[CANCER RESEARCH 61, 5885–5894, August 1, 2001]
Regulation of the Catalase Gene Promoter by Sp1, CCAAT-recognizing Factors,
and a WT1/Egr-related Factor in Hydrogen Peroxide-resistant HP100 Cells
Mitsuru Nenoi,1 Sachiko Ichimura, Kazuei Mita, Osami Yukawa, and Iain L. Cartwright
Radiation Hazards Research Group, National Institute of Radiological Sciences, Chiba 263-8555, Japan [M. N., S. I., O. Y.]; Department of Genome Research, National Institute
of Agrobiological Sciences, Tsukuba 305-8634, Japan [K. M.]; and Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267 [I. L. C.]
ABSTRACT
Reactive oxygen species play a critical role in the onset of apoptosis
induced by various extracellular stimuli, including ionizing radiation.
Therefore active regulation of reactive oxygen species-metabolizing enzymes may be one response to an apoptotic stimulus. In this regard,
HP100 cells, H2O2-resistant variants derived from human leukemia HL60
cells, display an interesting phenotype in which the activity of catalase is
constitutively high, whereas its mRNA is reduced after X-ray irradiation.
In the present study, we investigated the molecular mechanisms underlying this phenomenon. By combining analyses from nuclear run-on, reporter gene transient transfection, genomic footprinting, site-directed mutagenesis, electrophoretic mobility shift analysis, and Western blotting
experiments, we found that constitutively elevated catalase expression is
strongly regulated at the transcriptional level by both Sp1 and CCAATrecognizing factors and that much higher levels of nuclear Sp1 and NF-Y
are present in HP100 nuclei as compared with HL60 nuclei. In addition,
we demonstrated an X-ray-inducible association of a WT1/Egr-related
factor with an overlapping Sp1/Egr-1 recognition sequence located within
the core promoter of the catalase gene. This association may lead to
inactivation of the promoter by disturbing or competing with the transactivating ability of Sp1.
INTRODUCTION
There has been a significant accumulation of experimental data
showing that ROS2 such as the superoxide anion, hydrogen peroxide,
the hydroxyl radical, and singlet oxygen can play critical roles as
physiological mediators in the onset of apoptosis that occurs in
response to various extracellular stimuli (reviewed in Refs. 1 and 2).
ROS are thought to be involved in transition from the inducer phase
to the effector phase during the progression of apoptosis, where the
initial diverse signaling pathways converge into universal regulatory
events, including the mitochondrial permeability transition (1). Due to
subsequent generation in mitochondria, ROS are also considered to
function in triggering transition from the effector phase to the degradation phase by the release of cytochrome c and the activation of
caspases. Among the various ROS, the importance of H2O2 has been
emphasized. For example, the inhibitory capacity of catalase in stressinduced apoptosis has been demonstrated in mammalian lymphocytes
(3), fibroblasts (4), smooth muscle cells (5), neurons (6 – 8), and
leukemia cell lines (9, 10). In addition, several cell lines with acquired
resistance to apoptosis and an elevated activity of catalase have been
isolated after long-term treatment with various oxidative stresses
(11–15); at the same time, the activities of other ROS-metabolizing
Received 2/1/01; accepted 5/23/01.
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.
1
To whom requests for reprints should be addressed, at Radiation Hazards Research
Group, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku,
Chiba 263-8555, Japan. Phone: 81-43-206-3084; Fax: 81-43-255-6497; E-mail:
[email protected].
2
The abbreviations used are: ROS, reactive oxygen species; GSHPx, glutathione
peroxidase; 3AT, 3-amino-1,2,4-triazole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LM-PCR, ligation-mediated PCR; EMSA, electrophoretic mobility shift assay;
PMSF, phenylmethylsulfonyl fluoride; TBS-T, 20 mM Tris-HCl (pH 7.6), 150 mM NaCl,
and 0.1% Tween 20.
enzymes such as superoxide dismutase and GSHPx vary depending on
the cell line (see Table 1). These observations suggest the existence of
an antiapoptotic signaling pathway in which catalase functions in the
down-regulation of cellular levels of H2O2 produced in response to
proapoptotic stimuli. Thus, it is important to identify signaling molecules that cause the elevated expression of catalase in these cell lines.
Because one way that ionizing radiation kills cancer cells is by
inducing apoptosis, elucidation and potential control of the pathway
by which it elicits apoptosis are important goals for radiotherapy. The
tumor suppressor protein p53 has been shown to serve as a pivotal
component in radiation-induced apoptosis (16, 17). However, signaling pathways independent of p53 have also been revealed, including
those involving ceramide, which is generated at the plasma membrane
by sphingomyelinase (18 –20). There is evidence that H2O2 plays a
role in such ionizing radiation-induced apoptosis. For example, the
transcription factor Egr-1 functions in p53-independent radiationinduced apoptosis (21), and its transcriptional induction by X-rays
requires ROS (22). Furthermore, both X-ray and H2O2-dependent
induction of the Egr-1 gene have been shown to be mediated through
a CC(A⫹T-rich)6GG element situated in its 5⬘-flanking region (23).
The involvement of catalase in DNA damage-induced apoptosis has
recently been demonstrated through the use of a H2O2-resistant cell
line, HP100. This cell line, which was isolated from HL60 by repeated
exposure to H2O2, overexpresses catalase and displays significant
resistance to H2O2-induced apoptosis (11, 12). Production of H2O2 in
both HL60 and HP100 cells has been observed after treatment with a
DNA-damaging agent, but its generation and the subsequent activation of caspase 3, loss of mitochondrial transmembrane potential
(⌬⌿m), and DNA ladder formation were delayed in HP100 cells as
compared with HL60 cells (24).
In considering a critical function for H2O2 in ionizing radiationinduced apoptosis, regulation of the activity of H2O2-metabolizing
enzymes, such as catalase and GSHPx, is likely to be required. Thus,
it is interesting to note that catalase mRNA levels are decreased after
X-ray irradiation in HP100 cells.3 A reduction in catalase mRNA has
also been observed in mouse splenocytes after ␥-ray irradiation (25).
These observations strongly suggest that ionizing radiation-inducible
apoptosis is mediated, at least in part, by a signaling pathway involving the stabilization of H2O2 via a down-regulation of catalase.
With this in mind, we have investigated the molecular mechanism
for elevated expression of the catalase gene in HP100 cells, as well as
its down-regulation by ionizing radiation. We found that the elevated
catalase expression is strongly regulated at the transcriptional level by
both Sp1 and CCAAT-recognizing factors and that much higher levels
of nuclear Sp1 and NF-Y are expressed in HP100 nuclei as compared
with HL60 nuclei. In addition, we also demonstrated that association
of a WT1/Egr-related factor with the overlapping Sp1/Egr-1 recognition sequence at the core promoter of the catalase gene is induced by
X-rays; this association may lead to inactivation of the core promoter
by disturbing or competing with the transactivating ability of Sp1.
