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1 Gene Expression: Role of Estrogen Receptor-Sp1 Complex
Department of Veterinary Physiology and Pharmacology Texas A & M University College Station, Texas 77843-4466
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 (RAR1) gene
expression is induced by 17ß-estradiol
(E2) in estrogen receptor
(ER)-positive breast cancer cells, and the -100 to -49 region of the
RAR1 gene promoter was previously shown to be required for
E2-responsiveness. This region of the
RAR1 promoter was further analyzed using the following
oligonucleotides: -100 to -49 (RAR4); -79 to -56 (RAR3); -79
to -49 (RAR2); -100 to -58 (RAR1); and their derived promoter
reporter constructs (pRAR4, pRAR3, pRAR2, and pRAR1). In transient
transfection studies in MCF-7 human breast cancer cells, pRAR2 and
pRAR1 were E2-responsive; both of the RAR1
gene promoter inserts contained two GC-rich sites and bound Sp1 protein
in gel mobility shift assays. Using wild-type
[32P]RAR2 and oligonucleotides mutated in one
or both GC-rich sites, it was shown that ER enhanced Sp1 binding to
both sites, but a ternary ER-Sp1-DNA complex was not observed in gel
mobility shift assays. In transient transfection assays, each of the
GC-rich motifs were sufficient for E2-induced
transactivation. In ER-negative MDA-MB-231 cells transiently
transfected with pRAR2, E2 responsiveness was
observed only in cells cotransfected with wild-type ER or 11C-ER
containing a deletion of the DNA-binding domain but not with ER
variants that express activation function-1 (AF-1) or AF-2. Using a
similar approach, it was shown that the GC-rich sites in RAR1 were also
sufficient for ER activation. These results demonstrate that
interaction of a transcriptionally active ER/Sp1 complex with GC-rich
motifs is required for hormone inducibility of the downstream region of
the RAR1 gene promoter. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The three known subtypes of the RAR (, ß, and 
) bind
all-trans and 9-cis-retinoic acid (5, 6, 7, 9, 10, 11);
ligand-activated RARs are differentially expressed throughout
development and exhibit discrete and overlapping functions (5, 6, 7, 12, 13, 14, 15). Retinoids have been extensively used as antineoplastic agents
for treatment of epithelial- and mesenchymal-derived tumors (16, 17, 18, 19).
For example, retinoids inhibit basal and 17ß-estradiol
(E2)-induced proliferation and gene expression in human
breast cancer cell lines and carcinogen-induced mammary tumor
development and growth in female Sprague-Dawley rats (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38). RAR1
is highly expressed in estrogen receptor (ER)-positive breast cancer
cell lines and is required for retinoid-induced inhibition of human
breast cancer cell proliferation and E2-induced gene
expression. A recent report also showed that retinoids inhibit growth
of some ER-negative breast cancer cell lines that expressed RAR1
(26, 28).
Several studies have shown that E2 induces RAR1 gene
expression and reporter gene activity in human breast cancer cells
transiently transfected with constructs containing RAR1 gene
promoter inserts linked to reporter genes (20, 21, 24, 25, 27). Rishi
and co-workers (25) identified an estrogen responsive element (ERE)
half-site(N)10Sp1 motif
[GGTGA(N)10-GGCGGG] at -82 to -62 in the RAR
gene promoter that was responsible for E2-induced
transactivation in breast cancer cells. In contrast, Elgort and
co-workers (27) identified two E2-responsive regions in the
-491 to +36 sequence of the RAR gene promoter using HepG2 cells
cotransfected with the ER. The upstream sequence at -491 to -455 bp
contained two GGTCA half-sites and a GC-rich downstream region (-79 to
-49) which bound Sp1 protein. Neither of these promoter regions
directly bound to the ER in gel mobility shift assays.
