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INTRODUCTION |
Overexpression of the multidrug resistance gene (MDR1)
product P-glycoprotein (Pgp)1
is often correlated with chemotherapeutic failure (1, 2). Pgp is a
member of the large ATP-binding cassette transporter gene family (3).
In humans Pgp-mediated efflux of cytotoxic drugs is linked to only one
human gene (MDR1), whereas in rodents drug efflux is shared
between two mdr1 genes (pgp1/mdr1a and
pgp2/mdr1b). Whether the two rodent mdr1 genes
are functionally redundant with respect to the efflux of cytotoxic
chemotherapeutic agents remains controversial (4). There is evidence
that the two rodent mdr1 genes are differentially regulated.
For example, pgp1/mdr1a and pgp2/mdr1b show
divergent tissue regulation with some organs, such as murine intestine
and brain, expressing exclusively mdr1a while adrenal and
placenta express predominantly mdr1b (5, 6). In addition,
the rat pgp2/mdr1b and pgp1/mdr1a are divergently regulated during hepatocarcinogenesis in rats (7). In order to
extrapolate rodent studies to humans it is critical to determine the
molecular controls regulating the rat and human MDR1 genes.
Previous studies have suggested that human and rodent MDR1
genes are also distinctly regulated. For instance, in many cell lineages from rodents (liver, kidney, intestine, etc.) short exposures to chemotherapeutic agents lead to dramatic increases in
mdr1 expression, an effect that appears secondary to
specific increases in mdr1 transcription (8). In contrast,
transient exposure to these chemotherapeutic agents had no effect on
the expression of MDR1 in human cells (8). Other studies,
using primary cultured hepatocytes also showed a divergence in rodent
and human MDR1 expression after exposure to polycyclic
aromatic hydrocarbons (9-11). These studies suggest that fundamental
differences in MDR1 regulation exist between rodent and humans.
Recent studies suggest that events linked to transcription are
important in controlling MDR1 expression. For instance,
Scotto and co-workers (12) suggested that a conserved cis-element and binding proteins may be important in hamster Pgp1
transcriptional initiation in Pgp expressing cells. A different
conserved sequence in the mouse mdr1b gene and human
MDR1 gene, functionally important for both promoters
(13-15), was found to be activated in the MDR1 promoter by
a member of the C/EBP family (15). The human and rodent MDR1
promoters also contained a common stretch of GC-rich nucleotides. The
conservation of this GC region across species suggests that it is
important for regulation of the MDR1 genes. In the human
MDR1 promoter, Sp1 binds to a portion of the GC element and
activates the MDR1 promoter (16). This MDR1 GC
element also binds the transcription factor Egr-1 (17) and Egr-1
mediates 12-O-tetradecanoylphorbol-13-acetate activation of
the MDR1 promoter in K562 hematopoietic cells. More recent
studies have suggested that the human MDR1 promoter has multiple Sp1
sites that may not be functionally equivalent (18).
However, the importance in general of Sp1 and Egr1 to regulation of the
endogenous rat mdr1b gene and to its expression in tissues
that highly express this gene, such as liver, have not been examined.
These studies were undertaken to evaluate the functional importance of
Sp1 and Egr-1 to the regulation and expression of the rat
mdr1b gene in the rat H35 hepatoma cell line. In this report
we identify a Sp1/Egr-1 binding site in the promoter of pgp2/mdr1b that is similar to the Sp1/Egr-1 site in human
MDR1 (16). Using transfection and DNA binding assays of the
pgp2/mdr1b promoter and ectopic overexpression of the Egr-1
and Sp1 transcription factors these studies revealed regulation of
pgp2/mdr1b expression by Egr-1 and Sp1 that is unique from
what has been reported for regulation of human MDR1.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes, T4 polynucleotide kinase,
and T4 DNA ligase were from Life Technologies (Gaithersburg, MD),
Promega (Madison, WI), and New England Biolabs (Beverly, MA).
[
-32P]Deoxcytosine triphosphate and
[
-32P]ATP were from NEN Life Science Products Inc.
(Wilmington, DE). PCR reagents were from Perkin-Elmer (Norwalk, CT).
Cell culture medium and fetal bovine serum were from Biowhittaker
(Piscataway, NJ). G418 sulfate (geneticin) was from Life Technologies
(Gaithersburg, MD). Prime-It II kits were from Stratagene (La Jolla,
CA). Affinity-purified Sp1 was from Promega (Madison, WI). Polyclonal
antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and
the mdr1 (Ab-1) and actin antibodies were purchased from
Calbiochem (La Jolla, CA).
Construction of Vectors--
-250WT was generated by PCR
amplification using the sense oligonucleotide (
250 to
228)
5'-gaagatctATGCAACCGTGCACTATCCAGGT-3' and the antisense oligonucleotide
(+137 to +150) 5'-gaagatctCTCACGTAACACCTCCGGGTTTACCAT-3' using the promoter region of rat pgp2/mdr1b gene and
represents nucleotides
250 to +150 of the rat pgp2/mdr1b
gene. The PCR amplified product was digested with BglII and
subcloned into pGL2 Basic (Promega, Madison, WI). Orientation was
confirmed by restriction mapping and DNA sequence analysis.
