From the Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada
Received for publication, September 18, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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The mouse multidrug resistance gene
family consists of three genes (mdr1, mdr2, and
mdr3) encoding P-glycoprotein. We show that the expression
of mdr1 is increased at the transcriptional level upon
treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic
aromatic hydrocarbon 3-methylcholanthrene (3-MC). This increase
is not observed in the aromatic hydrocarbon receptor (AhR)-defective
TAOc1BPrc1 and the AhR nuclear translocator
(Arnt)-defective BPrc1 variants, demonstrating that the
induction of mdr1 by 3-MC requires AhR·Arnt. We
show that the mdr1 promoter ( Multidrug resistance
(MDR)1 is characterized by
cross-resistance of the cells to a large number of structurally and
functionally unrelated cytotoxic agents used in chemotherapy. In
cultured cells, MDR is frequently caused by the overexpression of
P-glycoprotein (Pgp), an integral membrane protein belonging to the
ATP-binding cassette superfamily of transporters and which functions as
an energy-dependent efflux pump of cytotoxic drugs (1, 2). Pgp is encoded by a small family of genes with two members in humans
(MDR1 and MDR2/MDR3) and three in
rodents (mdr1/mdr1b, mdr2, and
mdr3/mdr1a) (1, 2). Only one human gene
(MDR1) and two rodent genes
(mdr1/mdr1b and mdr3/mdr1a)
can confer MDR upon overexpression in drug-sensitive cells (1, 2).
The different mdr genes and Pgp isoforms are expressed in a
tissue-specific manner (1, 2). In the mouse, mdr1 is
expressed mostly in the adrenal cortex, kidney, and pregnant uterus,
mdr2 in the liver at the canalicular face, and
mdr3 in the intestine and to a lesser extent in the heart,
liver, lung, and capillaries of the brain (3). Pgps are localized on
the apical membrane of epithelial cells lining luminal spaces,
suggesting that they function in normal tissues as transporters of
toxic substances and/or specific endogenous cellular products (4).
Knockout mice experiments have demonstrated a role for the
mdr3 gene in the maintenance of the blood-brain barrier and
drug elimination and for the mdr2 gene in the transport of
phospholipids in the bile (5, 6). No physiological function has been
attributed to the mouse mdr1 gene so far, since knockout
mdr1 ( A number of factors have been found to modulate the level of
mdr gene expression in the liver. For example, high levels
of MDR1 RNA have been found in human hepatocarcinomas, and
overexpression of the mdr1 isoforms has also been observed
in rodent liver during cholestasis, during regeneration following
partial hepatectomy, during chemically induced hepatocarcinogenesis,
and following administration of various natural and synthetic
xenobiotics (1, 2). In particular, it has been shown that expression of
the rat mdr1b gene is increased in liver cells in response
to treatment with various polycyclic aromatic hydrocarbon (PAH)
compounds, including 3-methylcholanthrene (3-MC), and that this
increased expression occurs at the transcriptional level (9-11).
However, the precise molecular mechanisms involved in mdr1b
regulation in response to 3-MC are still unknown.
PAHs are carcinogenic compounds arising from the incomplete combustion
of organic matter and are widespread in the environment, including
tobacco smoke and tar. PAHs such as 3-MC and benzo(a)pyrene (B(a)P) as well as halogenated aromatic hydrocarbons such as
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are specific
inducers of genes coding for drug-metabolizing enzymes (DME), including
cyp1a1 and cyp1a2, that code for cytochromes P450
involved in metabolic oxidation (12). PAHs and TCDD bind in the
cytoplasm to the aromatic hydrocarbon receptor (AhR), a member of the
bHLH-PAS (basic helix-loop-helix Per-Arnt-Sim) family of transcription
factors (12, 13). The ligand-bound AhR translocates to the nucleus,
where it binds as a heterodimer with the AhR nuclear translocator
(Arnt; another bHLH-PAS protein) to specific cis-acting regulatory DNA sequences located in the promoter of its targets (known
as AH-, dioxin-, or xenobiotic-responsive elements (or AHRE, DRE, or
XRE, respectively)) to enhance their transcription (12, 13). Given that
mdr1b expression is increased in liver cells in response to
treatment with various PAHs, it was postulated that mdr1b
may be under the control of the AhR (9). However, studies failing to
show mdr1 induction in the liver of mice treated with TCDD,
one of the most potent agonists of the AhR, suggested that
mdr1 expression was not regulated by AhR (14). The
involvement of AhR in the regulation of mdr1 has so far
remained controversial.
The mouse hepatoma cell lines Hepa-1c1c7 (wild type),
TAOc1BPrc1 (AhR-defective), and BPrc1
(Arnt-defective) constitute a powerful experimental system to
investigate the transcriptional regulation of different AhR·Arnt targets in response to xenobiotics (12). The two mutant cell lines were
derived as B(a)P-resistant variants of Hepa-1c1c7 and were
identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment (15).
TAOc1BPrc1 cells have a decreased level of AhR (~10% of
wild-type cells) and therefore decreased induction of the
cyp1a1 promoter and lower aryl hydrocarbon hydroxylase
activity in response to TCDD and other AhR ligands (15-18).
BPrc1 cells have a normal cytosolic AhR, which fails to
accumulate in the nucleus because of a defective Arnt (15). They have
virtually no basal or inducible levels of cyp1a1 expression
and aryl hydrocarbon hydroxylase activity (15-17).
In the present report, we have used this panel of cell lines to
investigate the transcriptional regulation of the murine
mdr1 gene by 3-MC and other xenobiotic compounds. Our
results demonstrate that mdr1 is transcriptionally induced
by 3-MC and B(a)P and that this induction is mediated by p53
but also requires AhR·Arnt. A model for the AhR·Arnt- and
p53-mediated transactivation of mdr1 in response to
genotoxic stress is proposed.
Cell Culture--
Wild-type Hepa-1c1c7 and Hepa 1-6,
AhR-defective TAOc1BPrc1, and Arnt-defective
BPrc1 cells were obtained from the American Type Culture
Collection (ATCC; Manassas, VA) and maintained in culture under the
conditions recommended by the ATCC. Chinese hamster ovary LR73 cell
lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1, mdr2, or
mdr3 genes (LR73 mdr1, LR73 mdr2, and
LR73 mdr3, respectively; a gift from Dr. Philippe Gros, McGill University, Montréal, Canada) were grown as described elsewhere (19, 20). For inductions, cells at ~50% confluence were
exposed to different concentrations of xenobiotics for various periods
of time (the exact conditions for each experiment are indicated in the
figure legends). 3-MC, B(a)P, and daunorubicin were obtained
from Sigma, and TCDD was obtained from the Centre d'expertise en
analyze environnementale du Québec (Laval, Canada). Stock
solutions of 3-MC (5 mM) and B(a)P (25 mM) were prepared in Me2SO, and the
stock solutions of daunorubicin (1 mg/ml) were prepared in water. TCDD
was obtained in n-nonane at a concentration of 50 µg/ml
and was stored at room temperature. Stock solutions of 3-MC,
B(a)P, and daunorubicin were stored at RNA Preparation--
Total RNA was prepared from 3-MC-treated
and untreated hepatocytes as well as from the LR73 mdr1,
LR73 mdr2, and LR73 mdr3 cell lines by
homogenizing the cells in a solution containing guanidium hydrochloride
(6 M) followed by sequential ethanol precipitation, as
described previously (21).