This study reinvestigates E2-responsiveness of the more
proximal region in the RAR1 gene promoter. The RAR4 oligonucleotide
(-100 to -49) contained both the upstream Sp1(N)10ERE
half-site and downstream GC-rich motifs that were previously identified
as promoter sequences required for E2-induced
transactivation (25, 27). Deletion analysis of the -79 to -49 region
of the RAR1 gene promoter showed that the GC-rich Sp1
protein-binding sites at -68 to -62 and -59 to -52 were required
for E2-responsiveness. Analysis of the upstream sequence
(-100 to -58, RAR1) showed that ER did not bind to this region of the
promoter, and ER activation was associated with interaction of ER/Sp1
with GC-rich motifs. These results complement recent studies using a
consensus Sp1 oligonucleotide that first demonstrated that ER and Sp1
proteins physically interact to form a functional transcription factor
complex (39).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 gene promoter insert, there was a 3.3-fold increase in
chloramphenicol acetyltransferase (CAT) activity compared with cells
treated with solvent [dimethylsulfoxide (DMSO)] (Fig. 1
). Similar results were obtained using
pRAR2, which contained the -79 to -49 RAR1 gene promoter insert,
whereas pRAR3 (-79 to -56 insert) was not E2-responsive.
In MCF-7 cells transiently transfected with pRAR1, treatment with
E2 also significantly induced (3.4-fold) CAT activity.
E2 responsiveness of pRAR2 was compared with constructs
containing mutations in the GC-rich sites at -59 to -52 (pRAR2•m1)
and -68 to -62 (pRAR2•m2) (Fig. 2).
One nanomolar E2 did not induce CAT activity in cells
transiently transfected with both mutant plasmids (Fig. 2); however, 10
nM E2 caused a 3.4- and 5.2-fold induction
response using pRAR2•m1 and pRAR2•m2, respectively. Thus, each of
the GC-rich sites was sufficient for E2-responsiveness.
RAR1 contains GC-rich sites at -68 to -62 and -94 to -88 and an ERE
half-site motif at -82 to -78 (25). Previous studies reported that
E2-induced transactivation of pRAR1 required an intact ERE
half-site (25); however, pRAR1•m1, which is mutated in the ERE
half-site, was also E2-responsive (Fig. 2), suggesting that
only the GC-rich motifs are required for hormone inducibility in MCF-7
cells.
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compare the
binding of Sp1 protein (0, 5, 10, or 20 ng) to a consensus
[32P]Sp1 oligonucleotide with Sp1 binding to
[32P]RAR2, [32P]RAR2•m1, and
[32P]RAR2•m2. Formation of a retarded band (bound DNA)
was observed using all the oligonucleotides, indicating that the
GC-rich sites identified in the -79 to -49 region of the RAR1 gene
promoter bound Sp1 protein. The results also showed that
near-saturation of Sp1 binding to consensus [32P]Sp1 was
observed at the lowest amount of Sp1 protein, whereas only minimal or
nondetectable binding to the other oligonucleotides (lanes 6, 10, and
14) was observed using the same amount of Sp1 protein. Relative
intensities of the retarded band using the highest amount (20 ng) of
Sp1 (protein) and consensus [32P]Sp1,
[32P]RAR2, [32P]RAR2•m1, and
[32P]RAR2•m2 were 100, 20, 27, and 15, respectively.
Thus, the GC-rich sites within RAR2 bound Sp1 protein with lower
affinity than the consensus Sp1 oligonucleotide. Mobilities of retarded
bands using [32P]RAR2, [32P]RAR2·m1, or
[32P]RAR2·m2 gave a single retarded band with
comparable mobilities. These data suggest that of the GC-rich sites in
RAR2 initially bound only one Sp1 molecule/DNA under the conditions of
this assay, which was limiting in Sp1 protein. Incubation of
[32P]RAR2 with a large excess of Sp1 protein gave a
second band with decreased mobility (data not shown), suggesting that
binding can occur at both GC-rich sites.
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) showed that wild-type Sp1 protein (5
ng) bound consensus [32P]Sp1 oligonucleotide to form a
retarded band (lane 2, bound DNA), and the intensity of this band was
not decreased by competition with 400-fold excess of unlabeled mutant
Sp1 oligonucleotide (lane 4). The intensity of the
Sp1-[32P]Sp1 (oligo) retarded band was decreased by
coincubation with 0.5–5.0 pmol unlabeled RAR2 (lanes 5–7), RAR2•m1
(lanes 8–10), and RAR2•m2 (lanes 11–13) oligonucleotides. In
contrast, competition with RAR2•m3, which contains mutations in both
GC-rich sites, did not significantly decrease intensity of the retarded
band (data not shown). Thus, results in Figs. 3
and 4 demonstrate in
both direct and indirect binding gel mobility shift assays that GC-rich
sites within RAR2 interact with Sp1 protein.
