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Department of Veterinary Physiology and Pharmacology (R.D., W.P., I.S., S.S.) and Department of Biochemistry and Biophysics (C.V., M.K.) Texas A&M University College Station, Texas 77843
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Naphthoflavone, an aryl hydrocarbon receptor (AhR)
antagonist, blocked the inhibitory effects of TCDD; moreover, the
inhibitory response was not observed in variant Ah-nonresponsive MCF-7
cells, suggesting that the AhR complex was required for estrogen
receptor cross-talk. The E2-responsive sequence
(-1220 to -1155) in the c-fos gene promoter contains two
putative core pentanucleotide dioxin-responsive elements (DREs) at
-1206 to -1202 and -1163 to -1159. In transient transfection assays
using wild-type and core DRE mutant constructs, the downstream core DRE
(at -1163 to -1159) was identified as a functional inhibitory DRE.
The results of photo-induced cross-linking, gel mobility shift, and
in vitro DNA footprinting assays showed that the AhR
complex interacted with the core DRE that also overlapped the
E2-responsive GC-rich site (-1168 to -1161),
suggesting that the mechanism for AhR-mediated inhibitory effects may
be due to quenching or masking at the Sp1-binding site. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent studies in this laboratory have shown that induction of
c-fos expression in MCF-7 human breast cancer cells by
17ß-estradiol (E2) involves interaction of an estrogen
receptor (ER)/Sp1 complex with a distal GC-rich promoter element at
-1168 to -1161 (11). This novel pathway for ER action involves
binding of the ER to Sp1 protein and not DNA (29), and other
E2-responsive GC-rich motifs have been identified in the
retinoic acid receptor -1, cathepsin D, and adenosine deaminase gene
promoters (30, 31, 32). This pathway for DNA binding-independent ER action
is similar to results reported for ER/AP-1 interactions with promoters
containing AP-1 sites (33, 34, 35). Studies in this laboratory and others
have focused on the indirect antiestrogenic activity of aryl
hydrocarbon receptor (AhR) agonists and the mechanisms associated with
AhR-ER cross-talk (36, 37, 38, 39). Results obtained for at least two
E2-responsive genes, namely cathepsin D and pS2, have
identified GCGTG pentanucleotide sequences that are required for
AhR-mediated inhibition of ER action (38, 39). This motif corresponds
to the core-binding nucleotides required for a dioxin-responsive
element (DRE) (40) and have been designated as inhibitory DREs (iDREs)
(38, 39). This study demonstrates that in MCF-7 cells, the potent AhR
agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
inhibits E2-induced c-fos protooncogene
expression and reporter gene activity with constructs containing
c-fos gene promoter inserts. Deletion analysis of this
promoter has identified two GCGTG (core DRE) motifs in the -1220 to
-1155 region of the promoter, and only the downstream sequence at
-1163 to -1159 is a functional iDRE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In cells cotreated
with E2 (1 h) and 10 nM TCDD for 1, 2, 4, 12,
or 24 h, the hormone-induced mRNA levels were significantly
decreased within 2 h, and inhibition was observed for up to
24 h. pFC2 contains the -1400 to +41 region from the
c-fos gene promoter linked to a chloramphenicol acetyl
transferase (CAT) reporter gene in pBLCAT2. Ten nanomolar
E2 caused a more than 8-fold increase in CAT activity in
MCF-7 cells transiently transfected with pFC2, and in cells cotreated
with E2 and 10 nM TCDD, the hormone-induced
response was significantly decreased (Fig. 1B). The inhibitory
responses mediated by TCDD in MCF-7 cells were similar to those
observed for the antiestrogen ICI 182,780; however, the latter compound
was a more potent inhibitor (Fig. 1C).