RNase Protection Assay--
The plasmid constructed to detect
the mdr1 RNA consisted of a 165-bp BamHI fragment
isolated from the mdr1 cDNA (positions 1926-2090
relative to the ATG initiation codon (22)), blunt-ended with T4 DNA
polymerase, and cloned into plasmid pGEM-7Z (Promega, Madison, WI) at
the SmaI site, giving plasmid pmdr1-G7. This plasmid was
linearized with EcoRI and used as a template to synthesize an antisense mdr1 probe using SP6 RNA polymerase (Amersham
Pharmacia Biotech). The pKX10-3Z plasmid consisting of an
XbaI-KpnI mouse Nuclear Run-on Transcription Assay--
The run-on experiment
was performed essentially as described by Fisher et al.
(24). Nuclei were isolated from Hepa-1c1c7 cells treated with
Me2SO or with 3-MC (5 µM) for 48 h and
were used to label nascent RNAs with [ Slot Blot Analyses--
Slot blotting was performed as
previously described (21). RNA samples (10 µg) were denatured in 7×
SSC-7.5% formaldehyde for 15 min at 65 °C and applied to a nylon
membrane (Zeta-Probe). Detection of specific RNAs was performed by
hybridization at 65 °C in 0.5 M NaPO4, pH
7.2, 1 mM EDTA, 7% SDS, 1% bovine serum albumin, and 100 µg/ml salmon sperm DNA with 32P-labeled DNA probes. The
mdr1 probe was a 4.2-kb SphI-EcoRI
fragment overlapping the full-length mouse mdr1 cDNA,
isolated from plasmid pGEM7/mdr1 (a gift from Dr. Philippe Gros, McGill
University, Montréal); the cyp1a1 probe was a 1.2-kb
PstI fragment isolated from plasmid pmP1450-3'; and the
actin probe was a 245-bp XbaI-KpnI fragment
isolated from pKX10-3Z. The membranes were washed twice at 65 °C
with a solution containing 40 mM NaPO4, pH 7.2, 5% SDS, 1 mM EDTA, 0.5% bovine serum albumin and twice
with a solution containing 40 mM NaPO4, pH 7.2, 5% SDS, and 1 mM EDTA before autoradiography.
Chloramphenicol Acetyl Transferase (CAT) Expression
Plasmids--
Plasmid pMcat5.9 consists of a 482-bp DNA fragment
containing the dioxin-responsive elements of the cyp1a1 gene
cloned upstream of the mouse mammary tumor virus promoter and the CAT
gene (24) (kindly provided by Dr. Allan Okey, University of Toronto).
Plasmids pmdr1, p-452, p-245, p-141, and p-93 (previously referred to
as pSacICAT, pExo6CAT, pExo2CAT, pExo1CAT, and pAluCAT, respectively) have been described elsewhere (27). The mdr1 promoter
sequence in these constructs ends at position +84 with respect to the
transcription start site (27). To produce the p53 mutant constructs,
pM1 and pM2, plasmid pSBM13 was used. This plasmid consists of a 1.2-kb SacI-HindIII mdr1 promoter fragment
(positions Transient Transfections and CAT Assays--
Cells were plated at
a density of 8 × 105/60-mm plate and transfected on
the following day with 10 µg of plasmid DNA, using a standard calcium
phosphate precipitation method (28). After incubation with the DNA
precipitate for 16 h, the cells were washed twice with
phosphate-buffered saline and supplied with fresh medium containing the
different xenobiotics. After 48 h, the cells were collected. Cell
extracts were prepared, and protein concentrations were determined by
the Bradford method (29). CAT activities were assayed by standard
protocols as described previously, using 2 µg of proteins (27).
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared according to Schreiber et al. (30), with some
modifications. Cells were harvested in cold phosphate-buffered saline,
0.6 mM EDTA and collected by centrifugation. The cell
pellets were resuspended in 400 µl of ice-cold buffer A (10 mM Tris, pH 8.0, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM dithiothreitol)
containing 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 1 µg/ml pepstatin, and 5 µg/ml leupeptin and swelled on
ice for 15 min. Subsequently, 25 µl of 10% Nonidet P-40 were added,
and the tubes were vortexed vigorously. The nuclear pellets were
collected by centrifugation and resuspended in 100 µl of cold buffer
C (20 mM Tris, pH 8.0, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol) in the presence of protease inhibitors. The suspensions
were shaken vigorously at 4 °C for 1 h and centrifuged for 15 min at 4 °C, and the supernatants were frozen in aliquots at
Electrophoretic Mobility Shift Assay--
Oligonucleotides
overlapping the potential p53 binding site in the mdr1
promoter (5'-GAACACGTAAAGACAAGTCTAT) and the p53 consensus sequence in
the p21waf1/cip1 promoter
(5'-GAACATGTCCCAACATGTTGAG) (31) were end-labeled with
Western Blotting--
Total proteins from 3-MC- or
Me2SO-treated Hepa-1c1c7 and BPrc1 cells were
extracted in ice-cold buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 1%
sodium deoxycholate) containing 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 µM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. Total proteins (75 µg/sample) or nuclear extracts (30 µg/sample) were separated by
SDS-polyacrylamide gel electrophoresis on a 10% acrylamide gel,
transferred to a nitrocellulose membrane, and analyzed with the
monoclonal anti-p53 antibody pAb421 (32) (Calbiochem) at a
concentration of 5 µg/ml. Immune complexes were revealed by incubation with a goat anti-mouse IgG antibody coupled to alkaline phosphatase (Bio-Rad) and developed with
5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and
nitro blue tetrazolium chloride substrates as recommended by the
manufacturer (Life Technologies, Inc.).
Transcriptional Induction of the Mouse mdr1 Gene by 3-MC in
Hepatoma Cells--
We have used an RNase protection assay to study
the expression of mdr1 in the hepatoma cell line Hepa-1c1c7
upon exposure to 3-MC (Fig. 1). An
mdr1-specific riboprobe was prepared by cloning into
pGEM7-Zf a mouse mdr1 cDNA fragment overlapping the
linker region of the protein, this domain displaying the lowest
sequence homology among the three mouse mdr cDNAs (21).
