[CANCER RESEARCH 58. 4410-4416.
October 1. 1998]
Apurinic Endonuclease (Ref-1) Is Induced in Mammalian Cells by Oxidative Stress
and Involved in Clastogenic Adaptation1
Sabine (.roseli, Gerhard Fritz, and Bernd Kaiiur
Institute of Toxicology. Division of Applied Toxicology. University of Mainz, D-55Õ31 Mainz, Gertntmy
ABSTRACT
Apurinic endonuclease (APE; also known as Ref-1 protein) is a key
enzyme in base excision repair, cleaving apurinic sites that arise sponta
neously because of the activity of DNA glycosylases. To address the
question of whether APE can be modulated by genotoxic stress affecting
cellular protection, we analyzed the expression of APE in Chinese hamster
ovary (CHO) cells after treatment with various genotoxic agents. We show
that treatment of CHO cells with hydrogen peroxide (H2O2) or sodium
hypochlorite (NaOCI) increases the levels of APE mRNA and protein.
APE induction was observed 3-9 h after treatment and was accompanied
by an increase in APE activity. We also show that the cloned human APE
promoter transfected into CHO cells is stimulated by the oxidants, indi
cating transcriptional activation of the APE gene. When cells were pretreated with NaOCI, inducing APE, and then challenged with H2O2, the
clastogenic effect of the challenge dose was significantly reduced, suggest
ing clastogenic adaptation due to APE induction. To further prove the
involvement of APE in adaptation against induced chromosomal break
age, we transfected human APE cDNA driven by an inducible promoter
into CHO cells and observed that transient induction of APE reduced the
clastogenic effect of II ,<>,. Overall, the data demonstrate that the APE
gene can be activated by oxidative agents, resulting in a transient increase
in APE repair activity, which reduces the clastogenic response of cells to
an oxidative agent. The protection of cells from chromosomal aberrations
seen after prior exposure to oxidants is attributed to an adaptive response
to oxidative stress.
INTRODUCTION
APE1 (also designated Ref-1 protein) is a key enzyme in the BER
pathway. In concert with specific DNA glycosylases and DNA polymerase ß,as well as ligase III and XRCC1, it removes damaged
bases and spontaneously formed AP sites from DNA (1). The fre
quency of occurrence of these DNA lesions is considerably high:
—¿
10,000 depurinations have been estimated to arise spontaneously in
a human cell (2). Furthermore, many genotoxic exposures, such as
ionizing radiation, endogenously produced oxidative species, and
alkylating agents generate base damages that are removed by DNA
glycosylases, requiring APE to repair the resulting AP sites. Repair of
AP sites is essentially required for survival of cells because they block
DNA replication and, thus, may lead to cell death, mutations, and
chromosomal changes (1,2). It should also be noted that DNA strand
breaks induced by ionizing radiation and other kind of oxidative stress
produce .V-phosphoglycolate ends, which cannot be sealed by DNA
polymerase unless 3'-phosphoglycolate is removed by 3'-diesterase
activity of APE (3). Thus, both endonuclease and 3'-diesterase activ
ity of APE are important for repair of damaged DNA.
In addition to the repair activity, APE has a dual function in that it
regulates the redox state of various transcription factors, such as
activator protein-1, nuclear factor-«B, and myb. Reduction of activa
tor protein-1 (AP-1) by APE was shown to enhance its promoter
binding activity (4). Thus. APE is indirectly engaged in regulation of
gene expression. Moreover, APE/Ref-1 can regulate genes directly by
binding to the so-called negative calcium responsive element, which
was found to be present in the promoter region of various genes, such
as the parathyroid hormone gene and the APE gene itself. Upon
increase of intracellular calcium level, the activity of these genes
becomes down-regulated due to binding of the APE protein to the
corresponding promoter (5. 6). APE is essential for organismic sur
vival; mice with homozygous deletion of APE/Ref-1 are not viable
due to lethality of embryos in the early developmental stage (7).