The Sp1 mutation was introduced into the
36 to
60 region of the
Pgp2/mdr1b promoter by the overlap-extension method used previously (19) to generate
250MT. Briefly, two independent PCR
products spanning nucleotides
250 to
36 and
60 to +150 were made
that overlap at the mutated region. The 5'-half was made using the
sense oligonucleotide
250 to
228 (above) and the antisense
oligonucleotide (
60 to
36)
5'-CCGGCGGCCGAACTGTTGCCTAGC-3' with the
underlined base substitutions. The 3'-half was made using the sense
oligonucleotide (
60 to
36)
5'-GCTAGGCAACAGTTCGGCCGCCGG-3' and
antisense oligonucleotide (+137 to +150) (above). The two PCR products
were combined, denatured, and re-annealed at their common overlapping
24-bp sequence. Extension of this overlap followed by PCR amplification
using the two outer primers gave a recombined molecule with four
specific base changes in the GC region. This fragment was digested with
BglII, ligated into pGL2Basic, and the mutations confirmed
by DNA sequence analysis. The
250 Sp1mt/Egrmt
expression plasmid was developed by digestion of the
250MT plasmid with NgoMI followed by treatment with mung bean nucelase,
gel purification, and re-ligation.
The mammalian Sp1 expression vector was constructed by subcloning a
4.4-kilobase XbaI fragment encoding the full-length Sp1 cDNA from pBS-Sp1-fl (a gift of Dr. R. Tjian, University of
California, Berkeley, CA) into the XbaI site of the
pcDNA3 vector (Invitrogen, San Diego, CA) and orientation confirmed
by restriction mapping and DNA sequence analysis. The
Drosophila Sp1 expression construct (pPACSp1) and
COOH-terminal deletion (pPACSp1N539) (from R. Tjian) were subcloned
downstream of the Drosophila actin promoter in pPAC0 (20).
The wild-type (wtEgr-1) and mutant (mtEgr-1) expression vectors have
been described elsewhere (21, 22). The Egr-1-GST fusion was constructed
by subcloning Egr-1, from wtEgr-1 into the EcoRI site in
pGEX-5X-1 (Pharmacia, Milwaukee, WI).
Transient Transfections--
Reuber H35 hepatoma cells (American
Type Culture Collection) were maintained as described previously (19)
and were transfected by calcium phosphate co-precipitation with 10 µg
of QIAGEN (Qiagen Corp., Chatsworth, CA) purified plasmid DNA for
16-18 h. Cultures were then washed with serum-free medium, refed with
fresh medium, harvested after 24 h, and assayed for luciferase.
Drosophila SL2 cells were cultured in Schneider's Insect
Medium (Sigma) with 10% fetal calf serum and penicillin plus
streptomycin. Cells were transfected by calcium phosphate for 16-18 h
then refed with medium, and harvested 24 h later and assayed for luciferase.
Luciferase Assay--
Cells were washed twice in
phosphate-buffered saline, incubated for 15 min at room temperature
with agitation in 400 µl of Reporter Lysis buffer (Promega) and
scraped from the culture dishes. Luciferase activity was assayed on
20-40 µl of lysate using a luciferase kit (Promega, Madison, WI)
following the manufacturer instructions and utilizing a Opticomp
Luminometer (MGM Instruments, Hamden CT) with a 10-s counting window,
and normalized to protein.
Stable Transfections--
H35 cells were transfected with
Sp1-pcDNA3, Egr-1-pcDNA3, or pcDNA3 by the calcium
phosphate for 16 h, washed, refed, and after the 24-h expression
period, the cells were expanded and selected in medium containing 400 µg/ml G418. Individual G418-resistant colonies were isolated after
3-4 weeks drug selection. To obtain growth curves of the pcDNA3,
Sp1, and Egr-1 clones, cells were plated in duplicate on 60-mm dishes
and allowed to attach. The cells were then washed with
phosphate-buffered saline, scraped, and counted in a hemocytometer on
consecutive days. Population doubling times were calculated as
described previously (23, 24).
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from H35 cells plated on 100-mm tissue culture dish (19) and
analyzed by slot bot or Northern blot with a cDNA encoding Sp1 (20)
or a pgp2/mdr1b oligonucleotide (25). Prehybridization,
hybridization with random primer-labeled probes, and washing conditions
were as described previously (25). Blots were exposed to PhosphorImager
screens and band intensities were quantitated by the Image Quant
software or densitometry as described previously (25).
Western Immunoblot Analysis of Pgp in Cell Lysates and Egr-1 and
Sp1 in Nuclear Extracts--
For analysis of Pgp, cell lysates (35 µg) were analyzed by immunoblot with an anti-Mdr polyclonal antibody
(Ab-1) and developed as described previously (19). Intensity of bands
was quantified by densitometry and values were expressed relative to
untreated controls. For Egr-1 or Sp1, nuclear extracts from H35 cell
lines (prepared as below) were analyzed by immunoblot using specific polyclonal antibody directed against the carboxyl-terminal 14 amino
acids of Egr-1 or anti-Sp1 polyclonal PEP2 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA).
