Center Milan Molecular Pharmacology Laboratory and Laboratory for the Study of Arteriosclerosis (E.G.), Institute of Pharmacological Sciences, University of Milan, I-20133 Milan, Italy
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ABSTRACT |
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INTRODUCTION |
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The concentration of ER is an important determinant of cellular responsiveness to steroids, as suggested by the observation that in mammals, ER expression fluctuates in a spatial-temporal fashion in response to a variety of endocrine stimuli. Further support for this view is provided by studies at the cellular level, demonstrating that receptor expression above a specific concentration threshold is required for ER-mediated events to occur (11, 12).
Due to the involvement of estrogens in an increasing number of pathologies (13, 14), the study of receptor regulation may have relevant pharmacological and therapeutical consequences. To date, a limited number of studies have addressed the mechanisms of ER regulation, which remain elusive. It is known that ER intracellular content may be regulated by selected hormonal signals (e.g. the heterologous down-regulation induced by progesterone) (15). In the majority of animal cells and tissues studied, the ER concentration is under a control primarily exerted by estrogens through a process termed autologous down-regulation or autologous up-regulation depending on the tissue being considered (16, 17, 18). The experimental evidence obtained to date seems to indicate that in cells of cancer origin, the activated ER is an integral part of this regulatory process. Saceda et al. (19) showed that at least part of the autologous down-regulation is associated with a decrease in the cell content of ER messenger RNA (mRNA); this, in turn, results from a decreased ER gene transcription rate in a process that is cycloheximide resistant and hormone concentration dependent. The model devised to explain these findings invokes an interaction of the ER with its own gene, similar to the mechanism by which this same transcription factor regulates any other target gene (20, 21). The activated ER, therefore, would bind selected regions in the promoter of its own gene, preventing its further transcription. The promoter of the ER gene, however, lacks the canonical ERE; this suggests that a novel mechanism of ER-DNA interaction may be involved.
To study the effect of estrogen on ER turnover, we used antisense oligonucleotides (aODN) (22, 23) as specific blockers of ER mRNA translation (24, 25, 26). It is well known that many variables are involved in the mechanism of action of aODN, and systematic studies on these variables in eukaryotic systems are still lacking; it is, then, difficult to predict the best targets for aODN. To select the most active oligos, a series of molecules was synthesized and tested in a cell line expressing ER (MCF-7 cells). Contrary to our expectations, none of the antisense oligonucleotides used was able to block ER synthesis, whereas two of them caused a significant increase in the ER protein content. In addition, ER up-regulation was observed with oligos complementary (sense) to the aODNs tested.
These results indicated that the physiological machinery responsible for maintaining the intracellular ER levels could be offset by the presence of selected DNA sequences. This prompted us to further investigate the mechanisms underlying ER up-regulation mediated by aODNs.
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RESULTS |
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Western blot analysis using the H222 antibody as a
probe (Fig. 1B, right) confirmed the increase in ER protein
in MCF-7. Forty-eight hours after oligo2 treatment (right
lane), the intensity of the specific 67-kDa ER band
(arrow) was 5070% higher than that in controls
(left lane). After exposure to E2 (middle
lane), its intensity was 3040% lower than that in controls. As
the same amount of protein was subjected to immunoprecipitation, the
strongly reactive IgG bands (arrows) may be regarded as
loading and blotting controls. A number of bands, of lower
Mr, were detected in control and treated cell lysates.
These may correspond to either cross-reacting components or
intermediates in endocellular ER catabolism. The slight discrepancy in
the extent of up-regulation, as quantitated by EIA or by densitometric
scanning of Western blots, was attributed to both the different
sensitivities of the two techniques and to the fact that the
densitometric analysis had been restricted to the 67-kDa band.
To explain the ER intracellular accumulation caused by oligo1 and 2, we ruled out the hypothesis that this could occur via an interaction of the active oligos with the ER mRNA, reasoning that both sense and antisense had the same effect; therefore, a direct pairing with the mRNA sequence could not have occurred. An alternative explanation was provided by recent reports which postulated that intracellular receptors can control the transcriptional activity of their own genes by interacting with negative hormone-responsive elements located in the promoter or within the gene-coding sequence (20). If this were the case, the paradox effect observed with the oligonucleotides could have been due to the squelching of ER from the promoter of its own gene.