A role for APE in cellular defense against genotoxic agents was
indicated by the finding that down-regulation of APE expression by
transfection of antisense APE cDNA in rat glioma and HeLa cells
rendered them more sensitive to methylating and oxidative agents (8,
9). On the other hand, permanent overexpression of hAPE could not
be achieved by transfection of APE cDNA into CHO cells, presum
ably because of down-regulation of APE gene and enzyme activity in
stably transfected cells (10). This finding, however, does not argue
against the hypothesis that, in some cell types, APE repair activity
might be limiting in BER after genotoxic treatment. In support of this,
stable transfection of the yeast APE gene in CHO cells leads to an
increase in overall cellular APE repair activity, which renders cells
more resistant to DNA-damaging agents (10).
In view of the central role of APE in DNA repair and, presumably,
gene regulation under conditions of exposure of cells to genotoxic
stress, it is pertinent to ask the question whether APE gene activity is
modulated upon treatment of cells with genotoxic agents. Therefore,
we investigated the expression of APE on the RNA, protein, and
promoter levels after treatment of cells with various DNA-damaging
agents, including oxidative compounds. Here, we report that APE is
induced by oxidative stress. We also show that transient induction of
APE exerts a protective effect on the clastogenicity brought about by
oxidative treatment. Oxidative species are generated in cells by var
ious physiological processes, such as inflammatory reactions (11),
which may lead to DNA damage in the affected cells (12). Oxidative
stress-mediated induction of APE can, therefore, be considered to be
involved in the cellular defense against genotoxic attack of endog
enously produced radicals. It may also protect against oxidants in the
environment. This is the first report showing that APE induction by
oxidative treatments gives rise to clastogenic adaptation, i.e., reduc
tion in the frequency of challenge dose-induced chromosomal break
age.
Received 4/7/98; accepted 7/30/98.
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 lo indicate this fact.
1This work was supported by Deutsche Forschungsgemeinschaft Grants KA 724/4-3
and SFB 409/B4.
2 To whom requests for reprints should be addressed, at Abteilung fürAngewandte
Toxikologie. Obere Zahlhacher Strasse 67, D-55I31 Mainz. Germany. Phone: 49-06131173246; Fax; 49-06131-173421.
1The abbreviations used are: APE, apurinic endonuclease; BER, base excision repair:
AP. apurinic/apyrimidinic;
CHO. Chinese hamster ovary; hAPE. human APE; CAT,
chloramphenicol acelyltransferase; GDH. glyccraldehyde phosphodehydrogenase; MMS,
melhyl methanesulfonate.
MATERIALS
AND METHODS
Cell Culture and Plasmids. CHO-9 cells were grown in F12-Dulbecco's
medium, supplemented with 5% PCS (Life Technologies, Inc.) at 37°Cin an
atmosphere of 7% COi/air. The hAPE promoter was cloned by PCR from
HeLa genomic DNA using primers that were chosen according to the pub
lished sequence (13): primer 1, 5'-ACACGCATGCTTAGGAAGATGGAAGGC-3': and primer 2, S'-ACACGTCGACCCACTCACATCTAATCC3'. A 900-bp SphllNrul fragment was cloned into pCATBasie (Promega). For
4410
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INDUCTION
OF APE
stable transfection of hAPE cDNA, we made use of an ecdysone inducible
expression system (Invitrogen). Additionally, we cloned the c-myc-tagged
hAPE cDNA in sense orientation into the EcoRl/Xbal site of the pIND vector
(Invitrogen).
Transient Transfection Experiments and CAT Assay. Cells were transfected with hAPE promoter-CAT construct by calcium phosphate coprecipitation as described (14). In brief, 5 x IO5 cells were seeded per 10-cm dish and
treated 24 h later with 1 ml of DNA precipitate containing 10 /xg each of
plasmid and salmon sperm DNA. After overnight incubation, cells were treated
with DMSO (final concentration, 10%) for 30 min and incubated for another
24-h period. Then, they were treated with different concentrations of NaOCl or
AAPE00£
aCOJ=CO_c
o>•«t.c
-----
GDH
H2O2. After a further 48 h, cells were harvested for sonication. The amount of
protein in total cell extracts was determined (15), and CAT activity was
measured by CAT-ELISA according to the manufacturer's protocol (Boehringer Mannheim).