3
M. Akashi, personal communication.
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REGULATION OF THE CATALASE GENE IN H2O2-RESISTANT CELLS
Table 1 Sublines isolated after a long-term oxidative stress
Catalase
Cell line
Original line
Treatment
Activitya
Proteina
mRNAa
Ref.
HP100
OC14/OC5
O2R95
L⬘-Se(⫺)
H⬘-Se(⫺)
HL-NR6
HL60
HA1b
HA1
L1210c
HL60
HL60
100 mM H2O2, 150 days
50–800 mM H2O2, ⬎200 days
80–95% O2, ⬎200 days
Selenium deprivation, 140 days
Selenium deprivation, 140 days
250 mM DETA/NO,d several months
17.6-fold
20–30-fold
20–30-fold
⬎100-fold
10.9-fold
2.0-fold
ND
25-fold
25-fold
ND
10.4-fold
ND
16-fold
25-fold
25-fold
65-fold
5-fold
ND
11, 12
13
13
14
14
15
a
Relative activity or cellular content compared to original cells; ND, not determined.
Chinese hamster fibroblasts.
Murine lymphocytic leukemia.
d
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, NO donor that spontaneously releases NO radicals.
b
c
MATERIALS AND METHODS
Cell Culture, Apoptosis Detection, and X-ray Irradiation. Human HL60
promyelocytic leukemia cells and their H2O2-resistant variant cell line, HP100,
were a gift from Dr. M. Akashi. The cells were cultured in RPMI 1640 (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (JRH), 100
units/ml penicillin, and 100 ␮g/ml streptomycin (Life Technologies, Inc.) at
37°C under 5% CO2 in a humidified atmosphere. HP100 cells were routinely
maintained in the presence of 100 ␮M H2O2, although it was removed from the
medium 7 days before the experiments described here. When 3AT, a specific
inhibitor of catalase, was used, cells were incubated for 2 h in the presence of
25 mM 3AT (Sigma Chemical Co.) before treatment with H2O2. 3AT has been
used to modulate catalase activity in a wide variety of cell types, including
HL60 cells (26).
The induction of apoptosis was examined by DNA fragmentation (27). Cells
were treated with 100 ␮M H2O2 in the presence or absence of 3AT, followed
by DNA extraction using a kit supplied by Takara (ApopLadder EX Kit) that
can efficiently remove DNA fragments of high molecular weight.
X-rays were generated from a Pantak unit operating at 200 kilovolt peak
(kVp) and 20 mA, with a 0.5 mm copper plus 0.5 mm aluminum filter. A dose
rate of 1.4 Gy/min was used.
Nuclear Run-on Analysis. cDNAs for human catalase, GAPDH, and
GSHPx were amplified by reverse transcription-PCR and cloned into pCR2.1
(Invitrogen). Next, 5 ␮g of linearized and denatured plasmids were spotted and
fixed onto a GeneScreen membrane (New England Nuclear Life Science
Products).
Cells (5 ⫻ 107) were irradiated with 20 Gy of X-rays and then incubated for
various time intervals at 37°C. After washing with ice-cold PBS, cells were
suspended in 4.5 ml of ice-cold hypotonic buffer [10 mM Tris-HCl (pH 7.4),
10 mM KCl, and 3 mM MgCl2], left on ice for 30 min, and lysed by adding 0.5
ml of 5% NP40, followed by incubation on ice for 5 min (28). Subsequently,
nuclei were pelleted by centrifugation at 110 ⫻ g for 5 min at 4°C, washed
with 5 ml of 0.5% NP40, and resuspended in 200 ␮l of storage buffer [40%
glycerol, 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, and 0.1 mM EDTA]. mRNA
elongation was performed by mixing the nuclear suspension with 100 ␮l of
reaction buffer containing 340 mM KCl; 5 mM MgCl2; 10 mM Tris-HCl (pH,
8.0); 4.5 mM DTT; 0.75 mM each of ATP, CTP, and GTP; 36 units of RNase
inhibitor (Takara), and 200 ␮Ci of [␣-32P]UTP (3000 Ci/mmol; ICN Biomedicals), followed by incubation at 30°C for 30 min. After the DNA was digested
with 210 units of RNase-free DNase I (Takara), elongated mRNA was isolated
following the standard protocol of phenol/chloroform extraction.
Denatured RNA was hybridized with the membrane filter in hybridization
buffer [50% formamide, 3⫻ SSC, 10⫻ Denhardt’s solution, 10 mM phosphate
buffer (pH 7.0), 0.1 mg/ml salmon sperm DNA, 0.2 mg/ml yeast tRNA, and
0.1% SDS] for 72 h at 42°C. The membrane was washed sequentially with 2⫻
SSC at room temperature for 30 min, 2⫻ SSC at 42°C for 30 min, and 0.1⫻
SSC at 42°C for 30 min.
Nucleotide Sequence Analysis. The 5⬘-flanking region of the human catalase gene had been previously sequenced up to ⫺1527 bp with respect to the
major mRNA initiation site (29). Based on the nucleotide sequence reported
there, a further upstream region was cloned and sequenced in chromosomewalking fashion using a kit supplied by Clontech (Genome Walker kit). The
sequenced data were deposited in the DNA Data Bank of Japan, European
Molecular Biology Laboratory, and GenBank nucleotide sequence databases
under accession number AB034940.
Reporter Gene Construction and Transient Expression Analysis. Regions
containing the catalase gene promoter spanning nucleotides ⫺4526/⫹16,
⫺3113/⫹16, ⫺2379/⫹16, ⫺1518/⫹16, ⫺1226/⫹16, ⫺935/⫹16, ⫺733/
⫹16, ⫺404/⫹16, and ⫺176/⫹16 were PCR-amplified using the 5⬘ primers
5⬘-CAATGGTACCGTCTATGTCCACGTCCTTTGCTGC-3⬘, 5⬘-CAATGGTACCAAACACCAGATCAGTAGCGTGGC-3⬘, 5⬘-CAATGGTACCGAGTTCTGAAAATTGACTTCAGAGAACAGC-3⬘, 5⬘-CAATGGTACCTGTGGACTTTGGAGATGAACAGCTG-3⬘,
5⬘-CAATGGTACCGACACCAAATTACACAG CCAACAGCATC-3⬘, 5⬘-CAATGGTACC-AATCCTAGCACCTGAGGAGGTGTAG-3⬘, 5⬘-CAATGGTACCGAA GCCAATTTGGCAGTGTACCAGAG-3⬘, 5⬘-CAATGGTACCGCTGAGAAAGCATAGCTATG GAGCG-3⬘, and
5⬘-CAATGGTACCTATCTCCGGTCTTCAGGCCTCCTTC-3⬘, respectively,
and the common 3⬘ primer 5⬘-GTCAGATCTCAGCAGGCAAATCTGCCTGTTGC-3⬘; the underlined bases were introduced to facilitate cloning with KpnI
for 5⬘ primers and BglII for 3⬘ primers. LA Taq DNA polymerase (Takara) was
used to ensure high-fidelity amplification. The PCR fragments were ligated into
pGL3-Basic Photinus pyralis luciferase reporter vector (Promega). Recombinant
plasmids for each construct were extracted and purified by alkaline lysis and
ion-exchange chromatography (Genomed) following the manufacturer’s instructions. Site-directed mutagenesis was performed on the CAT-404 construct containing the region ⫺404/⫹16 by use of a kit supplied by Promega (GeneEditor in
vitro Site-Directed Mutagenesis System).
