From the Departments of Biochemistry and ** Urology,
Kyushu University School of Medicine, Maidashi, Higashi-ku, Fukuoka
812-8582, Japan, the ¶ Department of Molecular Biology, School of
Medicine, University of Occupational and Environmental Health,
Yahatanishi-ku, Kitakyushu 807, Japan, and the
Department of
Biochemistry, Molecular Biology, and Cell Biology, Northwestern
University, Evanston, Illinois 60208
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ABSTRACT |
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Expression of the DNA topoisomerase II
(topoII
) gene is highly sensitive to various environmental stimuli
including heat shock. The amount of topoII
mRNA was increased
1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer
cells to heat shock stress at 43 °C for 1 h. The effect of heat
shock on the transcriptional activity of the human topoII
gene
promoter was investigated by transient transfection of T24 cells with
luciferase reporter plasmids containing various lengths of the promoter
sequence. The transcriptional activity of the full-length promoter
(nucleotides (nt)
295 to +85) and of three deletion constructs (nt
197 to +85,
154 to +85, and
74 to +85) was increased ~3-fold
24 h after heat shock stress. In contrast, the transcriptional
activity of the minimal promoter (nt
20 to +85), which lacks the
first inverted CCAAT element (ICE1), the GC box, and the heat shock
element located between nt
74 and
21, was not increased by heat
shock. Furthermore, the transcriptional activity of promoter constructs
containing mutations in the GC box or heat shock element, but not that
of a construct containing mutations in ICE1, was significantly
increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h
after heat shock. No such change in factor binding was apparent with an
oligonucleotide containing the heat shock element of the topoII
gene
promoter. Finally, in vivo footprint analysis of the
topoII
gene promoter revealed that two G residues of ICE1 that were
protected in control cells became sensitive to dimethyl sulfate
modification after heat shock. These results suggest that transcriptional activation of the topoII
gene by heat shock requires the release of a negative regulatory factor from ICE1.
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INTRODUCTION |
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DNA topoisomerases are essential enzymes that participate in the
segregation of newly replicated chromosome pairs, in chromosome condensation, and in modification of the superhelical content of DNA
(1-3). Human topoisomerase II
(topoII)1 functions as a
homodimer by cleaving and opening one DNA duplex, passing a second
duplex through the opening, and then resealing the break (4-6). Two
topoII isoforms have been identified in mammals: 170-kDa topoII and
180-kDa topoII
(7). Although both enzymes are closely related in
structure, they differ in important biochemical and pharmacological
properties, including sensitivity to topoII-targeting drugs, cellular
localization, and regulation by the cell cycle (8). Whereas the amount
of topoII
remains relatively constant throughout the cell cycle, topoII
expression is coupled to the cell cycle (9, 10). topoII
is
of particular importance because of its association with DNA
replication, mitosis, and cell proliferation.
Expression of topoII is highly susceptible to environmental stimuli,
and such regulation is thought to be mediated at both the
transcriptional and post-transcriptional levels. The promoter region of
the topoII
gene contains various regulatory elements, including five
inverted CCAAT elements (ICEs), one GC box, and one heat shock element
(HSE) (11). Exposure of human colon cancer cells to glucosamine induces
down-regulation of topoII
, resulting in the development of
resistance to the topoII
-targeting epipodophyllotoxin, etoposide
(12). Development of resistance to such topoII
-targeting agents is
often associated with down-regulation of topoII
in various mammalian
cell lines (13, 14). In one etoposide-resistant cell line derived from
human head and neck cancer KB cells (15, 16), the transcription factor
Sp3 was implicated in the down-regulation of topoII
(17).
Introduction of the wild-type p53 tumor suppressor gene into murine
cells results in reduced expression of the topoII gene, and this
effect appears to be mediated by one of the ICEs in the topoII
gene
promoter (18). Apoptosis induced by adenovirus E1A protein in human KB
cells is associated with a marked decrease in the amount of topoII
that is due to accelerated degradation of topoII
by the ubiquitin
proteolysis pathway (19, 20). The amount of topoII
mRNA in late
S phase is ~15 times that during the G1 phase of the cell
cycle in human HeLa cells, apparently because of increased mRNA
stability in S phase (10). These observations indicate that topoII
expression is regulated by multiple mechanisms that operate at the
levels of transcription, mRNA stability, and protein
degradation.