Generation of Inducible APE Transfectants. We first stably transfected
the pVgRXR vector (Invitrogen) encoding the ecdysone receptor into CHO-9
cells. Transfection was performed by calcium phosphate coprecipitation. For
ty-eight h after transfection, cells were reseeded 1:4 and subjected to selection
with 250 /xM zeozin. After 10 days, zeozin-resistant clones were isolated and
checked in transient transfection experiments using the plasmid p!ND//ocZ for
inducibility mediated by muristerone A, which is an analogue of the steroid
hormone ecdysone. Clones were isolated that showed high levels of induction
of the transient transfected pIND//acZ gene after treatment with muristerone,
indicating expression of the ecdysone receptor. An individual ecdysone recep
tor transfected clone (designated pVgRXR-CHO-9) was used for transfection
of c-myc-APE pIND vector, which harbors, in addition to the human myctagged APE cDNA, the neomycin phosphotransferase gene for second-step
selection. In this case, after transfection, cells were selected with 1.5 mg/ml
G418 together with 250 /¿Mzeozin. Various transfected cell clones were
isolated, grown into 24-well plates, and tested for muristerone-induced expres
sion of c-myc-APE protein in Western blots.
Northern Blot Analysis. Cells grown on 10-cm plates were treated for
different times with NaOCl or H2O2. For the induction experiments with
inhibitors, cycloheximide, and anisomycin were added 30 min before H2O,
treatment. Thereafter, cells were washed twice with PBS and lysed in 5 ml of
STE buffer [ 100 mM NaCl, 20 mM Tris (pH 7.4), and 10 mM EDTA] containing
300 fig/ml proteinase K and 0.5% SDS. The suspension was incubated for l h
at 37°Cand then extracted with phenol/chloroform/isoamylalcohol
(24:1:1).
Poly(A)+ mRNA was isolated by the addition of oligo(dT)-cellulose (Boeh-
o
•¿o
10 15
Time (h)
B
o -C .C
ü co co
T-
20
25
CNJ
APE
GDH
ringer Mannheim) to the solution and incubation with gentle agitation over
night at room temperature. After elution of poly(A)* mRNA from oligo(dT)cellulose, mRNA was precipitated with ethanol. The dried pellet was
solubilized in distilled water. Poly(A)+ mRNA (7 /ng/lane) was loaded onto a
1% agarose gel in the presence of formamide. Gels were blotted onto Hybond
N+ (Amersham) membrane and hybridized with [32P]dCTP-labeled hAPE
cDNA or GDH cDNA (16).
Western Blot Analysis. Cells were treated for different times with NaOCl
and H2O2 or, for induction of myc-APE, with 5 /J.Mmuristerone. Thereafter,
they were washed with PBS, scraped off the plates for harvesting, and soni
cated in sonication buffer [20 mM Tris-HCl (pH 8.5). 1 mM EDTA, 1 mM
ß-mercaptoethanol, and 5% glycerin). Aliquots of 50 /xg of total protein
extract were electrophoretically separated by 12% SDS-PAGE and then electroblotted onto a nitrocellulose membrane (Schleicher & Schuell. Dassel.
Germany). The membrane was incubated overnight in 5% nonfat dry milk.
0.2% Tween 20, and PBS. Then the filter was incubated with either anti-hAPE
polyclonal antiserum from rabbit (generously provided by Dr. S. Mitra, Sealy
Center for Molecular Science, Galveston, TX) or anti-c-myc monoclonal
antiserum from mouse (Calbiochem) diluted in 5% dry milk, 0.2% Tween 20,
and PBS for 2 h. The filters were extensively rinsed with 0.2% Tween 20 in
PBS and incubated with peroxidase-conjugated
donkey antirabbit IgG (Dianova, Hamburg, Germany) or peroxidase-conjugated
sheep antimouse IgG
(Amersham), respectively, diluted 1:3000 in 5% dry milk, 0.2% Tween 20, and
PBS for 1 h. After extensive rinsing in 0.2% Tween 20 in PBS. proteinantibody complexes were visualized by ECL (Amersham)
manufacturer's protocol.
10
15
20
Time (h)
Fig. 1. Expression of APE mRNA in CHO cells after treatmem wilh NaOCl or H,O2.