When tested with RNA prepared from LR73 stable transfectants expressing
each of the three mouse mdr cDNAs, the mdr1
riboprobe was found to recognize the mdr1 RNA but not the
mdr2 or mdr3 RNA, thus confirming its specificity
(Fig. 1, top right). The mdr1 probe
was then used with RNA from Hepa-1c1c7 cells treated or not with 3-MC
(Fig. 1, top left). This experiment showed that
the amount of mdr1 RNA detected is very low in untreated
cells but is strongly increased in 3-MC-treated cells, demonstrating
that expression of the mouse mdr1 gene is induced by 3-MC
treatment. The use of an actin probe confirmed that equal quantities of
RNA were used in the assay (Fig. 1, bottom). A similar
experiment performed with mdr2- and mdr3-specific
riboprobes showed that the expression of these genes is not induced
under such conditions, demonstrating that the induction of
mdr1 expression by 3-MC is isoform-specific (data not
shown).
A nuclear run-on experiment was performed to determine whether
mdr1 induction by 3-MC occurs at the transcriptional level (Fig. 2). In addition to the mouse
mdr1 cDNA, cDNAs for the mouse cyp1a1
gene (known to be transcriptionally regulated by 3-MC (12)) and for the
actin gene were also included as positive and negative controls,
respectively. The data in Fig. 2 show that 3-MC induces an increase in
the rate of mdr1 mRNA synthesis, indicating that 3-MC
acts at the transcriptional level to induce mdr1 gene
expression in Hepa-1c1c7 cells.
AhR·Arnt-dependent Induction of mdr1 Expression by
3-MC--
To determine whether the increase in mdr1
expression in response to 3-MC exposure is AhR·Arnt-mediated, we
analyzed the mdr1 RNA levels upon 3-MC treatment in two
wild-type hepatoma cell lines Hepa-1c1c7 and Hepa 1-6 and in two
variant cell lines derived from Hepa-1c1c7, TAOc1BPrc1
(AhR-defective) and BPrc1 (Arnt-defective) (15) (Fig.
3). As controls, we also analyzed the
level of cyp1a1 and actin expression under the same
conditions (Fig. 3, middle and right,
respectively). This experiment showed that mdr1 is expressed
at low levels in the four cell lines in the absence of 3-MC induction
(Fig. 3, left panel). Upon 3-MC treatment, the
expression of mdr1 is induced in the two wild-type hepatoma
cell lines (by ~5-fold), this induction being completely abrogated in
the AhR-defective or in the Arnt-defective variants (Fig. 3,
left panel). The actin control probe confirmed
that equal amounts of RNA had been applied to the membrane (Fig. 3,
right panel). These data clearly demonstrate that
the induction of mdr1 in response to 3-MC requires an intact
AhR·Arnt complex, like cyp1a1 (Fig. 3, middle)
(12).
The Mouse mdr1 Promoter Confers 3-MC-regulated Expression in an
AhR·Arnt-dependent Manner--
To determine if
regulatory sequences responsible for mdr1 induction by 3-MC
are present in the promoter region of the gene, plasmid pmdr1,
consisting of a 1.2-kb SacI-HindIII DNA fragment overlapping the mdr1 promoter region (positions Two Putative XREs Located in the mdr1 Promoter Are Dispensable for
the Induction of mdr1 by 3-MC--
The AhR·Arnt transcriptional
complex binds to a specific DNA sequence, 5'-(A/T)NGCGTG, known as an
XRE to activate transcription (12). XREs render heterologous
promoters responsive to xenobiotics and function in a position- and
orientation-independent manner (33, 34). Examination of the
mdr1 promoter sequence indicated the presence of two
potential XREs in an inverted orientation in the distal portion of the
promoter at positions AhR·Arnt-dependent Induction of a p53·mdr1 Promoter
Complex by 3-MC--
Sequence comparison of the mouse mdr1
promoter with that of the rat mdr1b promoter revealed the
presence of a potential p53 binding site in the DNA region from
To assess p53 binding to the mdr1 promoter, we performed an
electrophoretic mobility shift assay (EMSA), using a
32P-labeled double-stranded oligonucleotide corresponding
to the p53 binding site of the mdr1 promoter and nuclear
extracts prepared from Hepa-1c1c7 and BPrc1 cells treated
or not with 3-MC. Nuclear extracts from both Hepa-1c1c7 and
BPrc1 cells gave rise to a complex (C1) (Fig.
6A, lanes
1, 3, and 4) that disappears only in
Hepa-1c1c7 cells upon 3-MC treatment (Fig. 6A,
lane 2). This result suggests that 3-MC treatment
abolishes the binding of an unknown protein to the mdr1
promoter and that this effect requires the presence of an intact
AhR·Arnt complex. It has been shown that the monoclonal antibody
pAb421, which binds within the C-terminal domain of p53, activates the
DNA binding activity of the protein (38). The addition of pAb421 to the binding reactions gave rise to the formation of a
p53·pAb421·mdr1 complex (C2), which was detected only in
Hepa-1c1c7 cells treated with 3-MC (Fig. 6A, lane
6). This complex was not formed when antibodies against the
transcription factors c-Jun and Skn-1 were used as a control,
confirming that C2 is specific for pAb421 and thus for p53 (Fig.
6B, lanes 6 and 8). Taken
together, these results demonstrate that 3-MC treatment of Hepa-1c1c7
cells induces binding of p53 to the mdr1 promoter and that
this induction requires AhR·Arnt.
An EMSA was also performed using the same nuclear extracts but this
time with a probe overlapping the high affinity p53 binding sites in
the p21waf1/cip1 promoter (5'-GAACATGTCCcAACATGTTg)
(31). As observed with the mdr1 probe, nuclear extracts from
Hepa-1c1c7 and BPrc1 cells gave rise to the C1 complex,
which was absent in Hepa-1c1c7 cells treated with 3-MC (Fig.
6A, lanes 9-12), indicating that the
protein factor present within C1 and which binds to the mdr1 promoter also binds to the p21waf1/cip1 promoter.
The addition of pAb421 to the binding reactions gave rise to the
formation of a p53·pAb421·p21 C2 complex of low
abundance in untreated Hepa-1c1c7 and BPrc1 nuclear
extracts, indicating that these cells express basal levels of
endogenous p53 (Fig. 6A, lanes 13 and
15). Moreover, the intensity of that complex was strongly
increased in Hepa-1c1c7 cells but not in BPrc1 cells
upon 3-MC treatment (Fig. 6A, compare lanes
14 and 16). The p53·pAb421 complex detected
with the p21 probe was more abundant than that detected with
the mdr1 probe (Fig. 6A, compare lanes 14 and 6), suggesting that the p53 binding site
of the p21waf1/cip1 promoter has a higher affinity
for p53 than the one present in the mdr1 promoter.