The observation that oligos promoting ER accumulation blocked
E2-induced down-regulation, whereas the inactive oligos did
not have any influence on the autologous down-regulation event (as
shown in Fig. 2), further indicated that the oligos
hindered the physiological mechanisms of ER autologous down-regulation.
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To further investigate the mechanism of aODN-dependent up-regulation, a series of experiments was undertaken with the most active of the oligos tested, oligo2.
Gel Mobility Assay Argues for a Direct Interaction between ER and
Oligo2
We first assessed by electrophoretic gel shift assay (EMSA)
whether a direct interaction between oligo2 and ER protein could occur.
The oligo2 retardation on gel, induced by ER, was compared with that of
the canonical target for ER, the ERE. All of the studies were performed
using MCF-7 nuclear extracts as the source of ER.
With oligo2 (Fig. 4; A, antisense; B, sense; C, double
strand), we observed several retarded bands; only one of these was
competed off by cold oligo2 (arrow). When other oligos, such
as those previously shown not to interfere with ER accumulation
(e.g. oligo4) or scrambled oligos, were used, none of the
retarded bands could be competed off (data not shown). We, therefore,
concluded that the binding between ER and oligo2 (sense and antisense)
was specific.
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To prove that the shifts of 32P-labeled oligo
observed were indeed due to the binding to ER, we checked whether the
band corresponding to the oligo-receptor complex was supershifted in
the presence of the specific anti-ER H222 antibody. As
shown in Fig. 5, with both dsoligo2 and
senseoligo2 the antibody caused a supershift of the complex
comparable to that observed with ER-ERE. With
antisenseoligo2 we observed a smear of the retarded band,
probably due to low affinity within the ER-antisenseoligo2
complex. The inactive oligo, oligo5, was not shifted in the presence of
ER. To further prove that ER was responsible for the supershift
reported above, we also tested nuclear extract from COS-1 cells,
transfected or not with ER cDNA. No retarded band could be observed
with extracts of COS-1 cells not expressing ER (see Fig. 8
).
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Oligo2-ER Complex Migration across a Transverse Urea
Gradient
The experiments described above proved the specific interaction of
both ss- and dsoligo2 with ER. We further compared the
characteristics of oligo2-ER and ERE-ER interaction by investigating
the electrophoretic behaviors of these complexes under denaturing
conditions.
The migration across a transverse urea gradient, as introduced by Creighton (29) correlates the unfolding of a protein molecule to a reduction in its electrophoretic mobility. The typical transition from the high to the low mobility form occurs over a defined interval of the chaotrope concentration, and the overall curve displays a sigmoidal shape. This technique may be applied to the study of interacting systems; different profiles of migration are observed depending upon the homo- or heteromultimeric nature of the complex (30). We, therefore, ran a series of experiments aimed at studying the behavior under denaturing conditions of the ss- and dsoligo2 as well as dsERE once complexed with ER.
Panels AC in Fig. 7 compare the profiles of migration
across increasing urea concentrations of ER-dsERE and the
complexes between ss- and dsoligo2 with ER. The end of the
radioactive tracing corresponds to the urea concentration at which
32P-labeled oligo and protein dissociate. This occurs at
urea concentrations of about 4 M for
[32P]dsERE-ER, 3 M for both
[32P]sssense2 and
[32P]dsoligo2-ER, and around 1 M
for [32P]ssantisense2-ER. These figures prove
the interaction between dsERE and ER to be much stronger
than that between oligo2 and ER, in agreement with the Kd
values calculated by EMSA.
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In the unfolding assay the various domains within the ER molecule appear to behave independently. These data, therefore, support the view that [32P]dsERE and oligo2 interact differently with the ER molecule. Moreover, the various domains appear to be differentially stabilized by the binding of their ligands. Examples of both features (ligand stabilization and stepwise unfolding) have been previously described for model systems (30, 31, 32, 33, 34).
Both the effective urea concentrations and the peculiar shapes of the dissociation/denaturation curves were evaluated in independent replicas of each run, and the behavior of each form of oligo2 was compared with that of ER-dsERE in experiments (middle section of AC) in which the reference and the test complexes were migrated in the same gel from two parallel trenches.