Oligonucleotides and Electrophoretic Mobility Shift Assays
(EMSA)--
The following double-stranded oligonucleotides were
used in EMSAs: pgp2GC,
5'-gaagatctGCGGGGGCAACAGGGCGGCCGCCGgagatctag-3'; mtpgp2GC,
5'-gaagatctGCGTAGGCAACAGTTCGGCCGCCGgagatctag-3';
Sp1mt/Egrmt,
5'-gaagatctGCGTAGGCAACAGTTCGGCCGXXXagatctag-3';
Egr, 5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3'; EgrMT,
5'-GGATCCAGCTAGGGCGAGCTAGGGCGA-3'; Sp1,
5'-ATTCGATCGGGGCGGGGCGAG-3'. The Xs in
Sp1mt/Egrmt indicate deletion of the
corresponding nucleotides. The pgp2GC, mtpgp2GC, and the
Sp1mt/Egrmt oligonucleotides were obtained from
Biosynthesis Inc. (Lewisville, TX). The consensus and mutant Egr
oligonucleotides were obtained from Santa Cruz Biotechnology Inc. and
the consensus Sp1 oligonucleotide was obtained from Promega.
All oligonucleotides were purified by agarose electrophoresis, annealed
with the complementary oligonucleotide, and then end labeled with
[
-32P]ATP and T4 polynucleotide kinase.
The labeled oligos were purified by Nuc-trap (Stragene, CA). EMSAs were
performed as described previously (26). Protein (either H35 nuclear
extract or affinity purified Sp1 or the recombinant Egr-1-GST fusion
protein) was incubated with 1 µg of poly(dI-dC) in 20 mM
Tris-HCl, 80 mM NaCl, and 1 mM dithiothreitol.
Unlabeled specific competitor was then added and specific radiolabeled
oligonucleotide was added last. Incubation was carried out at room
temperature for 20 min. The reaction mixture was electrophoresed on 4%
polyacrylamide gels with 0.25 × TBE as gel running buffer at room
temperature. Gels were dried and bands visualized by autoradiography.
Competitor oligo was added at 2-, 50-, or 200-fold excess and
concurrent with the radiolabeled probe. When antibody to Egr-1 was
used, the nuclear extract and antibody were preincubated at 4 °C for 25 min before the radiolabeled probe was added.
Nuclear Extracts and Expression of Egr-1-GST Fusion
Protein--
Nuclear Extracts were prepared from H35 cells essentially
as described by Latchman (27). Briefly, cells grown on 60-mm dishes were washed 1 × with phosphate-buffered saline, and then
harvested in 1 ml of chilled phosphate-buffered saline, spun down, and
resuspended in 50-100 µl of nuclear harvest buffer (20 mM Hepes, 450 mM NaCl, 0.5 mM
dithiothreitol, and 25% glycerol, 2 mM
phenylmethylsulfonyl fluoride). The suspension was then frozen and
thawed three times in a dry-ice ethanol bath, spun down at 10,000 × g at 4 °C for 10 min and the supernatant used in
gel-shift assays.
Recombinant Egr-1 was prepared using the Egr-1-GST fusion vector
according to the manufacturer's instructions (Pharmacia, Milwaukee,
WI). Briefly, an overnight culture of Escherichia coli transformed with the expression vector was diluted 1:10 into fresh LB
medium containing glucose and grown until an OD of 1-2 was reached.
Expression of the fusion protein was induced by adding isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.1 mM. After growing for an additional
2-6 h, the bacteria were pelleted, and resuspended in 1 × phosphate-buffered saline. Phenylmethylsulfonyl fluoride was added to 2 mM and the suspension sonicated on ice with six 30-s
bursts. Triton X-100 was then added to a final concentration of 10%.
The suspension mixed, allowed to sit on ice for another 30 min, spun
down, and the supernatant used in gel-shift assays.
In Vitro Transcription-Translation--
Egr1-pcDNA3, the
plasmid used in in vitro transcription-translation contains
Egr1 downstream of the T7 promoter in pcDNA3 (Invitrogen).
Egr-pcDNA3 was translated using a TNT T7 quick coupled transcription-translation system (Promega) under conditions recommended by the manufacturer. Preliminary studies determined that 1/10 to 1/50
of the in vitro translation mixture could be used in EMSA with a consensus Egr probe.
Short-term Drug Cytotoxicity Assays--
The proportion of total
cellular lactate dehydrogenase released into the culture medium was
used to measure cytotoxicity of various antitumor drugs and has been
previously described (28). 3-5 × 105 cells of each
of the H35 clones stably expressing Egr-1 or neo vector were plated
onto 60-mm dishes. Cells were then cultured for 48 h in the
presence of either 100 nM vinblastine or 100 µM fluorodeoxyuridine. After this interval aliquots of
medium were sampled. The medium was then aspirated and the remaining
attached cells lysed. Lactate dehydrogenase activity was measured in
the medium and cell lysate by using a Cytotoxicity Assay kit as
described previously (29). The proportion of lactate dehydrogenase
released into the medium as a fraction of the total lactate
dehydrogenase was then determined.
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RESULTS |
A Phylogenetically Conserved GC Box in MDR1
Genes--
Sequence comparison of the 5' promoter regions of the
rat, hamster, human, and murine MDR1 genes revealed within
each of the promoters a cluster of GC bases containing a consensus site
for Sp1 (Fig. 1). Because of the
phylogenetic conservation of the GC region in the MDR1 genes
and the fact that a number of transcription factors bind to GC-rich
regions (e.g. Sp1, Egr-1, and Ap-2) we hypothesized that the
interactions between these factors and Sp1 in this GC region are
important in basal regulation of the MDR1 promoters.