Increased p53 Levels in Hepa-1c1c7 Cells upon 3-MC
Treatment--
It has been shown that p53 levels are increased in
response to a variety of stresses, including DNA damage (39, 40). To determine whether the exposure of hepatoma cells to 3-MC results in
increased p53 levels, we analyzed by Western blotting total protein
extracts prepared from Hepa-1c1c7 and BPrc1 cells treated
or not with 3-MC, using antibody pAb421 (Fig. 7). This experiment showed that p53
expression is undetectable in total protein extracts from untreated
Hepa-1c1c7 and BPrc1 cells (Fig. 7A,
lanes 1, 3, and 5).
However, its expression was detected in Hepa-1c1c7 cells after 12 h of exposure to 3-MC and further increased after 48 h of
treatment (Fig. 7A, lanes 2 and
4). Increased p53 levels upon 3-MC treatment were not
observed in BPrc1 cells (Fig. 7A,
lane 5), demonstrating that an intact AhR·Arnt complex is required for p53 induction in response to 3-MC. Western blot
analysis of the nuclear extracts used in the EMSA showed that low
levels of p53 are present in the nuclei of the two untreated cell lines
(Fig. 7B, lanes 1 and 3)
and that p53 expression is increased upon 3-MC treatment in Hepa-1c1c7
but not in BPrc1 cells (Fig. 7B,
lanes 2 and 4, respectively). Taken
together, our results indicate that the appearance of the
p53·pAb421·mdr1 C2 complex detected in the EMSA (Fig. 6)
correlates with an AhR·Arnt-dependent change in p53
levels in the cells.
The p53 Binding Site in the mdr1 Promoter Is Required for the
Induction of mdr1 Transcription by 3-MC--
To determine whether p53
is responsible for conferring 3-MC-inducible expression to
mdr1, we abrogated p53 binding to the mdr1
promoter by introducing point mutations in the p53 binding site within
the context of the p53-mediated Induction of the mdr1 Promoter by Xenobiotic
Compounds--
Several metabolites of 3-MC possess high mutagenic
activity (41, 42) and thus could induce p53 through their DNA-damaging activity. To test this hypothesis, we have investigated the effect of
three additional xenobiotic compounds with different genotoxic activities on p53 activation and mdr1 transcriptional
induction: (i) B(a)P, a PAH and AhR ligand that, like 3-MC,
is not in itself genotoxic but that is oxidized to reactive species by
AhR·Arnt-regulated DME (12); (ii) TCDD, another AhR ligand that is
nongenotoxic and resistant to metabolic breakdown (12); and (iii) DN,
an anticancer drug and potent genotoxic agent that was recently shown to induce rat mdr1b expression via p53 (36).
Hepa-1c1c7 and BPrc1 cells were exposed to these three
compounds as well as to 3-MC, and the level of p53 was determined by Western blotting (Fig. 9A).
Treatment of Hepa-1c1c7 cells with B(a)P and DN resulted in
increased p53 levels, similar to those reached upon 3-MC treatment,
whereas TCDD had no effect on p53 expression (Fig. 9A,
left panel). As for 3-MC, the increased levels of
p53 in response to B(a)P required AhR·Arnt, since there
was no induction of p53 by this compound in BPrc1 cells
(Fig. 9A, right). Unlike 3-MC and
B(a)P, however, DN was able to activate p53 and
mdr1 in both cell lines. We then used a CAT assay to
determine the effect of these compounds on the transcriptional activity
of the mdr1 promoter (Fig. 9B). We found that in
Hepa-1c1c7 cells, B(a)P and DN were as efficient as 3-MC in
inducing mdr1 transcription (Fig. 9B,
left). TCDD, however, had no effect on mdr1
transcription, although the cyp1a1 promoter was induced
under the same conditions (data not shown). The absence of
mdr1 induction by TCDD is in agreement with the results
obtained by Teeter et al., which were unable to detect mdr1 induction in the liver of mice exposed to this compound
(14). The induction of mdr1 by 3-MC and B(a)P was
lost in BPrc1 cells, whereas DN was efficient at inducing
mdr1 transcription in the two cell lines (Fig.
9B). These results, showing that the levels of p53 correlate
with the induction of the mdr1 promoter, further confirm
that the induction of mdr1 is mediated by p53. Indeed, there
was no induction with any of the compounds tested when the pM2
mdr1 promoter construct carrying a mutation in the p53
binding site was used for the transfection (data not shown). Taken
together, these data suggest that the activation of p53 and the
subsequent induction of mdr1 are caused by a genotoxic stress resulting from the metabolism of 3-MC and B(a)P by
AhR·Arnt-regulated DME. The ability of intrinsically genotoxic DN to
induce p53 and mdr1 independently of the AhR·Arnt complex
together with the inability of nongenotoxic TCDD to induce p53 and
mdr1 further support this proposition.
In the present report, we have used a panel of wild-type, and AhR-
and Arnt-defective hepatoma cell lines to investigate the transcriptional regulation of the murine mdr1 gene by 3-MC
and other xenobiotic compounds. We show that mdr1 is induced
at the transcriptional level by exposure of the cells to 3-MC and that this induction depends upon a functional AhR·Arnt complex (Figs. 3
and 4). These characteristics are shared by the two phase I genes
cyp1a1 and cyp1a2 as well as by the four phase II
genes nmo1 (NAD(P)H:menadione oxidoreductase),
ahd4 (aldehyde dehydrogenase), ugt1a6
(UDP-glucuronosyltransferase), and gsta1 Ya (glutathione transferase) (43). Together, these genes form the so-called dioxin-inducible mouse Ah battery, which plays an important role in
environmental toxicity, cancer, and oxidative stress (43). These
findings are compatible with the proposition that mdr1 could belong to the Ah battery in the mouse. However, different observations indicate that this is not the case. First, TCDD, a prototypical AhR
ligand, does not induce mdr1 transcription (Fig. 9). Second, the kinetics of induction of cyp1a1 and mdr1 by
3-MC are different; while induction of cyp1a1 is readily
detectable after 3 h of treatment, induction of mdr1 is
only observed after 12 h of cell exposure to 3-MC (data not
shown), suggesting that the two genes are induced by different
mechanisms. Finally, our analysis of the mdr1 promoter deletions demonstrates that two potential XREs that were identified by
sequence analysis in the mdr1 promoter are not required for the induction of mdr1 by 3-MC (Fig. 5), consistent with an
indirect role for the AhR·Arnt complex in the induction process. It
is likely that 3-MC, upon binding to AhR, induces DME regulated by AhR·Arnt, leading to its own metabolism, and that one or more resulting metabolites are responsible for the subsequent induction of
mdr1 via an AhR·Arnt-independent pathway.
