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INTRODUCTION |
A major problem in cancer chemotherapy is the development of
resistance to a wide range of structurally and functionally unrelated anti-cancer drugs. One major mechanism in the development of multidrug resistance (MDR)1 is by
overexpressing MDR1 gene and its encoded
P-glycoprotein. It is generally believed that the overexpressed
P-glycoprotein facilitates the efflux of anticancer drugs from the
cytoplasm, thereby reducing the intracellular drug content to sublethal
level. In humans, there are two classes of MDR genes:
MDR1, which is involved in multidrug resistance, whereas
MDR2, in the lipid transport. There are three mdr gene
homologues in rodents, but only mdr1a and mdr1b confer multidrug
resistance; while mdr2 functions as a lipid transporter.
Levels of MDR1 expression is frequently elevated in human
hepatocellular carcinoma (HCC) (1, 2). Elevated expression of mdr gene
transcripts and their encoded P-glycoprotein is also seen in rodent HCC
(3, 4). However, mechanisms of the elevation of MDR
expression in HCC are largely unknown. In the present study, we investigated the mechanisms of elevated hepatic mdr1b expression in
rats induced by hepatocarcinogen 2-acetylaminofluorene (2-AAF). 2-AAF
is a hepatocarcinogen that has been frequently used in the development
of HCC in experimental animals. AAF is a genotoxic agent. Reaction of
electrophilic 2-AAF derivatives with nucleophilic DNA results in the
formation of DNA adducts (5). In the treated animals, DNA adducts are
proportional to dose in both target tissues, liver and bladder; whereas
tumor formation increases linearly with response to dose only in the
liver (6). It is believed that 2-AAF also induces liver cancers through
non-genotoxic effects, such as the promotion of cell proliferation (7).
We (4) and others (8, 9) have previously demonstrated that rat HCC
induced by 2-AAF exhibited elevated expression of mdr1b. Using a rat
hepatoma cell line H4-II-E, we demonstrate here that the induction of
mdr1b expression by 2-AAF is mediated by the activated NF-
B, which
recognizes a cis-acting element located upstream of mdr1b promoter. We
also demonstrated that the activation is mediated through oxidative
stress induced by 2-AAF, as evidenced by the observations that
induction of mdr1b expression can be regulated by the redox modulators.
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MATERIALS AND METHODS |
Reagents--
Reagents were purchased from the following
companies: Radioactive isotopes
[32P]dCTP, [32P]UTP, and
[14C]chloroamphenicol from ICN Biomedicals (Costa Mesa,
CA); poly(dI-dC)·(dI-dC) and acetyl-coenzyme A from Amersham
Pharmacia Biotech; oligonucleotides from SigmaGenosys Inc. (Houston,
TX); anti-I
B
, anti-I
B
, anti-IKK
, and anti-
-actin
antibodies from Santa Cruz Biotechnology Inc. (Santa Cruz, CA);
N-acetoxy-2-acetylaminofluorene (2-AAAF) was purchased from
NCI Chemical Carcinogen Reference Standard Repository (Kansas City,
MO). All other chemicals were purchased from Sigma.
Plamids--
Rat mdr1b-CAT constructs (
243RMICAT,
214RMICAT,
163RMICAT, and
243RMICAT-
m, were described previously (10-12).
245mdr1b-Luc was constructed by inserting a polymerase chain reaction
product between
245 and +123 bp of mdr1b gene into the
KpnI and HindIII sites of pGL3-luciferase vector
(Promega, Madison, WI). pNF
B-Luc and pFR-Luc were obtained from
Stratagene (La Jolla, CA). pcDNA1 was purchased from Invitrogen
(Carlsbad, CA). Antisense p65 (
p65) was a gift from Tom Maniatis
(13). Flag-tagged expression vectors pCMV4-F-I
B
and
pCMV4-F-I
B
were generously provided by S. Ghosh (14).
RNase Protection Assay--
The rat mdr1b and 18 S rRNA probes
were synthesized by in vitro transcription as described
previously (4). Either 20 or 1 µg of total RNA was used for mdr1b or
18 S rRNA, respectively. The protected RNA products were analyzed on a
7% denaturing PAGE gel and quantified using a densitometer (Molecular
Dynamics, Sunnyvale, CA).
Cell Culture, 2-AAF Treatment, and Transfection--
The rat
hepatoma cell line H-4-II-E (ATCC 1548) and human embryonic kidney cell
line 293 (CRL-1573) were obtained from the American Type Culture
Collection.
