Download Role of Topoisomerase II@3in the Resistance of 9-OH

[CANCER RESEARCH57, 4301-4308, October 1, 19971
Role of Topoisomerase II@3in the Resistance of 9-OH-ellipticine-resistant Chinese
Hamster Fibroblasts to Topoisomerase II Inhibitors'
Sophie
Dereuddre,
Charlotte
Delaporte,
URAJ47 Centre National de Ia Recherche
Roussy. 94805 Villejuif cedex, France
Scienufique,
and Alain
Physicochimie
Jacquemin-Sablon2
et Pharmacologie
ABSTRACT
In the Chinese hamster lung cell line DC-3F/9-OH-E, made resistant to
9-OH-ellipticine
and cross-resistant
to other topoisomerase II inhibitors,
the amount of topoisomerase
ha is 4—5-fold lower than in the parental
DC-3F cells. A mutation in position 1710 of topoisomerase IIfi cDNA,
generating a stop codon, completely abolishes the expression of this iso
form in DC-3F/9-OH-E
cells. To analyze the contribution
of the loss of
topoisomerase 110 to the resistance phenotype, DC-3F/9-OH-E cells were
cotransfected with two plasmids, one conferring the resistance to 0418,
the other carrying the topoisomerase 11(1cDNA. Among 200 0418-resist
ant clones, one was found to contain a topoisomerase
II@3activity similar
to that in the parental cells. These cells constitute an in vivo mammalian
model to study the pharmacological
transfected
cells,
different
resistance
reversion
levels
role of topoisomerase
of cleavable
were observed
complex
IIfi. In the
formation
with each topoisomerase
and
II inhibitor
examined. This work demonstrates that topoisomerase IIfi Is a pharma
cological target for 9-OH-ellipticine,
etoposide, or 4'-(9-acridinylamino)
methanesulfon-m-anisidide
and plays a role in the cytotoxicity of these
agents. Furthermore,
topoisomerase
II@3 is the preferential
target for
4'-(9-acridinylamino)methanesulfon-m-anisidide.
The loss of topoisomer
am ll@ activity in the DC-3F/9-OH-E cells is then in part responsible for
their resistance
to topoisomerase
II inhibitors.
INTRODUCTION
Type I! DNA topoisomerases are ubiquitous dimeric nuclear en
zymes that modulate the topological state of DNA by passing an intact
double helix through a transient double-strand break made in a second
helix (1 , 2). These enzymes play an essential role in a number of
cellular processes, including chromosome condensation and segrega
tion (3). Two distinct forms of topoisomerase II exist in mammals,
designated topoisomerase !!a (l70-kDa form) and topoisomerase I!@
(l80-kDa form; Ref. 4). These enzymes are encoded by two separate
genes with distinct but related sequences that, in human cells, are
located on chromosomes 17 and 3 for the a and (3 enzymes, respec
tively (5, 6). Transcription of the a gene varies as a function of cell
cycle position; it is 15-fold higher in late-S and 02-M phases than in
01 phase (7, 8). The level of the @3
form increases at mitosis, whereas
the enzyme diffuses in the cytosol (9). In addition to the molecular
masses of their subunits, topoisomerases Iks and (3also differ by some
of their biochemical and pharmacological properties and exhibit dif
ferent nuclear localizations and tissue expression specificity (7, 10—
15). It is then likely that both enzymes play different physiological
functions that have not yet been identified clearly.
Topoisomerases I! are a key target for many active anticancer
drugs, including anthracyclines, epipodophyllotoxins, acndines, and
ellipticines (16). An essential step in the mechanism of action of these
agents is the trapping of the covalent DNA-topoisomerase I! complex,
formed as an intermediate in the reaction catalyzed by topoisomerases
des Macromolecules
I Supported
in part
by
Association
pour
Ia Recherche
sur
Ic Cancer,
Villejuif.
whom
requests
for
reprints
should
be addressed,
at Unite
Institut Gustave
named
DC-3F/9-OH-E,
have been described
previously
(19). Briefly,
the DC-3F/9-OH-E cells exhibit three major phenotypic changes: (a)
cross-resistance to all known topoisomerase I! inhibitors; (b) cross
resistance to some other drugs through the expression of the MDR
phenotype; and (c) loss of tumorigenicity. Resistance to topoisomer
ase II inhibitors in these cells has been associated with a decreased
topoisomerase I! activity (approximately 10-fold) and a decreased
capacity of the inhibitors to induce the stabilization of the cleavable
complex (approximately 10-fold; Refs. 20 and 21). Further detailed
analysis showed that, in the parental DC-3F cells, the a enzyme was
approximately 20-fold more abundant than the @3
enzyme and thus
accounted for the overall topoisomerase H activity in these cells. In
the resistant cells, the amount of topoisomerase 1hz was about 4—5fold decreased, whereas the (3 enzyme was undetectable (22). Se
quencing of the topoisomerase 11(3cDNA revealed a mutation, in
position 1710, converting a TGG (Trp) codon to a TGA (stop) codon,
thus accounting for the loss of this isoform in the DC-3F/9-OH-E cells
(23).