Our results indicate that this AhR·Arnt-independent pathway involves
p53. Activation of p53 occurs in response to a number of cellular
stresses, including DNA damage, and leads to the activation of several
genes whose product trigger cell cycle arrest, apoptosis, or DNA repair
(44). Several PAH metabolites possess high mutagenic activity that can
induce p53 through their DNA-damaging activity (41, 42).
B(a)P treatment of murine 3T3 cells has been shown to result
in DNA damage associated with elevated levels of nuclear p53 (45).
Likewise, treatment of normal human fibroblasts with B(a)P-7,8-diol-9,10-epoxide, a highly reactive metabolite of
B(a)P, results in elevated p53 levels (46). Moreover,
B(a)P has been shown to activate the human p53 promoter
through induction of the transcriptional activator NF- Based on our present results, we can propose the following model for
the AhR·Arnt- and p53-mediated transactivation of mdr1 in
response to genotoxic stress (Fig. 10).
Treatment of a cell with the AhR ligands TCDD, 3-MC, and
B(a)P activates transcription of the genes coding for DME
regulated by the AhR·Arnt complex. Upon metabolic activation,
genotoxic metabolites of 3-MC and B(a)P are generated (3-MC*
and B(a)P*) that can cause DNA damage, such as
1-sulfooxy-3-MC and B(a)P-7,8-diol-9,10-epoxide, that can
form DNA adducts and thus act as mutagens (42, 52). TCDD, the
prototypical AhR ligand, is resistant to metabolic breakdown and is
nongenotoxic. Experiments in AhR knockout mice have shown that
TCDD-induced toxicity is mediated in most part by AhR, but the precise
mechanism(s) by which this toxicity is exerted are still unclear (53).
On the other hand, DN has intrinsic genotoxic activity and can induce DNA damage independently of the AhR·Arnt pathway. Upon DNA damage, p53 is activated and stabilized, resulting in the transcriptional induction of mdr1 (Fig. 10).
1165 to +84) is able to
activate the expression of a reporter gene in response to 3-MC in
Hepa-1c1c7 but not in BPrc1 cells. Deletion analysis
indicated that the region from
245 to
141 contains
cis-acting sequences mediating the induction, including a
potential p53 binding sequence. 3-MC treatment of the cells increased
the levels of p53 and induced p53 binding to the mdr1
promoter in an AhR·Arnt-dependent manner. Mutations in
the p53 binding site abrogated induction of mdr1 by 3-MC,
indicating that p53 binding to the mdr1 promoter is
essential for the induction. Benzo(a)pyrene, a polycyclic
aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by
metabolizing enzymes regulated by AhR·Arnt, also activated p53 and
induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand
resistant to metabolic breakdown, had no effect. These results indicate
that the transcriptional induction of mdr1 by 3-MC and
benzo(a)pyrene is directly mediated by p53 but that the
metabolic activation of these compounds into reactive species is
necessary to trigger p53 activation. The ability of the anticancer drug
and potent genotoxic agent daunorubicin to induce mdr1
independently of AhR·Arnt further supports the proposition that
mdr1 is transcriptionally up-regulated by p53 in response
to DNA damage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice display no obvious physiological
abnormalities (7). However, different experimental evidence indicates
that Pgp encoded by mdr1 can serve in the transport of
steroids (8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
-actin cDNA fragment
(positions 724-969 in the
-actin cDNA) cloned into pGEM-3Z at
the XbaI and KpnI sites (kindly provided by Dr.
Rashmi Kothary, Institut du cancer de Montréal, Montréal, Canada) was used to generate a control actin probe. pKX10-3Z was linearized with XbaI and used to synthesize an antisense
actin RNA probe with T7 RNA polymerase. The riboprobes were synthesized in the presence of [
-32P]UTP, and the RNase protection
assay was performed according to standard protocols (23).
-32P]UTP.
Plasmids pVT101-U/mdr1, carrying the full-length mouse mdr1
cDNA (25); pmP1450-3', carrying a 1.2-kb PstI cDNA
fragment overlapping part of the mouse cyp1a1 cDNA (26)
(obtained from the ATCC); and pKX10-3Z were linearized with
StuI, BamHI, and XbaI, respectively.
The linearized plasmids were denatured, immobilized in duplicate onto a
nylon membrane, and hybridized with the
[
-32P]UTP-labeled RNAs for 48 h at 65 °C. The
membranes were washed and exposed for 7 days with two intensifying screens.
1165 to +84) cloned into M13mp18. Single-stranded DNA was
prepared from pSBM13 and used as a template to perform site-directed
mutagenesis of the p53 binding site, using the mutant oligonucleotides
M1 5'-TACCTGAATACATAAAGACA and M2
5'-CGTAAAGATAAATCTATGTA (the base changes are
shown in boldface type). The resulting M1 and M2 mdr1
promoter fragments were then excised from pSBM13 with SacI
and HindIII, blunt-ended with T4 DNA polymerase, and cloned
into plasmid pCAT at the HindIII site also blunt-ended with
T4 DNA polymerase, yielding plasmids pM1 and pM2. The presence of the
mutations in the resulting constructs was confirmed by DNA sequencing.
80 °C. Protein concentrations were determined by the Bradford method (29).
-32P using T4 polynucleotide kinase and annealed to
their respective complementary oligonucleotides to generate
double-stranded probes. The EMSA was performed with 5 µg of nuclear
extracts in a binding mixture containing 10 mM Tris-HCl, pH
7.5, 50 mM NaCl, 2.5 mM dithiothreitol, 4%
Ficoll, 1 µg of poly(dI-dC), and 20,000 cpm of radiolabeled probe.
The binding reactions were carried out at room temperature for 15 min.
Where needed, 1 µg of the monoclonal anti-p53 antibody pAb421 (32)
(Calbiochem) or of the polyclonal anti-Jun or anti-Skn-1 antibodies
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and the
incubation was continued for an additional 15 min. The complexes were
separated on 5% nondenaturing polyacrylamide gels in 1× TBE (90 mM Tris, 65 mM boric acid, 2.5 mM
EDTA, pH 8.0) at 200 V. The gels were exposed to XAR films (Eastman
Kodak Co.) for 16 h with two intensifying screens at
80 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Increased mdr1 expression in
Hepa-1c1c7 upon 3-MC treatment. The expression of mdr1
was analyzed by RNase protection assay. Total RNAs (45 µg) from
Hepa-1c1c7 cells treated with 5 µM 3-MC (+MC)
or with Me2SO ( MC) for 56 h and from the
control cell lines LR73/mdr1, LR73/mdr2, and LR73/mdr3 were analyzed
with an mdr1 riboprobe, which protects a 169-nt fragment
within the mdr1 transcript, or with a
-actin riboprobe,
which protects a 245-nt actin transcript fragment. Autoradiography was
for 15 h with two intensifying screens (mdr1) or for
5 h without intensifying screens (actin).