-GCS overexpressing cell line H9 derived from H4-II-E
cells has been described elsewhere (15). For 2-AAF (or 2-AAAF)
treatment, 2 × 106 cells were plated in 10 ml of
growth medium on 10-mm Petri dishes 16 h prior to the addition of
drugs. Transfection of H4-II-E cells was performed by LipofectAMINE
Plus method (Life Technologies Inc., Rockville, MD), following the
manufacturer's manual. For stable transfection, 0.5 µg of
pcDNA3-Neo (Invitrogen, Carlsbad, CA) was mixed with 2.5 µg of
reporter plasmids. After transfection, cells were selected by G418
resistance according to the procedure described previously (15).
Luciferase Assay and CAT Assay--
Cells were lysed by reporter
lysis buffer supplied with the luciferase assay kits (Promega Co.,
Madison, WI). 20 µg of protein was used for both reporter assays. For
luciferase assays, the intensity of luminescence was measured by a
luminometer (Tuner Designs TD-20/20). CAT assay procedure was
previously reported (10). In transient transfection assays, pCMV
-gal was co-tranfected with reporter constructs and the results were
normalized by
-galactosidase activities.
Preparation of Cytoplasmic Extracts and Nuclear Extracts and Gel
Mobility Shift Assay--
1 × 107 cell pellets were
first washed with PBS and then lysed in 100 µl of hypotonic buffer
(10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml each of
aprotinin and leupeptin, and 0.5 mg/ml benzamidine). The supernatants
(cytoplasmic extracts) were saved for Western blots. The pellets were
resuspended in 40 µl of high salt buffer (20 mM HEPES,
400 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, and 0.5 mg/ml benzamidine) for 30 min on ice, with
occasional vortex. After centrifugation, the supernatants (nuclear
extracts) were saved for gel mobility shift assay.
Gel mobility shift assay was performed with 10 µg of nuclear proteins
in a total volume of 20 µl containing 10 mM Tris-HCl, 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol,
0.2% Nonidet P-40, 3 µg of poly(dI-dC)·poly(dI-dC) and
radiolabeled DNA probe (an 88-bp fragment containing 5 ×
B
sites of Ig
chain promoter).
Western Blotting Analysis of Endogenous and Transient Transfected
I
B
and I
B
--
To analyze endogenous I
Bs, 50 µg of
cell extracts from 2-AAF-treated H4-II-E cells were resolved on 10%
SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane
was sequentially probed with anti-I
B
, anti-I
B
, and
anti-
-actin antibodies. To analyze the transfected I
Bs, 6 µg of
pCMV4-F-I
B
or pCMV4-F-I
B
was transfected into 293 cells on
a 100-mm dish and 15 h after transfection, cells were split onto
6-well dishes and incubated for another 15 h. Then 100 µM 2-AAF was added and incubated for different times as
indicated in the figure legend. After washing with cold PBS for three
times, whole cell extracts were prepared and 20 µg were analyzed by
Western blotting, using anti-Flag M2 antibody and anti-
-actin sequentially.
Flow Cytometry Assay of ROS--
2 × 105 cells
were seeded on 6-well dishes 15 h prior to the experiment. 100 µM 2-AAF or 10 µM 2-AAAF was added to the
cell culture medium at different time points and cells were incubated for different periods of time as indicated in the figure legend. Cells
were washed three times with cold PBS and incubated with 8 µM dichlorofluorescein diacetate in serum-free phenol
red-reduced Dulbecco's modified Eagle's medium at 37 °C for 30 min. Cells were then washed with cold PBS for three times and scraped
from the dishes in 1 ml of cold PBS. The fluorescence intensity of the
dichlorofluorescein diacetate-labeled cells was measured on a FACcan
flow cytometer (Becton Dickson, San Jose, CA) and for each sample,
1 × 104 cells were analyzed using the green
fluorescene emission parameter. The mean fluorescent intensity values
were calculated from a four-decade logarithmic scale by the CellQuest
(Becton Dickson) software.
IKK Kinase Activity Assay--
Cells were washed twice with
ice-cold PBS and then lysed in lysis buffer (20 mM Hepes,
200 mM NaCl, 1% Triton X-100, 10 mM
-glycerophosphate, 1 mM NaF, 5 µg/ml each of aprotinin
and leupeptin, and 1 µg/ml benzamidine). 300 µg of total cell
lysate was incubated with 1 µg of IKK
antibody and 30 µl of
protein A-G-conjugated agarose beads (Santa Cruz Biotechology, Inc.) at
4 °C for overnight and then washed six times with wash buffer (20 mM Hepes, pH 7.4, 200 mM NaCl, and 0.5% Triton
X-100). Beads were resuspended in 20 µl of kinase mixture containing
2 µg of GST-I
B
, 0.5 µCi of [
-32P]ATP,
20 mM Hepes, 10 mg MgCl2, 2 mM
MnCl2, 2 mM dithiothreitol, 50 µM
Na3VO4, and 10 mM
-glycerophosphate at 30 °C for 30 min. 20 µl of 2 × SDS
loading dye was added to each reaction, boiled for 5 min, and one-third
of the total volume was separated on 10% SDS-PAGE gel. Gel was dried
and exposed to x-ray film.