Heat shock stress also affects the abundance of topoII mRNA in
mammalian cells. Exposure of human head and neck or colon cancer cells
to high nonpermissive temperatures results in an increase in expression
of the topoII
gene, apparent 6-12 h later, and consequent
sensitization to the cytotoxic effect of etoposide (21, 22). The same
heat shock stress markedly increases the abundance of the heat shock
protein HSP70 and induces a transient decrease in the amount of
topoII
mRNA and protein immediately after exposure to
hyperthermia (10, 22, 23). Whereas this early effect of heat shock
stress on topoII
expression appears to be mediated by increased
degradation of topoII
mRNA (10), the later up-regulation of
topoII
gene expression appears to be due to transcriptional
activation (22). We have now investigated which elements in the
5'-flanking region of the human topoII
gene are responsible for the
heat shock-induced activation of transcription.
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EXPERIMENTAL PROCEDURES |
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Materials--
Restriction enzymes and other nucleic
acid-modifying enzymes and reagents were obtained from Promega
(Madison, WI), Life Technologies, Inc., or Takara Shuzo (Kyoto, Japan),
unless indicated otherwise. Both [-32P]dCTP and
[
-32P]ATP were from NEN Life Science Products. Human
topoI cDNA was kindly provided by T. Andoh (Sohka University,
Tokyo, Japan), and human topoII
cDNA (pBS-hTOP2) was provided by
J. C. Wang (Harvard University, Boston, MA). Human HSP70 cDNA
was kindly given by R. T. N. Tjian (University of California,
Berkeley, CA). All cDNA fragments were separated from vector DNA by
agarose gel electrophoresis and labeled by random primer DNA
synthesis.
Cell Culture and Heat Shock Conditions-- The T24 cell line, established from human transitional cell carcinoma of the urinary bladder (24), was cultured at 37 °C under a humidified atmosphere of 5% CO2 in Eagle's minimal essential medium (Nissui Seiyaku, Tokyo) supplemented with 10% newborn calf serum (Sera-Lab, Sussex, United Kingdom), 1 mg/ml Bacto-peptone (Difco), 0.292 mg/ml L-glutamine, 100 units/ml penicillin, and 100 µg/ml kanamycin. For heat shock, culture plates were sealed with paraffin film and immersed in a water bath at 43 °C for 1 h.
Northern Blot Analysis-- Northern blot analysis was performed as described previously (17). Briefly, total RNA was extracted from T24 cells with the use of guanidine isothiocyanate (25), subjected (15 µg/lane) to electrophoresis on a 1% agarose gel containing formaldehyde, and transferred to a Hybond N+ membrane (Amersham International, Buckinghamshire, United Kingdom). The membranes were exposed to 32P-labeled cDNA probes for 18 h and washed twice at 42 °C in 2× SSC containing 0.1% SDS and twice at 42 °C in 0.2× SSC containing 0.1% SDS. Radioactivity was detected with a Fujix BAS 2000 image analyzer (Fuji Film, Tokyo).
Construction of topoII Plasmids--
We used the polymerase
chain reaction (PCR) to clone the human topoII
gene promoter (nt
295 to +85, relative to the major transcription start site) as
described previously (17). The 3'-end of all inserts was nt +85, 10 base pairs upstream of the translation initiation site. For the
construction of other deletion constructs, HindIII fragments
(nt
295 to +85) of the pTII
295 plasmid were digested with
BfaI (pTII
197), ScrFI (pTII
154), HphI (pTII
74), and SacI (pTII
20). The
digestion products were blunt-ended with the Klenow fragment of DNA
polymerase I, ligated to HindIII linkers, and cloned into
the HindIII site of the pGL2-Basic vector (Promega).