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-naphthoflavone (NF) inhibits formation of the
liganded nuclear AhR complex and thereby blocks inhibitory effects of
TCDD on E2-regulated responses (41). The results
illustrated in Fig. 1B
show that NF alone did not significantly
affect CAT activity in MCF-7 cells transfected with pFC2; however, in
combination with E2 plus TCDD, NF significantly blocked
the inhibitory effects of TCDD. In contrast, NF did not affect the
antiestrogenic action of ICI 182,780 and the inhibitory effects of ICI
182,780 were greater than those observed for TCDD (Fig. 1C). These data
illustrate the specificity of NF, and the results are consistent
with a role for the AhR in this inhibitory process. Benzo[a]pyrene
(BaP)-resistant MCF-7 cells are E2 responsive and
Ah-nonresponsive due to a defective AhR-Arnt heterodimer that does not
bind DREs (42). E2 induced CAT activity (3.6-fold) in
BaP-resistant MCF-7 cells transfected with pFC2, and TCDD alone did not
affect CAT activity compared with control cells (Fig. 1D). In cells
cotreated with E2 plus TCDD, the hormone-induced response
was not decreased, confirming that the inhibitory activity of TCDD
requires an intact nuclear AhR complex that binds DNA.
Characterization of a Functional iDRE in the c-fos Gene
Promoter
Previous studies have demonstrated that the GC-rich motif at
-1168 to -1161 is required for E2 responsiveness in MCF-7
cells, and pF1 contains a -1220 to -1155 fos gene promoter
insert with downstream GC-rich elements and an upstream imperfect
palindromic estrogen response element (ERE) that is required for
ER action in HeLa cells (43). E2 induced CAT in MCF-7 cells
transfected with pF1 (Fig. 2, A and B),
and this was similar to results of previous studies (11). The -1120 to
-1155 region of the fos promoter contains two
pentanucleotide GCGTG motifs that may function as iDREs, which have
been identified in the cathepsin D and pS2 gene promoters (38, 39).
pF1.d1m and pF1.d2m contain mutations in core DRE1 (-1206 to -1202)
and 2 (-1163 to -1159), respectively, and in transient transfection
assays in MCF-7 cells, both constructs were E2 responsive.
In contrast, TCDD significantly inhibited hormone-induced activity in
cells transfected with pF1.d1m but not pF1.d2m, indicating that the
downstream GCGTG sequence that overlaps the GC-rich motif is a
functional iDRE. In a separate experiment (Fig. 2B), ICI 182,780
inhibited E2-induced transactivation using the same
wild-type and mutant constructs (note: E2-responsiveness of
the constructs was somewhat variable between experiments).
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, lane 2); the intensity
of this band is enhanced by coincubation with AhR/Arnt (lane 5). Excess
unlabeled wild-type but not mutant Sp1 oligonucleotides decreased
binding (lanes 6 and 7), and cold DRE also slightly decreased binding.
This pattern of AhR/Arnt interaction with Sp1 and ER/Sp1 binding was
previously observed using a 32P-labeled
Sp1(N)4DRE(core) motif from the cathepsin D gene promoter
(44, 45) where the core DRE motif was required for enhanced Sp1-DNA
binding and transactivation. Core DRE2 (-1163 to -1159) in the
fos gene overlaps the GC-rich sequences (-1168 to -1161).
A T-G (at -1160) mutation to give fos4.m1 results in a key mutation of
the core DRE but [32P]fos4.m still binds Sp1 protein in a
gel mobility shift assay (Fig. 3B, lane 2), and this mutation did not
affect E2 responsiveness in transactivation (Fig. 2). ER
but not AhR/Arnt enhanced Sp1-DNA binding (lanes 3 and 4), and in
unlabeled oligonucleotide competition studies (lanes 6–8), Sp1 but not
mutant Sp1 or wild-type DRE competitively decreased Sp1-DNA binding.
These results demonstrate that the core DRE motif that overlaps the
GC-rich Sp1-binding site in [32P]fos4 is required for
modulation of Sp1-DNA binding, and these results are similar to
those previously reported for the
[32P]Sp1(N)4DRE(core) oligonucleotide from
the cathepsin D gene promoter (44, 45).
