fractionation step described in Ret".33 was modified as follows. The polymin-P precipitate and
Celile slurry were spun in a swinging bucket rotor at 2.500 rpm for 5 min. The pellet was
subject to three cycles tit washing and centritugation. each time the pellet was stirred I r
IO min with Buffer I plus 1?>0HIMKCI (33) before centrifugaron. The washed pellet w s
then extracted twice with Buffer I plus l M KCI. each time followed by centrifugaron
s
described, h. imniunobloumg of purified recomhinanl human DNA topoisornerase I r
fractions after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four conse ulive phosphocellulose peak fractions from each of two different preparations were loaded
on the gel; the ritjiitiHust lane contained prestained protein standards with molecular
masses of 205. 116. 80. and 49.5 kDa.
same temperature showed that, in the range 6.8 to 9. reduction of
relaxation activity was observed only near the high end. Assays of
decatenation of kinetoplast DNA by purified recombinuni human
DNA topoisomerase Ha yielded similar results. These cation concen
tration ranges are not significantly different from those observed for
human DNA topoisomerase 11«purified from HeLa cells (10. 25).
The purified human enzyme was highly active. Assays based on
decatenation of kinetoplast DNA indicated that the specific activity of
the purified enzyme was around 3 X |()5 units/mg. which was com
parable to values reported for fractions purified from HeLu cells (25).
To assess more directly the fraction of active enzyme molecules in the
human enzyme purified from yeast cells, relaxation of supercoiled
DNA by the enzyme under processive conditions was carried out. In
the experiment illustrated in Fig. 4. 2.4 jxg (7 pmol) of the recombi
nant human enzyme were mixed with 5 /ig (1.7 pmol) of predomi
nantly monomeric pBR322 DNA in a buffer containing 50 HIM
Tris-HCl. pH 7.4, 500 nisi KCI, 8 nisi MgCK. 5 HIM2-mercaptoethanol, 0.5 rnw EDTA. and 300 fxg/ml of bovine serum albumin. The
enzyme-DNA mixture was mixed with varying portions of the same
would predict the relaxation of only the first DNA added.
Drug Sensitivity of Human DNA Topoisomerase II«Purified
from Yeast Cells. Protein denaturant-induced cleavage of DNA by
the purified recombinant human enzyme was found to be stimulated
by amsacrine. As illustrated in Fig. 5. upon the addition of sodium
dodecyl sulfate to incubation mixtures containing supercoiled
pBR322, recombinant human DNA topoisomerase Ila. and varying
amounts of amsacrine, the amount of linear DNA was observed to
increase with increasing amounts of amsacrine up to 32 /^ig/ml of the
drug (Lunes 4 to 7). An additional 4-fold increase in drug concentra
tion did not increase the amount of linear DNA further, but relaxation
of the supercoiled DNA by the enzyme was inhibited (compare Lanes
2 and # in Fig. 5). oAMSA was found to stimulate DNA cleavage by
the recombinant human enzyme to a lesser extent relative to amsacrine
itself (compare Lanes 4 to 8 with Lanes 9 to 13 in Fig. 5). although the
«;v/;«-analog
appeared to be a more effective inhibitor of the relax
ation activity of the enzyme (compare Leines 7 and K with Lanes 12
and 13 in Fig. 5). These results are identical to those obtained with
human DNA topoisomerase Ha purified from HeLa cells (37). Ex-
Nicked
Supercoiled
Relaxed
Fig. 4. Relaxation of supercoiled pBR322 DNA by purified recombinant human DNA
topoisomerase Ila. The molar ratios of en/yme to DNA in the incubation mixtures were,
from right to left. 0. 0.6. 0.8. 1.0. 1.2. and 1.4. Electrophoresis of the samples was carried
out in 0.8r/f agarose in 100 mM Tris-honite. 2 tmt EDTA. and 0.6 ^ig/ml of ethidium
bromide.