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Fig. 1.
Comparison of the GC-rich region in
5'-flanking region of MDR1 among rodent and human family members.
A sequence comparison of the GC-rich region in promoters of the human,
rat, mouse, and hamster MDR1 genes. The numbers
are with respect to transcription initiation site. The accession
numbers of the genes are as follows: L16546, rat mdr1b;
M60348, mouse mdr1b; L03287, hamster pgp2;
L03286, hamster pgp1; S18971, mouse mdr1a;
L07624, human MDR1.
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Sp1 Binds to and Is a Positive Transcriptional Regulator of the
pgp2/mdr1b Promoter--
We first tested whether the GC region in the
pgp2/mdr1b promoter could bind Sp1 by preparing a wild-type
oligonucleotide (pgp2GC) and a mutant (mtpgp2GC) that contained a
2-base pair mutation in the GC box that we predicted would disrupt Sp1
binding. EMSA demonstrated that wild type olignucleotide was
effectively shifted by purified Sp1 and that the mutations in the
pgp2/mdr1b Sp1 site resulted in a complete loss of Sp1
binding (Fig. 2A).

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Fig. 2.
Sp1 activates and is essential for full
pgp2/mdr1b promoter activity. A, radiolabeled pgp2GC or
mtpgp2GC oligonucleotide was incubated with (+) and without ( )
affinity-purified Sp1 (one footprinting unit) and analyzed by EMSA. The
last lane with mtpgp2GC contains 5 footprinting units of Sp1.
B, the activity of 250WT promoter was compared with the
250MT pgp2/mdr1b mutant promoter that contained the
identical mutations as were found in the mtpgp2GC oligonucleotide. 10 µg of each of these plasmids were transiently transfected into H35
cells and luciferase activity measured. Results are expressed as
activity relative to the 250WT promoter (100%). C,
Drosophila Schneider cells were transiently transfected with
1 µg of the 250WT or 250MT plasmid along with indicated amounts
of pPacSp1 (wildtype Sp1 expression plasmid) or N539 (a COOH-terminal
Sp1 deletion mutant) and luciferase activity measured. Values in
B and C are the average of three separate
transfection experiments each performed in duplicate to quadruplicate.
The error bars represent the standard error of the mean.
Results are expressed as fold-activation relative to 250WT (1.0) or
250MT (1.0) co-transfected with empty vector.
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The functional significance of Sp1 mutation upon pgp2/mdr1b
constitutive promoter activity were assessed in vivo by
introducing the identical mutations of the mtpgp2GC oligonucleotide
into the pgp2/mdr1b reporter (
250MT). H35 hepatoma cells
were chosen for transfection because they express pgp2/mdr1b
(19), and maintain normal liver characteristics such as production of
bile and expression of cytochromes P450 (30). The introduction of two
point mutations in the pgp2/mdr1b promoter decreased basal
promoter activity of
250MT to approximately 40% of the activity of
250WT in transfected H35 cells (Fig. 2B).
The finding that Sp1 mutations in the mdr1b promoter
decreased its basal activity suggested Sp1 might play a role in
positively regulating pgp2/mdr1b. This possibility was
evaluated by transient transfection of
250WT or
250MT into
Drosophila Schneider cells (that are Sp1 negative) along
with various amounts of either a wild-type Sp1 expression vector or an
inactive COOH-terminal Sp1 mutant (N539) (20) (Fig. 2C). Sp1
(200-300 ng) readily increased pgp2/mdr1b promoter activity
from 7-25-fold. In contrast, cells co-transfected with N539, a
zinc-finger deletion mutant showed no dose-related increase in the
activity of the pgp2/mdr1b promoter. Finally, the
pgp2/mdr1b promoter with the mutated Sp1 site was not
appreciably activated by Sp1 (Fig. 2C) thus demonstrating that an intact Sp1 site is required for Sp1 dependent transactivation.
Induction of the Endogenous pgp2/mdr1b Gene by Enforced
Overexpression of Sp1 in H35 Rat Hepatoma Cells--
Although the
pgp2/mdr1b promoter requires Sp1 for full basal activity
(Fig. 2) it is unknown if ectopic overexpression of Sp1 can affect
basal Pgp expression. In preliminary studies, we found that
co-transfection of the cytomegalovirus-driven Sp1 expression vector
(see "Methods") and the
250WT plasmid into H35 cells lead to an
Sp1-dependent transactivation in H35 cells (not shown). This Sp1 expression vector was then transfected into H35 cells and
G418-resistant colonies were isolated. Several independent clones were
selected and evaluated for immunoreactive Sp1, the amount of Sp1 DNA
binding activity and the amount of immunoreactive Pgp. Compared with
the Neo (pc2) clone, immunoreactive Sp1 expression increased from
1.8-fold to almost 5-fold in Sp1 clones 20 and 9, respectively (Fig.
3A). Further analysis of
representative Sp1 clones revealed increased Sp1 DNA binding complexes
compared with the Neo cell line (Fig. 3B). The expression of
Pgp in Sp1 cell lines 9, 18, and 20 increased 3-6-fold above the Neo
control line (Fig. 3C). Finally, we have shown that the
transcriptional activity of the pgp2/mdr1b promoter
increased 4.3-fold in the Sp18 cell line, compared with its activity in
the Neo control line (not shown), a value comparable to the increase in
Sp1 expression. These studies are the first to forge a direct link
between enforced overexpression of Sp1 in cells and increased Pgp
expression.