Evaluation of the Size of the ER
[32P]dsoligo2 Complex
The data accumulated to date show that dsoligo2 and
ERE interact with ER differently. To determine whether accessory
proteins were participating in the establishment of the oligo2-ER
complex, we determined the size of the ER-oligo2 complex according to
the method of Ferguson (35). Samples were run in gels of different
polyacrylamide concentrations (T%) along with Mr markers,
as described in Materials and Methods. The larger the
protein size, the more pronounced was the decrease in protein mobility
due to the increasing sieving of the polyacrylamide matrix. The
log10 of protein mobility was plotted against the
corresponding gel concentrations, and the slope of the resulting
straight line, defined as KR, was computed. The molecular
size of the test sample, inversely proportional to KR, may
be estimated by comparing its mobility to that of a series of standards
(Fig. 8).
Extracts of COS-1 transfected with ER-cDNA (ER-COS) or MCF-7 used as a source of ER were incubated with either [32P]dsERE or [32P]dsoligo2. The analysis of both cell lines allowed cross-checking of the molecular size of the ER complexed with each oligo in cells constitutively expressing the hormone receptor (MCF-7 cells) or overexpressing its exogenous cDNA (COS-1 cells).
In untransfected COS-1 cells no retarded band could be observed (Fig. 8, middle section). With extracts from ER-COS and MCF-7, the
size of the ER-dsERE complex was evaluated at 158 kDa,
whereas that of the ER-dsoligo2 complex corresponded to 143
kDa (Fig. 8B
). The difference in size between the two complexes is
accounted for by the different lengths of the two test oligonucleotides
(33 nucleotides for dsERE vs. 18 nucleotides for
dsoligo2).
These figures, therefore, indicate that both oligonucleotides bound either the dimeric form of the activated ER or its monomeric form plus an accessory protein with a Mr around 67 kDa; no sign of different migrations of the complexes from the two cell lines could be detected.
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DISCUSSION |
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It could be hypothesized that the effects observed in the presence of oligonucleotides are linked to the mechanism of ER autologous down-regulation, which, according to Kaneko et al. (20) and Burnstein et al. (21), occurs via a transcriptional repression exerted by estrogen (or glucocorticoid) receptors on the ER (or glucocorticoid receptor) genes. This effect would be mediated by specific DNA sequences, acting similarly to the negative response elements well characterized for glucocorticoid and thyroid hormone receptors (37). In this perspective, the oligos we describe would compete with the target gene for ER, thereby decreasing the efficacy of ER repression. In agreement with this hypothesis is the finding that ER specifically binds oligos 1 and 6 (data not shown) and oligo2, which are active in determining the receptor up-regulation, and it does not interact with the inactive oligos (oligos 3, 4, and 5). However, a number of other observations argue against the hypothesis of ER acting as a repressor: 1) the oligos determining ER up-regulation have a sequence quite dissimilar from one another and certainly very different from any ERE described to date; 2) autologous down-regulation of the ER was observed with the ER mutant C241A/C244A, which is transcriptionally inactive; 3) the activity of a reporter gene controlled by a promoter containing oligo2 failed to show any negative transcriptional activity of ER. Indeed, the transcription from this promoter was slightly increased in the presence of ER and was hormone independent. ER, therefore, seems to modulate the transcription of its own gene via an alternative mechanism.
The gel shift analysis carried out under denaturing and nondenaturing conditions suggests that ER interacts differently with oligo2 and ERE. The migration across the urea gradient clearly shows that the binding to dsoligo2 prevents the change in ER conformation observed at about 0.5 M urea with the ER-ERE complex. In addition, ERE competes for binding to the receptor with all forms of oligo2, while the opposite does not apply to oligo2, even for very high concentration ratios (up to a 10,000-fold molar excess; data not shown). It is, therefore, conceivable that oligo2 and ERE do not bind the same ER domain; however, the binding to ERE hinders the oligo2-binding site.
On the basis of what we described above, it could be proposed that autologous down-regulation of ER occurs via a novel mechanism of receptor-DNA interaction that is unrelated to the mechanism of hormone-dependent transcriptional repression. This is further supported by the fact that full antagonists of the ER receptor, such as ICI 182,780, which allows the formation of transcriptionally unproductive ER-ERE complexes, are extremely efficient in causing ER down-regulation (38, 39).