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, lane 1).
Intensity of the cross-linked band was decreased after coincubation
with 100-fold excess wild-type consensus DRE (lane 2) or AhR antibody
(lane 4) but unaffected by 100-fold excess mutant DRE (lane 3) or
nonspecific IgG (lane 5). The competition with wild-type DRE and AhR
antibodies suggest that the 200-kDa cross-linked band contained
AhR/Arnt. However, effects of Arnt antibodies were not determined.
These results parallel previous photoinduced cross-linking studies with
functional iDREs in the cathepsin D and pS2 gene promoters (38, 39).
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summarizes in vitro
SssI DNA methylation footprinting of the c-fos
gene promoter in the region of the distal GC-rich (-1168 to -1161)
sites and the core pentanucleotide DRE motifs. SssI
methylates only the 5'-cytosine of a CG pair and therefore is useful
for footprinting promoters with GC-rich motifs. Incubation of pFC2-CAT
with Sp1 protein alone (lane 1) did not protect DRE1 or DRE2. Moreover,
coincubation with ER plus Sp1 proteins (lane 3) did not protect either
site. In contrast, incubation of pFC2-CAT with ER protein alone
protected DRE1 and DRE2, and protection of the former site is
consistent with an imperfect palindromic ERE that overlaps DRE1 (43).
In addition, an ERE half-site (-1178 to -1182) is adjacent to DRE2,
and we hypothesize that the protection of DRE2 by ER alone (lane 2) may
be associated with binding to this site. The failure to observe
protection after coincubation of ER with Sp1 is consistent with Sp1-ER
(protein-protein) binding (29) that competes with ER-DNA interactions.
The transformed AhR complex alone very weakly footprinted DRE2 (lane
4), whereas DRE1 was not protected (lane 4); however, after
coincubation with ER/Sp1 proteins, a greater than 2-fold increase in
binding to core DRE2 was observed (lanes 5 and 6), as evidenced by a
dose-dependent decrease in methylation with increasing amounts of
AhR/Arnt (3U and 6U, lanes 5 and 6, respectively). This enhanced
binding (decreased methylation) at core DRE2, but not DRE1, was
observed in replicate experiments and demonstrates direct binding of
the heterodimeric AhR complex to core DRE2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous studies have demonstrated that AhR agonists, typified by TCDD,
inhibit E2-induced responses in the rodent uterus and
mammary and in human breast cancer cells (reviewed in Refs. 36, 37).
For example, TCDD inhibited spontaneous and
7,12-di-methylbenzanthracene-induced mammary tumor development and
growth, and similar results were obtained in a mouse xenograft model
(46, 47, 48, 49). These studies have also led to development of relatively
nontoxic AhR-based compounds that have potential utility for treatment
of breast cancer (50, 51). This study utilizes c-fos
protooncogene as a model for understanding the molecular mechanism of
ER-AhR cross-talk in breast cancer. TCDD rapidly inhibits
E2-induced c-fos mRNA levels (Fig. 1A) and CAT
activity in MCF-7 cells transiently transfected with pFC2 containing
the -1400 to +41 region of the promoter (Fig. 1B), and results with
TCDD were comparable to those observed for the direct-acting
antiestrogen ICI 182,780 (Fig. 1C). Although TCDD totally inhibited
E2-induced c-fos gene expression, the inhibitory
response was lower in transient transfection experiments where ICI
182,780 was the more potent inhibitor.
The role of the AhR in mediating the inhibitory effects of TCDD on ER
action was confirmed in studies using NF, an AhR antagonist.