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RESULTS |
2-AAF and 2-AAAF Up-regulate Rat mdr1b mRNA Expression in
H4-II-E Cells--
We (4) and other (8, 9) have previously
demonstrated that expression of mdr1b in hepatoma cells and
in liver cancers can be induced by 2-AAF. To confirm that induction of
rat mdr1b mRNA could be seen in a cultured cell system, we measured
mdr1b mRNA expression by RNase protection assay in rat heptoma cell line H4-II-E, following 2-AAF treatment. Fig.
1 shows a concentration- and
time-dependent increase of mdr1b mRNA levels.
Densitometric analyses revealed that maximal levels (7.5-fold) of
induction were at 100 µM (panel A). Induction
of mdr1b mRNA was seen 3 h after treatment with 100 µM 2-AAF, reached maximum at 5 h, and declined
thereafter. Treatment of H4-II-E cells with 10 µM
2-AAAF, a major active metabolite of 2-AAF, also induces mdr1b
expression to similar levels, indicating that 2-AAAF is a much more
potent inducer than 2-AAF (data not shown).

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Fig. 1.
2-AAF induces rat mdr1b expression in H4-II-E
cells. Cells were treated with various concentrations of 2-AAF for
24 h (A) or with 100 µM 2-AAF for
different times as indicated (B). RNA was extracted and
subjected to RNase protection assay as described under "Materials and
Methods." The autoradiograph is representative of results from three
independent experiments.
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The NF-
B-binding Site Is Required for 2-AAF Induction of
mdr1b--
To determine the DNA sequences responsible for the
induction, a set of progressive deletion constructs
243 RMICAT,
214RMICAT, and
163RMICAT (11, 12) were stably transfected into
H4-II-E cells and their responses to 2-AAF treatment were measured by CAT assay. Mass cultures each consisted of more than 20 positive clones
were used. The reason for using stably transfected cells rather than
the transient transfection approach was because mdr1b gene expression
is sensitive to cellular stress and the transfection per se
is a stress inducing procedure. Moreover, the transfection efficiency
in H4-II-E cells is generally low. As shown in Fig. 2,
243RMICAT and
214RMICAT constructs
were responsive to 2-AAF treatment, but
163RMICAT completely lost it
responsiveness. Thus DNA sequences critical for the 2-AAF induction are
located between
214 and
163 bp, a region containing previously
identified binding sites of p53 and NF-
B (11, 12). To determine
whether the NF-
B site in this region is involved in the 2-AAF
induction, we made a stably transfected cell line containing a reporter
construct with mutation at this site. As shown in Fig. 2, mutation of
the NF-
B site abolished the induction of reporter expression by
2-AAF. These results thus identified that the NF-
B site located at
167 to
158 bp of the rat mdr1b promoter are responsible for the
induction of mdr1b expression by 2-AAF. Silverman and Hill (9) reported that the DNA sequence between
214 and
178 bp was important for the
basal and carcinogen inducible promoter activity. However, these
investigators failed to dissect the critical sequences that are
involved within this region.

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Fig. 2.
2-AAF induction of mdr1b promoter requires
intact NF- B motif. H4-II-E cells were
stably transfected with wild type or mutant reporter constructs as
indicated and CAT activity was measured in the absence or presence of
100 µM 2-AAF for 24 h. Values shown are average of
three independent experiments in which each group was tested in
duplicate. Error bars represent S.D. values. In the
schematic diagram, the position of NF- B site relative to the
deletion end points is indicated. Mutation of NF- B site is indicated
by "X".
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Down-regulation of NF-
B Abolishes the Induction of mdr1b-Luc by
2-AAF--
NF-
B usually consists of p50 and p65 subunits. In most
unstimulated cells, NF-
B is tightly controlled by a class of ankyrin containing inhibitors I
Bs, which bind to NF-
B subunits and
sequester them in the cytoplasm. To confirm the involvement of NF-
B
in 2-AAF induction of mdr1b, we established a stable cell line from 293 cells by transfecting
245 mdr1b-Luc. We then transiently transfected
recombinant plasmids encoding NF-
B inhibitors I
B
and antisense
p65, respectively, into this cells line. As indicated in Fig.
3, transfecting both antisense p65 and
I
B
expressing plasmids abolished the induction by 2-AAF. As a
control, no effect on 2-AAF induction was observed when the empty
vector was used. These results further demonstrated that NF-
B is
involved in the 2-AAF induction of mdr1b expression.

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Fig. 3.