Transient Transfection-- T24 cells (1 × 105) were transferred to 60-mm dishes, incubated at 37 °C for 48 h, and transfected with luciferase plasmid DNA (2.5 µg) by calcium phosphate precipitation as described previously (26). Four hours after transfection, the cells were washed, incubated at 37 °C for 24 h in fresh medium, and exposed to 43 °C for 1 h. The treated cells were then harvested immediately (0 h) or after further incubation at 37 °C for 1, 6, 12, or 24 h for determination of luciferase activity.
Luciferase Assays--
Cells were lysed in 200 µl of 25 mM Tris phosphate buffer (pH 7.5) containing 1% Triton
X-100 and subjected to centrifugation at 14,000 × g
for 15 s. The resulting supernatants were assayed for luciferase
activity with the use of a Picagene kit (Toyoinki, Tokyo); light
intensity was measured for 15 s with a luminometer (Model
TD-20/20, Promega). Cells were cotransfected with pSV2--GAL as a
control for transfection efficiency, and
-galactosidase activity was
measured with an Aurora GAL-XE kit (ICN, Costa Mesa, CA).
In Vivo Footprint Analysis--
Heat-treated or control T24
cells were exposed to dimethyl sulfate, and genomic DNA was then
extracted and cleaved as described (27, 28). Ligation-mediated PCR was
performed as described (27). Primer 1 (5'-CAGGCAGGACCCCACG-3', nt +46
to +31) was used for first-strand synthesis; primer 2 (5'-CCCGACCAAGCCGCTTCTCCAC-3', nt +22 to +1) was used for PCR
amplification; and primer 3 (5'-CCGACCAAGCCGCTTCTCCACAGACGCG-3', nt +21
to 7), which was labeled at the 5'-end with
[
-32P]ATP and T4 polynucleotide kinase, was used for
final detection of the DNA ladder. Samples were analyzed on a 6%
polyacrylamide sequencing gel.
Isolation of Stable Transfectants--
T24 cells (5 × 105) were transfected with a luciferase reporter vector
containing the topoII gene promoter (pTII
295; 10 µg) and
pRSV-neo (0.5 µg) with the use of Trans-it reagent (PanVera, Madison,
WI). After 8 h, the medium was replaced, and the cells were
incubated for 24 h. The cells were then incubated in selection medium containing G418 (0.8 mg/ml; Life Technologies, Inc.), and growing colonies (20-30/106 cells) were cloned, expanded,
and tested for luciferase activity.
PCR-- Unless indicated otherwise, PCR was performed in a final volume of 100 µl containing 1 ng of template DNA, a 100 pM concentration of each oligonucleotide primer, a 200 µM concentration of each deoxynucleotide triphosphate, 2.5 units of Taq DNA polymerase, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 0.01% (w/v) gelatin. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer) for 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, and polymerization at 72 °C for 2 min.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared as described previously (17). Briefly, T24 cells (4 × 107), subjected or not to heat shock at 43 °C for 1 h, were collected by exposure to trypsin; resuspended in 200 µl of an
ice-cold solution containing 10 mM Hepes-NaOH (pH 7.9), 10 mM KCl, 0.75 mM spermidine, 0.15 mM
spermine, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride; and incubated on ice for 15 min. The
cells were then lysed by passing 10 times through a 25-gauge needle
attached to a 1-ml syringe, and the lysate was centrifuged for 40 s in a microcentrifuge. The resulting nuclear pellet was resuspended in
100 µl of an ice-cold solution containing 20 mM
Hepes-NaOH (pH 7.9), 0.4 M NaCl, 0.75 mM
spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 25% (v/v) glycerol; incubated for 30 min on ice with frequent gentle mixing; and then centrifuged for 20 min at 4 °C in a microcentrifuge to remove insoluble material. The resulting supernatant (nuclear extract) was
stored at 70 °C, and its protein concentration was determined with
a protein assay kit (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)--
EMSAs were
performed as described previously (29). Briefly, 6 µg of nuclear