In an attempt to evaluate the relative contribution of both a and @3
enzyme modifications to the resistance phenotype, the DC-3F/9-OH-E
cells were transfected with a eukaryotic expression vector containing
either the topoisomerase lIes or @3
cDNA. In this study, we report the
selection and the phenotypic characterization of a transfected clone in
which a nearly normal topoisomerase 1I@3activity has been restored.
These cells are a new in vivo mammalian model to study the phar
macological role of topoisomerase !!f3.
MATERIALS AND METHODS
Cell Lines and Culture Conditions
The parental Chinese hamster lung fibroblast cell line DC-3F and the
9-OH-E-resistant
supplemented
subline
DC-3F/9-OH-E
(18) were grown
with 10% FCS, 100 IU penicillin,
and 50
in Eagle's
MEM,
@g/mlstreptomycin.
The resistant cells were permanently grown in the presence of 9-OH-E (0.6
p@g/ml).Before each experiment, the cells were grown for three passages in
absence of drug.
Drugs and Chemicals
m-AMSA, VP-l6, and genistein, all from commercial sources, were dis
solved in DMSO at 0.01 M and kept frozen at —20°C
as stock solution.
9-OH-E, provided by Dr. E. Lescot (Institut Gustave Roussy, Villejuif.
France), was dissolved in l0@ MHC1at 0.01 Mand kept frozen at —20°C
for
less
than
6 weeks.
and dissolved
intestinal phosphatase
NMHE
in H,O.
was
purchased
Restriction
from
enzymes,
Pasteur-Mérieux
(Lyon,
14 DNA ligase,
and calf
were purchased from Boehringer-Mannheim
(Meylan,
France). Escherichia coli DNA polymerase (Klenow fragment) was purchased
from New England Biolabs (Beverly, MA).
France.
andby LigucNationaleFrançaise
contreIcCancer,Paris,France.
2 To
Unite de Biochimie-Enzymologie,
I! and termed the cleavable complex ( 17). From the Chinese hamster
lung fibroblast cell line DC-3F, a variant resistant to the topoisomer
ase II inhibitor 9-OH-E3 has been isolated previously by exposure to
drug increasing concentrations (I 8). The properties of these cells,
France)
Received 3/21/97; accepted 8/1/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Biologiques,
3 The
abbreviations
used
are:
9-OH-E.
9-hydroxy-ellipticine;
NMHE,
2-N-methyl-9-
hydroxy-ellipticinium; VP- 16, etoposide; ns-AMSA, 4'-(9-acridinylamino)methanesul
fon-m-anisidide.
de Biochimie-Enzy
mologie, Institut Gustave Roussy, P.R.II, 94805 Villejuif cedex, France.
4301
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TOPOI5OMERASE II@3TRANSFECFION IN DRUG-RESISTANTCELLS
Western
Vectors
Topoisomerase
Blots
Freshly prepared nuclear extracts (100
IIfJ cDNA was cloned from a DC-3F cDNA library, con
@g)were fractionated on 7.5%
structed with the Zap cDNA synthesis kit, and ligated to the pBluescript SK±
SDS/polyacrylamide
plasmid following the manufacturer's protocol (Stratagene, La Jolla, CA; Ref.
22). The topoisomerase II@ cDNA was then transferred from this plasmid,
nitrocellulose membrane (Optitran BA-S 85; Schleicher and Schuell).
Membranes were incubated with the polyclonal anti-topoisomerase II an
named
pBhamTOP2/3,
to the eukaryotic
expression
vector
pSV-Sport
1. Ex
gels.
The
tibody A6. Bound antibody
proteins
were
was visualized
transferred
(4 mA/cm2)
with horseradish
to
peroxidase
pression of the cDNA inserted in this plasmid, pSV-hamTOP2I3, was tested in
conjugated goat anti-rabbit !gG and enhanced chemiluminescence detec
transient transfection experiments (Fig. 14). In this plasmid, a 250-bp fragment
is present between the promotor and the initiation codon (23). Therefore, the
tion (Amersham).
corresponding
transcript
migrates
in the gels with an apparent
size of approx
Topoisomerase
imately 5.9 kb, instead of 5.6 kb for the normal topoisomerase II@3mRNA.
Transfection of the Hamster Topoisomerase IIfi cDNA into DC
3F/9-OH-E Cells
The plasmids pSV-hamTOP2@and pSV-tk-neo-f3 (24), conferring resist
ance to the G4l8 antibiotic, were mixed (20 and 4 j.tg per dish, respectively).
DC-3F/9-OH-E
tated plasmids
transfection
cells were cotransfected
and carrier
medium
with the calcium phosphate-precipi
DNA by a standard
was replaced
with
procedure
fresh
(25). After 24 h, the
medium.