A, APE mRNA levels after treatment of cells with 0.05 mM NaOCl. assayed at various
time points. B, APE mRNA levels after treatment with 0.3 mM H2O2, assayed at various
time points. Filters were rehybridized with radiolabeled GDH cDNA to equalize the
amount of RNA onto the filter. Induction factors were calculated by densttomelric
measurements of the autoradiograms and set in relation to GDH. Data points were
expressed in relation to the control, which was set to 1.
according to the
APE Assay. The assay was performed as described previously (10). In
brief, we used a radiolabeled double-stranded DNA oligomer (35 bp) contain
by treating the oligomer with uracil DNA glycosylase generating a single
apurinic site in the duplex. This oligomer was incubated together with the
protein extract. APE within the extract is expected to cleave the oligomer 5' of
ing uracil in a defined position of the sequence. The uracil base was removed
the AP site. After different times of incubation, aliquots were removed from
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INDUCTION
OF APE
Increase in the level of APE mRNA and protein was accompanied by
an increase in APE enzyme activity, which was measured in extracts
o 'x
of cells treated under the same experimental conditions (Fig. 4).
To see whether induction of APE, as observed on RNA and protein
level,
is due to activation of the APE promoter upon oxidative stress,
o
o
M -5
we
cloned
the hAPE promoter and checked its inducibility in transient
C >»
transfection assays using a promoter-CAT construct (Fig. 5A). As
shown in Fig. 5, B and C, hAPE promoter activity is clearly stimulated
by oxidative treatments. APE promoter-driven CAT activity increased
c O
upon treatment of transfected cells with either NaOCI or H^O,, up to
o CM o evi
~6-fold. Methylating agents such as A'-methyl-W-nitro-A'-nitrosoguaü I
nidine and MMS did not significantly increase APE promoter-CAT
•¿
•¿
APE
expression. Also, the phorbol ester tumor promoter TPA was ineffec
tive in APE promoter activation under these experimental conditions
(data not shown).
Next, we analyzed whether induced APE expression exerts protec
GDH
tion of cells against the genotoxic effect of various agents. To this end,
we measured aberration frequencies in CHO cells exposed to H2O2,
with or without pretreatment with NaOCI. We should note that the
Induction factor: 3.1
o.9
concentration of NaOCI used for pretreatment, which was effective in
Fig. 2. Expression of APE mRN A after treatment of CHO cells with 0.3 mM H2O2 and
inducing APE, was nearly nontoxic in mass culture and only very
coincubalion with 10 /xg/nil cycloheximide and 100 /AManisomycin for 8 h. Filters were
weakly clastogenic ( 1% and 7% aberration frequency for nontreated
rehyhridi/ed with radiolabeled GDH cDNA to equalize RNA levels onto the filter.
Induction factors were calculated in relation to the nonpretrealed control.
control and 0.05 mM NaOCI pretreatment, respectively). However,
under these conditions of pretreatment, a significant effect on aber
ration frequency induced by a challenge dose of H2O2 was observed.
As shown in Fig. 6, H2O2-induced aberration frequency was clearly
the reaction mixture. The reaction was stopped by addition of formamide
sequencing butter. The samples were heated at 75°Cfor 3 min and then
reduced in the pretreated cell population, as compared with nonpreseparated on a 20% sequencing gel. Gels were dried and exposed to X-ray film
treated cells. Obviously, pretreatment of cells with NaOCI caused
overnight.
adaptation that rendered cells more resistant to the clastogenic effect
Chromosomal Aberrations. A total of 3 x IO5cells were seeded per 5-cm
of H2O2.
plate. After 24 h of incubation, cells were treated with 0.05 mM NaOCI or 5 /MM
In these experiments, the time interval between pretreatment and
muristerone for 9 h. Thereafter, the medium was replaced by fresh medium, to
challenge was 9 h, for which the level of APE protein induction
which H,O2 (final concentration 0.3 mM) or MMS (1.5 mM) was added.
reached a maximum. If the time span between adapting and challenge
Alternatively, cells were treated with X-rays (5 Gy) or UVC light (15 J/nr).
dose was shortened to 2 h, which is insufficient for increasing the APE
After further incubation for 17 h, colcemid (50 ng/ml) was added, and cells
expression level (Fig. 2 and data not shown), significant clastogenic
were incubated for another 2 h. Then, the cells were trypsinized and pelleted
by centrifugation. The cell pellet was resuspended in 75 mM KC1 and incubated
protection was not observed. The same was true if the time span
for 7 min at room temperature. After centrifugation, cells were resuspended
between pretreatment and challenge was extended to 24 h, at which
<D
SE
I«
<ü
o
üI
and fixed by slowly adding methanol/acetic acid (3:1). Cell suspensions were
centrifuged, again resuspended in methanol/acetic acid (3:1), and finally resuspended in freshly prepared methanol/acetic acid (2:1). Cell suspensions
were dropped onto cold and wet slides that were dried at room temperature.