Down-regulation of
NF- B abolishes the induction of mdr1b-Luc by
2-AAF. 293 cells stably expressing 245 mdr1b-Luc were
transiently transfected with pCDNA1, antisense p65, or pCMV
I B . 18 h later, cells either remained untreated or were
exposed to 100 µM 2-AAF for 24 h before luciferase
assays. Luciferase activity was normalized with -galactosidase
activity. The values shown are the average of three independent
experiments in which each transient transfection was performed in
duplicate. Error bars represent S.D. values.
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2-AAF Activates NF-
B DNA Binding Activity and NF-
B-mediated
Transcription--
We then examined whether induction of mdr1b
expression by 2-AAF is mediated by an increase of NF-
B DNA binding
activity. H-4-II-E cells were exposed to 2-AAF, nuclear extracts were
prepared. As shown in Fig. 4A,
DNA binding activity of NF-
B became detectable at 2 h,
continued to increase until it reached a maximum at 5 h
post-treatment and declined thereafter. The binding was verified by
virtue that the complex could be competed by an excess of unlabeled wild type mdr1b-
B oligonucleotide fragment, but not by a mutant mdr1b-
B oligonucleotide. The supershift by anti-p50 antibody and
partial reduction of binding using anti-p65 antibody also support the
binding specificity (Fig. 4B). The rise and fall of NF-
B
binding activity is generally consistent with the levels of mdr1b
mRNA as detected by RNase protection assay (Fig.
1B).

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Fig. 4.
2-AAF activates DNA binding activity and
transactivation potential of NF- B.
A, H4-II-E cells were treated with 100 µM
2-AAF for different times as indicated and nuclear extracts were
subjected to gel mobility shift assay (lanes 1-9).
B, the binding specificity was verified by
co-incubation with anti-p50 (Lane 11) or anti-p65
(Lane 12) antibodies, or 100-fold molar excess of unlabeled
NF- B site mutant oligos (Lane 13) or wild type oligos
(Lane 14). This figure is a representative of at least four
independent experiments. C, 2-AAF activates
NF- B mediated gene expression. pNF B-Luc, a luciferase reporter
gene carrying 5 × B binding element was stably transfected
into H4-II-E cells and were treated with vehicle, 20-100
µM 2-AAF for 24 h, or remained untreated. Values
shown are averages of three independent experiments in which each
treatment was performed in duplicate. Error bars represent
the S.D. values.
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We also examined the effect of 2-AAF on the transactivation activity of
NF-
B using an artificial luciferase reporter pNF
B-Luc, which
contains five copies of NF-
B consensus sequence. pFR-Luc, containing
five copies of Gal4 consensus sequence, was used as a control. These
two reporters were stably transfected into H4-II-E cells and exposed to
20-100 µM 2-AAF for 24 h. As shown in Fig. 4C, 2-AAF treatment led to a dose-dependent
induction of pNF
B-Luc, but not pFR-Luc. Taken together, these
results suggest that 2-AAF is able to activate both DNA binding and
transactivation activities of NF-
B in H4-II-E cells.
2-AAF Induces Degradation of I
B
, but Not
I
B
--
TNF-
and interleukin-1
, the two best characterized
NF-
B activators, activate NF-
B through
phosphorylation-dependent degradation of I
Bs, resulting
in the release and subsequent nuclear translocation of NF-
B (16,
17). To investigate how 2-AAF activates NF-
B, we performed Western
blotting analysis with I
B antibodies to examine whether 2-AAF causes
degradation of I
B proteins (Fig. 5A). Strikingly, no
degradation of I
B
was observed. In the same experiment, exposure
of H4-II-E cells to 1 nM TNF-
for 20 min led to nearly
complete degradation of I
B
, indicating that the phosphorylation
and proteolysis mechanisms were functional in these cells. The same
membrane was stripped and re-probed with I
B
antibody. As shown in
Fig. 5A, after exposure to 2-AAF, I
B
became partially
degraded at 2 h and still remained so after 5 h, following a
time course compatible with that for the activation of NF-
B DNA
binding activity. In several independent experiments, we were able to
obtain reproducible results.

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Fig. 5.
2-AAF induces degradation of
I B , but not
I B .
A, H4-II-E cells were treated with 100 µM 2-AAF for different times as indicated. As a control,
H4-II-E cells were treated with 1 nM TNF- for 20 min.
The same membrane was subjected to Western blot analysis sequentially
with antibodies against I B , I B , and -actin. This figure
is representative of four independent experiments.
B, 2-AAAF induces degradation of transiently
expressed F-I B , but not F-I B . F-pCMV4-I B , or
F-pCMV4-I B was transiently transfected into 293 cells and 24 h after 2-AAAF treatment equal protein amount of cell lysates were
subjected to Western blot analysis using anti-Flag M2 antibody and
anti- -actin antibody sequentially.