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show
that [32P]RAR2 binds Sp1 protein (lane 3) but not ER
protein (lane 2); incubation of [32P]RAR2 and Sp1 protein
plus 200, 400, or 800 fmol ER (lanes 4–6) resulted in a
concentration-dependent increase in formation of the
Sp1-[32P]RAR2-retarded band (bound DNA,
upper). Intensity of the retarded band was increased by
2.8-fold at the highest concentration of ER (lane 6). Intensity of the
ER-enhanced retarded band (lane 6) was competitively decreased by
coincubation with 400-fold excess unlabeled consensus Sp1 and RAR2
oligonucleotides (lanes 7 and 8) but not by mutant consensus Sp1
oligonucleotide (lane 9). Incubation of [32P]ERE and ER
gave a retarded ER-ERE band (bound DNA, lower) (lane 11),
which was more mobile than the Sp1-DNA complexes.
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show
that wild-type [32P]RAR2 bound Sp1 protein to form a
retarded band (lane 2), and coincubation with ER (200–800 fmol)
enhanced intensity of the retarded band (lanes 3–5). Incubation of Sp1
protein alone with [32P]RAR2•m1 (lane 7) or
[32P]RAR2•m2 (lane 12) resulted in retarded band
formation, and coincubation with ER (200–800 fmol) resulted in a
concentration-dependent increase in retarded band intensity (lanes
8–10 and 13–15, respectively).
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). [32P]RAR1
binds Sp1 protein (lane 3) but not ER protein (lane 2); however,
coincubation of [32P]RAR1, Sp1, and ER resulted in
enhanced (1.7-fold) intensity of the [32P]RAR1-Sp1
retarded band (lanes 4–6, bound DNA). These results show that ER did
not directly bind [32P]RAR1 but ER enhanced binding of
[32P]RAR1 to the Sp1 protein.
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) and also showed that transfection with
19C-ER and 15C-ER, which contain only activation function-2 (AF-2) and
AF-1, respectively, did not result in E2-induced
transactivation. These results show that the GC-rich sites at -68 to
-62 and -59 to -52 in the RAR1 gene proximal promoter region are
primarily responsible for E2-responsiveness via
interactions with a transcriptionally active ER/Sp1 complex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 gene expression, and at least two
regions of the RAR1 gene promoter at -491 to -455 and -100 to
-49 were sensitive to E2-induced transactivation (27).
Contributions of the two E2-responsive regions of the
RAR1 gene promoter may also be dependent on cellular context. In
transient transfection studies in HepG2 cells, the upstream region
(-491 to -455) was the more important contributor to E2
responsiveness (27), whereas the downstream sequence (-100 to -49)
played the major role in E2-mediated transactivation in
breast cancer cells (25). Analysis of the downstream region of the
promoter by transient transfection and gel mobility shift assays gave
conflicting results. Rishi and co-workers (25) showed that an
ERE(1/2)(N)10Sp1 (-100 to -58) motif bound the ER
and Sp1 proteins in gel mobility shift assays and was
E2-responsive in transient transfection studies. These data
were consistent with other reports showing that an
ERE(1/2)(N)xSp1 sequence also played a role in
ER-mediated transactivation of c-myc, creatine kinase B,
cathepsin D, and heat shock protein 27 gene expression (40, 41, 42, 43, 44, 45). In
contrast, Elgort and co-workers (27) showed that
E2-responsiveness of the downstream RAR1 gene promoter
sequence was localized in the -79 to -49 region, which contained two
GC-rich sequences but not the ERE(1/2).