Transfection was carried out by electroporation. Cells grown to a density of
approximately 106 cells/ml were washed three times with serum-free RPMI
1640. Cells (2 ⫻ 107) were mixed with 35 ␮g of test plasmid DNA and 1–2
␮g of pRL-SV40 (Renilla reniformis luciferase under the control of SV40
early enhancer/promoter; Promega) as a transfection efficiency control in 240
␮l of serum-free RPMI 1640 and electroporated with the Gene Pulser
(Bio-Rad) in a 0.4-cm electroporation cuvette at 250 V with a capacitance of
960 microfarads. Cell lysates were prepared 48 h after transfection by adding
100 ␮l of Passive Lysis Buffer (Dual-Luciferase Reporter Assay System;
Promega) to the cell pellet. The luciferase activity was measured with an
analytical luminometer (model LB9506; Berthold), and the promoter activity
was evaluated as relative light units, which is defined as the ratio of the light
intensity produced by Photinus luciferase (test plasmid) to that produced by
Renilla luciferase (control plasmid).
Genomic Footprinting Analysis. HL60 and HP100 cells grown to a density of approximately 106 cells/ml were irradiated with various doses of X-rays
and then incubated for 1 h at 37°C. After washing with ice-cold PBS, 5 ⫻ 106
cells were permeabilized by treatment with lysolecithin (Sigma Chemical Co.;
0.002% for HL60 and 0.001% for HP100) in ice-cold permeabilization buffer
[35 mM HEPES (pH 7.4), 5 mM potassium phosphate (pH 7.4), 80 mM KCl, 5
mM MgCl2, 0.5 mM CaCl2, and 150 mM sucrose] for 2 min. After washing with
digestion buffer [35 mM HEPES (pH 7.4), 5 mM potassium phosphate (pH 7.4),
80 mM NaCl, 5 mM MgCl2, 2 mM CaCl2, and 150 mM sucrose], the permeabilized cells were treated with DNase I (Takara; 4.0 units/ml for HL60 and 11
units/ml for HP100) in digestion buffer at 25°C for 5 min to partially digest the
DNA within chromatin. The reaction was stopped by adding 0.1 volume of 6%
SDS and 250 mM EDTA (pH 8.0). DNA was purified by a standard phenol/
chloroform extraction method. A naked DNA control was prepared by cleaving
high molecular weight genomic DNA isolated previously from HL60 and
HP100 cells with 0.09 unit/ml DNase I at 25°C for 2 min in digestion buffer.
The DNase I cleavage conditions described above resulted in a broad distri-
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REGULATION OF THE CATALASE GENE IN H2O2-RESISTANT CELLS
bution of fragment sizes ranging from shorter than 100 bp to longer than 10 kb
as determined by agarose gel electrophoresis.
The DNase I digestion pattern in the promoter region of the catalase gene
was assessed by a LM-PCR method (30 –33) with the addition of an extension
product capture step (34). The unidirectional double-stranded linker DNA,
which was blunted at one end, was prepared by annealing a pair of complementary oligonucleotides, L25 (5⬘-GCGGTGACCCGGGAGATCTGAATTC3⬘) and L11 (5⬘-GAATTCAGATC-3⬘). 5⬘-Biotinylated primers (primer 1,
5⬘-ATAGCGTGCGGTTTGCTGTGCAGAAC-3⬘) complementary to the upper strand at site ⫹75/⫹50 were annealed to heat-denatured DNase I-treated
genomic DNA (5 min at 95°C, 30 min at 60°C) and extended using Vent DNA
polymerase (New England Biolabs) for 10 min at 76°C in 30 ␮l of the
first-strand mixture containing 40 mM NaCl, 10 mM Tris-HCl (pH 8.9), 5 mM
MgSO4, 0.01% gelatin, 0.2 mM each deoxynucleotide triphosphate, 0.3 pmol
of primer, 2 ␮g of DNase I-treated DNA, and 1.2 units of Vent polymerase.
After diluting the reaction mixture with 20 ␮l of Vent dilution buffer [110 mM
Tris-HCl (pH 7.5), 18 mM MgCl2, 50 mM DTT, and 0.125 mg/ml DNase-free
BSA], the linker DNA was ligated to DNase I-cleaved sites (where ligatable
blunt ends were created by the primer extension) by the addition of 25 ␮l of
ligation solution (10 mM MgCl2, 20 mM DTT, 3.75 mM ATP, 0.05 mg/ml
DNase-free BSA, 4 pmol of linker DNA, and 16 units/ml T4 DNA ligase). The
ligation reaction was carried out overnight at 16°C, followed by precipitation
with ethanol. A 175-␮g portion of Dynabeads M-280 streptavidin (Dynal) was
washed twice and incubated with ligation products in 70 ␮l of binding buffer
[10 mM Tris-HCl (pH 7.7), 1 mM EDTA, and 1 M NaCl] for 15 min at room
temperature. After washing the beads with 10 mM Tris-HCl (pH 7.7), 1 mM
EDTA, and 2 M NaCl, the nonbiotinylated template strand was eluted from the
beads by incubation at 37°C for 10 min in 35 ␮l of 0.15 N NaOH. The eluted
DNA was neutralized and precipitated with ethanol. The fragments of interest,
which contained the promoter region of the catalase gene, were PCR-amplified
using linker primer (L25) and a second site-specific primer (primer 2, 5⬘TGTGCAGAACACTGCAGGAGGCCTC-3⬘), which anneals at ⫹59/⫹35
just upstream of the primer 1 annealing site. The reaction was carried out for
18 cycles (1 min at 95°C, 2 min at 68°C, and 3 min at 76°C) in an amplification
mixture containing 40 mM NaCl, 20 mM Tris-HCl (pH 8.9), 5.2 mM MgSO4,
0.009% gelatin, 0.09% Triton X-100, 0.2 mM each deoxynucleotide triphosphate, 10 pmol of primer 2, 10 pmol of L25, and 3 units of Vent polymerase.
