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Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466
Address all correspondence and requests for reprints to: Dr. Stephen H. Safe, Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormone responsiveness of ADA has not previously been reported; however, it has been shown that 17ß-estradiol (E2) induces several enzymes involved in purine, pyrimidine, and DNA synthesis in MCF-7 human breast cancer cells, and this is accompanied by increased [3H]thymidine uptake and cell proliferation (15, 16, 17, 18, 19). Results of this study demonstrate that ADA, an enzyme that decreases intracellular pools of adenosine and deoxyadenosine, is E2 responsive in MCF-7 human breast cancer cells. This may represent a feedback loop that ultimately inhibits the hormone-induced proliferative response. Analysis of the proximal region of the ADA gene promoter shows that only one of the GC-rich sites (IV) is required for estrogen receptor (ER) activation via formation of an ER/Sp1 complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligonucleotides derived from the ADA gene promoter and a consensus Sp1 oligonucleotide were synthesized by the Gene Technologies Laboratory, Texas A&M University (College Station, TX). RT-PCR primers were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Structures of these oligonucleotides (sense strands) are summarized below, and the putative GC-rich sites are underlined. Mutations incorporated in the mutant oligonucleotides are denoted by an asterisk.
Cloning
The inserts encoding the wild-type hER, HE11, HE15, and HE19 ER
deletion variants were removed by digesting the appropriate plasmids
with EcoRI. The inserts were then religated into pCDNA3-Neo
(Invitrogen, Carlsbad, CA), which had been linearized with
EcoRI and treated with calf intestinal alkaline
phosphatase. The ligation products were transformed into DH5a
cells and clones were verified by sequencing. In vitro
transcription/translation of these genes was periodically determined
using the rabbit reticulocyte lysate system and
[35S]methione followed by SDS-PAGE and quantitation of
radiolabeled proteins by densitometry. Results showed that levels of
immunoreactive wild-type and variant ER were not significantly
different. The ADA81m oligonucleotide was cloned into the pBLCAT6
vector (ATCC, Manassas, VA) at the HindIII and
BamHI site to give the pADA81m. 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 containing complementary 5'-overhangs was
then inserted into the corresponding sites. ADASp1.4 and ADASp1.4m
oligonucleotides were cloned into the pBLTATA-CAT vector at the
HindIII and BamHI sites to give the pADASp1.4,
pADASp1.4m constructs, respectively.
RT-PCR
RNA was extracted from the cells treated with dimethylsulfoxide
(control) or E2, tamoxifen, ICI 182,780 by using the
RNAZol-B kit (Tel-Test, Friendswood, TX), and dissolved in
nuclease-free water (Promega Corp., Madison, WI). RT-PCR
was performed using a GeneAmp, RNA PCR kit (Perkin-Elmer Corp., Roche Molecular Biochemicals,
Branchburg, NJ). The reaction mixture consisted of 5 mM
MgCl2, 2 µl 10x PCR buffer II, ribonuclease (RNase)-free
water, deoxyribonucleoside triphosphates (dATP, deoxythymidine
triphosphate, dGTP, and deoxycytidine triphosphate) at a final
concentration of 1 mM, 1 U/µl RNase inhibitor, and 2.5
µM murine leukemia virus reverse transcriptase in a final
volume of 20 µl. After incubation for 10 min at 25 C, the reaction
mixture was reverse transcribed at 42 C for 25 min, denatured at 99 C
for 5 min, and cooled at 4 C for 5 min. For the PCR reaction
MgCl2 was adjusted to 2 mM and PCR buffer.
RNase-free water, primer (200 ng), and AmpliTaq DNA polymerase 0.5 µl
(2.5 U/100 µl) were added to a final volume of 50 µl. PCR
conditions included an initial denaturing for 3 min, and then 25 cycles
as follows: denaturing for 1 min at 95 C and anneal-extend for 1 min at
62 C. A final cycle for 10 min at 72 C concluded the PCR. After
amplification, products were separated on a 7% (wt/vol) polyacrylamide
gel, visualized by autoradiography using Kodak XAR film (Eastman Kodak Co., Rochester, NY), and quantitated by densitometry using
the Molecular Dynamics, Inc. Zero-D software
package (Sunnyvale, CA) and a Sharp JX-330 scanner (Mahwah, NJ).
Intensities of ADA transcripts were normalized to the ß-actin
internal control.