The following
dishes were split 1 to 10, and G418 selection was started at 400
maintained
with
twice
weekly
changes
of medium.
day,
@g/mland
0418-resistant
clones
developed after about 15 days. Clones were picked up with sterile pipettes,
transferred to 35-mm-diameter Petri dishes, and grown in the presence of 0418
at the same concentration.
The cells were then transferred to 60-mm diameter
Petri dishes and grown in the absence of antibiotic prior to being frozen.
Transient
expression
of pSV-hamTOP2/3
was determined
in NIH3T3
cells
transfected with the vector in the same conditions as above. The transfection
medium
was replaced
transfected
with fresh medium
gene was analyzed
after 24 h, and the expression
of the
Decatenation of Kinetoplastic DNA. Topoisomerase II catalytic activity
was quantified by decatenation of 3H-labeled Trypanosoma crusi kineto
plastic DNA. Approximately 0.25 @gof DNA was incubated with nuclear
extracts (4.5 @.tg/ml) in the topoisomerase
II reaction buffer [40 mM
Tris-HC1 (pH 8), 100 mM KC1, 10 mM MgC12, 0.5 mM EDTA, 1 mM ATP,
0.5 mMDTT, 30 @g/ml
BSA, and 0.01% Triton X-l00J for different times
at 37°C.The reaction was stopped by the addition of NaCl and EDTA at
final concentrations of 1 and 0.01 mM, respectively. Samples were filtered
through a Millipore filter, and a l00-pJ aliquot was counted in scintillation
buffer Instagel (Packard).
Topoisomerase 11-mediated Cleavage Reactions. pSP 65 DNA (0.2 @tg)
was incubated with approximately 5 @g
of nuclear extracts from the different
cell lines, in the presence or absence of drug, in topoisomerase II buffer for 15
mm at 37°C.The cleavage reaction (15 @l)
was terminated by the addition of
SDS and proteinase K to final concentrations of 0.4% and 0. 1 mg/rn!, respec
tively, and the mixture was incubated for an additional 30 mm at 50°C.After
the addition of 5 p3 of loading buffer (0.4% bromphenol blue, 0.4% xylene
cyanol, and 50% glycerol), the products of the reaction were fractionated by
48 h later.
electrophoresis
cDNA Probes
The probe PH1 is a Pstl-HindIII fragment (1991 bp) of the hamster cDNA
isolated from the pBhamTOP2@3 plasmid. The actin DNA probe was kindly
provided by Dr. F. Dautry (Institut Gustave Roussy, Villejuif, France). These
probes were labeled by random priming with [a-32P]dCTP using the Multi
prime
labeling
kit from
Amersham
International
(Buckinghamshire,
32P]dATP
from
both human
in ThE
(89 mM
@g/mlof ethidium
with UV light (300
scanning (Chromoscan
3; Joyce-Loebl,
Gateshead, England) of
of Specific
DNA Cleavage
in the Presence
of VP-16
and
m-AMSA. For preparation of 3'-end labeled pSP65 DNA fragment, circular
pSP65 DNA was first linearized with EcoR! and then labeled with [a-
The rabbit polyclonal antibody (designated A6) was described previously
the a and (3 enzymes
16 h (2.5 V/cm)
nm) and were photographed. DNA cleavage stimulation was quantified by
Pattern
United
(22). This antibody, raised against a 837-amino acid fragment of the human
recognizes
gels for about
negative films.
Anti-DNA Topoisomerase II Antibodies
ha,
on 1% agarose
Trisborates, 2.5 mM EDTA, pH 8.3) containing 0.25
bromide. DNA bands were visualized by transillumination
densitometer
Kingdom).
topoisomerase
11-mediated Reactions
using
the large
fragment
of E. co!i DNA
polymerase
I (Klenow
fragment) at 20°Cfor 20 mm. The labeled DNA was purified by two cycles of
ethanol precipitation and was further cut with Hindffl restriction endonuclease.
The 3'-end labeled DNA fragment (6000 cpm Cerenkov) was then incubated
with nuclear extracts of the different cell lines in cleavage buffer [10 mM
Tris-HC1 (pH 7.5), 50 mM MgCl2, 50 mM KCL, 1 mM EDTA, 15 @tg/mlBSA,
and
hamster origins.
and 0.01% Triton X-lOO],in the absence or presence of the drug (final volume,
Northern
Blots
15 s.d) at 28°Cfor 6 mm. The reaction was terminated by addition of SDS,
Na3EDTA, and proteinase K at final concentrations of 1%, 2 mM, and 0.4
Five @.tgof RNAs, extracted by the guanidine thiocyanate technique (26),
were fractionated
by electrophoresis
on 1.2% (w/v) agarose
gels containing
7%
formamide and transferred to Hybond-N nylon membrane (RPN 3050; Am
ersham)
in 150 mM ammonium
acetate.