Chromosomes were stained by Giemsa (5% in Sörensenbuffer). Per treatment
level, 100 metaphases were scored under the microscope (1000-fold magnifi
Coü.c
-e
mCM—
m
j;
cation). Percentages of aberrant metaphases were compared statistically using
the x2 test.
—¿
Induction
factor : 1
RESULTS
First, we measured the APE mRNA level in cells after treatment
with oxidi/.ing agents. CHO-9 cells were exposed to either NaOCI or
H->O->,and mRNA was extracted various times after the beginning of
treatment. As shown in Fig. \A, increases in APE mRNA level were
observed 3-9 h after addition of NaOCI to the medium. For H2O2,
increases in the amount of APE mRNA were observed 6 and 9 h after
treatment. Thereafter, the amount of APE mRNA returned to control
level (Fig. Iß).Thus, induction of APE mRNA by oxidative stress is
transient. Increase in APE mRNA level after H2O2 treatment was
inhibited by cycloheximide/anisomycin
(Fig. 2), indicating that de
novo protein synthesis was required for eliciting the response.
Induction of APE mRNA resulted in an increase in the amount of
APE protein, which is shown in Fig. 3 for NaOCI and H-,0-,. Peak
levels of induction (up to 4-fold) were observed 11 and 15 h after
treatment with NaOCI and H2O2, respectively, which occurred
slightly later than the maximum of induction observed on RNA level.
•¿c^£
3.1 3.9 1.1 0.9
B
ü_c
CO.C CM^
CD.cO)£in£•<t
'*~112.22.14.03.0 —¿ —¿
Induction
factor :o
Fig. 3. Induced expression of APE protein as delected by Western blot analysis. CHO
cells were treated with either 0.05 m\i NaOCI (A) or 0.3 HIMH2O, (B) for different time
periods. After treatment, cells were harvested and sonicated, and 50 ¿igof total protein
extraci were loaded per lane. The amount of APE protein was quantified by densitometric
measurements of band intensities and related to the mock-treated control, which was set
to 1. Results of one representative experiment are shown.
4412
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INDUCTION
clastogenic dose of H2O2, chromosomal aberration frequency was
significantly reduced, as compared to the mock-pretreated control
(Fig. 8ß).From this, we infer that a transient increase in APE level,
as observed in experiments with NaOCl- and H2O2-pretreated
cells, caused clastogenic adaptation, which was specifically di
rected against oxidative agents.
Control
.E
o
.E
T-
.E .E
E
E
e
E
e
E
co •¿*
*-
Ã-
°
OF APE
A)
Hindlll
(-768)
H202
.E .E .E .E
EEEEE^EE
Ot-
COrftt)
(+114)
AsP'
Sphl
5' l
.E
^
-|
2
ÕS
I
Nrul
•¿Â§
ATF/CREB
AP2SPI
B)
M
C)
ÃœSF AP1
7,
1
6
B
5
Contro!
H202
5.
5
Ii 4
4
•¿o
O
3
3
2
E
m
e
o
U
NaOCl
Time (min)
Fig. 4. Activity of APE in control and H2O2-treated cell extracts. A, the activity assay
was stopped after 1-20 min. and samples were separated by gel electrophoresis. Upper
hand, uncleaved 35-mer substrate; lower band, converted 15-mer radiolabeled oligonucleotide. Al zero time, the oligonucleotide was mixed with cell extract, and the reaction
was stopped immediately after the incubation was begun. B, quantification of the radio
grams shown in A by densitometric analysis. The amount of conversion was set in relation
to the uncleaved substrate.
&
1
o
U
e
m
u>
ö
i».
o
H202
Fig. 5. Transcriptional activation of the hAPE promoter hy oxidative treatment.