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Fig. 2.
Nuclear run-on experiment. Nuclei were
isolated from Hepa-1c1c7 cells treated with 5 µM 3-MC
(+MC) or with Me2SO ( MC) for
48 h. Nascent RNAs were radiolabeled with
[
-32P]UTP and used to probe duplicate nylon membranes
on which denatured cDNAs for mdr1, cyp1a1,
and actin had been immobilized. The membranes were washed and exposed
for 7 days with two intensifying screens.
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Fig. 3.
AhR·Arnt-dependent induction of
mdr1 expression by 3-MC. Total RNAs (10 µg)
from wild-type Hepa-1c1c7 and Hepa 1-6, AhR-defective
TAOc1BPrc1, and Arnt-defective BPrc1 cells
treated (+MC) or not treated ( MC) with 3-MC at
5 µM for 56 h were applied onto a nylon membrane.
The membrane was hybridized sequentially with an mdr1
(left), a cyp1a1 (middle), and a
-actin (right) probe. Autoradiography was for 18 h
(mdr1 and cyp1a1) or for 2 h (actin) with
two intensifying screens.
1165 to
+84 with respect to the transcription start site (27)) fused to the
CAT reporter gene, was analyzed in transient transfection
experiments. Plasmid pMcat5.9, which consists of a 482-bp fragment
derived from the cyp1a1 promoter fused to the mouse mammary
tumor virus promoter and to the CAT gene (24), as well as the empty
pCAT vector were also included as positive and negative controls,
respectively. The three plasmids were transiently transfected into
Hepa-1c1c7 and BPrc1 cells. The cells were treated with
3-MC or with Me2SO for 48 h, and the cellular extracts
were prepared and assayed for CAT activity. This experiment showed that
the mdr1 promoter is transcriptionally active in Hepa-1c1c7
cells and BPrc1 cells, since it can drive the expression of
the CAT gene in both cell lines, albeit at low levels (Fig.
4). This result is consistent with the
basal level of expression of mdr1 detected by slot blot
analysis in these cells (Fig. 3). 3-MC treatment of the Hepa-1c1c7
cells transfected with pmdr1 resulted in a 10-fold induction in CAT
activity as compared with untreated cells, reaching levels of CAT
activity similar to those detected in the Hepa-1c1c7 pMcat5.9
transfectants upon 3-MC treatment. However, this induction was
completely abrogated in BPrc1 cells (Fig. 4), consistent
with the lack of mdr1 induction at the RNA level observed in
the slot blot assay (Fig. 3). Similar results were obtained upon
transfection in TAOc1BPrc1 cells (data not shown). These
results, showing that the mdr1 promoter is able to activate
the expression of the reporter gene in response to 3-MC in Hepa-1c1c7
but not in BPrc1 and TAOc1BPrc1 cells,
demonstrate that (i) the mdr1 promoter is able to confer 3-MC-mediated transcriptional activation; (ii) this activation requires
a functional AhR·Arnt complex; and (iii) the sequences mediating this
induction are located between positions
1165 and +84 in the
mdr1 promoter.
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Fig. 4.
AhR·Arnt-dependent induction of
the mdr1 promoter by 3-MC. Plasmids pCAT (no
promoter), pmdr1 (mdr1 promoter from position 1165 to
+84), and pMcat5.9 (pMcat; 482-bp fragment from the cyp1a1
promoter fused to the mouse mammary tumor virus promoter) were
transiently transfected into Hepa-1c1c7 and BPrc1 cells by
the calcium phosphate method. The cells were then treated with 3-MC (5 µM) or Me2SO for 48 h. Total cellular
extracts were prepared, and equal quantities of proteins (2 µg) were
assayed for CAT activity. A, autoradiogram of a
representative CAT assay, showing the activity of plasmids pCAT, pmdr1
and pMcat in Hepa-1c1c7 and BPrc1 cells treated (+) or not
treated (
) with 3-MC (MC). The position of the
[14C]chloramphenicol (CM) and of its
acetylated products (AcCM) is indicated on the
left. B, quantitative analysis of CAT activities.
The percentage of conversion of [14C]chloramphenicol to
its acetylated derivatives was quantitated by liquid scintillation
counting. Open bars,
MC;
filled bars, +MC. The results
presented are the averages of three independent transfections performed
in duplicate. S.D. values are represented by the bars.
1129 and
620 (5'-CACGCAT and 5'-CACGCAA,
respectively). To identify the cis-acting sequences
responsible for the induction of mdr1 by 3-MC and to investigate the possible involvement of these putative XREs, we analyzed the transcriptional activity of a series of mdr1
promoter 5'-deletion CAT constructs after transient transfection into
Hepa-1c1c7 and treatment of the resulting transfectants with 3-MC (Fig.
5A). 3-MC treatment of
Hepa-1c1c7 cells transfected with plasmids p-452 or p-245 resulted in a
level of CAT induction similar to that observed in cells transfected
with plasmid pmdr1 carrying the full-length promoter, indicating that
sequences located within positions
1165 to
245 are dispensable for
the induction of mdr1 by 3-MC, including the two putative
XREs as well as a potential AP-1 binding site (5'-TGACTCA; positions
265 to
255 (35)) (Fig. 5, B and C). However,
further deletion of a 104-bp region down to position
141 (p
141) was
found to greatly diminish the induction of CAT activity by 3-MC (Fig.
5, B and C), demonstrating that sequences
important for the induction are located between positions
245 and
141. CAT activity in the absence of 3-MC was reduced in the p
141
transfectants when compared with the p
245 transfectants, indicating
that sequences between positions
245 and
141 are also involved in
the basal transcriptional activity of the mdr1 promoter in
hepatoma cells. Finally, we found that a low level of induction was
still detectable in the p
141 but not in the p
93 transfectants,
suggesting that sequences located between positions
141 and
93 may
be required for maximal induction of mdr1. However, this
residual induction was not seen in Hepa 1-6 cells in which deletion of
sequences down to position
141 completely abolished the induction of
CAT activity by 3-MC (data not shown). Taken together, these results
indicate that (i) the two potential XREs present in the mdr1
promoter are not required for its 3-MC-regulated expression, thus
suggesting an indirect role for the AhR·Arnt complex in the induction
process, and that (ii) the region from
245 to
141 contains
cis-acting sequences mediating the 3-MC-inducible
mdr1 expression in hepatoma cells.