Previous studies have demonstrated that 1 µM NF blocks
formation of the nuclear AhR complex and some of the suppressive
effects of TCDD including inhibition of E2-induced cell
proliferation (41). Similar results were observed in the present
study where NF blocks the effects of TCDD on E2-induced
transactivation using pFC2 (Fig. 1B), whereas NF does not affect the
antiestrogenic activity of ICI 182,780 (Fig. 1C). Further confirmation
for the role of a functional AhR in mediating the inhibitory effects of
TCDD was obtained using BaP-resistant MCF-7 cells that express a
defective nuclear AhR complex that does not bind DNA (42).
E2 induces CAT activity in BaP-resistant MCF-7 cells,
whereas TCDD does not inhibit this response, and this is consistent
with results of previous studies showing that TCDD does not inhibit
other E2-induced responses in this cell line (42).
The rapid inhibition of E2-induced fos gene
expression in MCF-7 cells (Fig. 1A) suggests that this may be related
to direct genomic effects of the AhR complex as previously reported for
inhibition of E2-induced cathepsin D and pS2 expression
(38, 39). In both of these studies, core iDREs (GCGTG) were identified
as functional inhibitory cis-elements required for
AhR-mediated activity, and this involved weak interactions with the AhR
complex that could be detected only by photoinduced cross-linking
studies. Two pentanucleotide GCGTG sequences at -1206 to -1202 and
-1163 to -1159 were identified in the -1220 to -1155 region of the
c-fos gene promoter that also contains the GC-rich region
(-1168 to -1161) required for E2 responsiveness. The
results illustrated in Fig. 2 compare the antiestrogenic action of TCDD
and ICI 182,780 in MCF-7 cells transfected with pF1 or constructs
mutated in the upstream or downstream core DREs (pF1.d1m and pF1.d2m).
All three constructs were E2 inducible, and ICI 182,70
inhibited more than 90% of the hormone-induced response. In contrast,
the inhibitory effects of TCDD were observed only for wild-type pF1 and
pF1.d1m, whereas no significant inhibition was observed in cells
transfected with pF1.d2m. These results clearly distinguish between the
direct antiestrogenic effects of ICI 182,780 and the indirect
inhibitory effects of TCDD that were dependent on an intact core DRE2
that overlaps the GC-rich Sp1-binding site at -1168 to -1161.
Previous studies on the cathepsin D gene promoter have demonstrated
that Sp1 and the AhR complex physically interact, and estrogen
responsiveness of the -145 to -119 promoter region was dependent on
an Sp1(N)4DRE (core pentanucleotide) motif (44). The
GC-rich site was necessary but not sufficient for ER/Sp1 action, and
hormone-induced transactivation was dependent on cooperative
interactions between proteins bound to the GC-rich and core DRE sites.
Subsequent studies showed that the nuclear AhR complex (in the absence
of endogenous ligand) cooperatively interacted with ER/Sp1 complex or
Sp1 protein to modulate hormone-induced or basal gene expression,
respectively (44, 45). The cooperative ER/Sp1-AhR/Arnt interactions
also decreased with increasing distance between the GC-rich and core
DRE-binding sites (45). The downstream core DRE motif in the
c-fos gene promoter overlaps the GC-rich sequence at -1168
to -1161 and therefore differs from the Sp1(N)4DRE(core)
sequence in the cathepsin D gene promoter. Mutation of core DRE2 (to
give pF1.d2m) did not significantly affect basal or
E2-inducibile responses compared with those observed for
pF1 (Fig. 2), and therefore the overlapping core DRE2 does not play a
critical role in ER/Sp1 action at the GC-rich motif (-1168/-1161). In
contrast, both basal and inducible responses were decreased by >85%
by mutation of the core DRE in the cathepsin D-derived construct (44),
suggesting that the overlap between the GC-rich and core DRE elements
may have functional significance (45).