.3594
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USE OF YEAST IN THE STUDY
1
2
34
5
OF ANTICANCER
6
7
8
DRUGS
9
10
11
12
13
Nicked or
Relaxed
Linear
Supercoiled
Fig. 5. Dependence of the recombinant human DNA topoisomerase Ila-mediated DNA cleavage on amsacrine (Lanes 4 to 8) and its orrto-analogue (Lanes 9 to 13). Sixteen jil
of a mixture containing 5.6 /xg ( 1.9 pmol) of pBR322 DNA in 217 u.1of buffer (see below) were removed for a no-enzyme control, and the remaining solution was mixed with 7 ^il
of a purified fraction containing 2.7 /ig (7.9 pmol) of the recombinant human DNA topoisomerase Ma. Sixteen-^il aliquots of this mixture were placed into each 1.5-ml conical centrifuge
tube, to which 2 fil of H;:O. 30% dimethyl sulfoxide or various concentrations of amsacrine or its ortho analogue in 30% dimethyl sulfoxide. to he specified below, were added. After
mixing and standing at room temperature for several minutes, 2 (¿Iof H) HIMATP were added to each tube. The final reaction mixtures contained 50 m.\i Tris-HCI, pH 7.4. 100 IÕIM
KC1, 10 HIMMgCU, 5 m\i 2-mercaptoethanol. 0.5 msi EDTA. 1 nisi ATP. and 300 fig/ml of bovine serum albumin. The samples were incubated at 37°Cfor IO min. and 4 fil of a mixture
containing 5% sodium dodecyl sulfate and 2 mg/ml of proteinase K were added to each sample. Incubation of the samples at 37°Cwas continued for 30 min. after which each sample
was mixed with 2 jil of a gel-loading stock solution and analyzed by electrophoresis in a 1.0% agarose slab in 100 msi Tris-borate:2 HIMEDTA. Lane I. no-enzyme control: the solvent
or drug added to the reaction mixtures loaded in ÕMttt's2 to 13 was as follows: Lane 2. H^O: Lane 3, dimethyl sulfoxide: Lanes 4 to tf, 0.5. 2. 8. 32. and 128 fig/ml of amsacrine. (final
concentration in reaction mixture), respectively: Lanes 9 to 13. 0.5. 2. 8. 32. and 128 /Ag/ml of oAMSA (final concentration in reaction mixture), respectively.
periments with etoposide (VM-26) and merbarone similarly showed
that inhibition of the recombinant human enzyme by these drugs was
similar to that reported for human DNA topoisomerase lia purified
from HeLa cells (data not shown).
described here for human DNA topoisomerase Ha can be readily
extended to the study of human DNA topoisomerase Ilß.
REFERENCES
I.
DISCUSSION
We describe in the section above the expression of a recombinant
human DNA topoisomerase Ila in yeast. In this enzyme, the first 28
amino acids of the 1531-amino-acid-long wild-type human enzyme
are replaced by the first five amino acids of yeast topoisomerase II.
Tests with two temperature-sensitive yeast iop2 mutants show that
expression of the recombinant human enzyme can rescue the lethal
phenotype of these mutants at 35°C.This complementation strongly
suggests that the human enzyme is catalytically active and biologi
cally functional in yeast. It appears, however, that the human enzyme
is less apt in yeast as the wild-type yeast enzyme; whereas the ex
pression of yeast DNA topoisomerase II in yeast lop! top2 ts strains
restores cell growth at the nonpermissive temperature, cell growth is
suboptimal at the nonpermissive temperature when human DNA topo
isomerase Ila is expressed in the same strains.
Although a high cellular level of the human or yeast type II topo
isomerase is detrimental to yeast cells, overexpression of human DNA
topoisomerase Ila in yeast can be readily accomplished through the
use of an inducible promoter. The yield of the present expression
system, 0.35 mg of purified enzyme per liter of yeast culture, is
adequate for the preparation of the human enzyme for biochemical
and pharmacological studies: the purification procedure is also sim
pler than those for the purification of the enzyme from cell lines (25).