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Fig. 3.
Pgp and Sp1 levels and Sp1 DNA binding in H35
clones stably expressing Sp1 (Sp1-9, 10, 18, or 20) or Neo vector
(pc2). A, analysis of Sp1: 50 µg of nuclear extract
protein from the indicated cell lines was analyzed by immunoblot with
anti-Sp1 IgG. B, a radiolabeled consensus Sp1
oligonucleotide was incubated with nuclear extracts prepared from H35
clones stably expressing Sp1 or pcDNA vector and analyzed by EMSA.
C, analysis of Pgp: 35 µg of total cell lysate protein
from H35 clones was analyzed by immunoblot.
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Egr-1 Binds the pgp2GC Site but Negatively Regulates the pgp2/mdr1b
Promoter--
To first test whether the pgp2GC region interacts with
Egr1, H35 cell extracts were prepared. EMSA analysis demonstrated a specific DNA-protein complex formed with pgp2GC that was specifically reduced by incubation with either unlabeled consensus pgp2GC or Egr
consensus and not by unrelated oligonucleotides such as Oct-1 and Sp1
(Fig. 4A).

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Fig. 4.
Pgp2/mdr1b binds authentic Egr-1 and Egr-1 in
H35 Cells. A, 32P end-labeled pgp2GC
oligonucleotide was incubated with H35 nuclear extract in the presence
or absence (NC, no competitor) of excess unlabeled pgp2GC,
Sp1, Egr, or Oct-1 oligonucleotide (see "Experimental Procedures"
for amounts) and analyzed by EMSA. B,
32P-labeled pgp2GC or Egr consensus oligonucleotides were
incubated with in vitro translated Egr1 in the presence or
absence (NC, no competitor) of various competitor
oligonucleotides. The molar excess of competitors was as follows: Oct-1
(200-fold), pgp2GC (100-fold), EgrWT (100-fold), and EgrMT
(200-fold).
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To evaluate whether pgp2GC bound Egr-1, we in vitro
translated Egr-1 and then performed EMSA analysis utilizing either
radiolabeled pgp2GC or Egr as probes. Egr-1 readily bound the pgp2GC
and Egr probes. The nonspecific competitor Oct-1 did not displace bound Egr-1 from either pgp2GC or Egr probes. In contrast, pgp2GC readily displaced Egr-1 as did the 100-fold excess wild-type Egr competitor. A
small displacement was seen with 200-fold excess mutant Egr competitor.
To evaluate the biological importance of Egr-1 in regulating the
pgp2/mdr1b promoter, we co-transfected H35 cells with
250WT and either a wild-type Egr-1 expression plasmid (wtEgr-1) or a mutant Egr-1 that has a zinc-finger deleted and is incapable of binding
DNA (mtEgr-1) (Fig. 5). Egr-1 suppressed
the
250WT promoter in a dose-dependent manner reaching
nearly complete suppression at 5 µg of Egr-1. Egr-1 suppression of
the pgp2/mdr1b promoter was specific and not secondary to
squelching because Egr-1 did not repress thymidine kinase promoter
activity (not shown). Thus, the potent suppression of
pgp2/mdr1b by wtEgr-1 but not mtEgr-1 indicates that an
intact Egr-1 DNA-binding domain is required. Furthermore, Egr-1
suppressed the pgp2/mdr1b promoter in both human colon
carcinoma cells (LS180) and in a human hepatoblastoma cell line (HepG2)
(not shown). Thus, Egr-1 potently transrepressed the
pgp2/mdr1b promoter regardless of cell context.

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Fig. 5.
Egr-1 specifically represses the Pgp2/mdr1b
promoter in H35 cells. 250WT (5 µg) was co-transfected into
H35 cells with the indicated amounts of a wild-type (wtEgr-1) or mutant
(mtEgr-1) cytomegalovirus-Egr-1 expression vector. The amount of
cytomegalovirus promoter was kept constant by co-transfecting in empty
parent vector pCB6+. Cells were harvested and luciferase activities
were measured. Values represent the mean ± S.D. of two different
experiments from a total of four independent determinations per
experiment.
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Egr-1 Displaces Sp1 from Its Site on the GC-rich Promoter Element
in the Rat pgp2/mdr1b Gene--
Because Egr-1 and Sp1 have opposing
effects on the pgp2/mdr1b promoter activity (Figs. 2 and 5)
we assessed by EMSA whether Egr-1 and Sp1 might competitively interact
at the pgp2GC site (Fig. 6). The
Egr-1·pgp2GC complex migrates between Sp1 and the free probe. As a
control for the specificity of the complexes, the Egr-1·DNA complex
was competed with excess pgp2GC and the Sp1·DNA complex was competed
with a consensus Sp1 oligonucleotide (not shown). In preliminary EMSA
studies using limiting amounts of radiolabeled pgp2GC, we determined
that recombinant Egr-1 and Sp1 do not simultaneously bind to pgp2GC
because a third slower migrating complex was not observed when the two
proteins were added together and then compared with separate Sp1 or
Egr-1 complexes individually (not shown). To test whether Egr-1 and Sp1
share an overlapping site, the pgp2GC probe was incubated with
increasing concentrations of recombinant Egr-1 in the presence of a
fixed amount of Sp1. As the concentration of Egr-1 was increased, the amount of Egr-1 and pgp2GC complex increased while the Sp1·pgp2GC complex decreased (Fig. 6). However, when the amount of recombinant Egr-1 was held constant and Sp1 changed incrementally, the
Egr-1·pgp2GC complex showed only minimal displacement. Clearly, the
inability to displace Egr1 is not due to excess probe because in the
adjacent Egr1 versus Sp1 competition experiment (left
panel) the free probe is at a slightly higher amount (Fig. 6).