Evidence for interaction of the ligand-transformed AhR complex with the
core DRE2 site was further investigated by gel mobility shift,
photoinduced cross-linking, and SssI DNA methyltransferase
footprinting assays. Direct binding of the AhR complex to
[32P]fos4 was not observed in gel mobility shift assays
(data not shown), and these results were consistent with previous
studies showing that AhR/Arnt binding in this assay requires not only
the core DRE but additional flanking sequence (38, 39, 40). Interactions of
the 200-kDa AhR complex with core DRE2 were confirmed by cross-linking
studies (Fig. 4) to give a specifically bound cross-linked AhR complex,
and these results were comparable to cross-linking studies using core
DREs from the cathepsin D and pS2 gene promoters (38, 39). In
vitro SssI DNA footprinting showed consistent interactions
of the transformed nuclear AhR complex with core DRE2 (but not DRE1)
only in the presence of ER and Sp1 proteins (Fig. 5). These results are
consistent with the inhibition of E2-induced responses by
the liganded AhR/Arnt complex, whereas TCDD alone did not significantly
affect basal responses (e.g. Fig. 1, B–D, and Fig. 2).
Ongoing studies will further extend applications of the SssI
DNA footprinting assay for determining site- specific binding of
specific nuclear proteins and nuclear extracts from breast cancer cells
to hormone-regulated gene promoters.
Previous gel mobility shift studies using 32P-labeled
Sp1(N)4DRE(core) (cathepsin D gene promoter) demonstrated
that although the nuclear AhR complex did not directly bind the
oligonucleotide, the on-rate and Bmax value for Sp1-DNA
complex formation were significantly increased after coincubation with
the AhR complex (44, 45). The results in Fig. 3, A and B, utilizing
[32P]fos4 and core DRE mutant ([32P]fos4.m)
show that the transformed AhR complex also enhanced Sp1-DNA complex
formation (>2-fold) using the wild-type oligonucleotide without
forming a ternary supershifted complex. Enhanced Sp1 binding to
[32P]fos4 by ER and AhR/Arnt was determined in the
presence of E2 and TCDD, respectively; however, similar
effects can be observed using other GC-rich oligonucleotides in the
presence or absence of ligand (11, 29, 30, 32, 44, 45). The failure of
the AhR complex to induce formation of a supershifted ternary
AhR/Sp1-DNA complex is not unprecedented since it has also been
reported that other nuclear proteins, including human T-cell leukemia
virus, Type-I Tax, sterol-regulatory element-binding protein, and
cyclin D1 enhanced binding of bZIP, Sp1, and ER to their cognate
enhancer elements (52, 53, 54). Sp1-DNA binding was not enhanced by the
transformed AhR complex using [32P]fos4.m (mutant DRE2),
showing that enhanced binding was dependent on both the AhR complex and
an intact core DRE. These results are consistent with repressors and
activators that can co-occupy overlapping DNA sequences and suggests
that AhR-mediated inhibition of ER/Sp1 action in the fos
gene promoter may be due to quenching or masking at the distal GC-rich
site (55).
Interestingly, the inhibitory core iDREs identified in the c-fos (present study), cathepsin D (38), and pS2 (39) gene promoters modulate E2-responsiveness via different pathways. The iDRE in the cathepsin D gene promoter is located between the Sp1(N)23ERE (half-site), and results of in vitro studies suggest that the nuclear AhR complex-iDRE interaction prevents formation of the functional ER/Sp1 complex. This may be due to steric interactions since the iDRE is located within the Sp1(N)23ERE (half-site) motif required for ER/Sp1-DNA complex formation. The functional iDRE in the pS2 gene promoter is required for AhR-AP-1 protein