Transient transfection assay
MCF-7 cells were transfected using the calcium phosphate method
with 10 µg of ADA gene promoter-derived constructs and 1 µg of
wild-type or variant ER expression plasmids; in the absence of
cotransfected wild-type ER, no hormone responsiveness was observed, and
this was due to overexpression of the ADA promoter-derived constructs.
ß-Galactosidase-lacZ plasmid (5.0 µg) obtained from
Invitrogen (Carlsbad, CA) was cotransfected in studies
determining differences in basal CAT activities with constructs
containing ADA gene promoter inserts; activities are corrected for
transfection efficiencies. Previous studies have also shown the
requirement for cotransfection of hER expression plasmid using other
E2-responsive constructs in MCF-7 cells (20, 21, 22, 23, 24, 25, 26, 27, 28).
pCDNA3-Neo (Invitrogen, Inc., Carlsbad, CA) was used as an
empty vector (control) and was also added in some experiments to
maintain uniform levels of added DNA. Transfection efficiency was high
and no additional shock was required. After 18 h, media were
changed and cells were treated with Me2SO (0.2% total
volume), E2, tamoxifen, ICI 182,780, or their combinations
in Me2SO for 44 h. Cells were then 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. Acetylated products were visualized
and quantitated using a Betagen Betascope 603 blot analyzer
(Intelligenetics, Mountain View, CA). CAT activity was
calculated as fraction of that observed in cells treated with
Me2SO alone (arbitrarily set at 100), and results are
expressed as means ± SD. At least three separate
experiments were carried out for each treatment group.
Electrophoretic mobility shift assays
Gel electromobility shift assays were performed using Sp1
protein and different amount of ER protein. E2 was added to
the reaction at a final concentration of 20 nM and then
incubated on ice for 15 min. Sp1 and 32P-labeled
oligonucleotides were then added to the reaction mixtures in the
presence of 1 µg of poly d(I-C) and incubated for 15 min at 25 C. In
competition experiments, different amounts of unlabeled
oligonucleotides were also included in the incubation mixture. 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-Labeled DNA and DNA-protein bands were visualized
by autoradiography and quantitated by densitometry using the
Molecular Dynamics, Inc. Zero-D software package and a
Sharp JX-330 scanner. For some of these studies, relative band
intensities are presented as means ± SD for three
separate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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show
that E2 induced a rapid increase in ADA mRNA levels that
was maximal after 6 h (>3.76-fold induction) and persisted for up
to 24 h. In a separate experiment, E2 and tamoxifen
induced a 3.17- and 1.5-fold increase in ADA mRNA levels, whereas ICI
182,780 decreased mRNA levels. In cells cotreated with E2
plus ICI 182,780, there was a significant decrease in the
hormone-induced response; there was also a small decrease in ADA mRNA
levels in cells cotreated with tamoxifen plus E2,
suggesting that tamoxifen exhibited partial ER agonist and antagonist
activities.
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summarizes results of experiments that
examine the role of GC-rich sites within the proximal region of the ADA
gene promoter in mediating basal and E2 responsiveness.
Results of initial studies showed that basal CAT activity in MCF-7
cells transiently transfected with pADA211 (arbitrarily set at 100),
pADA81, pADA56, and pADA81m was 100, 98 ± 11, 56 ± 5.2, and
14 ± 0.9, respectively (Fig. 2A). The significant decrease in
basal activity of pADA56 vs. pADA81 was previously reported
in MOLT-4 and Raji cells (10). These results coupled with the low
activity of pADA81m (containing a mutation in site IV) confirms the
importance of the site IV GC-rich element for basal activity of the
proximal region of the ADA gene promoter in MCF-7 cells. E2
induced a significant 2.35- and 2.36-fold increase in CAT activity in
MCF-7 cells transfected with pADA211 and pADA81, whereas only a
1.57-fold increase (not significant) was observed for pADA56 (Fig. 2B).