We have demonstrated that human DNA topoisomerase Ha can be
studied as the target of anticancer drugs in yeast. In the case of
amsacrine. the availability of yeast top2 mutants resistant to the drug
has made it possible to show that the human enzyme is its cellular
target. In mammalian cells, the existence of two closely related type
II DNA topoisomerases Ha and Ilßhas complicated pharmacological
studies of drugs acting on such enzymes at a molecular level. The
expression of each of the individual human enzyme variants in yeast
provides a way of studying the effects of drugs on one variant at a
time. We and others have recently cloned and sequenced full-length
human DNA topoisomerase IlßcDNA (12, 38), and the approaches
Potmesil, M., and Ross, W. E. (eds.). First conference on DNA topoisomerase in
cancer chemotherapy. In: NCI Monographs. No. 4. Bethesda: National Cancer Insti
tute. 1987.
Liu. L. F. DNA topoisomerase poisons as antituinor drugs. Annu. Rev. Biochem.. 58:
351-375, 1989.
Andoh. T.. Ikeda. H.. and Oguro. M. (eds.). Molecular Biology of DNA Topoi
somerases and Its Application to Chemotherapy. Proceedings of the International
Symposium on DNA Topoisomerases in Chemotherapy. Nagoya. Japan. 1991. Boca
Raton. FL: CRC Press. 1993.
Liu. L. F. (ed.). DNA Topoisomerases and Their Applications in Pharmacology.
Orlando. FL: Academic Press, in press. 1993.
Drlica. K.. and Franco. R. J. Inhibitors of DNA topoisomerases. Biochemistry. 27:
2253-2259. 1988.
Hsiang. Y-H.. Hen/berg. R.. Hecht. S., and Liu. L. F. Camptothecin induces proteinlinked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem.. 260:
14873-14878. 1985.
Nitiss, J.. and Wang. J. C. DNA topoisomerase-targeting
antitumor drugs can be
studied in yeast. Proc. Nati. Acad. Sci. USA. 85: 7501-7505. 1988.
Eng. W.-K.. Faucette. L.. Johnson. R. K.. and Sternglanz. R. Evidence that DNA
topoisomerase I is necessary for the cytotoxic effects of camptolhecin. Mol. Pharmacol., 34: 755-760. 1988.
Jaxel. C.. Kohn, K. W.. Wani. M. C.. Wall, M. E.. and Polmmier. Y. Structure-activity
study of actions of camptothecin derivatives on mammalian topoisomerase I: evi
dence for a specific receptor site and a relation to antitumor activity. Cancer Res., 49:
1465-1469, 1989.
10.Chung. T. D. Y.. Drake. F. H.. Tan. K. B., Per. S. R.. Crooke, S. T., and Mirabelli. C.
K. Characterization and immunological identification of cDNA clones encoding two
human DNA topoisomerase II isozymes. Proc. Nati. Acad. Sci. USA. 86: 9431-9435.
1989.
Austin. C. A., and Fisher. L. M. Isolation and characterization of a human cDN A clone
encoding a novel DNA topoisomerase II homologue from HeLa cells. FEBS Lett..
266: 115-117. 1990.
Austin. C. A.. Sng. J-H.. Palei. S.. and Fisher, L. M. Novel HeLa topoisomerase II is
the Ilßisoform: complete coding sequence and homology with other type II topoi
somerases. Biochim. Biophys. Acia, 1172: 283-291. 1993.
Hsieh, T.-S. Mechanistic aspects of type-II DNA topoisomerase. In: N. R. Cozzarelli
and J. C. Wang (eds.). DNA Topology and Its Biological Effects, pp. 243-263. New
York: Cold Spring Harbor Laboratory. 1990.
Wang. J. C. DNA topoisomerases. Annu. Rev. Biochem.. 54: 665-697. 1985.
Wang. J. C. DNA topoisomerases: why so many? J. Biol. Chem.. 266: 6659-6662.
1991.
Drake. F. H.. Hoffmann. G. A.. Mong. S-M.. Bartus. J. O.. Hettzberg. R. P.. Johnson.
R. K.. Mattem. M. R., and Mirahelli. C. K. In \iiro and intracellular inhibition of
topoisomerase II by antitumor agent merbarone. Cancer Res.. 49: 2578-2583. 1989.