interactions that modulate ER action at a downstream imperfect palindromic ERE, and the mechanisms of these interactions are unknown (39). Thus, inhibition of E2-induced transactivation by the transformed AhR/Arnt heterodimer is complex and gene promoter specific. Moreover, recent studies in this laboratory have identified other E2-responsive genes that are also inhibited by AhR agonists via core iDRE-independent pathways (our unpublished results) and these are currently being investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oligonucleotides derived from the c-fos protooncogene promoter and other oligonucleotides were synthesized by the Gene Technologies Laboratory, Texas A&M University (College Station, TX). The complementary strands were annealed, and the 5'-overhangs were used for cloning. The structures of these oligonucleotides are summarized below, and the GC-rich Sp1 site is underlined. Pentanucleotide core DREs are indicated in bold letters. Mutations incorporated into mutant oligonucleotides are denoted by an asterisk. fos1 (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGT GGC ACG-3' fos2.d1m (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA T*A*T* TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGT GGC ACG-3' fos1.d2m (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGG* GGC ACG-3' BrdU-F2 (-1131/-1182) (antisense) 5'-TGG GGA GGC AAG GTG CTC CAG AGT GTG CCA CGC CCC CCA ATC TCC TGC CCT GCT-3' fos4 5'-AGC TTG GAG ATT GGG GGG CGT GGC ACA CG-3' fos4.m 5'-AGC TTC GAG ATT GGG GGG CGG* GGC ACA CG-3' Consensus Sp1 oligonucleotide 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3' Mutant Sp1 oligonucleotide 5'-AGC TTA TTC GAT CGA* A*GC GGG GCG AGC G-3' Wild-type DRE (antisense) 5'-GAT CTC CGG TCC TTC TCA CGC AAC GCC TGG GG-3' Mutant DRE (antisense) 5'-GAT CTC CGG TCC TTC TA*C* A*T*C AAC GCC TGG GG-3' Primer sequence used for BrdU-F2 5'-AGC AGG GCA GGA GAT-3'
Cloning
The pBLTATA-CAT plasmid was made by digesting the pBLCAT2 vector
with BamHI and XhoI to remove the thymidine
kinase promoter; the double-stranded E1B oligonucleotide (29)
containing complementary 5'-overhangs was then inserted into the
corresponding sites. The fos1, fos1.d1m, and fos1.d2m oligonucleotides
were cloned into the pBLTATA-CAT vector at the HindIII and
BamHI sites to give the pF1, pF1.d1m, and pF1.d2m
constructs, respectively, as previously described (29).
Northern Blot Analysis
Plasmids containing c-fos and ß-tubulin genes were
purchased from ATCC. RNA was extracted from MCF-7 cells
treated with DMSO (control), E2, and/or TCDD using the
acidic guanidinium thiocyanate extraction procedure followed by
separation on a 1.2% agarose gel electrophoresis and transfer to a
nylon membrane. The membrane was then exposed to UV light for 5 min to
cross-link RNA to the membrane and baked at 80 C for 2 h. The
membrane was prehybridized in a solution containing 0.1% BSA, 0.1%
Ficoll, 0.1% polyvinylpyrollidone, 10% dextran sulfate, 1% SDS, and
5x SSPE (0.75 M NaCl, 50 mM
NaH2PO4, 5 mM EDTA) for 18–24 h at
65 C and hybridized in the same buffer for 24 h with the
32P-labeled DNA probe (106 cpm/ml). DNA probes
were labeled with [-32P]dCTP using the random-primed
DNA labeling kit (Roche Molecular Biochemicals,
Indianapolis, IN). The resulting blots were quantitated using a Betagen
Betascope 603 blot analyzer (Intelligenetics, Inc.,
Mountain View, CA) and visualized by autoradiography. c-fos
mRNA levels were standardized against ß-tubulin mRNA.
Transient Transfection and CAT Assay
Cultured MCF-7 and MCF-7BaPr cells were transiently
transfected utilizing the calcium phosphate method with 10 µg of the
pFC2-CAT plasmid and 10 µg of wild-type hER expression plasmid.