These data indicate that Sp1 and Egr-1 do not simultaneously bind to
pgpGC and that these two transcription factors competitively interact
at this site. It is also obvious under these conditions that Sp1 is not
very effective in displacing Egr1, a finding that likely depends upon the relative binding affinity of these two factors for the site.

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Fig. 6.
Competitive displacement of Sp1 binding to
the 36 GC-rich region in the Pgp2/mdr1b promoter by Egr-1. EMSA
was performed on 32P end-labeled pgp2GC oligonucleotide
(left panel) incubated in the presence of a constant amount
of Sp1 (1 footprinting unit/incubation) and increasing amounts of
bacterially expressed Egr-1-GST fusion protein, or (right
panel) incubated with a fixed amount of recombinant Egr-1-GST (2 µl/reaction) and increasing amounts of recombinant Sp1 (1-10
footprinting units).
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Diminished Egr-1 Binding Up-regulates the Basal Pgp2/mdr1b Promoter
in H35 Cells--
To address the role of Egr-1 in
pgp2/mdr1b promoter activity, a 4-bp deletion was introduced
in the Sp1 mutant oligonucleotide (mtpgp2GC) to generate
Sp1mt/Egrmt. The Sp1 mutant, mtpgp2GC
oligonucleotide, readily bound the recombinant Egr-1 (Fig.
7). This finding is consistent with
evidence that Egr-1 does not bind consensus Sp1 sites. However,
equivalent amounts of Egr-1 and even 2.5 times this amount of
recombinant Egr-1 protein showed significantly diminished Egr-1 binding
to the Sp1mt/Egrmt oligonucleotide (Fig.
7A).

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Fig. 7.
Decreased Egr-1 binding results in
up-regulation of the basal pgp2/mdr1b promoter. A,
32P-labeled mtpgp2GC or Sp1mt/Egrmt
oligonucleotide was incubated with (+) or without ( ) equivalent
amounts of recombinant Egr-1 or two times the amounts of Egr-1-GST
(farthest right hand lane, panel A) and analyzed by EMSA.
B, H35 cells were transiently transfected with 10 µg of
either wild-type ( 250WT) pgp2/mdr1b promoter-luciferase
plasmid, or mutant pgp2/mdr1b promoter-luciferase plasmids,
250MT or 250 Sp1mt/Egrmt and luciferase
activities measured. The values represent the average of two
independent experimental determinations with each value derived from
the average of a duplicate sample. Results are expressed as activity
relative to the 250WT promoter (1.0) and represent results from
experiments separate from Fig. 2B.
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In order to evaluate the effect of Egr-1 on the
pgp2/mdr1b promoter independent of Sp1, we
engineered the same 4-bp deletion (bp
39 to
36) into the putative
Egr-1 site using as template the
250MT construct that contains
disruptions of the Sp1 binding motif to generate
250
Sp1mt/Egrmt. Like the experimental results
depicted in Fig. 2B, disruption of the Sp1 binding motif
alone (
250MT) decreased pgp2/mdr1b transcriptional activity (Fig. 7B) in H35 cells. Despite the loss in Sp1
binding, the additional disruption of the Egr-1-binding site (
250
Sp1mt/Egrmt) led to a greater than 4-fold
increase in basal promoter activity compared with
250MT (Fig.
7B). Moreover, this promoter was more active than the
plasmid containing the unaltered wild type pgp2/mdr1b promoter (
250WT). These studies show that in the absence of an Sp1
site, the pgp2/mdr1b promoter activity is increased by
disruption of the Egr-1 DNA-binding site and that the decreased Egr-1
binding correlates with an up-regulation of pgp2/mdr1b
promoter activity. Therefore, it is most likely that the loss of
activity of
250MT promoter (Fig. 2C) is due to an overall
negative effect of Egr1 in these cells. To additionally prove that
Egr-1 repressed the pgp2/mdr1b promoter through the Egr1
site alone, we attempted to selectively disrupt the Egr-1 site but were
unable to successfully generate this deletion.