We have reinvestigated the E2-responsiveness of the
proximal promoter region of the RAR1 gene in transient transfection
studies utilizing constructs containing the -100 to -49 (pRAR4),
-100 to -58 (pRAR1), -79 to -49 (pRAR2), and -79 to -56 (RAR3)
RAR1 gene promoter inserts. The insert in pRAR4 encompasses both the
ERE(1/2)(N)10Sp1 (RAR1) and GC-rich (RAR2) promoter
sequences and, not surprisingly, was E2-responsive (Fig. 1). Both E2-responsive RAR1 (-100 to -58) and RAR2 (-79
to -49) regions of the RAR1 gene promoter contain two GC-rich
sites. Recent studies in this laboratory have demonstrated that
Sp1-binding sites are potentially hormone-responsive via formation of
ER/Sp1 protein-GC rich (DNA) complexes (39). Therefore, this study
reexamined the role of GC-rich elements in the RAR1 gene promoter as
mediators of ER activation. Rishi and co-workers (25) previously
reported that [32P]RAR1 bound ER in a gel mobility shift
assay and that the ERE half-site was required for this binding and for
E2-induced transactivation of pRAR1.
E2-responsiveness of pRAR1 was also observed in our study
(Fig. 1); however, the results also showed that [32P]RAR1
did not directly bind the ER in a gel mobility shift assay (Fig. 7) and
pRAR1·m1, which is mutated in the ERE half-site, was
E2-responsive (Fig. 2). Thus, only the GC-sites in RAR1
were required for ER activation, and results of gel mobility shift
assays showed that [32P]RAR1 bound Sp1 protein, and
unlabeled RAR1 and Sp1 oligonucleotides competitively decreased binding
of Sp1 protein to [32P]RAR1 (Fig. 7). Moreover, ER
enhanced Sp1 binding to [32P]RAR1 (Fig. 7), indicating
that the GC-rich Sp1-binding sites are critical elements for ER
activation of RAR1.
Elgort and co-workers (27) previously reported that RAR2 region of the
promoter (-79 to -49) contained two GC-rich sites that bound Sp1 but
not ER protein and was E2-responsive in transient assays.
Therefore, we investigated the role of one or both of the GC-rich sites
within RAR2 in mediating E2-responsiveness. pRAR2·m3 is
mutated in both downstream GC-rich sites and was not active in
transient transfection studies (data not shown); however, as previously
reported (27), hormone responsiveness was observed in transient
transfection studies using pRAR2 (-79 to -49) (Figs. 1 and 2)
suggesting that the Sp1 sites are required for inducibility.
E2 induced CAT activity in transient transfection studies
with constructs containing mutations in the -68 to -62 (pRAR2·m2)
or -59 to -52 (pRAR2·m1) sites (Fig. 2), suggesting that either of
the two GC-rich sites are sufficient for ER-mediated transactivation of
pRAR2.
Gel mobility shift assays clearly showed that RAR2, RAR2·m1,
RAR2·m2, and RAR1 bound Sp1 protein to form a retarded band or
competitively decreased Sp1-[32P]Sp1 in competition
assays (Figs. 3, 4, and 7). Although ER and Sp1 physically interact
(39), coincubation of both proteins with a consensus
[32P]Sp1 oligonucleotide resulted only in formation of an
Sp1-[32P]Sp1 complex in which ER enhanced the rate of
complex formation and retarded band intensity (39). Similar results
were obtained in this study using [32P]RAR2,
[32P]RAR2•m1, [32P]RAR2·m2, and
[32P]RAR1 (
Figs. 5–7). The failure of ER to supershift
the Sp1-DNA complex but to enhance Sp1-DNA binding has previously been
observed in other studies showing that HTLV-1 Tax, SREPB, and cyclin D1
enhanced binding of bZip, Sp1, and ER to their respective enhancer
elements, respectively (46, 47, 48). The results with the RAR2 (-79 to
-49) region of the RAR1 gene promoter demonstrate that both GC-rich
sites bind Sp1 protein in gel mobility shift assays, and intensities of
both retarded bands were enhanced by coincubation with ER, which is
consistent with results of transactivation assays (Fig. 2).