extract protein were incubated for 30 min at room temperature in a
final volume of 20 µl containing 10 mM Tris-HCl (pH 7.5),
50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 8% glycerol, 1 mM dithiothreitol, 0.1 µg of poly(dI-dC), and 1 × 104 cpm of
32P-labeled oligonucleotide probe (1 ng) in the absence or
presence of various competitors. The reaction mixtures were then
applied to a nondenaturing 5% polyacrylamide gel and separated by
electrophoresis at 100 V for 3 h in a buffer containing 50 mM Tris, 380 mM glycine, and 2 mM
EDTA. The gel was exposed to x-ray film with intensifying screens. The
following oligonucleotides were used for EMSAs: topo-ICE1 (5'-GAGTCAGGGATTGG CTGGTCTGCTTCGGGC-3', nt 77 to
48 of the
topoII
gene), topo-HSE (5'-GGGCTAAAGG AAGGTTCAAGTGGAGCTCTC-3', nt
47 to
18 of the topoII
gene), and HSP70-HSE
(5'-GA AACCCCTGGAATATTCCCGACC-3', nt
114 to
91 of the human
HSP70 gene). For supershift assays, 2 µg of antibodies to
heat shock factor HSF1 or HSF2 (30) were incubated with nuclear extract
for 30 min at room temperature before addition of
32P-labeled oligonucleotide probe.
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RESULTS |
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Effects of Heat Shock Stress on the Abundance of topoI, topoII,
and HSP70 mRNAs--
Consistent with our previous observations
with human head and neck or colorectal cancer cells (22, 23), Northern
blot analysis revealed that exposure of T24 cells to 43 °C for
1 h resulted in an initial small decrease in the amount of
topoII
mRNA, which was followed by an increase in transcript
abundance that was maximal (~3-fold) at 24 h (Fig.
1). The amount of HSP70 mRNA was increased immediately after heat treatment, reaching a
maximum (~18-fold induction) at 1 h. In contrast, the amount of
topoI mRNA was not affected by heat stress. The HSP70
and topoII
genes thus showed characteristics of immediate-early and
late genes, respectively, in response to heat shock.
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Basal Transcriptional Activity of the topoII Gene
Promoter--
We measured the basal transcriptional activity of the
topoII
gene promoter in T24 cells transiently transfected with
various luciferase reporter plasmids (Fig.
2). Maximal luciferase activity was
obtained with the reporter construct with the pTII
295 insert, which contains four ICEs, the GC box, and the HSE between nt
295 and
+85 of the topoII
gene. Stepwise deletion of ICE3, ICE2, and the
combination of ICE1, GC box, and HSE from the 5'-end of the promoter
resulted in marked -fold decreases in luciferase activity, in general
agreement with previous results (11).
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Effect of Heat Shock Stress on the Transcriptional Activity of the
topoII Gene Promoter--
Exposure at 43 °C for 1 h of T24
cells transiently transfected with the reporter construct containing
pTII
295 resulted in an initial ~80% decrease in luciferase
activity, followed by an increase that was maximal (3-fold) 24 h
after heat treatment (Fig. 3). This
experiment was repeated with two T24 cell lines stably transfected with
the pTII
295 luciferase construct. Again, luciferase activity was
decreased immediately after heat treatment, but then showed a
time-dependent increase that was maximal (3-4-fold) after 24 h (data not shown).
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Effects of Mutations in the topoII Gene Promoter on Heat Shock
Sensitivity--
The roles of ICE1, the GC box, and the HSE in heat
induction of topoII
gene promoter activity were investigated in T24
cells transiently transfected with luciferase reporter plasmids
containing promoter sequences with specific mutations in these
elements: GGATTGGCT in ICE1 was converted to GGAAAAACT
(pTII
295m5), GGGCGGG in the GC box to AAAAAAG (pTII
295m6),
and GGAAGGTTCAAGTG in the HSE to GAAAGGAAAAAATG (pTII
295m7) (Fig.