Tanabe, K.. Ikegami. Y.. Ishida. R.. and Andoh. T. Inhibition of topoisomerase II by
antitumor agents bis(2.6-dioxopiperazine) derivatives. Cancer Res., 5/: 4903—4908.
1991.
IX. Ishida. R.. Miki. T.. Narita. T.. Yui. R., Sato. M.. Utsumi. K. R.. Tanabe. K.. and
3595
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1993 American Association for Cancer Research.
USE OH YEAST IN THE
STUDY
Andoh. T. Inhibition of inlracellular lopoisomerase I] by antilurnor bis(2.6-dioxopipcrazine) derivatives: mode ot cell growth inhibition distinct troni that of cleavable
complex-forming type inhibitors. Cancer Res.. 51: 4909-4916. 1991.
19. D'Arpa. P.. Machlin. P. S.. Ralrie III. H. R.. Rothfield. N. F.. Cleveland. D. W.. and
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Karnshaw. W. C. cDNA cloning ot human DNA topoisomerase I: catalytic activity of
a 67.7-kDa carboxyl-lerminal fragment. Proc. Nati. Acad. Sci. USA. 85: 2543-2547.
19X8.
Juan. C.-C.. Hwang. J.. Liu. A. A.. Whang-Peng. J.. Knutsen. T.. Huebner. K.. Croce.
C. M.. Zhang. H.. Wang. J. C, and Liu. L. F. Human DNA inpoisomera.se I is encoded
by a single-copy gene that maps to chromosome region 2(>q12-13.2. Proc. Nati. Acad.
Sci. USA. US: 8910-8913. 1988.
Tsai-Plluglelder. M.. Liu. L. F.. Liu. A. A.. Tewey. K. M. Whang-Pheng. J.. Knutsen.
T.. Huchncr. K.. Croce. C. M., and Wang. J. C. Cloning and sequencing of cDNA
encoding human DNA topoisomerase II and locali/ation of the gene to chromosome
region l7q2l-22. Proc. Nati. Acad. Sci. USA. 85: 7I77-7I8I.
1988.
Tan. K. B.. Dormán.T. E.. Falls. K. M.. Chung, T. D. Y.. Mirabelli, C. K.. Crooke. S.
T.. and Mao, J. Topoisomerase Ha and lopoisomerase Ilßgenes: characterization and
mapping to human chromosomes 17 and 3. respectively. Cancer Res.. 52: 231-234.
1992.
Bjomsti. M-A.. Benedetti. P.. Vigliami. G. A., and Wang. J. C. Expression of human
DNA lopoisomerase 1 in yeast cells lacking yeast DNA lopoisomerase I: restoration
of sensitivity of the cells to the antnumor drug camplothecin. Cancer Res.. 49:
6318-6323. 1989.
Benedetti. P.. Tsai-Pflugtelder. M.. and Wang. J. C. The use of yeast and yeust strains
expressing human DNA topoisomerases in the study of anlicancer drugs. In: T.
Tsuruo. M. Ogawa. and S. K. Carter (eds.l. Bristol-Myers-Squibh Symposium on
Cancer Research. Vol. 14. pp. 129-145. San Diego. CA: Academic Press. 1992.
Miller. K. G.. Liu. L. F.. and Englund. P. T. A homogeneous type II DNA topo
isomerase from HeLa cell nuclei. J. Biol. Chem.. 25(5: 9334-9339. 1981.
Giaever, G. N.. Snyder. L.. and Wang. J. C. DNA supercoiling in vivo. Biophys.
Chem.. 2V: 7-15. 1988.
Lindsley. J. E., and Wang, J. C. Proteolysis patterns of epitopically labeled yeast DNA
topoisomerase II suggest an allosteric transition in the en/yme induced by ATP
binding. Proc. Nati. Acad. Sci. USA. H8: 10485-10489. 1991.