Prehybridization
was performed
mg/ml respectively. After an additional 20-rain incubation at 45°C,the loading
buffer (5 @d)was added, and the samples were fractionated by agarose gel
electrophoresis
for 2 h
at 42°Cin 40% formamide, 5X SSC (1X SSC is 0.15 MNaCl, 15 mMsodium
citrate), 50 mMphosphate buffer (pH 6.8), 5X Denhardt's solution, and 0.1%
SDS. Hybridizationwas thenperformedfor 20 h in the same buffercontaining
the 32P-labeledprobe. The membrane was washed twice at room temperature
for 15 mm in 0.1% SDS, 2X SSPE (I X SSPE is 10 mt@iNaH2PO4,0.13 M
NaCI, and 1 mMEDTA) and twice at 50°Cfor 30 mm in 0. 1% SDS, I X SSPE.
After autoradiography at —70°C,
the autoradiograms were obtained on Fuji
RX film with Hyperprocessor(Amersham).
(1.2% agarose,
0.1% SDS in ThE)
for 16 h at 1—5V/cm. After
electrophoresis, the gels were dried on a 3MM paper sheet and autoradio
graphed with Fuji X-ray film.
Nuclear Extracts
Nuclear extracts were prepared from l0@cells as described by Riou et a!.
(27). Freshly prepared protease inhibitors (phenylmethylsulfonyl fluoride, ben
zamidine, aprotinin, and soybean trypsin inhibitor) were added to all buffers.
Protein concentration in the nuclear extracts was determined as described
previously (28). The extracts were used immediately.
Cell Survival
The in vitro colony formation assay was used to determine the cell survival
fraction after 3 h ofdrug exposure. Exponentially growing cells (2 X l0@)were
grown
into 60-mm
diameter
Petri dishes
at 37°Cbefore drug treatment.
(Falcon;
Becton
Dickinson)
for 18 h
After 3 h of exposure to 9-OH-ellipticine,
VP-16, or m-AMSA, the cells were trypsinized, and appropriate dilutions were
made to seed 250 treated cells into 60-mm diameter Petri dishes. Colonies were
stained and counted 8—10
days later. Because of its limited solubility in water,
it was not possible
to use this technique
to measure
the toxicity
of NMHE
on
the resistant cells. In this case, we counted the number of residual cells after
exposure of 2 X 10―DC-3F and 5 X l0@ DC-3F/9-OH-E
drug concentrations for 72 h.
cells to increasing
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TOPOISOMERASE II@TRANSFECTION IN DRUG.RESISTANT CELLS
RESULTS
w
Transfection of the Topoisomerase Hfi cDNA in DC-3F19OH-E Cells.
@
Cloning and sequencing
of the topoisomerase
SV4O origin
and early promoter
Sma I
Ampicillin
hamTOP2@3R
U-.
CY)
u2
4@dr_5,9
Kb
“@5,5kb
—
Fig. 2. Northem blot analysis of pSV-hamTOP2@3
cDNA expression in DC-3F/9-OH
E-transfected cells. Total RNAs were fractionated by agarose (I .2%) gel electrophoresis,
transferred to Hybond-N membrane, and hybridized with the PHI probe. Loading of the
gel was controlled by hybridization with a f3-actinprobe.
However, the expression of the transfected gene in clone 11 turned
out to be unstable and was markedly decreased after approximately 12
passages. Selection, growth, and analysis of the 041 8-resistant clones
required 7 to 9 passages before freezing the cells. Therefore, all of the
following experiments were carried out on cells grown only for one or
two passages after thawing.
Stimulation of DNA Cleavage by Topoisomerase II Inhibitors.
The primary effect of antitumor agents, such as ellipticines, VP-l6, or
m-AMSA, is to increase the number of cleavable complexes formed
between the DNA and topoisomerase I!, either by inhibiting the
religation of the cleaved DNA or by increasing the rate of DNA
cleavage without inhibiting the religation (29). Multiple studies have
demonstrated the effects of these agents on topoisomerase !!a (re
viewed in Ref. 30). The effects of these drugs on topoisomerase 1113
are much less well documented. The topoisomerase 1113transfected
cells described in this report provides a convenient experimental
system to study the pharmacological role of this enzyme.