It has also been reported that both wild-type ER and 11C-ER (but not
19C-ER or 15C-ER) enhance Sp1 binding to GC-rich elements in gel
mobility shift assays, confirming that the effect of ER does not
require DNA binding (39). In ER-negative MDA-MB-231 cells,
E2 induced CAT activity in cells cotransfected with pRAR2
and wild-type ER or 11C-ER, which does not contain the DNA-binding
domain of the ER (Fig. 8). In contrast, no induction response was
observed in cells cotransfected with 15C-ER or 19C-ER, which express
AF-1 or AF-2 domains of the ER, respectively. These results were
comparable to previous studies using gel mobility shift assays and
constructs containing a consensus Sp1 oligonucleotide insert and
further support hormone-induced transactivation via ER/Sp1 interactions
with GC-rich Sp1-binding sites (39).
Results of this study demonstrate that the GC-rich motifs in the
proximal region of the RAR1 gene promoter are functional
E2-responsive enhancer sequences in which ER-mediated
transactivation is independent of ER-DNA interactions. Recent studies
in the laboratory have identified Sp1-binding sites in the
c-fos protooncogene promoter that are also functional
enhancer elements for ER/Sp1-mediated gene expression in MCF-7 cells
(49). These hormone-induced responses do not require interaction of the
ER with DNA and are similar to ER-AP1 interactions (50). Sp1-binding
sites are common motifs in promoters of diverse cellular and viral
genes, most of which are hormone-independent. The reasons for
differential sensitivity of Sp1-binding sites in gene promoters are
unknown but could be due to specific interactions with other nuclear
proteins including coactivators. Current studies in this laboratory are
focused on identifying other E2-responsive GC-rich motifs
within gene promoter sequences and determining their cell-, promoter
region-, and ligand-dependent functionality.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]ATP (3000 Ci/mmol) and
[14C]chloramphenicol (53 mCi/mmol) were purchased from
NEN Research Products (Boston, MA). Poly deoxy-(inosinic-cytidylic)
acid [poly d(I-C)], restriction enzymes (HindIII and
BamHI), and T4-polynucleotide kinase were purchased from
Boehringer Mannheim (Indianapolis, IN). The human estrogen receptor
(hER) expression plasmid was kindly provided by Dr. Ming-jer Tsai
(Baylor College of Medicine, Houston, TX). The hER deletion mutants
11C-ER, 15C-ER, and 19C-ER were kindly provided by Dr. Pierre Chambon
(Strasbourg, France). Sp1 protein and bacculovirus-expressed hER
proteins were purchased from Promega (Madison, WI), and Panvera
(Madison, WI), respectively. Plasmid preparation kit was purchased from
Qiagen (Santa Clarita, CA); 40% polyacrylamide was obtained from
National Diagnostics (Atlanta, GA). All other chemicals and
biochemicals were the highest quality available from commercial
sources. DNA oligonucleotides (Table 1) were
synthesized by the Gene Technologies Laboratory, Texas A & M University
(College Station, TX).
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1
promoter listed above were cloned into the pBL/TATA-CAT at the
HindIII and BamHI sites to give pRAR1,
pRAR2, pRAR3, pRAR4, pRAR1·m1 pRAR2·m1, pRAR2·m2, and pRAR2·m3
plasmids, respectively. All ligation products were transformed into
DH5-competent Escherichia coli cells, plasmids were
isolated, and correct clonings were confirmed by restriction enzyme
mapping and DNA sequencing using the Sequitherm cycle sequencing kit
from Epicentre Technologies (Madison, WI). Plasmid preparation for
transfection was carried out by alkaline lysis followed by cesium
chloride gradient centrifugation (2x) or by using a Qiagen Plasmid
Mega Kit.