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Fig. 5.
Deletion analysis of the mdr1
promoter. A, schematic representation of the
different 5' deletion fragments of the mdr1 promoter fused
to the CAT reporter gene. The mdr1 transcription start site
is shown (arrows) (27). The positions of the consensus
binding sequences for the transcription factors NF-Y (64), p53 (36),
AP-1 (35), and AhR·Arnt (XRE1 and XRE2) (33) are indicated
(top line). B, autoradiogram of a
representative CAT assay showing the activity of the 5' deletion
constructs in Hepa-1c1c7 cells treated (+) or not ( ) with 3-MC (5 µM) for 48 h. The positions of the
[14C]chloramphenicol (CM) and of its
acetylated products (AcCM) are indicated on the
left. C, quantitative determination of the CAT
activities. The percentage of conversion of
[14C]chloramphenicol to its acetylated derivatives was
quantitated by a PhosphorImager using a low energy screen and
ImageQuant software. Open bars,
MC;
filled bars, +MC. The results are the
averages of three independent transfections performed in duplicate.
S.D. values are represented by the bars.
245
to
141 of the mouse promoter (5'-GAACAcGTaaAGACAAGTCT; positions
197 to
178). The p53 binding site in the rat mdr1b
promoter has been shown to mediate the induction of mdr1b by
the anticancer drug daunorubicin (DN) (36). The p53 binding site in the
mouse promoter contains three mismatched nucleotides (lowercase
letters) when compared with the p53 consensus sequence, which consists
of two copies of the 10-bp motif 5'-RRRC(A/T)(T/A)GYYY (37).
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Fig. 6.
Binding of p53 to the mdr1
promoter. A, an EMSA was performed with nuclear
extracts prepared from Hepa-1c1c7 (Hepa) and
BPrc1 (BPr) cells treated (+) or not
treated ( ) with 3-MC (5 µM) for 48 h. The nuclear
extracts were assayed for binding to 32P-labeled
double-stranded oligonucleotides overlapping the p53 binding site in
the mdr1 (lanes 1-8) or in the
p21waf1/cip1 (31) (lanes
9-16) promoters. The binding reactions were carried out in
the presence (lanes 5-8 and 13-16)
or in the absence (lanes 1-4 and
9-12) of the anti-p53 mAb pAb421. The complexes were
separated on a native polyacrylamide gel. The gel was dried and exposed
for 16 h with two intensifying screens. The C1 and C2 complexes
are indicated on the left. The position of the free probe is
also indicated (bracket). B, nuclear extracts
from Hepa-1c1c7 cells treated (+) or not treated (
) with 3-MC (5 µM) for 48 h were assayed for binding to the
mdr1-labeled probe in the absence (lanes
1 and 2) or in the presence of the
-p53
antibody pAb421 (lanes 3 and 4), of an
-Jun (lanes 5 and 6) or an
-Skn-1 (lanes 7 and 8)
antibody.
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Fig. 7.
3-MC Increases p53 levels in an
AhR·Arnt-dependent manner. A, total
protein extracts were prepared from Hepa-1c1c7 (Hepa) and
BPrc1 (BPr) cells treated with 5 µM 3-MC (+) or with Me2SO ( ). Cells were
treated for 12 or 48 h (Hepa-1c1c7) or for 48 h
(BPrc1). Protein extracts (75 µg) were separated by
SDS-polyacrylamide gel electrophoresis on a 10% acrylamide gel and
transferred to a nitrocellulose membrane. The membrane was analyzed
with the anti-p53 monoclonal antibody pAb421. The molecular mass
standards (kDa) are shown on the left. B, nuclear
extracts were prepared from Hepa-1c1c7 (Hepa) and
BPrc1 (BPr) cells treated with 5 µM 3-MC (+) or with Me2SO (
) for 48 h.
Nuclear extracts (lanes 1, 3 and
4, 32 µg; lane 2, 16 µg) were
analyzed as described for A. The position of p53 is
indicated on the right of each membrane. The star at the
right of B shows a nuclear protein of ~75 kDa
detected by the pAb421 antibody.
1165 mdr1 promoter CAT construct pmdr1.
The C and G nucleotides of each half-site, known to be essential for
binding of p53 to its DNA consensus sequence (37) were mutated to T and
A, respectively, generating the two p53 mutant mdr1 promoter
constructs pM1 and pM2 (Fig.
8A). These two constructs were
transiently transfected into Hepa-1c1c7 cells along with the
appropriate controls, and the transfectants were treated with 3-MC or
with Me2SO for 48 h. The cellular extracts were
prepared and assayed for CAT activity. This experiment showed that both
the basal and inducible levels of CAT activity conferred by the
wild-type mdr1 promoter were completely abrogated (pM2) or
strongly reduced (pM1) by mutations in the p53 binding site, resulting
in levels of CAT activity similar to those conferred by the minimal
mdr1 promoter in the p-93 transfectants (Fig. 8, B and C). Taken together, our results clearly
demonstrate that the p53 binding site in the mdr1 promoter
is essential for the basal and 3-MC-inducible expression of
mdr1 in hepatoma cells.
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Fig. 8.
The p53 binding site mediates induction of
the mdr1 promoter by 3-MC. A,
schematic representation of the wild-type mdr1 promoter
fused to the CAT gene (pmdr1). The double mutations introduced within
the p53 consensus sequence in mutants pM1 and pM2 are indicated
below. B, autoradiogram of a representative CAT
assay showing the activity of the pmdr1, pM1, pM2, p-93, and pCAT
plasmids in Hepa-1c1c7 cells treated (+) or not treated ( ) with 3-MC
(5 µM) for 48 h. The position of the
[14C]chloramphenicol (CM) and of its
acetylated products (AcCM) is indicated on the
left. C, as in Fig. 5C.
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Fig. 9.
AhR·Arnt-dependent induction of
p53 and mdr1 by different xenobiotics.