The
5500pb
Sv 40 t intron
9
o@)
I!@ cDNA
from DC-3F/9-OH-E cells revealed a mutation in position 1710 that
converts a ftp codon to a stop codon, and thus accounts for the loss of
topoisomerase Hf3 activity in these cells (23). To restore normal
enzyme activity, the DC-3F/9-OH-E cells were cotransfected with the
pSVhamTOP2@ plasmid, which contains the entire coding region of
the Chinese hamster topoisomerase !!@3cDNA from DC-3F cells
(hamTOP2f3; Fig. 1), and the pSVtk-neo-f3 plasmid, which confers the
resistance to the antibiotic 0418. In the transfected cells, first selected
for the resistance to 041 8, the expression of the transfected ham
TOP2IJ was then detected by Northern blot analysis. Two hundred
G41 8-resistant clones, selected from three independent transfection
experiments, were analyzed. The transfected topoisomerase I!@
cDNA was expressed at a high level in one of them, clone 11. As
expected from previous experiments (22), the endogenous topo
isomerase ll@ transcript (5.6 kb) was undetectable in the resistant cells
(Fig. 2). However, in clone I 1, a topoisomerase II@ transcript, with
approximately the size expected for the transfected topoisomerase !!j3
insert (5.9 kb), was expressed at the same level as the endogenous
transcript in the DC-3F cells. Low amounts of the 5.9-kb transcript
were also detected in three other clones, which were not further
analyzed (data not shown).
The amount of topoisomerase 11(3in the nuclear extracts from the
transfected cells was then determined by immunoblot analysis using
the A6 antibody that recognizes both the a and (3isoforms (22). In the
untransfected DC-3F/9-OH-E cells and the seven transfected clones
shown on Fig. 3, the amount of topoisomerase 1hz was approximately
5-fold lower than in the parental DC-3F cells. Topoisomerase II@3was
undetectable in all of the resistant cell lines but clone 11. In this clone,
the intensity of the additional band at Mr 180,000 indicated that
topoisomerase !!/3 was present in these cells at a level comparable to
that in the DC-3F cells.
Topoisomerase I! catalytic activity in the nuclear extracts from the
different cell lines was determined by the kinetoplastic DNA decat
enation assay, which measures both the a and f3 isoforms. Fig. 4
shows that the residual topoisomerase !! activity in the untransfected
DC-3F/9-OH-E cells was about 10% of that in the DC-3F cells. The
enzyme activity in clone 11 was increased up to approximately
20—30%of that in the parental DC-3F cells. Considering the relative
amounts of topoisomerases Ha and j3 in the DC-3F cells (22), these
data are compatible with the restoration of a nearly normal topo
isomerase H@3activity in clone 11.
plasmid
pSP65
DNA
was
used
as a substrate
to analyze
the
stimulation of DNA cleavageafter incubation with nuclear extracts
and polyadenylation
Hind 111
Apa I
TAA
Fig. 1. Plasmid pSV-hamTOP2fJ. Topoisomerase II@cDNA isolated from DC-3F cells
(hamTOP2I3)is inserted into the multiple cloning site of pSV-Sport I plasmid.
from the different cell lines in the presence of increasing drug con
centrations. Fig. 5, A and B, shows that, in agreement with our
previous results (20, 2 1), the amount of cleavable complex formed in
the presence of VP- 16 and m-AMSA with nuclear extracts from
DC-3F/9-OH-E cells was approximately 15% of that formed with the
extracts from DC-3F cells. With the nuclear extract from clone 11,
stimulation of DNA cleavage with VP-l6 and m-AMSA was in
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TOPOISOMERASE lI@ TRANSFECTION IN DRUG-RESISTANT CELLS
Analysis of DNA Cleavage Sites. The previous experiments
showed that, despite a comparable stimulation of DNA cleavage,
m-AMSA was much more cytotoxic on clone 11 than VP-l6. One
possibility was that each drug might stabilize the enzyme-DNA in
teractions at different sites, the sites recognized in the presence of
m-AMSA resulting in a more toxic effect than those recognized in the
presence of VP-16. To further support this hypothesis, we analyzed
the cleavage sites stimulated on a 3'-end labeled pSP6S DNA frag
ment after incubation with nuclear extracts from either DC-3F/9OH-E or clone 11 cells in the presence of VP-16 or m-AMSA. Fig. 7
shows that three cleavage sites of interest can be identified: A and C
are cleavage sites only induced by m-AMSA in the presence of
extracts from both untransfected DC-3F/9-OH-E and clone 11 cells,
which confirms the existence of drug specific sites; and B is a
cleavage site sensitive to both drugs, which is only detected in clone
11, indicating that this is a 13specific site associated with the expres
sion of the transfected enzyme.
k Da
1
2
180
A
170'
0
k Da
4)
0
0
U
@- -
@- ‘@ .@
@I
DISCUSSION
@‘i
170
Resistance to topoisomerase
Fig. 3. Immunoblot
analysis of pSV-hamTOP2l3
expression
in transfected
cells.
Experimental conditions are described in “Materials
and Methods.―
One hundred @.sg
of
nuclear extracts were loaded in each lane. A, transient transfection of NI}13T3cells. Lane
1, NIH3T3; Lane 2, NIH3T3 transfected with pSV-hamTOP2(3. B. stable transfection of
DC-3F/9-OH-E cells. DC-3F, DC-3F/9-OH-E, and clone 11 are indicated; other lanes
correspond to G4l8-resistant clones with no expression of topoisomerase 1Ij3.Topo
@
isomerase ha and
I! inhibitors may involve different
B biochemical
alterations
(reviewed
inRef.