The fact that ER uses two different mechanisms of DNA interaction to induce the transcription of target genes and the repression of its own is not totally surprising. It is quite possible that, once activated by the cognate hormone, ER binds the responsive elements to modulate the transcription of the target genes, which are then inactivated by still undescribed modifications. In this transcriptionally inactive form, however, the receptor could acquire the capability to interact, directly or via specific adapter proteins, with its own gene to hinder its own transcription. Supporting this view is the observation that the phenomenon of down-regulation requires a longer time than transcription to start. Our study did not allow us to prove that accessory proteins take part in binding of the ER to oligo2; in the migration study performed according to the method of Ferguson (35), no distinguishable difference in size for the complexes ER-oligo2 and ER-ERE was observed regardless of the ER protein source (MCF-7 or COS-1 transfected with ER). We hypothesize, therefore, that a homodimer of the receptor is responsible for binding the ER gene; however, we cannot rule out the hypothesis that in the complex with oligo2, ER heterodimerizes with a protein with a Mr very close to 67 kDa. Other reports on steroid receptors (estrogen or progesterone) suggest that they can modulate transcriptional events in a conformation that does not require the presence of the DNA-binding domain (40, 41).
A better understanding of this phenomenon might have important pharmacological consequences, as certain drugs could be devised to induce the specific conformation aimed at carrying out a selected task.
The squelching assay with short oligonucleotide sequences homologous to the 5'-segment of the ER gene here described can be used to identify the targets of ER in autologous regulation and might be relevant for clarification of the mechanism of ER activity.
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MATERIALS AND METHODS |
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Cell Culture
MCF-7 and COS-1 cells were grown in RPMI 1640 without phenol red
(Sigma, Milan, Italy) supplemented with 10% charcoal-stripped FBS
(DCC-FBS) (43). The cells were grown in 100-mm petri dishes containing
10 ml culture medium. About 1 x 106 cells were
seeded/dish. The medium was replaced every 4 days. Once a week, at
semiconfluence, the cells were split by treatment with trypsin (0.5
g/liter trypsin and 0.2 g/liter EDTA) for 5 min at 37 C and
resuspension in fresh medium at a 1:20 dilution.
To test aODN activity, the cells were plated in 2.5-cm wells (0.3 x 106 cell/well) and grown in phenol red-free medium supplemented with 10% DCC-FBS. To limit the degradation of the oligonucleotides by serum nucleases, the FBS was heat inactivated at 65 C for 45 min (44). As some degradation still occurred, as evaluated by PAGE, 14 µM nucleotides, dissolved in water, were added every 24 h. MCF-7 cells were treated in parallel with several oligonucleotides for 48 h. E2 was diluted in phenol red-free RPMI at a final concentration of 10-8 M/0.01% ethanol; 0.01% ethanol was added to controls. ER quantitation was performed in duplicate on high salt buffer extracts as previously described (26). ER content was quantified immunoenzymatically (ER-EIA kit, Abbott Laboratory, North Chicago, IL) and normalized for the protein concentration, as evaluated by Bradfords method (45).
Transient Transfection Assay
E2-Induced Down-Regulation in COS Cells Transfected
with ER
Twenty-four hours before transfection, 2 x 105 cells
were plated in 2.5-cm wells containing 3 ml phenol red-free RPMI 1640
medium supplemented with 10% DCC-FBS. Six hours before addition of the
CaPO4-DNA mixture, the medium was replaced with DMEM with
10% DCC-FBS containing 1 nM E2 or its solvent.
In a typical experiment, the cells were transfected using the following
DNA concentrations: 200 µl of a suspension of 0.05 mg/ml ER cDNA (or
its mutants), 0.05 mg/ml of the gene pUHC-ßgal (as control for
transfection efficiency), and 0.12 mg/ml carrier DNA (pGEM3z) in 1.8 ml
medium. Sixteen hours after addition of the precipitate, the medium was
discarded and, after a few washes with RPMI 1640, replaced with phenol
red-free RPMI 1640 containing 1% DCC-FBS (with or without
E2). Forty-eight hours later, the medium was removed, the
cells were washed several times with PBS, and cell extracts were
prepared according to the method of Patrone et al.
(46). ßGal activity was measured as previously reported (47). The protein content was measured according to the method of Bradford (45). Each experiment was repeated at least three times on duplicate samples.
Assessment of ER Transactivation Activity of oligo2-pGL2B
For plasmid construction, the oligo2-pGL2B-luciferase reporter was
generated by insertion of dsoligo2 into the SmaI
site of the pGL2B plasmid (Promega).
Transfections
Cells were plated at high density, as described above (HeLa cells were
grown in Modified Eagles Medium). Cells were transfected with 1.5
µg oligo2-pGL2B-luciferase (or pGL2B-luciferase), 0.5 µg
pCMV-ßgal, and 1 µg ER expression plasmid or mock plasmid. All
transfections were performed in triplicate. After a 2-h transfection,
the cells were washed, and fresh medium containing either 1
nM E2 or its solvent was added. After 48-h
incubation, luciferase and ßgal were measured as previously described
(48).