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To further strengthen the observation that I
B
but not I
B
is
degraded in response to 2-AAF treatment, we transfected Flag-tagged I
B
and I
B
expressing plasmids into 293 cells and examined the effect of 2-AAF on their degradation, using anti-Flag M2 antibody. The reason for using 293 cells for transient transfection was because
the transfection efficiency of H4-II-E cells was very poor (1-2%),
whereas 293 generally gave high (70-80%) transfection efficiency.
Moreover, as human cells usually lack certain metabolic enzymes to
convert 2-AAF into its active metabolite, we used 2-AAAF instead of
2-AAF in this experiment. As shown in Fig. 5B, 2-AAAF treatment had no effect on the stability of F-I
B
, but caused degradation of F-I
B
. The time course of the induced F-I
B
degradation was earlier than that found in Fig. 5A. This is
probably due to the fact that different cell lines (H4-II-E
versus 293), different carcinogens (2-AAF versus
2-AAAF), and different I
Bs (endogenous versus exogenously
transfected) were used. These experiments suggest that the signal
initiated by 2-AAF causes preferential degradation of I
B
.
2-AAF Induces mdr1b through Generating Intracellular
ROS--
NF-
B has long been regarded as an important sensor of
oxidative stress (18, 19). Expression of NF-
B can be induced by a
variety of excellular influences, including growth factors, UV
irradiation, heat shock, and anti-tumor drugs. Many of these inducers
are also known for the induction of MDR gene expression (20-23).
To determine whether oxidative stress plays an important role in
up-regulation of mdr1b expression, we first applied a strong oxidant
H2O2 to H4-II-E cells stably transfected with
mdr1b-Luc, pNF
B-Luc, and pFR-Luc recombinants, respectively. Six
hours after H2O2 exposure, both mdr1b-Luc and
pNF
B-Luc were highly activated in a dose-dependent
manner, but H2O2 had no effect on pFR-Luc (Fig.
6A). These results suggest
that the mdr1b promoter, which contains a NF-
B binding site, is
sensitive to oxidative stress.

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Fig. 6.
2-AAF induces mdr1b through generating
ROS. A, H2O2 activation of
mdr1b-Luc and pNF B-Luc. H4-II-E stable cells expressing 245
mdr1b-Luc, pNF B-Luc, and pFR-Luc were treated with
H2O2 and luciferase activity was measured
6 h later. B, inhibition of mdr1b-Luc and pNF B-Luc
by NAC. H4-II-E stable cells expressing 245 mdr1b-Luc and pNF B-Luc
were treated with increasing doses of NAC and 100 µM
2-AAF for 24 h. Fold of induction was normalized with protein
concentrations. C, inhibition of 2-AAF induction of mdr1b
mRNA by overproduction of GSH. H9 cells, which overexpress
-glutamylcysteine synthetase, and H4-II-E cells were treated with
increasing doses of 2-AAAF for 24 h. Total RNA was subjected to
RNase protection assay. The density of mdr1b protected band was
normalized with the density of 18 S rRNA of corresponding samples. Fold
of induction was calculated relative to untreated controls.
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To further demonstrate the role of oxidative stress in the regulation
of NF-
B-mediated mdr1b expression, we next investigated whether
antioxidants would down-regulate AAF-induced mdr1b expression using
stably transfected cells containing mdr1b-Luc, pNF
B-Luc, and
pFR-Luc. N-Acetylcysteine (NAC) is a thiol reducing
agent, which is commonly used as an anti-oxidant to block the
ROS-induced stress. The transfected cells were treated with 100 µM 2-AAF in the absence or presence of increasing
concentrations of NAC for 24 h and luciferase activity was
determined. As shown in Fig. 6B, NAC at concentrations of
1-15 nM was able to partially abolish the 2-AAF induced
expression of mdr1b-Luc and pNF
B-Luc, but 2-AAF and NAC had no
effect on pFR-Luc. These results support the role of ROS in the
regulation of mdr1b expression.
GSH is an important physiological antioxidant in mammalian cells and
high level of GSH could be achieved by overexpressing the heavy subunit
of
-glutamylcysteine synthetase, a rate-limiting enzyme of GSH
synthesis. A
-glutamylcysteine synthetase overexpressing cell line
H9 was established from H4-II-E cells and was previously shown to have
elevated levels of intracellular GSH (15, 24). We treated H9 and
H4-II-E cells in parallel with different doses of 2-AAAF and compared
their steady-state mdr1b mRNA levels by RNase protection assay. As
indicated in Fig. 6C, the induction levels of mdr1b mRNA
by 2-AAAF were significantly lower at each concentration in H9 cells
than that in H4-II-E cells. In H4-II-E cells, 2-AAAF treatment induced
mdr1b mRNA by up to 7.3-fold, however, in H9 cells the maximal
induction was only 3-fold. Taken together, these results
indicated that 2-AAF (or 2-AAAF) up-regulates mdr1b expression through
generation of ROS and inhibition of intracellular ROS was able to block
the induction of mdr1b mRNA by 2-AAF.