30).
After
along
period
of
selection in the presence of increasing drug concentrations, the 9-OH
ellipticine-resistant cell line DC-3F/9-OH-E displays a complex phe
notype resulting from the accumulation of multiple genetic defects.
Alterations, identified in these cells as specifically related to resist
ance to topoisomerase I! inhibitors, include a decreased amount of
topoisomerase Ha and the absence of topoisomerase 1113 (19).
Complementation of either one of these defects, by transfection of the
DC-3F/9-OH-Ecells with either the topoisomeraseI!a or 13cDNA,
was used as an approach to analyze their relative contribution to the
resistance phenotype. In this work, we describe the properties of a
DC-3F/9-OH-E clone in which a normal topoisomerase H3 activity
has been restored and then constitutes a new in vivo mammalian
model to study the pharmacological role of this enzyme.
The selection of cells expressing the transfected topoisomerase 1113
cDNA at a high level was very difficult. Among 200 clones selected
for G4l8 resistance from three independent transfection experiments,
proteins were identified with the A6 antibody.
creased up to approximately 40% and 50% of the DC-3F control,
respectively. Fig. 5, C and D, shows that, with the ellipticine deriv
atives 9-OH-E and NMHE, the level of cleavable complexes, which
were undetectable on DNA incubated with the nuclear extracts from
DC-3F/9-OH-E cells, was raised with clone 11 up to approximately
10—12%of that observed with the DC-3F cells.
In conclusion, although at different levels, the four tested topo
isomerase II inhibitors were able to induce the formation of topo
isomerase I!13-mediated cleavable complexes.
Drug Resistance. The cytotoxicity of 9-OH-E, VP-16, and m
1500
AMSAin DC-3F,DC-3F/9-OH-E,and clone 11 cells,determinedby
colony formation assay after 3 h exposure to the drug, is shown on
Fig. 6. A 10% increase in the amount of 9-OH-ellipticine-induced
cleavable complex was associated with an approximately 10% rever
sion of the drug resistance in clone 11 as compared to the DC-3F/9-
1000
OH-E cells (Fig. 6A).The sensitivityof clone 11 to NMHE,another
ellipticine derivative, was also examined. However, because of the
limited water solubility of this compound, the clonogenic assay, after
3 h drug exposure, could not be used. Instead, the toxicity of 9-OH-E
and NMHE was determined by counting the residual cell number after
72 h exposure to increasing drug concentrations. In these conditions,
a similar level of resistance reversion was observed with both drugs
(data not shown).
Although VP-16 and m-AMSA were inducing the cleavable com
plex formation in the presence of nuclear extracts from clone 11 at a
comparable level (40 and 50% of the control DC-3F cells, respective
ly), the resulting sensitivity of the transfected cells to these agents was
very different; the resistance to VP-l6 was approximately 40% lower
than in the DC-3F/9-OH-E cells, in proportion of the level of cleav
able complex formation (Fig. 6B). Meanwhile, the resistance to m
AMSA was decreased by 90% (Fig. 6C).
500
0
0
10
TIME
20
30
@flNUTE@
Fig.4. TopoisomeraseII activityin the hamTOP2-transfected
cells.Nuclearextracts
(4.5 @g/ml)from DC-3F (X), DC-3F/9-OH-E (•),and clone 11 (@) were incubated with
0.25 @agof 3H-labeled Trypanozoma cruzi kinetoplast DNA. Aliquots were taken at the
indicatedtimes,and the amountof decatenatedDNA was measuredas describedin
“Materials
and Methods.―The double experimental points correspond to the results of two
independentexperiments.
4304
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TOPOISOMERASE II@TRANSFECTION IN DRUG-RESISTANTCELLS
A
B
20
60
50
15
40
0
r@.
0
30
10
20
5
a.
10
0
0
50
100
150
0.4.8
1.6
3.2
VP-16 (@tM)
6.4
12.8
m-AMSA(@tM)
C
D
40
50
40
30
0
r@.
30
0
20
20
10
10
0
0
0
1
2
3
4
5
7.5
10
0
9-OH-E
(@sM)
Fig. 5. Effect of9-OH-E, NMHE, VP-l6, and ,n-AMSA on DNA cleavage stimulation in the presence ofnuclearextracts
2
4
6
8
10
NMII!:
from hamTOP2 transfected cells. Five
@g
of nuclear extracts
fromDC-3F(X), DC-3F/9-OH-E
(•),
andclone11(Li)cellswereincubatedwith0.2 @.&g
of pSP6SDNAin thepresenceof the indicatedconcentrationsof VP-16(A).m-AMSA(B).