5A). The pTII
295m5 construct showed increased basal transcriptional activity, but luciferase activity was not increased further by heat shock (Fig. 5B). In contrast, heat shock increased the transcriptional
activities of pTII
295m6 and pTII
295m7 ~3-fold; the
transcriptional activities of these two plasmids were ~30 and 10%,
respectively, of that of the wild-type plasmid. Thus, a factor that
binds to ICE1 might negatively regulate basal promoter activity, and
ICE1 appears to play a key role in heat-induced activation of the
topoII
gene promoter. Whereas the GC box and HSE appear to
contribute to basal promoter activity, they do not appear to be
directly responsible for heat-induced promoter activation.
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EMSA Analysis-- We next investigated the effects of heat shock on the ICE (Y-box) binding proteins and HSFs with the use of EMSAs. A marked decrease in Y-box binding activity was apparent 3, 6, 12, and 24 h after heat shock (Fig. 6A). Formation of the complex was inhibited in the presence of either excess unlabeled oligonucleotide.
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In Vivo Genomic Footprint Analysis--
We examined the effects of
heat shock on the dimethyl sulfate methylation patterns in the promoter
region of the topoII gene by in vivo genomic footprint
analysis. Both G
64 and G
65 in ICE1 were
protected in untreated cells, but protection was markedly reduced 3, 6, and 24 h after heat shock (Fig. 7).
Methylation patterns of the GC box, HSE, and other elements in the
topoII
promoter region (nt
295 to +85) were not substantially
affected by heat shock stress (data not shown).
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DISCUSSION |
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We have previously shown that expression of the topoII gene is
increased 6-24 h after exposure of human head and neck or colorectal
cancer cells to heat shock stress (22, 23). In the present study, we
have shown that heat stress also induced activation of topoII
gene
expression in human urinary bladder cancer cells. This heat-induced
up-regulation of topoII
gene expression appeared to be mediated
through an ICE or Y-box located between nt
74 and
21 on the basis
of the following results. (i) The luciferase activity of T24 cells
transfected with reporter constructs containing pTII
295,
pTII
197, pTII
154, or pTII
74 was increased ~3-fold by
heat shock stress, whereas that of cells transfected with a construct
containing pTII
20 was not increased by heat treatment. (ii)
Introduction of mutations into ICE1 of the topoII
gene promoter
virtually eliminated the heat shock-induced increase in transcriptional
activity, whereas mutation of the GC box or HSE had no such effect.
(iii) EMSA analysis with nuclear extracts revealed a marked decrease in
ICE1-binding activity 3-24 h after heat shock, consistent with the
time course of the heat shock-induced increase in promoter activity,
whereas HSE-binding activity was not affected by heat stress. (iv)
In vivo genomic footprint analysis revealed a specific
change in the methylation pattern of ICE1 induced by heat shock stress.
Members of the ICE-binding (YB-1) family of proteins are expressed in a
wide range of cell types and function as important regulators of
growth-associated and other genes (31-34). The expression of genes
encoding the epidermal growth factor receptor (35), proliferating cell
nuclear antigen (36), DNA polymerase (37), and thymidine kinase
(38) is regulated in a positive manner by ICEs. In contrast, such
elements mediate down-regulation of the expression of genes encoding
serum albumin, estrogen-dependent very low density
lipoprotein apolipoprotein II, aldolase B, and class II major
histocompatibility complex (39-41). In the present study, deletion of
nt
197 to
155, with contain ICE3, reduced basal promoter activity
to about half of that apparent with the topoII
gene promoter
constructs pTII
295 and pTII
197. Further deletion of nt
154
to
75, containing ICE2, and of nt
74 to
21, containing ICE1,
reduced basal promoter activity to ~10 and 2%, respectively, of that
apparent with pTII
295. Consecutive deletion of the five ICEs from
the topoII
gene promoter was also previously shown to reduce basal
promoter activity in a stepwise manner (11, 18). Thus, the ICEs in the
promoter of the human topoII
gene appear to play an important role
in basal transcriptional activity.