Enforced Expression of Egr-1 in H35 Hepatoma Cells Leads to
Decreased Pgp Levels--
Although H35 cells express Egr-1 (31) to
assess the possible dominant role of Egr-1 upon Pgp expression, H35
cells were stably transfected with an Egr-1 expression vector and
clonal cell lines selected (Fig. 8). We
selected this approach over chemical treatments known to affect Egr-1
(e.g. 12-O-tetradecanoylphorbol-13-acetate or
thapsigargin) (17) because these agents might affect other signaling
pathways in H35 cells. Western blot analysis of these lysates revealed
that Egr-1 expression in Egr-Neo clones increased significantly
compared with the Neo cell line (Fig. 8). It is interesting to note the
level of immunoreactive Sp1 also increased, but not in direct
proportion to Egr-1 expression. The steady-state levels of
pgp2/mdr1b mRNA among these same clones was inversely related to Egr-1 expression and decreased an average of 3-fold in the
Egr-1 overexpressing cell lines compared with either the Neo2 or a cell
line that contained Egr-1 in the reverse orientation (not shown).

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Fig. 8.
Enforced expression of Egr-1 in H35 hepatoma
cells affects mdr1 expression. H35 cells were transfected with
either the Egr-1 expression vector or pcDNA3, placed under G418
selection and Egr-Neo and Neo clones isolated, expanded and
characterized by Western analysis for Egr-1 and Sp1 expression. The
loading control was a nonspecific band that appears equivalent in all
samples.
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To determine how pgp2/mdr1b promoter activity was affected
by Egr1 overexpression,
250WT and
250MT were transiently
transfected into Neo and Egr-Neo#6 cells and the ratio of
250WT:
250MT promoter activity was determined. For the Neo cells the
ratio was 2.2, a value consistent with the findings in Figs.
2B and 7B showing decreased basal activity of
250MT. In contrast, in Egr-Neo#6 cells the ratio was only 1.2, a
value reflecting suppression of the
250WT promoter in the Egr1
overexpressing cells (not shown).
Increased Levels of Egr-1 Correlates with Reduced Drug
Resistance--
The functional consequence of increased Egr-1
expression on drug sensitivity was measured by 5-fluorodeoxyuridine and
vinblastine sensitivity. Because in some cell types, Egr-1
overexpression is linked to an increased susceptibility to apoptosis
(32), we also evaluated the Egr-1 cell lines (three additional Egr-1 expressing cells were also analyzed) for altered sensitivity to 5-fluorodeoxyuridine, a fluorinated nucleoside analog and a non-Pgp substrate. Enhanced expression of Egr-1 correlated with decreased Pgp
protein (Fig. 9A) and a
selective increase in vinblastine sensitivity (Fig. 9B). In
contrast, the 5-fluorodeoxyuridine sensitivity was almost identical
among the cell lines (Fig. 9C). Furthermore, the enforced
expression of Egr-1 had no gross affect upon the doubling time of the
Egr-1 clones (Egr-1 = 27.4 ± 3.0 and neo 24.9 ± 4.3),
thus indicating that the selective difference in vinblastine
sensitivity among Egr-1 cells lines was not secondary to alterations in
cell proliferation. These studies show that enforced up-regulation of
Egr-1 is correlated with decreased Pgp expression and a specific
sensitization to vinblastine, a known Pgp substrate.

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Fig. 9.
Egr-1 overexpression in H35 hepatoma cells
decreases Pgp levels and leads to a selective increase in vinblastine
cytotoxicity. A, total cell lysates (35 µg) from
Egr-1 H35 clones or vector Neo H35 clones (Neo2) were analyzed by
immunoblot for Pgp or actin expression. B and C,
3-5 × 105 cells of each of the indicated Egr-1 and
Neo H35 clones were plated on 60-mm dishes. 24 h later the dishes
were washed and treated with either (A) vinblastine (100 nM) or (B) 5'-fluorodeoxyuridine (100 µM). After a 48-h treatment, lactate dehydrogenase
released into the medium and remaining in attached cells were
independently measured and lactate dehydrogenase in the media expressed
as a percent of total cellular lactate dehydrogenase.
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DISCUSSION |
We have identified a GC-rich region (pgp2GC) in the rat
pgp2/mdr1b promoter that is highly conserved among the human
and rodent MDR1 promoters (Fig. 1). This region has a
consensus Sp1-binding site. An intact Sp1 site is required for full
activity of the pgp2/mdr1b promoter as defined by the
following criteria. 1) Transient transfection of an Sp1 expression
vector results in the activation of the pgp2/mdr1b promoter
in Sp1 negative cells, and 2) mutation of the Sp1-binding site
abrogates Sp1 binding and produces a dramatic reduction in promoter
activity. The in vivo relevance of Sp1 was shown by
increased Pgp expression in H35 cells stably overexpressing Sp1.
Further studies of pgp2GC revealed that this 13-bp GC region in
pgp2/mdr1b promoter bound recombinant Egr-1, however, unlike Sp1, co-transfected Egr-1 suppressed the pgp2/mdr1b
promoter. Moreover, a 4-bp mutation in the Egr-1 site decreased Egr-1
binding and correlated with the up-regulation of the
pgp2/mdr1b promoter in the absence of a functional Sp1 site.
Ectopic overexpression of Egr-1 decreased Pgp expression in H35 cells
and was correlated with a selective increase in vinblastine
sensitivity, thus identifying Pgp as a downstream target of Egr-1.
Finally, EMSA revealed that Sp1 and Egr-1 compete and cannot
simultaneously bind to the pgp2GC element in the pgp2/mdr1b
promoter. In total, the concordance between the effects of ectopic
expression of Sp1 and Egr1, transient co-transfection with these
factors and EMSA with Sp1 and Egr-1 on pgp2/mdr1b suggest
that the interactions between Sp1 and Egr1 at the pgp2/mdr1b
promoter defined here are functionally significant in
vivo.