The APE promoter-CAT
construct was transfected into CHO cells, which were
subsequently treated with either NaOCl or H2O2 for 48 h with the doses indicated.
Cells were harvested, and CAT activity was measured by CAT-EL1SA assay. Induc
tion factors were given in relation to untreated control transfections. A. map of hAPE
promoter fragment indicating various restriction sites and putative transcription factor
binding sites, as revealed from consensus sequences obtained by a computer search.
B, induction of APE promoter-CAT construct after treatment of transfected cells with
different concentrations of NaOCl. C. induction after treatment with different con
centrations of H2O2. Columns, means of three independent experiments; bars. SD.
The mock-trealed control was set to 1.
time APE returned to control level (Table 1). Clastogenic adaptation
following APE-inducing treatment was reproducibly observed for
H2O2. It was not found when cells were challenged with a clastogenic
dose of MMS or UV light (Fig. 7).
without pretreatment
Although the dose of NaOCl eliciting clastogenic adaptation
induced increase in APE expression, one might argue that other
functions that were possibly coinduced with APE were responsible
for the adaptive response. To provide supportive evidence for
involvement of APE in clastogenic adaptation, we generated CHO
cells stably transfected with an inducible APE expression vector,
using an ecdysone-inducible
promoter system. First, we generated
parental CHO cells (clone pVgRXR-CHO-9) that express the ec0.2
0.4
0.6
0.8
dysone receptor. These cells were stably transfected with an ecdysone promoter-APE
cDNA expression construct that can be
H2O2 (mM)
stimulated by muristerone. As shown in Fig. 8/4, ecdysone proFig. 6. Frequency of chromosomal aberrations in CHO cells as a function of dose of
moter-APE-transfected
cells responded to treatment with muris
H2O2. Cells were pretreated with 0.05 mM NaOCl for 9 h followed by challenge with
terone with a clear increase in APE level. If these cells were
H,O2. The recovery lime after the challenge was 18 h. For each dala point. 100
challenged 9 h after addition of ecdysone to the medium with a metaphases were evaluated.
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INDUCTION
Table I HtO^-indltced
chromosomal aberration frequencies
OF APE
in CHO-9 cells with and without NiiOCl treatment before challenge with H2Ot
Exponentially growing cells were treated with agents that were added from a freshly prepared stock solution directly into the medium. Mitoses were scored with a recovery time
after the challenge of 18 h. Aberrations were mostly of chromatid type and consisted of chromatid breaks and the various forms of translocations. Percentage of mitoses with aberrations
(aberration ratei and the average total number of aberrations per cell (aberration yield) were determined per 100 metaphases, which were scored per measure point. For calculation of
the corrected challenge dose-induced aberration frequency, aberration frequency of pretreated cells was subtracted from that obtained after the challenge. The difference in aberration
frequency between pretreated and nonpretreated cells that were challenged 9 h after pretreatment was significant (P < 0.01).
(%)Control
Aberrations
H2O2challenge1473WithH2O2challenge40311735Challengeaberration
H,0,challenge0.010.040.080.03With
rate(corrected)39271032Aberrations/cellWithout
H2O2challenge0.870.990.731.01Challengeaberration
yield(corrected)0.860.950.650.98
pretreatment)2-h
(without
pretreatment9-h
NaOCI
pretreatment24-h
NaOCI
NaOCI pretreatmentWithout
-NaOCI
+NaOCI
that APE is highly regulated both on transcriptional and protein level.
Presumably, long-term increase in the basal level of APE/Ref-1 in
cells not exposed to a genotoxic agent is not tolerable. However,
under conditions of oxidative stress, a transient increase in the level of
APE protein and APE activity appears to be advantageous giving rise
to protection of cells. This is reflected by our finding that, after
pretreatment of CHO cells with a low subtoxic dose of NaOCI
inducing APE, followed by challenge with a clastogenic dose of
4-*OüJI
.Ct
o>£
Control
H2Û2
MMS
UV
Fig. 7. Frequency of chromosomal aberrations in CHO cells after treatment with
different agents with or without pretreatment with NaOCI. Exponentially growing cells
were pretreated with 0.05 mM NaOCI ( +NaOCI) for 9 h or left untreated (-NaOCI).