Overexpression of E2-responsive constructs containing
promoter inserts from the progesterone receptor, cathepsin D, pS2,
retinoic acid receptor 1, and heat shock protein 27 genes requires
cotransfection with hER (11, 29, 30, 31, 32, 38, 39, 56, 57, 58, 59). After 18 h,
the media were changed, and cells were treated with DMSO (0.2% total
volume), 10 nM E2, 10 nM TCDD, or
their combinations in DMSO for 44 h. Cells were washed with PBS
and scraped from the plates. Cell lysates were prepared in 0.15 ml of
0.25 M Tris-HCl (pH 7.5) by three freeze-thaw-sonication
cycles (3 min each). Protein concentrations were determined using BSA
as a standard, and analysis for CAT activity in cell lysates used a
constant amount of protein from each treatment group. Lysates were
incubated at 56 C for 7 min to remove endogenous deacetylase activity.
CAT activity was determined by incubating aliquots of the cell lysates
with 0.2 mCi
d-threo-[dichloroacetyl-1-14C]chloramphenicol
and 4 mM acetyl-CoA. Acetylation was allowed to proceed to
less than 20–25% completion (linear range), and acetylated
metabolites were analyzed by TLC. After TLC, acetylated products were
visualized and quantitated using a Betagen Betascope 603 blot analyzer.
CAT activity was calculated as fraction of that observed in cells
treated with DMSO alone (arbitrarily set at 100), and results are
expressed as means ± SD. The experiments were carried
out at least in triplicate. The TLC plates were subjected to
autoradiography using X-Omat film ( Eastman Kodak Co.,
Rochester, NY).
Electrophorectic Mobility Shift Assays
Pure Sp1 protein was purchased from Promega Corp.
(Madison, WI). Expression plasmids for hER, AhR, and Arnt were used
to in vitro translate proteins in 1x binding buffer
(20 mM HEPES, 5% glycerol, 100 mM potassium
chloride, 5 mM magnesium chloride, 0.5 mM
dithiothreitol, and 1 mM EDTA in a final volume of 25
µl). Equal volumes (1 µl) of lysate containing the AhR and Arnt
complex were transformed with 20 nM TCDD for 2 h at 25
C. The hER (1 µl) was transformed with 20 nM
E2 for 15 min on ice. Sp1 (2.5 ng) and
32P-labeled oligonucleotides (60,000 cpm) were then added
to the reaction mixtures in the presence of 1 µg poly [d(I-C)] and
incubated for 15 min at 25 C. In competition experiments, different
amounts of unlabeled oligonucleotides were also incubated in the
incubation mixtures. Aliquots of these mixtures were loaded onto a 4%
polyacrylamide gel (acrylamide/bisacrylamide ratio, 30:0.8) and run at
110 V in 0.09 M Tris/0.09 M borate/2
mM EDTA (pH 8.0). [32P]DNA and DNA-protein
bands were visualized by autoradiography and quantitated by
densitometry using the Zero-D software package (Molecular Dynamics, Inc., Sunnyvale, CA) and a JX-330 scanner (Sharp
Electronics Corp., Mahwah, NJ).
UV-DNA Cross-Linking
For cross-linking studies, 10 pmol of the synthetic
oligonucleotide (c-fos-DRE) were annealed to 10 pmol of a
cross-linked primer sequence. The annealed template was end-filled with
the Klenow fragment of DNA polymerase in the presence of 0.1
µM dGTP, dATP, and BrdU and 1 mM
[32P]dCTP as previously described (38, 39) and was
designated the BrdU-substituted DRE oligonucleotide. Nuclear extracts
(10 µg) from MCF-7 cells treated with appropriate chemicals were
incubated with the BrdU-DRE for 15 min at 20 C after a 15-min
incubation at 20 C with 400 ng of poly(dI-dC) in HEGD buffer
(2.5 mM HEPES, 1.5 mM EDTA, 10% glycerol, 1.0
mM dithiothreitol, pH 7.6) for 10 min followed by a 5-min
incubation at 20 C with unlabeled excess competitor. Incubation
mixtures were irradiated using a UV transilluminator (Fotodyne, Inc., New Berlin, WI) at more than 205 nm for 30 min at 20 C.