After amplification was completed, 2.3 pmol of 32P-labeled primer (primer 3,
5⬘-TGTGCAGAACACTGCAGGAGGCCTCGGC-3⬘), whose annealing site
overlaps with that of primer 2 with an additional three bases extending to the
3⬘ end) were added, and two cycles of primer extension were carried out (1 min
at 95°C, 2 min at 71°C, and 10 min at 76°C). The reaction products were
purified by phenol/chloroform extraction, precipitated with ethanol, and subjected to 6% PAGE. The gels were dried, and the bands were visualized by
autoradiography. The site specificity of the primers used in this experiment
was confirmed in advance by observing a single specific band when LM-PCR
was carried out with genomic DNA digested with a restriction enzyme as the
template.
Preparation of Nuclear Extracts from Cells. Cells (108) grown to a
density of approximately 106 cells/ml were irradiated with 20 Gy of X-rays and
then incubated for 1 h. After sequential washing with ice-cold PBS and
hypotonic buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM
PMSF, and 0.5 mM DTT], cells were incubated in 320 ␮l of hypotonic buffer
for 10 min on ice. Cells were then homogenized with a Dounce homogenizer.
Subsequently, nuclei were collected by centrifugation and suspended in an
equal volume of extraction buffer [20 mM HEPES (pH 7.9), 25% (v/v)
glycerol, 1.5 mM MgCl2, 0.6 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5
mM DTT]. After incubation for 30 min on ice, nuclear extracts were dialyzed
against a dialysis buffer [20 mM HEPES (pH 7.9), 20% (v/v) glycerol, 100 mM
KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT] for 2 h. After
centrifugation at 25,000 ⫻ g for 20 min, supernatant was stored at ⫺80°C.
Protein concentrations were measured by the Bradford method using a kit
supplied by Pierce (Coomassie Protein Assay Reagent Kit).
EMSA. Double-stranded DNA probes were prepared by annealing oligonucleotides spanning ⫺113/⫺80 (5⬘-GGCCTGCCTAGCGCCGAGCAGCCAATCAGAAGGC-3⬘) and ⫺78/⫺45 (5⬘-GTCCTCC CGAGGGGGCGGGACGAGGGGGTGGTGC-3⬘), and they were end-labeled with [␥-32P]ATP
(4,000 Ci/mmol; ICN Biomedicals) and T4 polynucleotide kinase (Takara).
Nuclear extracts, prepared as described above, were mixed with 10,000 cpm
(0.5 ng) of the probe in 20 mM HEPES (pH 7.9), 70 mM KCl, 5 mM MgCl2,
0.05% NP40, 1 mg/ml BSA, 0.5 mM DTT, 0.1 mg/ml poly(deoxyinosinicdeoxycytidylic acid), and 12% glycerol and incubated for 20 min at 25°C. The
reaction mixture was subjected to electrophoresis on a 6% polyacrylamide gel
with 0.25⫻ Tris-borate EDTA running buffer. The gels were dried, and the
bands were visualized by autoradiography. In competition studies, either
double-stranded Egr-1 consensus oligonucleotide (5⬘-GGATCCAGCGGGGGCGAGCGGGGGCGA-3⬘) or Egr-1 mutant oligonucleotide (5⬘-GGATCCAGCTAGGGCGAGCTAGGGCGA-3⬘; Santa Cruz Biotechnology) was added
at a 100-fold excess over the radiolabeled probe. In supershift studies, polyclonal
antibodies against either NF-Y (AHP298; Oxford Biomarketing) or Sp1 (PEP2;
Santa Cruz Biotechnology) were used. When either competitor oligonucleotides or
antibodies were used, they were preincubated with the nuclear extracts at 4°C for
25 min before the addition of the radiolabeled probe DNA.
Western Blotting. A 4-␮g sample of nuclear proteins prepared as described above was separated on a 10% SDS polyacrylamide gel and electrotransferred to a Hybond-P polyvinylidene difluoride membrane (Amersham
Life Science). The membrane was then incubated sequentially with 2% BSA
in TBS-T for 1 h, with either 0.067 ␮g/ml anti-Sp1 antibody (PEP2) or 1 ␮g/ml
anti-NF-Y antibody (AHP298) in TBS-T for 1 h, and with a 1:1000 dilution of
horseradish peroxidase-conjugated secondary antibody (Amersham Life Science) in TBS-T for 1 h. The bands were visualized by enhanced chemiluminescence using reagents supplied by Amersham Life Science (enhanced
chemiluminescence Western blotting detection reagent).
RESULTS
Critical Role of Catalase in Resistance of HP100 to H2O2. The
HP100 cell line is a stable variant isolated from HL60 by repetitive
treatment with 100 ␮M H2O2 for 5 months. The effective dose of
H2O2 for killing 50% of the cell population of HP100 (2.4 mM) was
reported to be much higher than that of HL60 (7 ␮M; Ref. 11).
Because HP100 overexpresses catalase, with the enzymatic activity
being 18-fold higher than that of HL60 (12), it appears likely that the
elevated catalase inhibits the induction of apoptosis via its enhanced
decomposition of H2O2. However, the possibility that other apoptotic
pathway signaling components may be disrupted in HP100 cannot be
excluded. We therefore examined the effects of a catalase-specific
inhibitor, 3AT, on the sensitivity of HP100 to H2O2. Fig. 1A shows
that HP100 cells were resistant to 100 ␮M H2O2 and grew with a
doubling time similar to that of untreated cells, but that the growth of
HP100 cells was completely inhibited if 25 mM 3AT was added 2 h
before the addition of H2O2; treatment with 3AT alone did not affect
the growth of these cells. The observed growth inhibition resulted
from apoptosis because DNA fragmentation was only observed when
the cells were treated with H2O2 in the presence of 3AT (Fig. 1B).
This is in contrast to HL60 cells, which readily die by apoptosis in 2
min after treatment with 20 ␮M H2O2 alone (35). These results suggest
that the H2O2-induced apoptotic signaling pathway is intact in HP100
cells and that the overexpressed catalase plays a decisive role in the
resistance of HP100 cells to H2O2. Regulation of catalase expression
is therefore critical for induction of apoptosis in HP100 cells.
Transcriptional Regulation of the Catalase Gene. By Northern
analyses, it has been determined that the level of catalase mRNA is
16-fold higher in HP100 cells than in HL60 cells (12) and that this
level decreases after X-ray irradiation.3 A possible mechanism for this
phenomenon involves regulation of the catalase gene at the transcriptional level. However, the 3⬘-untranslated region of the human catalase gene is highly A/T rich, and the presence of four ATTTA
sequences (36) suggests the possibility of a regulated degradation of
its transcripts. Actually, the half-life of catalase mRNA has been
shown to be variable in the lung of rats when exposed to an oxidative
stress (37). In addition, it has been reported that the catalase gene is
amplified in HP100 cells (12), implicating a mRNA elevation caused
by the increased gene dosage. Therefore, we performed a transient
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Fig. 1. The effect of a catalase inhibitor on the resistance of HP100 cells to H2O2.