The importance of GC-rich site IV for E2 responsiveness was
confirmed using pADASp1.4 in transfection assays; E2
caused a 2.8-fold induction of CAT activity. In contrast, pADASp1.4m
and pADA81m, which contain mutations in site IV, were not
E2 responsive (data not shown). Previous studies in this
laboratory using E2-responsive constructs containing either
a consensus Sp1 insert or GC-rich elements from the c-fos or
heat shock protein 27 gene promoter showed that induction of reporter
gene activity in breast cancer cells was observed after cotransfection
with wild-type hER or variant HE11 (DNA-binding domain-deficient ER)
expression plasmids (25, 26, 28). The results in Fig. 3A demonstrate that in MCF-7 cells
treated with 10 nM E2 and cotransfected with
pADA81 and hER or HE11, there was a 4.65- and 3.77-fold increase in CAT
activity. In contrast, cotransfection with variant ER expression
plasmids containing C-terminal (HE15) or N-terminal (HE19) deletions
did not result in induction of CAT activity by E2, and this
was consistent with results of previous studies using
E2-responsive plasmids containing GC-rich elements (25, 26, 28). The results summarized in Fig. 3B demonstrate that in cells
transfected with pADA211, the effects of E2, tamoxifen, ICI
182,780, and E2 plus tamoxifen or ICI 182,780 on CAT
activity were similar to those observed for gene expression (Fig. 1
).
Both E2 and 4'-hydroxytamoxifen induced CAT activity (4.15-
and 3.45-fold, respectively), ICI 182,780 alone did not induce CAT
activity but significantly inhibited E2-induced activity.
CAT activity in cells cotreated with 4'-hydroxytamoxifen plus
E2 was lower than observed after treatment with
E2 alone; however, this decrease was not statistically
significant, suggesting that 4'-hydroxytamoxifen exhibited minimal ER
antagonist activity.
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demonstrate
that [32P]Sp1.4 [contains site IV (-86 to -65) GC-rich
sequence] and consensus [32P]Sp1 oligonucleotides bind
purified Sp1 protein (lanes 2–6) whereas mutant
[32P]Sp1.4m did not form a retarded band (lanes 7 and 8).
In competition experiments (Fig. 4B), [32P]Sp1.4 bound
Sp1 protein (lane 2) and competition with excess unlabeled consensus
Sp1 (lanes 3 and 4) or unlabeled Sp1.4 (lanes 5–7) oligonucleotides
decrease retarded band intensity, whereas competition was not observed
for Sp1.4m (lane 8). The effects of ER on Sp1-DNA binding were
determined using [32P]Sp1.4 plus Sp1 protein alone (Fig. 4C, lane 2) or in combination with 0.2, 0.4, or 0.8 pmol ER (lanes
3–5). The results showed that coincubation with ER resulted in a 2.14-
to 3.75-fold enhancement of Sp1-[32P]Sp1.4 retarded band
intensity; however, supershifted ternary ER/Sp1-DNA complexes were not
observed, and similar results using ER and Sp1 protein with other
GC-rich elements have previously been reported (25, 26, 28). In a
parallel study, it was also shown that [32P]Sp1(1, 2, 3),
which contains three Sp1 sites (I–III) in the -60 to -31 region of
the ADA gene promoter, also bound Sp1 protein (Fig. 5A, lanes 2 and 3), and ER also enhanced
intensity (1.76- to 2.15-fold) of the Sp1(1, 2, 3)-DNA retarded band (Fig. 5B, lanes 2–5). Thus, although the Sp1(1, 2, 3) sequence containing Sp1
binding sites I–III did not confer E2 inducibility on
pADA56 (Fig. 2B) or pADA81m, the overlapping GC-rich element bound Sp1
protein and ER enhanced retarded band formation in gel mobility shift
assays (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and this cell line has been extensively used as a model for
investigating the effects of estrogens and antiestrogens on cell growth
and gene expression (35). E2 induces expression of multiple
genes in MCF-7 or other hormone-responsive cells including pS2,
progesterone receptor, cathepsin D, retinoic acid receptor, heat shock
protein 27, c-fos, myc, mucin 1, and transforming growth
factor- (20, 21, 22, 23, 24, 25, 26, 27, 28, 36, 37, 38, 39, 40, 41, 42, 43, 44). Lippman and co-workers previously reported
that the mitogenic activity of E2 in breast cancer cells is
accompanied by increased DNA synthesis and also increased expression or
activity of several genes that are involved in purine, pyrimidine, and
DNA synthesis (15, 16, 17, 18, 19). The effects of E2 and
tamoxifen/4'-hydroxytamoxifen on genes required for DNA synthesis were
somewhat variable and dependent on growth conditions; however, in some
studies, it was shown that E2 increased thymidine kinase,
dihydrofolate reductase, carbamoylphosphate synthetase, aspartate
transcarbamylase, orotidine pyrophosphorylase, orotidine and
thymilidate synthetase gene expression, or enzyme activities (15, 16, 17, 18, 19).