Gel Retardation Assay
The EMSA was performed as described by Bettini et al.
(49). The oligonucleotides were labeled by incorporation of
[-32P]ATP (Amersham, Aylesbury, UK) with
T4 kinase (Promega) to a specific activity of about
108 dpm/µg DNA. The 32P-labeled oligos
(
10,000 cpm/lane) were incubated with the nuclear extract (58 µg
protein) in the presence of 1 µg HaeIII-cut pBR322 for
about 15 min at 4 C before adding the competing cold nucleotides. The
receptor concentration was chosen to ensure a ratio between the oligo
bound and total oligo lower than 10% as required by Munson and Rodbard
(28). The binding reaction was then carried to equilibrium with an
incubation time of 20 min at room temperature. Addition of the DNA dye
\[50% (vol/vol) glycerol, 0.25% (wt/v) bromophenol blue, and 0.25%
(wt/v) xylene cyanol\] ended the reaction. The entire incubation
mixture (10 µl) was loaded onto nondenaturing polyacrylamide gels
(6% T and 2.5% C). The gels were run with TBE buffer (90
mM Tris-HCl, 90 mM boric acid, and 10
mM EDTA) for about 2 h. Gels were then dried with a
gel dryer (Slab Dryer, Savant, Holbrook, NY) and exposed to x-ray film
(Kodak RP, Eastman Kodak, Rochester, NY) for about 16 h. The gel
retardation analysis was performed on several samples to which the same
unlabeled competitor was added in various concentrations (4
nM to 1 µM) in the presence of a fixed
concentration of [32P]oligo. After densitometric scanning
of the autoradiograph, the results were evaluated as homologous
competition models by the program Ligand (to calculate the apparent
affinity of ER for the DNA) (28).
Bandshift Assay
The labeled probes (10,000 cpm/lane) were incubated with 2 µg
MCF-7 nuclear extract in a buffer consisting of 10 mM HEPES
(pH 7.9), 100 mM KCl, 1 mM dithiothreitol, 0.5
mM EDTA, 2.5 mM MgCl2, 6%
glycerol, and 2% Ficoll. The reaction was allowed to proceed at room
temperature for 10 min. The addition of the antibody (100 ng) was made
before mixing with the labeled probe. The samples were subjected to
electrophoresis (150 V at room temperature) in a nondenaturing 5% T
polyacrylamide slabs in 0.5 x Tris-borate-EDTA for 4 h.
Electrophoresis across a Transverse Urea Gradient
The complexes between hormone-saturated ER and the four
oligonucleotides were run across 0- to 8-M urea gradients
according to the method of Creighton (29). The polyacrylamide matrix
had a concentration of 6.5% T and 4% C in TBE buffer. The gels were
cast against GelBond PAG foils (FMC, Rockville, MD) in a 0.5-mm thick
cassette (181013-74, Pharmacia, Uppsala, Sweden); one or two parallel
sample application trenches were shaped with 170 x
2.5-mm2 strips of embossing tape glued to the glass plate.
A 4-ml concentration plateau of the 8-M urea mix was poured
first, followed by an 8-ml urea gradient delivered from a two-chamber
mixer (181019-87, Pharmacia) and a further concentration plateau from
the solution without urea. The gels were run in a horizontal
electrophoresis chamber (Multiphor, LKB Pharmacia). To minimize
radioactive contamination, according to the suggestion of Kleine
et al. (50), 1% agarose strips in 4 x TBS (5 x
15 x 250 mm3) were substituted for electrodic
solutions and paper wicks. Under a constant voltage of 100 V/11 cm and
at a temperature of 15 C, the run of a single sample lasted 80 min.
When the behavior of a different oligonucleotide complex was compared
with that of ERE, the latter was applied first in the more cathodal
trench, whereas the second sample was loaded after a 30-min run, once
the free oligonucleotide had migrated past the more anodal trench. The
sample volume was 130 µl, containing 20 µg of nuclear proteins (in
20 µl), 75 µl EMSA buffer (48), 1 µg HaeIII-cut pBR322
(in 1 µl), 2 µl [32P]oligo to a specific activity of
about 10-8 dpm/µg DNA, and 13 µl H2O; the
mixture was incubated for 30 min at room temperature, then added with
22 µl DNA dye \[50% (vol/vol) glycerol, 0.25% (wt/vol) bromophenol
blue, and 0.25% (wt/vol) xylene cyanol\].