HP100 cells were either treated or not treated with 100 ␮M H2O2 in the presence or
absence of 25 mM 3AT, a specific inhibitor of catalase. When 3AT was incorporated, cells
were preincubated for 2 h in the presence of 25 mM 3AT before the addition of H2O2. A,
growth of HP100 cells cultured under conditions with no treatment (E), with H2O2 alone
(‚), with 3AT alone (F), or with both H2O2 and 3AT (Œ). Cell number was counted with
a Coulter Counter (Coulter Electronics). B, DNA ladder formation (characteristic of
apoptosis) was examined 6 h after the addition of H2O2.
expression analysis of luciferase gene constructs and a nuclear run-on
analysis to determine whether catalase gene expression is regulated at
the transcription level.
For the transient expression analysis, we examined the region up
to ⫺4526 bp from a major catalase transcription start site located
⫺73 bp upstream of the initiation codon (29). As shown in Fig. 2,
an enhanced promoter activity (3.4 – 8.3-fold) was observed in
HP100 cells compared with that in HL60 cells for every construct
containing the 5⬘-flanking region (anywhere from ⫺176 to
⫺4526). We conclude that elevated catalase mRNA in HP100 cells
is due to an enhanced transcription of individual genes and that the
majority of cis-regulatory elements causing its elevation are located in the region from ⫺176 to ⫹16 bp.
Next we performed transient transfections followed by irradia-
tion of transfectants with X-rays. However, no convincing downregulation of the catalase gene promoter in HP100 cells was seen.
Because the luciferase protein encoded by pGL3-Basic vector is
artificially modified to be stabilized in mammalian cells, we speculate that any transcriptional down-regulation may have been
masked by long-lived luciferase proteins that had accumulated
before X-ray irradiation. In a further attempt to address this issue,
we performed a nuclear run-on experiment. As shown in Fig. 3A,
a drastic and transient decrease in transcription of the catalase gene
in HP100 cells after irradiation with 20 Gy of X-rays was revealed.
When normalized to the transcriptional activity of the GAPDH
gene, transcription of the catalase gene after 1 h of incubation was
reduced to one-tenth of that in unirradiated HP100 cells (Fig. 3B).
It can therefore be concluded that the reduction in catalase mRNA
levels after X-ray irradiation in HP100 cells is regulated primarily
at the transcriptional level.
The transcriptional activity of the catalase gene in unirradiated
HP100 cells was 20-fold higher than that in HL60 cells (Fig. 3B), a
figure that was similar to the previously reported fold increase in
mRNA (12). However, it was larger than that observed in the transient
expression analysis (5.6-fold for the CAT-4526 construct in Fig. 2).
This apparent discrepancy may be attributable to the amplification of
the catalase gene that occurs in HP100 cells.
In Fig. 3, no difference in the transcriptional activity of the GSHPx
gene, which encodes another H2O2-decomposing enzyme, can be
observed between HL60 and HP100 cells, which is consistent with the
observation by Kasugai and Yamada (38) that both cell lines show
comparable GSHPx enzymatic activity. Furthermore, no change in the
transcriptional activity of the GSHPx gene was observed after X-ray
irradiation.
DNA-Protein Interactions in the Regulatory Region. To elucidate the molecular mechanisms behind the transcriptional regulation
of the catalase gene, we attempted to identify the cis-regulatory
elements that caused its elevated transcription in HP100. For this
purpose, we analyzed DNA-protein interactions in the upstream region of the catalase gene by DNase I genomic footprinting. Fig. 4A
shows the DNase I cleavage pattern on the upper strand in a region
encompassing the transcription start site to approximately ⫺200 bp,
which fully covers the region ⫺176 to ⫹16 where the cis-regulatory
elements causing elevated transcriptional activity in HP100 cells had
been determined to reside based on the transient expression analysis
(Fig. 2). Throughout the region examined, the DNase I cleavage
pattern was very similar between HL60 and HP100 cells as well as
Fig. 2. Transient expression of the luciferase
gene constructs. HL60 and HP100 cells were transfected with different CAT-x constructs (left),
which consist of the 5⬘-flanking region of the catalase gene spanning nucleotides ⫺x/⫹16 linked to
the Photinus luciferase gene in the pGL3 Basic
vector or with the promoterless pGL3 vector together with a Renilla luciferase expression vector
(pRL-SV40) as a transfection efficiency control.
Cells were lysed 48 h after transfection, and luciferase activity was measured with an analytical
luminometer. Promoter activity was evaluated in
relative light units, which is the ratio of light intensity produced by Photinus luciferase (CAT-x) to
that produced by Renilla luciferase. Error bars
represent the means ⫾ SD of at least four experiments in duplicate.
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Fig. 3. Nuclear run-on analysis. HL60 and HP100 cells were either untreated or
irradiated with 20 Gy of X-rays, followed by incubation at 37°C for 1, 2.5, and 4 h. Nuclei
were isolated, and mRNA was elongated for 30 min at 30°C in the presence of
[␣-32P]UTP. Purified mRNA was hybridized to cDNAs for catalase, GAPDH, and
GSHPx, as well as to the pCR2.1 vector used to clone the cDNAs. A, autoradiography of
the blots. The bottom panel shows the position of the blotted cDNAs and vector. B, kinetic
changes in the transcriptional activity of the catalase and GSHPx genes normalized to that
of GAPDH.
between irradiated HP100 cells and unirradiated HP100 cells. However, in the vicinity of the transcription start site, strongly increased
sensitivity to DNase I in HP100 cells was observed (HS1 and HS2),
which suggests that this region has an open chromatin conformation in
this cell type. In addition, clear footprints (FP1, FP2, and FP3) were
observed in both HL60 and HP100 cells in the region from ⫺100 bp
to ⫺50 bp, where the band intensities were obviously lower compared
with that in the naked DNA lane (Lanes N). Fig. 4B shows a magnified portion of the region around these observed footprints. Three
bands at approximately ⫺99, ⫺98, and ⫺69 bp (arrows) that are more
intense in HP100 cells as compared with HL60 cells can be clearly
observed, as well as two bands at approximately ⫺101 and ⫺75 bp
(arrowheads designated X) that show an X-ray-dependent reduction in
intensity in HP100 cells. In the naked DNA, differential DNase I
sensitivity was observed for bases at approximately ⫺101 and ⫺75 bp
when comparing HL60 and HP100 cells. It appears that these discrepancies are not due to a point mutation in HP100 DNA because we
were able to confirm the identity of the nucleotide sequence in HL60
and HP100 cells across the whole region investigated. It is supposed
that when the naked DNA was isolated, removal of nuclear proteins
may have been insufficient at these locations, potentially causing the
differential base sensitivity to DNase I attack.