Introduction of point mutations into ICE1 of the topoII gene
promoter alleviated the inhibition of topoII
gene expression by
wild-type p53 (18). Fraser et al. (42) showed that the topoII
gene promoter is activated at an early stage during monocytic differentiation of human leukemia cells induced by phorbol ester or
sodium butyrate and that this sodium butyrate-dependent
up-regulation of topoII
gene expression is mediated by the promoter
region between nt
90 and +90, which contains ICE1. In contrast,
inhibition of topoII
gene promoter activity in confluence-arrested
cells appears to be mediated through interaction of the CCAAT-binding factor CBF/NF-Y with ICE2 (43).
In the present study, deletion or mutation of ICE1 in the topoII
gene promoter prevented the heat shock-induced increase in
transcriptional activity. Moreover, both EMSA and in vivo
genomic footprint analysis indicated that nuclear ICE1-binding activity was decreased after heat shock stress. These observations indicate that
ICE1 negatively regulates the human topoII
gene and that heat shock
stress reverses this effect, possibly by inducing the dissociation of
negative regulatory factors from ICE1. The Y-box binding protein YB-1
has been shown to inhibit interferon
-induced activation of class II
major histocompatibility complex genes (41). In contrast, activation of
the human MDR1 gene in response to heat shock, DNA-damaging
anticancer agents, or ultraviolet light is mediated by interaction of a
Y-box binding protein with an ICE in the promoter of this gene (29,
44-47). Expression of YB-1 is also increased in response to genotoxic
stress, suggesting that the promoter of the YB-1 gene itself
is also sensitive to cytotoxic environmental stimuli (32, 48). ICEs
thus appear to mediate either negative or positive regulation of
specific genes in response to exogenous stimuli. Brandt et
al. (21) recently showed that c-Myb activated the human topoII
gene promoter via a Myb-binding site at nt
16 to
11 in human
leukemia cells. In the present study, the basal promoter activity of
pTII
20 was only 1.6% of that of pTII
295, and heat shock did
not increase the transcriptional activity of this construct. It is thus
unlikely that the Myb-binding site at
16 to
11 plays an important
role in the heat activation of promoter activity of the topoII
gene.
Heat shock induces the expression of heat shock-related genes in
mammalian cells, and this activation is mediated by HSFs (49-53). HSFs
bind to HSEs, which consist of contiguous arrays of the pentanucleotide
motif 5'-NGAAN-3' present in alternating orientations in the promoter
regions of heat shock genes. Most heat-inducible genes, including
HSP genes, contain an HSE consisting of four or more
pentanucleotide motifs and respond to heat treatment within 1 h
concomitant with marked fluctuations in nuclear HSF content (27, 30,
54, 55). Our data confirm that HSF1, but not HSF2, binds to the HSE of
the human HSP70 gene immediately after heat shock. However,
the HSE of the topoII gene consists of only two pentanucleotide
motifs, and heat shock-induced transcriptional activation of the
topoII
gene was not apparent until 6-24 h after heat treatment.
Furthermore, no increase in the binding of nuclear factors to the HSE
of the topoII
gene after heat treatment was apparent by EMSA or
in vivo footprint analysis. It is thus unlikely that the HSE
in the topoII
gene promoter is responsible for the heat-induced
activation of this gene.
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ACKNOWLEDGEMENT |
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We thank Takanori Nakamura (of our laboratory), Dr. Katsuhiko Hidaka (Saga Medical School), and Dr. Akira Nakai (Kyoto University) for fruitful discussion and Tomoko Matsuguma for help in preparing the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science, Sports, and Culture of Japan, and from the Ministry of Health and Welfare of Japan, and by the Fukuoka Anticancer Research Fund.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.
§ To whom correspondence should be addressed. Tel.: 81-92-642-6098; Fax: 81-92-642-6203; E-mail: mana{at}biochem1.med.kyushu-u.ac.jp.
1 The abbreviations used are: topoII, topoisomerase II; ICE, inverted CCAAT element; HSE, heat shock element; PCR, polymerase chain reaction; nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; HSF, heat shock factor.
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REFERENCES |
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