9-OH-E(C). and NMHE(D). Afteragarosegel electrophoresisseparation,the amountof linearDNAwasquantifiedby densitometricscanning.The doubleexperimentalpoints
correspond to the results of two independent experiments.
only four were found to express the transfected gene by Northern blot
analysis. In one of them, clone 11, the amount of enzyme protein and
the catalytic activity were comparable to that in the parental DC-3F
cells. In transient transfection experiments, a high expression of the
hamTOP2f3 cDNA was observed (Fig. 2A), indicating that the diffi
culty to select stable topoisomerase IIJ3—transfectedclones was not due
to a defect in the construct. Furthermore, the expression of the
transfected topoisomerase 1113gene in clone 11 was found to be
unstable and was greatly decreased after approximately 10—12pas
sages. Cells completely devoid of topoisomerase H13activity are then
perfectly viable, indicating that this enzyme is not specifically re
quired for any essential function in the cell. However, continuous
overexpression of this enzyme could be deleterious to the cells. In
different cell types transfected with the human topoisomerase Ha
cDNA, such as yeast (31) or DC-3F/9-OH-E cells,4 a comparable
toxicity was also observed. Furthermore, in cells transfected with a
topoisomerase 1hz cDNA under the control of an inducible promoter,
the overexpression of the transfected gene was only observed either at
a very low level (32) or for a short period of time (33). These data
indicate that, in the parental DC-3F cells and in other cell types as
well, accurate regulation mechanisms limit the expression of each
topoisomerase II isoform to a maximum level that cannot be exceeded
in the transfected cells.
Topoisomerase II is the target for some of the most frequently used
anticancer agents. However, the role of the individual a and (3
isoforms is still unknown. In previous in vitro studies, recombinant
topoisomerase ha was equally effective in promoting DNA cleavage
induced by teniposide, etoposide, and m-AMSA (34). Topoisomerase
11(3produced higher levels of cleavage than a in the presence of
m-AMSA and, surprisingly, o-AMSA was also able to promote DNA
cleavage specifically with this isoform (34). Using nuclear extracts
4 T. Khélifa, B. René. J. Markovits,
H. Jacquemin-Sablon.
and A. Jacquemin-Sablon.
from DC-3F/9-OH-E cells complemented with the a enzyme, we also
Expression of human topoisomerase Ha in 9-OH-ellipticine resistant Chinese hamster
observed a similar cleavage level with etoposide and m-AMSA.4 In
fibroblasts does not reverse the resistance to 9-OH-ellipticine but restores partial sensi
the present work, when plasmid DNA was incubated with nuclear
tivity to some other topoisomerase II inhibitors. manuscript in preparation.
4305
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@
1
TOPOISOMERASE 11@3
TRANSFECTION IN DRUG-RESISTANTCELLS
A
extracts
from
clone
11inthepresence
ofellipticine
derivatives,
etoposide, or m-AMSA, an increased amount of cleavable complex
100
was observed
as compared
with the extracts
from the untransfected
DC-3F/9-OH-E cells. However, the level of complex formation was
highly dependent on the drug, m-AMSA, and to a lesser extent VP-16,
being much more efficient than ellipticines. We may hypothesize that
the molecules that inhibit the religation step of the catalytic cycle, like
m-AMSA or VP-16, might be more efficient in forming the ternary
drug-enzyme-DNA complex than the ellipticine derivatives assumed
@
@
@
@
@
10
to be acting
@
@
@
@
through
another
mechanism
(29). Taken
together,
all of
these in vitro studies show that both topoisomerase Ila and (3 can
promote DNA cleavage in the presence of a variety of inhibitors and
are potential targets for these agents. However, each enzyme does not
display the same sensitivity to the different drugs, and some drugs are
interfere more efficiently with either one isoform. These differences
I
are
likely
to play
a part
in the
cellular
response
to topoisomerase
II
inhibitors.
0
5
10
15
20
Recently,
topoisomerase
1113was shown
to complement
a temper
ature sensitive topoisomerase II mutation in the yeast Saccharomyces
cerevisiae (35). This yeast in vivo system was used to compare the
9-OH-E (pM)
B
effects
ofvarious
topoisomerase
IIinhibitors
ontransformants
bearing
the individual human isoforms. In this system, m-AMSA was found to
100
produce
similar
cell killing
with a and (3 enzymes,
whereas
etoposide
@
produced a higher degree of cell killing with a than with (3 topo
isomerase II (35). The consequences of the cleavable complex for
mation induced by these agents in mammalian transformants were
@
very different.