Transient Transfection and CAT Centrifugations
Cultured MCF-7 and MDA-MB-231 cells were seeded in 5%
charcoal-stripped DME/F12 medium in 100-mm plates for 16 h and
then transiently transfected by the calcium phosphate method with 10
µg reporter plasmid and 5 µg of wild-type hER or variant ER
expression plasmids in pCDNA3-Neo (InVitrogen, Inc., Carlsbad, CA). The
empty vector pcDNA3.1 (InVitrogen) was used to bring the total DNA
content to 15 µg. E2 responsiveness in MCF-7 cells was
observed only after cotransfection with WT-ER (or 11C-ER), and this has
been observed in other studies using E2-responsive
constructs due to overexpression of the plasmids (39, 40, 41, 44). After
incubation for 14–16 h, media was changed and cells were treated with
the appropriate chemicals in DMSO for 44 h. Cells were then washed
with PBS and harvested by scraping, and then lysed in 200 µl of 0.25
M Tris-Cl (pH 7.6) by three cycles of freeze (1.5 min)-thaw
(1.5 min) sonication (3 min). Cell debris was pelleted and protein
concentration was determined by the method of Bradford using BSA
as standard. An aliquot of cell lysate was brought to 120 µl with
0.25 M Tris-Cl (pH 7.6) and incubated with 1 µl
[14C]chloramphenicol (53 mCi/mmol) and 42 µl of 4
mM acetyl coenzyme A for an appropriate time at 37 C. The
reaction was stopped by vortexing with 300 µl ethyl acetate. After
vortexing for 30 sec and centrifuging at 16,000 x g
for 1 min at 20 C, a 250-µl aliquot of ethyl acetate was evaporated
in vacuo, resuspended in 20 µl ethyl acetate, spotted on a
TLC plate (Whatman Ltd., Maidstone, England), and developed using a
95:5 chloroform-methanol solvent mixture. The percent protein
conversion into acetylated chloramphenicol was quantitated using the
counts/min obtained from the Betagen Betascope 603 blot analyzer
(Tritech, Annapolis, MD). CAT activity was calculated as the percentage
of that observed in cells treated with DMSO (arbitrarily set at 100).
TLC plates were subjected to autoradiography using a Kodak X-Omat film
(Eastman Kodak, Rochester, NY) for 20 h.
Electrophoretic Mobility Shift Assays
Oligonucleotides were annealed and labeled at the 5'-end
using T4-polynucleotide kinase and [-32P]ATP. Gel
electrophoretic mobility shift assays were performed by incubating
0–20 ng pure Sp1 protein (Promega, Madison, WI) in 25 µl of 1
x binding buffer (6% glycerol, 1 mM MgCl2,
0.5 mM EDTA, 0.5 mM dithiothreitol, 50
mM NaCl, 10 mM Tris-HCl, pH 8.0), 0.1 mg/ml of
BSA. After incubation for 10 min at 4 C, 32P-labeled
oligonucleotides (50,000 cpm) were added to the reaction mixture in the
presence of 0.5 µg poly d(I-C) and incubated for an additional 15 min
at 25 C. Excess unlabeled DNA for competition studies was added before
the addition of 32P-labeled oligonucleotides. The following
procedure was used for ER-enhanced Sp1 binding studies: 1) 200–800
fmol pure hER protein in 1 x binding buffer containing 40
mM E2 and BSA was incubated for 15 min at 4 C;
2) 1–5 ng Sp1 protein was added to the mixture and incubated on ice
for 5 min; 3) 32P-labeled oligonucleotides (50,000 cpm)
were added to the reaction mixture in the presence of 0.5 µg poly
d(I-C), and the mixture was incubated for an additional 15 min at 25 C.
Samples were loaded onto a 5% polyacrylamide gel
(acrylamide-bisacrylamide ratio, 30:0.8) and run in 1 x TBE
buffer (0.09 M Tris, 0.09 boric acid, and 2 mM
EDTA, pH 8.3) at 110 V. Protein-DNA binding was visualized by
autoradiography and quantitated by densitometry using the Zero-D
software package (Molecular Dynamics, Sunnyvale, CA) and a Sharp JX-330
scanner (Sharp Corp., Mahwah, NJ) and subjected to autoradiography
using a Kodak X-Omat film for the appropriate time at -80 C.
Statistical Analysis
Statistical significance was determined by ANOVA and Scheffe’s
test, and the levels of probability are noted. Results are expressed as
means ± SD for at least three separate
experiments.
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FOOTNOTES |
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This work was supported by NIH Grant CA-76636, the Robert A. Welch Foundation, and the Texas Agricultural Experiment Station. S.S. is a Sid Kyle Professor of Toxicology.
Received for publication October 17, 1997. Revision received February 11, 1998. Accepted for publication February 25, 1998.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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pS2 gene expression in breast carcinoma cells. Cancer Res 52:3938–3945This article has been cited by other articles:
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