Thereafter, cells were challenged with either 0.03 mM H2O2, 1.5 HIMMMS. 5-Gy X-ray,
15 J/m2 UV light (UV), or left untreated (Control) for 17 h. Colcemid was added for 2 h,
(O£Si—
_ _
—¿-^—myc-APE
B
and cells were harvested for chromosomal preparation. For each treatment level. 100
metaphases were scored. The difference between aberration frequencies of pretreated and
nonpretrealed cells in the case of H2O2 was significant (*. P < 0.05).
DISCUSSION
Here, we aimed to elucidate whether APE can be induced upon
exposure of cells to genotoxic stress. We showed that the endogenous
level of APE mRNA and protein as well as APE enzyme activity was
enhanced in CHO cells upon oxidative treatment. Induction was
dependent on de novo protein synthesis because cycloheximide/anisomycin prevented accumulation of APE mRNA. Furthermore, we
showed that the cloned hAPE promoter can be induced by oxidative
agents such as H2O2 and NaOCI, indicating that induction of APE
occurs on transcriptional level. In a previous study by other authors,
regulation of APE promoter activity was investigated upon treatment
of cells with various other agents. The APE promoter was found not
C«
be induced by dexamethasone, the tumor promoter 12-O-tetradecanoN
O
O
ylphorbol-13-acetate, dimethyl sulfate, paraquat, and bleomycin (17).
M
O
N
O
It was also shown that, upstream of the hAPE promoter, a specific
sequence (negative calcium responsive element) is located to which
APE can bind, leading to gene repression (5, 17), and that APE gene
without muristerone
9 h muristerone
is subject to negative autoregulation (6, 18).
Fig. 8. Response of cells transfected with a muristerone-inducible
promoter-APE
Transcriptional activation of the APE gene appears to be a specific
expression vector. A, induction of the c-myc-tagged APE protein after treatment of
response of cells to oxidative stress. We should note that the enzyme
c-myc-APE-pIND-transfected
CHO cells with 5 fia muristerone for different time peri
activity in treated cells did not increase to the same extent, as expected
ods. The amount of induced APE protein was detected in a Western blot with anti-c-myc
from the observed increase in protein level. This could be due to antiserum and peroxidase-conjugated sheep antimouse IgG as secondary antibody. Upper
hand, c-myc-tagged APE protein; lower band, nonspecific. B. chromosomal aberration
posttranslational modification of the APE protein. In line with this,
frequency in c-myc-APE-pIND-transfected
CHO cells not induced and induced by muris
stable transfection of an APE cDNA expression vector into CHO-9
terone and treated with H2O2. respectively. Muristerone pretreatment occurred for 9 h.
Thereafter, cells were challenged with 0.3 mM H2O, or left untreated (control). Colcemid
cells did not yield cell clones expressing higher APE activity than did
was added 17 h later (for 2 h), and cells were harvested for chromosomal preparation. The
the nontransfected control, although the transfectants displayed a difference in aberration frequency between muristerone pretreatment and the nonpre
treated control was significant (*. P < 0.05).
higher level of APE mRNA and protein ( 10). We, therefore, suppose
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INDUCTION
H2O2, the challenge dose-induced aberration frequency was signifi
cantly reduced when compared with the nonpretreated control. This
phenomenon was initially observed for methylating agents and called
clastogenic adaptation (19). In this particular case, the adaptive re
sponse may be speculated to be due to poly(ADP) ribosylation, which
has been shown to become activated under these experimental con
ditions (20). Clastogenic adaptive responses have been found in
different other experimental systems for a variety of agents (21, 22),
notably ionizing radiation (23). However, until now, the molecular
basis of most of these adaptive responses relating to a genotoxic end
point such as chromosomal aberrations remained unknown. Here, we
provide evidence for induction of a particular DNA repair activity that
is related to clastogenic adaptation upon oxidative treatment. In ex
periments with NaOCl-pretreated cells, the period of pretreatment
eliciting an adaptive response coincided with the time at which
maximal induction of APE protein was found. This supports the
conclusion that reduction in challenge dose-induced aberration fre
quency is due to increase in APE expression. This was further sup
ported by experiments with cells stably transfected with hAPE cDNA
controlled by a muristerone-inducible promoter. Cells that were pretreated with muristerone exhibited an enhanced APE protein level and
displayed a significantly lower challenge dose-induced chromosomal
aberration frequency. Taken together, it is reasonable to conclude that
induced APE is involved in clastogenic adaptation to oxidative stress.