The Ferguson Plot
The molecular size of ER-oligonucleotide complexes was evaluated
on polyacrylamide gels (4% and 8% T), cast in TBE buffer according to
the method of Ferguson (34). The 250 x 125 x
0.5-mm3 slabs, supported on GelBond PAG foils, were
polymerized in halves at the two gel concentrations. Sample and marker
proteins (-lactalbumin, carbonic anhydrase, and BSA plus ovalbumin,
which, however, exhibited an anomalous behavior in the presence of
borate ions; all markers from Sigma Chemical Co., St. Louis, MO) were
dissolved in high salt buffer; 7 µl were applied/lane, corresponding
to 7 µg of the markers and 58 µg of the nuclear proteins. The
gels were run at 15 C for 90 min in a Multiphor chamber (Pharmacia) at
100 V/11 cm. The marker proteins were stained with Coomassie blue R,
whereas the ER-oligonucleotide complexes were detected by
autoradiography.
Western Analysis of the ER Protein
Sample preparation was the same for MCF-7 and COS-1 cells,
except that MCF-7 lysates were first subjected to an
immunoprecipitation step, as previously reported (51). Thirty
micrograms of proteins were immunoprecipitated, then denatured and
loaded onto a 10% SDS-polyacrylamide slab gel. After electrophoretic
migration, the proteins were transferred to Hybond-C Extra
nitrocellulose (Amersham) in a Trans-Blot apparatus (Bio-Rad, Hercules,
CA). Blots were stained with Red Ponceau (Sigma, St. Louis, MO) to
assess the efficiency of protein transfer. The filters were saturated
for 16 h with 5% milk proteins and 0.2% Tween-20 in TBS (50
mM Tris and 150 mM NaCl) before incubation with
the anti-ER monoclonal antibody H222 (Abbott Laboratories,
North Chicago, IL). Immunoreactive proteins were detected after
incubation with a peroxidase-conjugated secondary antibody (rabbit anti
rat-IgG antibody, Vector Laboratories, Burlingame, CA) through ECL
reaction (Amersham) and exposure to Hyperfilm-MP (Amersham).
Semiquantitative Analysis of ER mRNA by reverse
transcription-PCR
Total cell RNA was isolated with the Bio/RNA-X Cell kit
(Bio/Gene, Kimbolton Cambs, UK) using 1 ml RNA-X reagent for 510
x 106 cells. One microgram of RNA was reverse transcribed
using oligo(deoxythymidine)12-18 and Moloney murine
leukemia virus reverse transcriptase (HT Biotechnology, Cambridge, UK)
as previously described (46). One tenth of the reaction of cDNA was
amplified in a 100-µl mixture containing 2.5 U DynaZyme-DNA
polymerase (Finenzyme OY, Espo, Finland), the buffer provided by
Finenzyme, 0.2 µM deoxy-NTP, and 100 pmol PCR primers,
the 18-mers 5'-AGC GTG TCT CCG AGC CCG-3' and 5'-TGC ACA GTA GCG AGT
CTC-3' for human ER. Fifty picomoles of a set of primers for the
constitutively expressed enzyme GAPDH (the 24-mers 5'-CCA CCC ATG GCA
AAT TCC ATG GCA-3' and 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3') were used
in each reaction as an internal control for the amount of mRNA
transcribed and amplified. After denaturing at 94 C for 2 min, PCR
amplification was performed for 32 cycles (94 C for 15 sec, 48 C for 20
sec, and 72 C for 30 sec), followed by a final extension step (72 C for
3 min). The duration and temperature of the PCR cycles were
experimentally optimized to fall into the exponential phase of the
amplification (52).
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ACKNOWLEDGMENTS |
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The authors express their gratitude to Roberto Maggi for his important contribution with the analysis of ER-DNA binding data, Malcolm Parker for kindly providing the mouse ER mutants, and Ms. Monica Rebecchi and Ms. Simona Bennici for excellent technical and secretarial support.
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FOOTNOTES |
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This work was supported by Italian National Council of Research (Strategic Project Antisense Oligonucleotides) and the Italian Association for Cancer Research.
Received for publication September 27, 1996. Revision received December 16, 1996. Accepted for publication February 3, 1997.
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REFERENCES |
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