A, Western blot analysis of p53 expression upon xenobiotic
treatment. Nuclear extracts were prepared from Hepa-1c1c7
(left) and BPrc1 (right) cells
treated with 3-MC (5 µM), TCDD (10 nM),
B(a)P (1 µM), DN (3.5 µM), or
Me2SO (NT) for 36 h. Nuclear extracts (15 µg) were analyzed as described in the legend to Fig. 7. The position
of p53 is indicated on the right. The 75-kDa protein
detected by pAb421 is indicated by a star. B,
autoradiogram of a representative CAT assay, showing the activity of
plasmid pmdr1 ( 1165 to +84) upon transient transfection into
Hepa-1c1c7 (left) and BPrc1 (right)
cells. The cells were treated for 48 h with the different
compounds as indicated in A.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (47). The
induction of p53 upon B(a)P bears biological significance,
since p53 has been shown to be required for the efficient repair of
B(a)P-7,8-diol-9,10-epoxide adducts in human cells and to
play a protective role against the teratogenic effects of
B(a)P in mice (48-50). Our data showing that p53 activation
by 3-MC and B(a)P requires an intact AhR·Arnt complex
demonstrates a pivotal role for AhR in the activation of p53 by PAHs.
It also suggests that, in addition to its own direct targets, AhR
indirectly regulates different p53 transcriptional targets in response
to PAHs. The recent demonstration that c-Ha-ras activation
by B(a)P requires an intact AhR further substantiates the
widespread consequences of AhR activation upon cell exposure to PAHs
(51).
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Fig. 10.
Proposed model for the AhR·Arnt- and
p53-mediated transactivation of mdr1. 3-MC* and
B(a)P* represent genotoxic metabolites of 3-MC and
B(a)P, respectively, and p53 represents activated p53
(for details, see "Discussion").
The combined results from the EMSA and Western blots yield interesting information about the mechanisms regulating mdr1 transcription. On the one hand, the EMSA shows that under normal conditions, an unknown protein within the C1 complex is bound to the p53 binding site in the mdr1 promoter but that this complex dissociates upon 3-MC treatment (Fig. 6A). This dissociation depends upon AhR·Arnt, since it occurs in Hepa-1c1c7 cells and not in BPrc1 cells, suggesting that the metabolic activation of 3-MC is required for the dissociation. Similar results were obtained with the p21waf1/cip1 probe, showing that the formation and dissociation of a C1 complex is not restricted to mdr1 but also extends to another p53 target. On the other hand, we observed in our Western blots that pAb421 detects, in addition to p53, a nuclear protein of ~75 kDa (Figs. 7 and 9). It is possible that this protein corresponds to p73, a recently discovered homologue of p53 (54). Indeed, it has been shown that pAb421 recognizes p73 in immunoprecipitation experiments (55). Interestingly, the intensity of the 75-kDa band was found to decrease upon treatment of the cells with 3-MC (Fig. 7 and 9) in a manner that correlates with the decrease in the C1 complex upon 3-MC treatment. This observation, together with the ability of p73 to bind canonical p53 DNA binding sequences (54), suggests a link between p73 and the protein present within the C1 complex. However, the inability of pAb421 to supershift the C1 complex indicates that this is not the case. Also, p73 has been shown to be up-regulated upon DNA damage, not down-regulated (56, 57). Studies aimed at identifying the C1 and p75 protein are currently under way.
The transcription factor NF-B mediates the cellular response to
various stimuli, including oxidative stress, UV irradiation, DNA
damage, and viral infections (58). NF-
B binds to DNA sequence elements in the promoter of its target genes to activate their transcription (5'-GGGN2-4CC). It has been recently
reported that the expression of the rat mdr1b gene is
induced by the hepatocarcinogen 2-acetylaminofluorene through the
generation of reactive oxygen species that activate NF-
B signaling
and binding to the mdr1b promoter (59). The metabolism of
B(a)P also generates reactive oxygen species, and it has
been shown that B(a)P activates transcription of the human
p53 and cyclooxygenase-2 genes by inducing the transcriptional activity
of NF-
B (47, 60). It is therefore likely that the NF-
B pathway is
also induced in hepatoma cells upon B(a)P exposure, in
addition to the AhR and p53 pathways. Sequence analysis indicates the
presence of a putative NF-
B binding site in the mouse
mdr1 promoter (5'-GGGAACC; positions
163 to
157).
Interestingly, this site is adjacent to the p53 binding site and is
located within the region conferring B(a)P induction of the
mouse mdr1 promoter. It was recently shown that NF-
B and
p53 cooperatively bind to the p53 promoter to activate its
transcription (61), suggesting that these two sites in the
mdr1 promoter could also cooperate to regulate
mdr1. However, it has been reported that p53 and NF-
B also have antagonizing effects on transcription, most likely by competing for the transcriptional coactivator p300/CBP (62). Experiments are under way to assess a potential role for NF-
B in the
regulation of mdr1 in response to PAHs.
What is the functional significance of mdr1 up-regulation in
response to genotoxic stress? Western blot analysis of Pgp in Hepa-1c1c7 treated with 3-MC showed that increased mdr1
transcription results in increased levels of Pgp in these cells (data
not shown), indicating that Pgp can mediate some biological effects of
p53 activation. A characteristic feature of Pgp is its ability to interact with a wide variety of structurally and mechanistically diverse compounds (8). It is therefore possible that mdr1 is activated by p53 as part of a global detoxification mechanism. In such
a case, Pgp would function to export xenobiotic compounds from the
cells, thus preventing their accumulation and eventual cellular damage.
However, the demonstration that Pgp does not affect intracellular
accumulation of B(a)P indicates that this is not the case,
at least for this particular compound (63). Alternatively, it is
possible that the biological effects carried out by Pgp upon genotoxic
stress may be related to a yet unidentified function of the protein.
Thus, the physiological consequences of p53-mediated Pgp induction
in response to DNA damage remains to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank Philippe Gros, Allan Okey, and Rashmi Kothary for the gift of plasmids and Alan Anderson for comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by a research grant from the Cancer Research Society Inc. (to M. R).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a studentship from the Medical Research Council of
Canada. Present address: Dept. of Biological Sciences, Bio-Mega Research Division, Boehringer Ingelheim (Canada) Ltd., Laval, Québec H7S 2G5, Canada.
§ Supported by a scholarship from Le Fonds de la recherche en santé du Québec. To whom correspondence should be addressed: Institut de recherches cliniques de Montréal, 110 Pine Ave. W., Montréal, Québec H2W 1R7, Canada. Tel.: 514-987-5770; Fax: 514-987-5764; E-mail: raymonm@ircm.qc.ca.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008495200
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ABBREVIATIONS |
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The abbreviations used are: MDR, multidrug resistance; Pgp, P-glycoprotein; 3-MC, 3-methylcholanthrene; B(a)P, benzo(a)pyrene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; DN, daunorubicin; CAT, chloramphenicol acetyl transferase; AhR, aromatic hydrocarbon receptor; Arnt, AhR nuclear translocator; EMSA, electrophoretic mobility shift assay; DME, drug metabolizing enzymes; PAH polycyclic aromatic hydrocarbon, XRE, xenobiotic response element; bp, base pair(s); kb, kilobase pair(s).
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