Samples were then mixed with 10 µl of an SDS loading buffer, heated
to 95 C for 5 min, and then subjected to electrophoresis on
SDS-polyacrylamide gels. Molecular weights of UV cross-linked nuclear
ligand-AhR complexes were calculated from 14C-methylated
standards obtained from Amersham Pharmacia Biotech
(Arlington Heights, IL). Immunodepletion of the AhR was carried out by
incubating 10 µg of nuclear extract with 1 µg of either AhR
antibody or nonspecific mouse IgG for 1 h at 25 C. The
immunodepleted extract was used in the UV cross-linking studies as
described above.
In Vitro SssI Footprinting
Fifty micrograms of plasmid pFC2-CAT (which contains the region
of the human c-fos promoter from -1400 to +41) were
restricted with XbaI and diluted to a concentration of 10
ng/µl. One microliter of the diluted plasmid was incubated with human
recombinant Sp1 (Promega Corp.), ER proteins (PanVera
Corp., Madison, WI), and in vitro translated Ah and Arnt,
and transformed with TCDD, both Sp1 and ER proteins, and varying
concentrations of in vitro translated and TCDD-transformed
Ah and Arnt in the presence of both ER and Sp1 proteins. Binding
reactions were carried out in 1x TNS binding buffer [0.02
M HEPES, 0.1 M KCl, 0.005 M
MgCl2, 0.004 mM EDTA, 5% glycerol, 4% TNT
lysate (Promega Corp.), 50 mM SAM] in a
volume of 25 µl. The binding reactions were incubated on ice for 5
min and then equilibrated to room temperature for 20 min. Two
microliters of 1:4 dilution of purified SssI (New England Biolabs, Inc., Beverly, MA) were added to the
equilibrated reactions, which were then incubated at 30 C for 15 min.
After 15 min at 75 C, 10 µl of freshly made deamination denaturation
buffer (0.9 N NaOH, 25 mM EDTA, 0.2 mg/ml of
sheared salmon sperm DNA) were added. After 5 min at 98 C, 200 µl of
a saturated solution of sodium metabisulfite were added, and the
samples were processed as described by Kladde and co-workers (60). The
primers used to amplify from the purified deaminated plasmid DNA were
cfosb1 (5'-AAACCCAAAAAATAAAAAAAAAA-AAAC-3') and cfosb2
(5'-GTTTTAGGGGTAGGGAGTGTGAG-3'). PCR products were purified and cleaned
using the Wizard PCR prep kit from Promega Corp. Purified
PCR products were sequenced with radiolabeled cfosb1 primer in the
presence of a 5 µM solution of dATP, dCTP, and dTTP using
50 µM cddGTP as the stop nucleotide. Sequitherm 10X
buffer and Sequitherm Thermostable SNA Polymerase (Epicentre
Technologies, Madison, WI) were used for the sequencing reactions.
Sequencing reactions were run on 5% PAGE-urea sequencing gels.
The dried gels were exposed to a phosphor screen for 12 h and
analyzed on a Storm 860 (Molecular Dynamics, Inc.).
|
FOOTNOTES |
|---|
This work was supported by the NIH Grants ES-04176 and ES-09106, the Welch Foundation, and the Texas Agricultural Experiment St ation. S. Safe is a Sid Kyle Professor of Toxicology.
Received for publication February 11, 1999. Revision received May 4, 1999. Accepted for publication May 25, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 h. B, Inhibition of
E2-induced CAT activity by TCDD. MCF-7 cells were
transiently transfected with pFC2-CAT (-1400 to +14) and
treated with various hormones/chemicals, and CAT activity was
determined as described in Materials and Methods.
Significant (P < 0.05) induction was observed for
E2 alone (
), and this response was inhibited by
TCDD (ET) (
); however, the inhibitory effect was reversed by 
2-fold decrease), and this was observed in replicate
experiments. One microliter of in vitro translated AhR
and Arnt proteins was arbitrarily defined as 1 U. AhR/Arnt was
transformed with TCDD before incubation.