2-AAF and 2-AAAF Increase Intracellular ROS and Overexpression of
-Glutamylcysteine Synthetase Reduces ROS--
To substantiate that
2-AAF or 2-AAAF generate ROS in H4-II-E cells and to investigate
whether overproduction of GSH suppresses the generation of ROS, we
performed flow cytometric assay to measure intracellular ROS levels.
Both H4-II-E and H9 cells were treated with either 100 µM
2-AAF or 10 µM 2-AAAF for time intervals ranging from 20 min to 6 h and ROS levels were measured using a non-fluorescent dye dichlorofluorescein diacetate, which becomes highly fluorescent upon oxidation by intracellular ROS. A representative set of histograms from untreated controls and 100 µM 2-AAF 1 h
treatments was shown in Fig.
7A. The average values of the
mean fluorescence intensity from triplicate treatments were plotted in
Fig. 7B. In H4-II-E cells, the ROS level was rapidly
increased and reached a peak at ~1 h after treatment. Levels of ROS
were then decreased, but reached a second peak at ~4 h. This two-wave
profile was reproducible. The underlying mechanism of this ROS wave is
unclear at present. In contrast, the increase of ROS in H9 cells was
much less dramatic, with a maximum only 50% of that of H4-II-E cells.
When 2-AAAF was used, we also obtained a similar result (data not
shown), indicating that in terms of both mdr1b induction and ROS
generation, 2-AAF and 2-AAAF are nondistinguishable.

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Fig. 7.
2-AAF generates intracellular ROS and
overexpression of -glutamylcysteine synthetase
reduces ROS. A, a representative ROS
flow-cytometric assay result. H4-II-E (left panel) and H9
cells (right panel) were left untreated (open
profile) or treated with 100 µM 2-AAF for 1 h
(filled profile). ROS (fluorescence intensity) was measured
by flow cytometry using dichlorofluorescein diacetate as a dye. The
linearized mean fluorescence intensity values for 2-AAF treated
(a) and untreated (b) (background fluorescence)
are indicated. B, H4-II-E (solid line) and H9
cells (broken line) were treated with 100 µM
2-AAF for different times as indicated. This figure is representative
of at least four independent experiments and each point is the average
of the mean fluorescence intensity of triplicate treatments.
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2-AAF Activates NF-
B DNA Binding through Generating
ROS--
Since mdr1b is a NF-
B-regulated gene and NF-
B is an
important oxidative stress responder, we then compared 2-AAF induced NF-
B DNA binding activity in H4-II-E (Fig.
8A) and GSH overproducing H9
cells (Fig. 8B). Nuclear extracts were obtained from both
cell lines treated with 100 µM 2-AAF for different times
and gel mobility shift assay was performed. 2-AAF activated NF-
B DNA
binding in both cell lines, but the maximal induction level in H9 cells
were about 40% lower than that in H4-II-E cells. This result indicates that suppression of ROS was correlated with suppression of the 2-AAF
induced activation of NF-
B signaling.

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Fig. 8.
Down-regulation of ROS inhibits the
activation of NF- B DNA binding by 2-AAF.
H4-II-E and H9 cells were treated with 100 µM 2-AAF for
various times as indicated and NF- B DNA binding activity was
measured by gel mobility shift assay. NF- B DNA-protein complexes
were quantified by densitometry and fold induction was calculated
relative to untreated controls.
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2-AAF Activates IKK through Generating ROS--
Phosphorylation
and ubquitinization dependent degradation of I
B proteins is one of
the most common mechanisms through which various extracellular stimuli
activate NF-
B (25-27). IKK kinase complex has been shown to
phosphorylate I
Bs in cells treated with these inducers. To address
the questions whether IKK activation is involved in 2-AAF induction of
mdr1b and whether suppression of ROS also down-regulates IKK kinase
activity, we performed in vitro kinase assay to measure IKK
activity in H4-II-E and H9 cells treated with 2-AAF. As shown in Fig.
9, treatment of H4-II-E cells with 2-AAF
increased IKK activity 1.3-1.8-fold with a kinetics and induction
level compatible with the NF-
B DNA binding activity. In a parallel
experiment, IKK activity remained unchanged after 2-AAF treatment in H9
cells. The specificity of the phosphorylation of recombinant
GST-I
substrate was confirmed by the absence of phosphorylation
of GST (panel C in Fig. 9). In addition, the induction of
IKK activity in H4-II-E cells was not due to elevation of IKK
protein synthesis, since Western blotting analysis with IKK
antibody
failed to reveal significant increase of IKK
protein. Thus, we
conclude that induction of mdr1b expression by 2-AAF is mediated
through the generation of ROS that activate IKK kinase activity and the
downstream NF-
B signaling.