Because the FP1, FP2, and FP3 footprints were not altered between
HL60 and HP100 cells or by X-ray irradiation, it can be concluded
that nuclear proteins are stably bound to these sites. However, we
observed that the cleavage intensities of several bases at the edges of
the footprints were altered between HL60 and HP100 cells (indicated
by arrows in Fig. 4B), which indicates that some change in the fine
structure of the DNA-protein interaction occurs between these two
cell types. Such changes may be caused by an altered interaction
between the bound proteins, possibly mediated by a non-DNA binding
coactivator or alternatively by a substitution of the bound proteins
with some others. This result strongly suggests that the cis-regulatory
elements that cause elevated transcription of the catalase gene in
HP100 cells are located either at the observed footprints or in their
immediate flanking regions.
The cis-regulatory elements responsible for down-regulation of the
catalase gene in HP100 cells after X-ray irradiation were not mapped
directly by transient transfection analysis in this study (see above).
However, Fig. 4B shows an X-ray-dependent change in the DNase I
sensitivity of bases (indicated by arrowheads designated X) that are
only a few nucleotides removed from those bases whose in vivo
DNase I sensitivity differed between HL60 and HP100 cells. These
results suggest that specific DNA-protein interactions thought to play
a role in the elevated catalase transcription of HP100 cells are further
modified by X-rays and strongly imply that a common cis-regulatory
element functions in both the elevated transcription in HP100 and its
down-regulation by X-rays.
Fig. 4C depicts the nucleotide sequence around the observed footprints. There are numerous motif sequences of transcription factors.
Among these, clustered CCAAT boxes (CCAAT-42, CCAAT-92,
CCAAT-121, and CCAAT-130) and tandemly repeated Egr-1 recognition sequences (Egr-1–57 and Egr-1–70; Ref. 39), one of which is
overlapped by a Sp1 recognition sequence (Sp1– 67; Ref. 40), characterize this region. These elements are important in regulation of
redox-sensitive genes. The CCAAT-92 sequence is located at the FP1
footprint, and the Sp1– 67, Egr-1–70, and Egr-1–57 sequences are
colocated at the FP3 footprint. These motifs are certainly candidates
for the functional elements that cause the elevated transcription of the
catalase gene in HP100 cells as well as its reduced transcription after
X-ray irradiation.
Identification of the Functional cis-Regulatory Element. To
identify the functional cis-regulatory element(s) responsible for elevated transcription of the catalase gene in HP100 cells, mutations
were introduced into the luciferase construct CAT-404 (Fig. 2) by
site-directed mutagenesis. As shown in Fig. 5A, the CCAAT sequences (CCAAT-42, CCAAT-92, CCAAT-121, and CCAAT-130) and
the overlapping recognition sequence for Egr-1 and Sp1 (EgrSp-70)
were substituted with sequences containing recognition sites for restriction enzymes (Mut-42, Mut-92, Mut-121, Mut-130, and Mut-70,
respectively) to facilitate screening of the mutated constructs. These
constructs were transfected into HL60 and HP100 cells, and the
luciferase activity was measured 48 h after transfection. As shown in
Fig. 5B, a mutation at EgrSp-70 resulted in loss of most of the basal
activity of the promoter, clearly demonstrating that EgrSp-70 plays a
critical role in transcription of the catalase gene. The GC box in
EgrSp-70 may therefore function as the core promoter in the TATAless catalase gene. A mutation at CCAAT-92 had no influence on
promoter activity in HL60 cells, although it drastically reduced promoter activity in HP100 cells to a level almost identical to that in
HL60 cells. These results suggest that CCAAT-92 functions as a
transcriptional enhancer in HP100 cells. In contrast, a mutation at
CCAAT-130 did not significantly affect promoter activity in either
HL60 or HP100 cells, indicating a nonessential role for CCAAT-130.
Interestingly, a mutation at either CCAAT-42 or CCAAT-121 led to
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Fig. 4. Genomic footprinting analysis. A, HL60 and HP100
cells were either left untreated or irradiated with the indicated
dose of X-rays, followed by incubation at 37°C for 1 h. Cells
were permeabilized and treated with DNase I to partially digest
the chromatin in vivo. The DNase I cleavage pattern on the upper
strand in the 5⬘-flanking region of the catalase gene was analyzed by the LM-PCR method, followed by electrophoresis on a
6% denaturing polyacrylamide gel. Lanes N show the DNase I
cleavage pattern for naked DNA isolated from HL60 or HP100
cells before digestion with DNase I in vitro. End-labeled DNA
fragments from MspI-digested pBR322 were used as a molecular
weight marker (lane marker). The footprints observed in HL60
and HP100 cells are indicated by FP1–FP3, and the DNase
I-hypersensitive sites observed in HP100 cells are indicated by
HS1 and HS2. B, the gel was run for twice as long to obtain a
higher resolution, and the region around the observed footprints
was magnified. Bands whose intensity was reduced in HP100
cells depending on the X-ray dose are indicated by arrowheads
designated X, and bands that were more intense in HP100 cells
than in HL60 cells are indicated by arrows. C, nucleotide sequences around the observed footprints FP1–FP3 and the location of binding motifs found therein. The arrows and arrowheads indicate bases whose sensitivities to DNase I attack were
altered as described in B.
increased promoter activity in HL60 cells but to decreased promoter
activity in HP100 cells. This opposing function may be due to recognition by different factors in HL60 and HP100 cells or, alternatively, to binding factors being modified differently in these cells. In
summary, it can be concluded that the GC box in EgrSp-70 functions
as a core promoter for the catalase gene and that CCAAT-42,
CCAAT-92, and CCAAT-121 function as modulators of transcriptional activity. CCAAT-92 functions as an enhancer element in
HP100 cells, whereas both CCAAT-42 and CCAAT-121 function as
either negative or positive modulators in HL60 and HP100 cells,
respectively. All these elements are necessary for the elevated transcription of the catalase gene in HP100 cells.
Identification of the trans-acting Factor. To identify the factors
binding to the cis-regulatory elements, we performed EMSA analyses.
Because we observed that CCAAT-92 functions as a transcriptional
enhancer in HP100 cells (Fig. 5), EMSA analysis was first performed
with a probe containing the CCAAT-92 element (Fig. 6A). As shown
in Fig. 6B, multiple complexes were formed with nuclear extracts
from both HL60 and HP100 cells. However three additional shifted
bands (S1, S2, and S3) were observed when nuclear extracts from
HP100 cells were used. Incorporation of anti-NF-Y antibodies caused
a disappearance of S3, demonstrating that S3 represents a complex
containing NF-Y. Because these complexes were not observed when
nuclear extracts from HL60 cells were used, at least some of the
factors composing these complexes are likely to be responsible for the
elevated transcription of the catalase gene in HP100 cells. However,
because the band pattern was not changed when HP100 cells were
irradiated with X-rays, these factors are probably not involved in
down-regulation of the catalase promoter by X-rays.