Our results do not entirely exclude the possibility that the adaptive
response, as observed on chromosomal level, is due to stimulation of
gene activity other than APE, which is mediated by the redox function
of the APE protein (Ref-1 ). Thus, one could speculate that various as
yet undefined genes involved in defense against oxidative stress can
be induced via transcription factors that are subject to APE/Ref-1
mediated regulation, thus reducing the clastogenic effect of agents
such as H2O2. Despite this uncertainty, the results presented here
show that APE can be induced by oxidants, giving rise to transient
increase in APE activity and concomitant reduction of the chromo
some breakage-inducing effect of an oxidizing agent. Clastogenic
adaptation brought about by APE induction appears to be specific, i.e.,
directed against agents producing oxidative DNA damage. It did not
pertain to the clastogenic effect of agents such as MMS and UV light.
In the case of MMS, this might be surprising because repair of DNA
damage induced by this agent also requires BER using APE. It should
be noted, however, that methylated and oxidized purines such as
7-methylguanine and 8-oxoguanine are removed from DNA by dif
ferent DNA glycosylases, such as A'-methylpurine-DNA glycosylase
and 8-oxoguanine-DNA glycosylase, which, depending on expression
level and substrate specificity, may produce different amounts of AP
sites after mutagen treatment. Thus, a given basal level of APE might
be saturating in the repair of methylation lesions, whereas the same
level may be limiting in the case of 8-oxoguanine-DNA glycosylasemediated BER. The effect of APE overexpression on cellular resist
ance may also be cell type specific and, therefore, hard to predict.
Also, APE exhibits 3'-phosphoesterase activity (3), which is impor
tant for repair of DNA breaks generated by oxidants but is likely not
as important for repair of lesions induced by alkylating agents.
Adaptation of bacteria to oxidative treatments is a well-known
phenomenon caused by induction of various genes that are under
coordinated control (24). Transient adaptation to oxidative stress as
measured by the end point cell killing has also been described for
mammalian cells (25). Whether in eukaryotes a similar complex
regulatory network that becomes activated by oxidative stress does
exist as in bacteria remains to be elucidated. It is reasonable to
speculate that mammalian cells need an efficient defense system
against oxidative stress because oxidative species are generated in
many physiological processes such as wounding, inflammation, and
OF APE
infection. Thus, stimulation of neutrophiles gives rise to an oxidant
burst that serves as an antimicrobial shield. At the same time, how
ever, it may cause damage to the surrounding tissue (12, 26). In this
process, H-.O-, and hypochlorite formed from H,O, by the action of
myeloperoxidase play major roles (27, 28). It is, therefore, especially
interesting to see that both agents are able to induce repair function(s)
in mammalian cells, protecting them from the genotoxic effect of
H2O2, which is a powerful inducer of DNA damage (29). It should
also be noted that hypochlorite ions are widely used as a bactericidal
agent, e.g., for disinfection of drinking water. Therefore, the data
obtained could impact various human health aspects.
In mammalian cells, only a few DNA repair genes have been
demonstrated to be inducible by genotoxic stress. Thus, the repair
gene MGMT was shown to become activated by DNA-damaging
treatments, causing protection specifically directed against alkylating
agents generating O6-alkylguanine in DNA (30, 31). Ligase I is
induced by UVC irradiation; the biological role of this response is
unclear (32). Recently. DNA polymerase ßexpression level was
shown to be enhanced by oxidative treatments, and this enhancement
was accompanied by increased cell survival (33). During submission
of this study, a report appeared demonstrating APE induction by
oxidants that was related to increase in survival (34). Here, we provide
an example of cellular response directed against the clastogenic effect
of oxidative DNA damage in mammalian cells, which is based on
induced expression of the DNA repair gene APE by oxidative stress.
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Apurinic Endonuclease (Ref-1) Is Induced in Mammalian Cells
by Oxidative Stress and Involved in Clastogenic Adaptation
Sabine Grösch, Gerhard Fritz and Bernd Kaina
Cancer Res 1998;58:4410-4416.
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