Hinds. M.. Deisserroih. K., Meyers. J.. Allschuler. E.. Jensen, R.. Ledley. F. D.. and
/welling. L. A. Identification of a point mutation in the topoisomerase II gene from
a human leukemia cell line containing an amsacrine-resistant form of lopoisomerase
II. Cancer Res., 52: 4729-2732. 1991.
Nitiss. J. L., Liu. Y-X., Harhury. P.. Jannatipour. M.. Wasserman. R.. and Wang. J. C.
OF ANT1CANCKR
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
DRUGS
Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topo
isomerase II. Cancer Res.. .52: 4467-4472. 1992.
Brill. S. J., DiNardo, D.. Voelkel-Meiman. K.. and Stcmglanz. R. Need for DNA
topoisomerase activity as a swivel for DNA replication and for transcription of
rihosomal RNA. Nature (Lond.l. .(26: 414-416. 1987.
Goto, T.. and Wang. J. C. Cloning of yeast TOPI, the gene encoding DNA topo
isomerase I. and construction of mutants detective in both DNA topoisomerase I and
DNA topoisomerase II. Proc. Nati. Acad. Sci. USA. K2: 7178-7182. 1985.
Carón. P. R.. and Wang. J. C. DNA topoisomerases as targets of therapeutics: a
structural overview. In: T. Andoh. H. Ikeda. and M Oguro (eds.l. Molecular Biology
of DNA Topoisomerases and Its Application to Chemotherapy. Proceedings of the
International Symposium on DNA Topoisomerases in Chemotherapy. Nagoya. Japan,
1991. pp. 1-18. Boca Raton. FL: CRC Press. 1993.
Worland. S. T.. and Wang. J. C. Inducible overexpression. purification, and active site
mapping of DNA lopoisomerase 11from the yeast Sticfhantniyct's cercvisiuc. J. Biol.
Chem.. 264: 4412-4416. 1989.
Thrash. C.. Bankier. A. T.. Barrel!. B. G.. and Sternglan/. R. Cloning, characteri/ation. and sequence of the yeast DNA topoisomerase I gene. Proc. Nati. Acad. Sci.
USA, 82: 4375-4378, 1985.
Uemura. T. and Yanagida. M. Isolation of type I and II DNA topoisomerase mutants
from fission yeast: single and double mutants show different phenotypcs in cell
growth and chromatin organization. EMBO J.. .<: 1737-1744. 1984.
Uemura. T. Morino. K.. Uzawa, S.. Shinzaki. K.. and Yanagida. M. Cloning and
sequencing of Sclli-n.'i(iccll(initn\ct'\ poinhc DNA topoisomerase I gene, and effect of
gene disruption. Nucleic Acids Res.. /5: 9727-9739. 1987.
Nelson. E. M.. Tewey. K. M.. and Liu. L. F Mechanism of antilumor drugs. Poisoning
of mammalian DNA lopoisomerase II on DNA by an antilumor drug m-AMSA. Proc.
Nail. Acad. Sci. USA. HI: 1361-1365. 1984.
Jenkins. J. R.. Avion. P.. Jones. T. Davies. S. L.. Simmons. D. L., Harris, A. L.. Sheer.
D.. and Hickson. 1. D. Isolation of cDNA clones encoding the ßiso/yme of human
DNA lopoisomerase II and localisation of the gene to chromosome 3P24. NucleicAcids Res.. 20: 5587-5592. 1992.
Botstein. D.. Falco, S. C., Stewart. S. E.. Brennen. M.. Scherer. S., Stinchcomb. D. T..
Slruhl. K.. and Davis. R. W. Stérilehost yeasts (SHY): a eukaryoiic system of
biological containment for recomhinant DNA experimenls. Gene. <V:17-24. 1979.
Sherman. F. Guide to yeasl genetics and molecular biology. In: C. Gulhrie and Ci. R.
Fink leds.i. Methods in En/ymology. Vol. 194. pp. 3-21. New York: Academic Press.
1991.
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Use of Yeast in the Study of Anticancer Drugs Targeting DNA
Topoisomerases: Expression of a Functional Recombinant
Human DNA Topoisomerase IIα in Yeast
Robin A. Wasserman, Caroline A. Austin, L. Mark Fisher, et al.
Cancer Res 1993;53:3591-3596.
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