In preliminary screening assays designed to identify
E2-induced genes associated with DNA synthesis in MCF-7
cells, we observed that ADA mRNA levels were induced by E2
in MCF-7 cells. ADA is primarily a catabolic enzyme in the purine
salvage pathway, and hormone responsiveness of this gene was surprising
since E2 induces enzymes involved in purine and pyrimidine
biosynthesis (15, 16, 17, 18, 19). The role of ADA in modulating hormone-induced
DNA synthesis is unknown and is currently being investigated. The
antiestrogen 4'-hydroxytamoxifen significantly induced ADA mRNA levels
(Fig. 1) and reporter gene activity (Fig. 3B), and the magnitude of the
latter response was similar to that observed for E2 alone.
In cells cotreated with E2 plus 4'-hydroxytamoxifen, there
was a significant decrease in ADA mRNA levels (Fig. 1) but not CAT
activity (Fig. 3B) compared with E2 alone, suggesting the
4'-hydroxytamoxifen was a response-specific ER antagonist. In
contrast, ICI 182,780 did not significantly induce ADA mRNA levels or
reporter gene activity but inhibited both hormone-induced responses
(Figs. 1 and 3B). This profile of ligand-dependent ER activation of ADA
is similar to that observed for other E2-regulated genes in
MCF-7 cells, and the molecular mechanisms of this response were further
investigated by analysis of the ADA gene promoter.
Dusing and Wiginton (10) previously investigated the proximal region of
the ADA gene promoter (-211 to +11) and identified six GC-rich
elements (I–VI) (see Fig. 2) that bound Sp1 protein in gel
electrophoretic mobility shift assays. Moreover, in deoxyribonuclease I
footprinting using recombinant Sp1 protein or MOLT 4 lymphoid cell
extracts, all six Sp1 sites were protected from deoxyribonuclease I
digestion. Recent studies in this laboratory have demonstrated that the
ER and Sp1 protein physically interact and E2-induced
transactivation can be observed by ER/Sp1 binding to GC-rich elements
in which only Sp1 protein binds promoter DNA (25, 26, 28). GC-rich
elements in the distal region of the c-fos protooncogene
promoter were also identified as target sequences for ER activation via
ER/Sp1 interactions (28). These results suggested that one or more of
the six GC-rich site in the proximal region (-211 to +11) ADA gene
promoter may be required for E2 responsiveness. Results of
initial studies (Fig. 2A) demonstrated that basal CAT activity in MCF-7
cells associated with constructs containing promoter inserts from the
proximal region of the ADA gene promoter was strongly associated with
site IV, and the results were similar to those reported in MOLT-4 and
Raji cells (10). The results in Fig. 2 demonstrate that in transient
transfection studies with pADA211, pADA81, and pADA56, which contain
Sp1 sites I-VI, I-IV and I-III, respectively, only the former two
constructs were E2 responsive. The differences in
E2-induced CAT activity observed for pADA81, pADA81m, and
pADA56 suggested that site IV was primarily responsible for ER/Sp1
activation. Moreover, this was supported by the E2
responsiveness of pADASp1.4, which contains the site IV oligonucleotide
insert linked to a bacterial CAT reporter gene.
The results in Fig. 3B also show that both 4'-hydroxytamoxifen and
E2 exhibited ER agonist activity and ICI 182,780 is
primarily an antagonist in MCF-7 cells transiently transfected with
pADA211. These results are consistent with effects of the same ligands
on ADA mRNA levels (Fig. 1) and further support the role of the -211
to +11 promoter sequence in mediating ER action. Previous studies with
constructs containing a consensus GC-rich element or GC-rich sites
derived from the heat shock protein 27 or c-fos gene
promoters showed that E2-dependent induction of reporter
gene activities in breast cancer cells was observed only after
cotransfection with wild-type hER or HE11 that contains AF1 and AF2,
but not the DNA-binding domain of the ER (26, 28). Similar results were
also obtained using pADA81 (Fig. 3A), suggesting that transactivation
is observed without direct binding of ER to specific responsive
elements. As reported earlier (25, 26, 27), cotransfection with variant ERs
expressing AF-1 (HE15) or AF-2 (HE19) did not result in a
hormone-induced response.