The results of our genomic footprinting analysis suggest that a
common cis-regulatory element functions in both the elevated transcription of the catalase gene in HP100 cells and its down-regulation
by X-rays. In this regard, it is interesting to note that Egr-1 has been
shown to down-regulate the rat Pgp2/mdr1b gene (41) and the mouse
adenosine deaminase gene (42) promoters by competing with Sp1 for
binding to an overlapping sequence motif. In addition, Egr-1 is an
immediate early gene responding to ionizing radiation and is inducible
less than 1 h after irradiation (21, 22). Accordingly, we hypothesize
that Egr-1 is induced by X-rays in HP100 cells and that competition
with Sp1 for binding to the core promoter in EgrSp-70 results in
reduced promoter activity. To test this hypothesis, we performed
EMSA analyses with the probe containing EgrSp-70 sequence
(Fig. 7A). As shown in Fig. 7B, we were able to observe three shifted
bands (S1–S3) when 3.3 ␮g of nuclear protein per reaction were used.
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Fig. 5. Site-directed mutagenesis analysis. Mutations were introduced into luciferase reporter
construct CAT-404 (Fig. 2) that contains the region
⫺404/⫹16 of the catalase gene linked to the Photinus luciferase gene. A, mutant sequences (Mut130, Mut-121, Mut-92, Mut-42, and Mut-70) used
to replace the native motif sequence-containing
regions (CCAAT-130, CCAAT-121, CCAAT-92,
CCAAT-42, and EgrSp-70, respectively). B, HL60
and HP100 cells were transfected with CAT-404 or
with the mutant constructs CAT-404/Mut-42,
CAT-404/Mut-70, CAT-404/Mut-92, CAT-404/
Mut-121, and CAT-404/Mut-130, as illustrated on
the left panel. Promoter activity was evaluated as
described in the Fig. 2 legend. Error bars represent
the means ⫾ SD of at least two experiments in
duplicate.
We found, however, that an additional band (S4) appeared when a low
concentration of HP100 nuclear proteins (0.10 – 0.41 ␮g/reaction) was
used. This observation can be explained by assuming that the nuclear
factor that forms the S4 complex acquires the ability to interact with
itself or other proteins after binding to DNA and that when a high
concentration of nuclear proteins was used, the binding of multiple
proteins to the S4 complex retarded its migration on the gel, giving
rise to an accidental comigration with one of the S1–S3 bands. To
avoid complications, we carried out the EMSA experiments described
below under conditions in which S4 was observable (0.22 ␮g nuclear
protein/reaction).
When anti-Sp1 antibodies (PEP2) were incorporated, the intensity
of S1 obviously decreased, and S2 and S3 disappeared (Fig. 7C); this
disappearance was accompanied by the appearance of the supershifted
bands SS1 and SS2. These results demonstrate that S1, S2, and S3
represent complexes containing Sp1. Combining this result with the
finding that the GC box in EgrSp-70 functions as a core promoter and
the previously proposed model that Sp1 functions in recruiting RNA
polymerase II by tethering the preinitiation complex to TATA-less
promoters (43, 44), it appears that Sp1 plays an essential role in
transcription of the catalase gene by binding to the GC box in
EgrSp-70.
On the other hand, the addition of an Egr-1 consensus competitor
oligonucleotide, but not an Egr-1 mutant oligonucleotide, at a 100fold molar excess obviously reduced the S4 band intensity (Fig. 7D),
demonstrating that S4 contains a member of the WT1/Egr protein
family. In addition, Fig. 7, B–D, reveals that the S4 band intensity was
increased after X-ray irradiation, whereas a significant change was not
observed for S1, S2, and S3. Therefore, we conclude that the WT1/
Egr-related factor is X-ray inducible.
When EMSA was carried out with nuclear extracts of HL60 cells at
the same concentration (0.22 ␮g/reaction), bands S1 and S4 were
observed only at very low intensity (Fig. 7C). This demonstrates an
extremely low binding activity of both Sp1 and the WT1/Egr-related
factor in HL60 cells, similar to the observation with NF-Y made in
Fig. 6. We further investigated the nuclear content of Sp1 and NF-Y
by Western blotting analysis. Fig. 8A shows Coomassie Blue staining
of nuclear proteins separated by SDS-PAGE, revealing similar
amounts of proteins loaded on the gel. When probed with anti-Sp1
antibodies (PEP2), two specific protein species were observed, as
shown in Fig. 8B, the lower of which is considered to be a degradation
product of Sp1. It is evident that both Sp1 and NF-Y are quite
abundant in the nuclei of HP100 cells as compared with the nuclei of
HL60 cells.
In summary, it seems that remarkably high levels of Sp1 are
expressed in HP100 nuclei, which leads to activation of the core
catalase promoter in cooperation with CCAAT-recognizing factors.
Moreover, by analogy with the observation that members of the
WT1/Egr family are capable of repressing a wide range of mammalian
Fig. 6. EMSA analysis. Nuclear factors bound to a DNA fragment containing the
CCAAT-92 element were analyzed. A, the sequence of the probe DNA. B, EMSA was
performed using 8.0 ␮g of nuclear extracts per reaction from unirradiated or irradiated (20
Gy) HP100 and HL60 cells. A supershift assay was carried out using anti-NF-Y antibody
(AHP298).
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Fig. 7. EMSA analysis. Nuclear factors bound to a DNA fragment containing the EgrSp-70 element were analyzed. A, sequences of the probe DNA and the Egr competitors. The
EgrSp-70 sequence in the probe and the Egr-1 binding motif in the competitors are boxed. The point mutations introduced into the Egr mutant competitor are denoted by asterisks.
B, EMSA was performed using the indicated amount of nuclear extracts per reaction from unirradiated or irradiated (20 Gy) HP100 cells. C, EMSA was performed using 0.22 ␮g of
nuclear extracts per reaction from unirradiated or irradiated (20 Gy) HP100 and HL60 cells. A supershift assay was carried out using anti-Sp1 antibody (PEP2). D, EMSA was performed
using 0.22 ␮g of nuclear extracts per reaction from unirradiated or irradiated (20 Gy) HP100 cells. Competition was carried out using either Egr consensus competitor (Lanes C) or
Egr mutant competitor (Lanes M).
promoters (45– 48), an X-ray-inducible WT1/Egr-related factor,
which forms the S4 complex, appears to function by inactivating the
core promoter in competition with Sp1. However, because the binding
activity of Sp1 does not appear to change after X-ray irradiation, the
WT1/Egr-related factor may also act by modulating the transactivation ability of Sp1.
DISCUSSION
HP100 cells are remarkably resistant to H2O2-inducible apoptosis.