These studies were undertaken to determine whether in liver cells a
conserved GC region in the pgp2/mdr1b promoter, similar to a
corresponding GC region in human MDR1, influences
pgp2/mdr1b transcriptional activity. Our studies revealed
that loss of the Sp1 site leads to a dramatic reduction in
pgp2/mdr1b basal activity in H35 cells. Moreover, the Sp1
site is required for Sp1-dependent transcriptional
activation of the pgp2/mdr1b promoter. These findings underscore the significance of Sp1 and the conserved GC region for
MDR1 (16, 18) and mdr1b expression (this study)
and extend previous findings by determining that ectopic overexpression
of Sp1 affects endogenous mdr1 expression. While a previous
study with a drug-selected cell line showed a concurrent increase in MDR1 and Sp1 (33), undoubtedly, cellular changes during drug selection (e.g. Fos (34), Ras (35), or p53 (29)) could have affected MDR1 expression in that study. Enforced
overexpression of Sp1 in a defined cell system provides more direct
evidence that increased Sp1 expression correlates with increased Pgp
levels in H35 cells. Although the cells lines with increased Sp1 have corresponding changes in Pgp expression, we cannot completely exclude
an indirect mechanism, i.e. Sp1 may activate other genes that affect Pgp expression. Regardless, the current studies predict that factors that either increased Sp1 or Sp1 function (e.g.
viral infection increases the amount of Sp1 (36)) would lead to
increased Pgp expression and correlate with the outgrowth of
multidrug-resistant cells. Conversely, diminishing either Sp1 DNA
binding or amount (e.g. phosphorylation by casein kinase II
(37)) might be expected to decrease Pgp expression and lead to
increased drug sensitivity.
The mdr1b/pgp2 promoter Egr-1 site is not typical, however,
it contains a single consensus Sp1 site. The interaction described by
EMSA with recombinant Egr1 and Sp1 depicts Egr-1 displacing Sp1 leading
to only Egr-1 binding. This is recapitulated in H35 nuclear extracts
because Egr1 seems to be the predominant protein binding to pgp2GC.
However, EMSA studies, alone, do not indicate how this interaction is
relevant to the pgp2/mdr1b promoter activity in cells. In
some reports genes that contain overlapping Egr-1 and Sp1 sites are
positively regulated by both factors. For instance, despite Egr-1 and
Sp1 competition for an overlapping site in the adrenal
phenylethanolamine N-methyltransferase gene, both
transcription factors activate this promoter (38). Similar findings
were reported for the human MDR1 promoter (16). Based upon
these precedents we might have predicted that both Sp1 and Egr-1 would
activate the pgp2/mdr1b promoter. Unexpectedly, Egr-1
repressed the pgp2/mdr1b promoter, while Sp1 activated it.
Moreover, the up-regulation of the pgp2/mdr1b promoter by
decreased Egr1 binding is consistent with its role as a transrepressor
of pgp2/mdr1b. Thus, the opposing functional interaction
between Sp1 and Egr-1 at the pgp2/mdr1b promoter in H35
cells could not have been predicted from the DNA binding assays in this
system. Furthermore, only in cases where Sp1 either dramatically
increased or Egr1 decreased would pgp2/mdr1b transcription
be expected to increase.
Currently it is unknown why Egr-1 activates the human MDR1
promoter and not the rat pgp2/mdr1b promoter. We do not
believe these differences are due to position effects since the
Egr-1/Sp1 site in pgp2/mdr1b is essentially in equal
position with respect to transcriptional initiation of MDR1.
Nor do we believe that Egr-1 transpression of pgp2/mdr1b is
due to cell context because Egr-1 repressed the
pgp2/mdr1b promoter in human colon (LS180) and
hepatoblastoma (HepG2) cells. A more likely explanation is that the
human MDR1 promoter is simply transactivated by Egr-1. In
support of this possibility we have obtained preliminary evidence that
the human MDR1 promoter (between
137 and +30) is
transactivated by co-transfected Egr-1 in HepG2 cells and that enforced
expression of Egr-1 leads to up-regulation of endogenous
MDR1 (not shown).
The decrease in pgp2/mdr1b expression in H35 cells
ectopically overexpressing Egr-1 identifies pgp2/mdr1b as a
downstream target of Egr-1 in H35 cells. While it cannot be ruled out
that Egr-1 could affect other transcription factors that act in concert to down-regulate Pgp expression, it seems unlikely for the following reasons. 1) The pgp2/mdr1b promoter contains a site
specifically bound by Egr-1. 2) The pgp2/mdr1b promoter is
readily and specifically repressed by Egr-1. 3) The pgp2/mdr1b promoter
is down-regulated in cells that ectopically overexpress Egr1. Current
studies are evaluating whether chemical treatments
(12-O-tetradecanoylphorbol-13-acetate, thapsigargin) or
other stresses (e.g. ionizing radiation) reported to produce
alterations in Egr-1 result in similar changes in Egr-1 in our system.
Nevertheless, it is clear in H35 cells that Sp1 and Egr-1 have distinct
biological roles in regulating pgp2/mdr1b as Sp1 can
activate while Egr-1 suppresses the pgp2/mdr1b promoter.