@
@
revealed a 10 and 40% reversion of the initial resistance of the
DC-3F/9-OH-E cells, respectively, in proportion to the level of cleav
@
0
able complex
@
@
@
@
Treatment
formation.
of clone
With
1 1 with either ellipticines
m-AMSA,
the reversion
or VP-16
of the resist
ance phenotype was approximately 90%. Although the slightly higher
amount of cleavable complex formed in the presence of m-AMSA
may contribute to the toxicity of this drug on clone I 1, it is likely that
other parameters have to be considered to explain the difference with
VP-16. In agreement with previous reports (36), Fig. 7 shows that
1
m-AMSA
0
5
15
10
at different
and VP-16
DNA
stabilize
sites,
suggesting
the topoisomerase
that
selective
11(3-DNA complex
DNA
damage,
and
VP-16(MM)
1234567
C
100
A—
10
B
C—
0
5
10
15
20
m-AMSA(ji M)
Fig. 6. Cytotoxicity of 9-OH-E (A), VP-16 (B), m-AMSA in DC-3F cells (X),
Fig. 7. DNA double-strand break cleavage pattem induced by nuclear extracts of
DC-3F/9-OH-E and clone I I in the presence of VP-l6 and m-AMSA. A 3-end labeled
pSP65 DNA fragment was reacted with 4 @igof nuclear extracts from DC-3F/9-OH-E
DC-3F/9-OH-E cells (•),and clone 11 (A). Exponentially growing cells were treated with
cells (Lanes 2, 4, and 6) and clone I I (Lanes 1, 3. an 5) in the absence (Lanes S and 6)
the drug for 3 h at 37'C. Cells were trypsinized, counted, and seeded. Six to 8 days later,
colonies were stained and counted. Bars, SE of at least three independent determinations.
or presence of 50 @iM
VP-l6 (Lanes I and 2) or 6.4 @M
m-AMSA (Lanes 3 and 4). Lane
7, DNA alone. A. B, and C, three particular cleavage sites defined in the text.
4306
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TOPOISOMERASE 11f3TRANSFECTION tN DRUG-RESISTANTCELLS
differential cellular responses to these damages as well, may contrib
ute to differential cytotoxic activity. Recently, using a new set of
monoclonal and polyclonal antibodies, Meyer et al. (9) reexamined
the cellular localization of topoisomerases in mammalian cells. They
essentially found that topoisomerase 11(3was exclusively present in
the extranuclear nucleoplasm throughout interphase, whereas a frac
tion of the a isoform was linked to the nucleoli. In mitosis, topo
isomerase Hj3 diffused completely in the cytosol, whereas topoisomer
ase Ha remained chromosome bound. These differences, which are
likely to be associated with different physiological functions, might
also account for differences in pharmacological properties of each
isozymes.
Pharmacological differences of the a and (3 isoforms were first
described by Drake et a!. (10), who observed that agents such as
teniposide and merbarone displayed some selectivity for the a iso
form, both for inhibition of the catalytic activity and formation of the
cleavable complex. Since then, several reports have addressed the
question of the relationship between the expression of topoisomerase
H isoforms and the toxicity of topoisomerase II inhibitors with diver
gent results. Studies on breast cancer (37) and leukemia (38) cell lines
concluded that the sensitivity to etoposide was correlated with the
level of expression of the topoisomerase 11(3protein and that sensi
tivity to m-AMSA was correlated to the level of the topoisomerase Ila
protein. Analysis of the DNA cleavage sites induced by the a or (3
enzymes in the presence of m-AMSA showed that both proteins form
similar ternary complexes with the drug and DNA, thus indicating that
both enzymes are potential cellular targets for this drug (39). Finally,
Qiu et al. (40) showed that, in cells pretreated with teniposide or
merbarone, topoisomerase Ila forms complexes with nascent DNA,
whereas the (3 isoform is bound to bulk DNA. Formation of stabilized
complexes of topoisomerase ha with nascent DNA appeared to be
essential to the toxicity of teniposide. In DC-3F/9-OH-E cells, trans
fected with the pSV-hTOP2 plasmid and containing a nearly normal
topoisomerase ha activity, VP-16 and m-AMSA were able to induce
about 75% of the cleavable complex formation observed in the DC-3F
cells.4 Cross-resistance of the transfected cells to both drugs was
70—75% decreased as compared to the resistance in the DC-3F/9OH-E cells. Therefore, these experiments suggested that, in cells
devoid of any topoisomerase 11(3protein, both drugs were interfering
with the a enzyme with an equal efficiency. Alternatively, in cells
containing a low amount of topoisomerase lIa and a normal amount
of topoisomerase H(3, m-AMSA was much more cytotoxic than VP
16, indicating that in vivo the (3 enzyme is the preferential target for
this drug. This conclusion is consistent with previous data showing
that the resistance to m-AMSA in a small cell lung carcinoma cell line
(GLC4) is specificallymediatedby a topoisomeraseH(3alteration
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Role of Topoisomerase IIβ in the Resistance of
9-OH-ellipticine-resistant Chinese Hamster Fibroblasts to
Topoisomerase II Inhibitors
Sophie Dereuddre, Charlotte Delaporte and Alain Jacquemin-Sablon
Cancer Res 1997;57:4301-4308.
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