In this study, we have demonstrated that the overexpression of catalase plays a decisive role in this phenotype (Fig. 1). We then investigated the molecular mechanisms responsible for the elevated expression of catalase in HP100 cells and for its down-regulation by X-rays,
and we found that: (a) the expression of catalase is regulated primarily
at the transcriptional level; (b) the GC box located ⫺70 bp from the
major transcriptional start site of the catalase gene functions as the
core promoter element; (c) much higher levels of Sp1 are expressed in
HP100 cells than in HL60 cells, associating with the overlapping
Sp1/Egr-1 recognition sequence at ⫺70 bp; (d) a WT1/Egr-related
factor is induced in response to 20 Gy of X-ray irradiation and
associates with the overlapping Sp1/Egr-1 recognition sequence at
⫺70 bp; (e) the X-ray-inducible WT1/Egr-related factor does not
seem to compete with Sp1 for binding to their overlapping recognition
sequence; (f) the CCAAT element at ⫺92 bp strongly enhances
transcription in HP100 cells; (g) higher levels of NF-Y are expressed
in HP100 cells than in HL60 cells, associating with the CCAAT
element at ⫺92 bp; and (h) both the inverted CCAAT sequence at
⫺42 bp and the CCAAT sequence at ⫺121 bp regulate transcription
negatively in HL60 cells and positively in HP100 cells.
Based on these findings, we suggest that a mechanism such as that
shown in Fig. 9 could be the means by which promoter regulation of
the catalase gene occurs. Fig. 9A illustrates the catalase gene promoter
in HL60 cells, where the association of factors/complexes (F2 and F3)
with the CCAAT-92 and EgrSp-70 sequences is revealed by genomic
footprinting (Fig. 4). In addition, the involvement of factors (F1 and
F4) recognizing the CCAAT-121 and CCAAT-42 sequences is also
expected because these CCAAT elements work to repress the promoter activity (Fig. 5B). The presence of DNase I-sensitive sites
observed in the genomic footprinting (Fig. 4) suggests an interaction
among factors. Because of the low nuclear content of Sp1 and NF-Y
in HL60 cells, the transcription initiation complex containing RNA
Fig. 8. Expression of Sp1 and NF-Y in nuclei of HL60 and HP100 cells. A, 4-␮g
samples of nuclear proteins extracted from unirradiated or irradiated (20 Gy) HL60 and
HP100 cells were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie Blue. B, a Western blotting analysis was performed with anti-Sp1 (PEP2) or
anti-NF-Y (AHP298) antibody. Multiple protein species detected with both antibodies are
considered to be caused by protein degradation during preparation.
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REGULATION OF THE CATALASE GENE IN H2O2-RESISTANT CELLS
Fig. 9. A possible mechanism for promoter-based regulation of the catalase gene in
HL60 and HP100 cells. DNI and FP indicate DNase I-sensitive sites and footprints,
respectively, which were observed in the genomic footprinting analysis. Egr represents the
WT1/Egr-related factor found to be a component of the S4 complex in the EMSA
experiment. Other symbols are as described in the text.
polymerase II may not be very effectively recruited to the transcriptional start site. In HP100 cells, on the other hand, displacement of
CCAAT-92-bound factors (F2) by a complex containing NF-Y (F2⬘/
NF-Y) may have occurred (Fig. 6), resulting in the appearance of
DNase I-sensitive sites at approximately ⫺98 and ⫺99 bp (Fig. 4B).
Modification or replacement of the F1 and F4 factors may also be
important because the CCAAT-42 and CCAAT-121 elements function in enhancing the promoter activity in this cell. Binding of Sp1 to
the EgrSp-70 element might now result in an assemblage of factors
that allows efficient entry of the initiation complex at the transcriptional start site (Fig. 9B). NF-Y bound to the CCAAT-92 sequence
may cooperatively stimulate this process, as has been reported for the
transcription of the type A natriuretic peptide receptor gene (49) and
the rat fatty acid synthase gene (50). When HP100 is irradiated with
X-rays, induction of the WT1/Egr-related factor may somehow lead to
complex disassembly, resulting in the initiation complex being inhibited from entry (Fig. 9C). The identification and precise characterization of factors postulated in Fig. 9 remain the subject of future study.
In particular, identification of the WT1/Egr-related factor is of great
interest in relation to the potential involvement of catalase in ionizing
radiation-inducible apoptosis.
It is natural to believe that the overexpression of catalase confers
resistance of HP100 cells to H2O2-inducible apoptosis because H2O2
is thought to be rapidly metabolized in these cells. This was confirmed
in the present study by showing that resistance to H2O2-inducible
apoptosis is lost when an inhibitor of catalase, 3AT, is added to the
HP100 culture. However, in cell lines established by long-term treatment with oxidative stresses other than H2O2, elevated catalase activity is also consistently observed (Table 1). This suggests that H2O2
may play a critical role in the apoptotic pathway inducible by a wide
range of oxidative stresses. Thus, our finding that Sp1 and CCAATrecognizing factors participate in the activation of catalase in HP100
cells marks these transcription factors as key molecules in the regulation of oxidative stress-inducible apoptosis.
Catalase has been thought to play a central role in the protection of
cells against oxidative stress by converting H2O2 to O2 and H2O. The
observation that catalase is induced by H2O2 stimulation in bacterial
cells (51) and in Schizosaccharomyces pombe (52) provides strong
evidence for this hypothesis. However, in mammals, an objection can
be raised based on observations that catalase is down-regulated by
H2O2 (29), lipopolysaccharides [Ref. 37; which cause an oxidative
stress by generating O2⫺ and H2O2 (53)], and ionizing radiation (25),
the cytotoxic effects of which are primarily mediated by the production of ROS. It may be hypothesized that the predominant role of
catalase in multicellular organisms is not to protect cells against H2O2
but to regulate apoptosis by controlling cellular levels of H2O2. This
hypothesis is supported by a large body of data demonstrating the
antiapoptotic effects of catalase (3–15). Furthermore, the promoter
structure of the mammalian catalase gene is quite distinct from that of
yeast catalase in that it has no TATA box, multiple CCAAT boxes,
GC boxes, and multiple transcription start sites (29, 54 –57). In this
context, the down-regulation of catalase gene transcription in HP100
cells by X-rays provides a model system to study the regulation of
apoptosis by antioxidant enzymes. Whether the onset of apoptosis can
be modulated by artificially inhibiting the pathway for induction of
the WT1/Egr-related factor after X-ray irradiation remains to be
determined.
ACKNOWLEDGMENTS
We thank Drs. M. Akashi and M. Hachiya (National Institute of Radiological Sciences, Chiba, Japan) for providing us with the HL60 and HP100 cell
lines and for helpful suggestions on this work. We also thank Drs. H. Ishihara
and T. Nakajima (National Institute of Radiological Sciences) for valuable
advice and stimulating discussion.
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5894
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2001 American Association for Cancer
Research.
Regulation of the Catalase Gene Promoter by Sp1,
CCAAT-recognizing Factors, and a WT1/Egr-related Factor in
Hydrogen Peroxide-resistant HP100 Cells
Mitsuru Nenoi, Sachiko Ichimura, Kazuei Mita, et al.
Cancer Res 2001;61:5885-5894.
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