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Fig. 9.
2-AAF activates IKK in H4-II-E cells, but not
in H9 cells. H4-II-E and H9 cells were treated with 100 µM 2-AAF for various times as indicated. IKK activity
(upper panel) and IKK protein abundance (bottom
panel) were measured by IKK kinase assay and Western blot
respectively as described under "Materials and Methods." In
C, the immunoprecipitated IKK from 2- and 5-h 2-AAF treated
H4-II-E cells was incubated with purified GST protein.
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DISCUSSION |
Activation of NF-
B Signaling and Induced mdr Gene Expression by
2-AAF in Rat Hepatoma--
Previous studies have demonstrated that
mdr1b expression is frequently up-regulated in HCC developed by various
hepatocarcinogenetic programs. However, the mechanisms of this
activation are largely unknown. In this study, we studied mechanisms of
mdr1b up-regulation in rat hepatoma cells induced by 2-AAF.
We identified that the NF-
B-binding site on the mdr1b promoter is
necessary for the induction by 2-AAF. We further demonstrated that
treatment of rat hepatoma cells with 2-AAF increases DNA binding
activity of NF-
B through activation of IKK, which degrades I
B
but not I
B
. Our present study reveals a sequence of events
involved in the activation of mdr1b by 2-AAF as shown in Fig.
10. This is the first demonstration
that activation of mdr1b expression by a hepatocarcinogen is mediated
by the NF-
B signaling.

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Fig. 10.
Putative mechanisms of 2-AAF induction of
mdr1b expression in rat hepatocarinogenesis. 2-AAF generates
intracellular ROS and then ROS activates IKK complex, which leads
to degradation of I B . The degradation of I B causes nuclear
translocation of NF- B and activation of mdr1b expression which
is regulated by NF- B.
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In most cases, NF-
B activation involves phosphorylation and
degradation of I
B
, which in turn releases NF-
B and leads to the nuclear translocation of NF-
B. However, we were not able to
observe I
B
degradation after 2-AAF treatment, but instead I
B
was degraded. These observations were also supported using recombinant DNA containing Flag-tagged I
B
and I
B
in
transfection assays followed by 2-AAF treatments. To the best of our
knowledge, this is the first observation showing that activation of
NF-
B pathway is mediated by I
B
but not I
B
degradation.
Unlike I
B
, I
B
itself is not regulated by NF-
B and thus
it is not rapidly resynthesized after degradation. For this reason, I
B
degradation is thought to associate with prolonged activation of NF-
B (14, 28). In agreement with this notion, our data showed
that 2-AAF caused a relatively slow and prolonged degradation of
I
B
and activation of NF-
B DNA binding, in comparison with that
caused by cytokines, e.g. TNF-
. The activation of IKK in the 2-AAF-treated cells could be demonstrated by the in
vitro kinase assay experiments, suggesting that the mechanisms of
preferential degradation of I
B
may lie downstream from the IKK
activation. At present it is unclear why I
B
is preferentially
degraded, but possibilities can be offered: One possibility is that
there may exist a 2-AAF-inducible inhibitor which preferentially
shields I
B
from phosphorylation by IKK. Alternatively, an
I
B
-specific kinase which preferentially recognizes and
phosphorylates I
B
but not I
B
may be induced by 2-AAF.
Recent studies have identified an I
B
-specific interacting protein
(29). Moreover, activation of NF-
B by multiple distinct I
B kinase
complexes has been demonstrated (30). There results suggest that there
may be additional I
B-interacting proteins and/or kinases involved in
the activation of NF-
B by 2-AAF. Another possibility is that both
I
B
and I
B
are phosphorylated, but their subsequent
proteolytic degradation is differentially regulated. Further studies on
the kinetics and extent of I
B
and I
B
phosphorylation as
well as their degradation will be helpful to differentiate these possibilities.
Recent studies have demonstrated that multiple pathways are responsible
for activation of NF-
B. In addition to the mechanism reported here
which involves the degradation of I
B
, other alternative mechanisms may also play a role in the NF-
B activation in the absence of I
B
degradation. Beraud et al. (31) found
that hypoxia, reoxygeneration, and the pervanadate-induced I
B
phosphorylation at tyrosine 42 and this led to NF-
B activation
independent of I
B
proteolysis. They observed that the regulatory
subunit of phosphatidylinositol 3-kinase could recognize
tyrosine-phosphorylated I
B
and sequester it from binding to
NF-
B. Alternatively, several reports have shown that NF-
B
subunits p65 and p50 are phosphorylated and phosphorylation increases
NF-
B DNA binding activity or transactivation potential (32, 33).
Moreover, the degradation of other NF-
B inhibitors including
I
B
, I
B
, and p105 may also contribute to NF-
B activation
by 2-AAF without degradation of I
B
. To define these
possibilities, more detailed studies are warranted.
Redox Regulation of NF-
B Signaling and mdr Gene
Expression--
We also presented evidence showing that ROS levels are
elevated in H-4-II-E cells treated with 2-AAF. Induction of ROS is much
reduced in the GSH overproducing H9 cells. These results are consistent
with our previous report demonstrating that overproduced antioxidant
GSH in cultured cells suppressed oxidative stress induced by cytotoxic
agents, including TNF-
, phorbol ester, and okadaic acid (24).
Concomitantly, we found that induction of IKK activity, NF-
B DNA
binding activity, and mdr1b expression is retarded in the H9 cells as
compared with the parental H-4-II-E cells. These results strongly
suggest that, like MRP1 (15), expression of mdr1b can be
regulated by redox conditions. The identification of IKK as an upstream
redox sensor of NF-
B activating signal by 2-AAF is consistent with
those reported by Chen et al. (34) demonstrating that the
activity of I
B kinase
(IKK
) was significantly elevated in
cells exposed to proxidant vanadate. However, our results may differ
from those reported by Li and Karin (35). These investigators reported
that induction of IKK activity by TNF-
(and therefore I
B
phosphorylation/degradation) was not affected in cells treated with NAC
(35).
Aside from the involvement of IKK as a target of redox regulation,
oxidative stress may cause protein conformational changes. Particularly, oxidation of methionyl residues may render proteins susceptible to proteasomal degradation (36). In this regard, our
observation that 2-AAF triggering ROS formation raises an additional
possibility that ROS might modulate the degradation of I
Bs by direct
oxidation. It is possible that I
B
, because of its minor
difference in amino acid composition compared with I
B
, is more
sensitive to oxidative alterations and thus prone to proteolysis.
Further investigations on the redox control of NF-
B signaling in
different cell settings with different prooxidants, in combination with
domain swapping between I
B
and I
B
, may allow us to
elucidate all these possibilities.
ROS are regarded as having carcinogenic potential and have been
associated with tumor promotion (37, 38). In addition, ROS are also
pivotal factors in the genesis of heart disease (37). There is a fine
balance between ROS and endogenous antioxidants, and any disturbance of
this balance may cause cancer and heart diseases. For this reason, many
natural or synthetic antioxidants have been used to prevent
carcinogenesis and cardiovascular problems. Our results that carinogen
2-AAF causes increase of ROS and GSH blocks the 2-AAF-initiated ROS
signaling, might have implications in the development of novel cancer
therapeutic and preventive interventions. In fact, it has been reported
that oral administration of the thiol NAC produced a significant
decrease of mitochondrial DNA adduct, in the liver of 2-AAF-treated
rats and in the lung and liver of rats exposed to cigarette smoke
(39).
Activation of NF-
B Signaling and
Hepatocarcinogenesis--
Liver cancers in experimental animals can be
induced by many different protocols including chemical carcinogens,
steroid hormones, dietary intervention, and viral infections (40).
Because these different agents have different cellular targets and
models of cytotoxicity, it is likely that multiple pathways are
involved in the initial events but then converged during
hepatocarcinogenesis. The consistent observations of
mdr gene up-regulation in the various hepatocarcinogenetic programs strongly suggest an overlapping, if not
common, pathway between the induction of mdr gene
expression and the development liver cancers. The discovery that
NF-
B signaling is involved in the activation of mdr1b expression in
AAF-treated rat hepatoma cells raises an important scenario suggesting
that NF-
B may plays a role in liver cancer development as well.
Several lines of evidence support the involvement of Rel/NF-
B
signaling in liver cancer. (i) The retroviral v-rel
oncogene acutely induces tumors in birds and mammals (41). (ii)
Overexpression or constitutive activation of the Rel/NF-
B gene
family has been noted in many human hematopoietic and solid tumors (42,
43). (iii) NF-
B signaling can be activated by a wide variety of
stimuli, including genotoxins and nongenotoxins. As liver is the major
detoxification reservoir of xenobiotics, many of these excellular
stimuli are known to induce liver cancers in experimental animals. (iv)
Evidence has accumulated that ROS plays an important role in
hepatocarcinogenesis in animal models and suppression of ROS retards
liver cancer progression in these models (37, 44). Greater than 10-fold
increases of mdr1b expression in Fisher rats can be induced
by 2-AAF (4). On the other hand, liver neoplastic lesions can be
chronically induced in these animals by this hepatocarcinogen. The
results described in this article thus provide a molecular basis for
further investigation on the roles of NF-
B signaling in
hepatocarcinogenesis and the induction of mdr1b gene
expression in the process. These experiments are currently being
investigated in this laboratory. These studies may eventually lead to a
better understanding on the mechanisms of liver cancer development and
the evolution of drug resistance in this devastating disease.