It has previously been shown (26) that although both ER and Sp1
physically interact in coimmunoprecipitation and
glutathione-S-transferase pulldown assays, ER does
not supershift an Sp1-[32P]GC-rich retarded band in gel
mobility shift assays. However, ER enhances the intensity of the
retarded band by increasing the rate of formation (on rate) of this
complex (26). Similar results were also observed in this study using
[32P]Sp1.4 (site IV) in gel mobility shift assay.
[32P]Sp1.4 specifically binds Sp1 protein (Fig. 4A) and
competitively decreases binding of Sp1 protein to a consensus
[32P]Sp1 oligonucleotide (Fig. 4B). Moreover,
coincubation of Sp1 protein, [32P]Sp1.4 with different
amounts of ER, resulted in a more than 3.5-fold increase in formation
of the retarded band, and this is consistent with results of previous
studies using GC-rich oligonucleotides (26, 27). Enhancement of protein
DNA-retarded band formation in gel mobility shift assays has been
observed with other proteins in studies showing that human T cell
leukemia virus type 1, sterol-regulatory element-binding protein, and
cyclin D1 enhance bZIP, Sp1, and ER binding to their respective
enhancer sequences without forming ternary supershifted complexes
(45, 46, 47). Thus, E2-responsive Sp1.4 bound Sp1 protein to
form a retarded band, and coincubation with ER enhanced retarded band
intensity (Fig. 4). However, these in vitro binding
properties in gel mobility shift assays were not necessarily diagnostic
of functional activity since the Sp1(1, 2, 3) oligonucleotide (contains
GC-rich sites I–III) also bound Sp1 protein to form a retarded band
(Fig. 5A), and coincubation with ER enhanced retarded band intensity
(Fig. 5B). However, in transactivation assays with constructs
containing only sites I–III (pADA56 and pADA81m), E2 did
not induce reporter gene activity. In contrast to results observed in
this study showing that only one of six GC-rich sites was responsible
for ER action in the ADA gene promoter, a recent study showed that all
three GC-rich elements in the proximal region of retinoic acid receptor
1 gene promoter contributed to E2-induced
transactivation (48).
Mammalian and viral gene promoters contain multiple GC-rich elements
that bind Sp1 protein to play an important role in basal transcription
of these genes (49, 50). Activator protein-1 (AP-1) sites are also
important promoter elements for gene regulation, and recent studies
have now shown that both AP-1 and GC-rich elements in some gene
promoters are E2 responsive via ER-AP-1 and ER-Sp1 protein
complexes (26, 27, 51, 52). However, since only a small number of
mammalian genes are E2 responsive, relatively few AP-1 or
GC-rich elements will function as enhancer elements for ER activation.
The results of this study demonstrate that in the proximal region of
the ADA gene promoter, only one of the six GC-rich elements (site IV)
is required for E2 responsiveness. Interestingly, this same
element was also important for Sp1-dependent basal activity of the
-211 to +11 region of this promoter (Fig. 2A) (10). The reasons for
selective ER/Sp1 action at site IV could be associated with
preferential cell-specific interactions with other nuclear factors such
as coactivators or corepressors, and this is currently being
investigated.
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Footnotes |
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2 Sid Kyle Professor of Toxicology.
Received June 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene in human breast carcinoma cells
is mediated via an imperfect half-palindromic estrogen response element
and Sp1 motifs. Cancer Res 55:4999–5006 and its messenger RNA in human breast
cancer: its regulation by estrogen and its possible functional
significance. Mol Endocrinol 2:543–5551 gene expression: role of estrogen
receptor-Sp1 complex. Mol Endocrinol 12:882–890 and ERß at AP1 sites. Science 277:1508–1510This article has been cited by other articles:
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D. Lopez, M. D. Sanchez, W. Shea-Eaton, and M. P. McLean Estrogen Activates the High-Density Lipoprotein Receptor Gene via Binding to Estrogen Response Elements and Interaction with Sterol Regulatory Element Binding Protein-1A Endocrinology, June 1, 2002; 143(6): 2155 - 2168. [Abstract] [Full Text] [PDF]
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J. L. Brockman, M. D. Schroeder, and L. A. Schuler PRL Activates the Cyclin D1 Promoter Via the Jak2/Stat Pathway Mol. Endocrinol., April 1, 2002; 16(4): 774 - 784. [Abstract] [Full Text] [PDF]
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J. Shimada, Y. Suzuki, S.-J. Kim, P.-C. Wang, M. Matsumura, and S. Kojima Transactivation via RAR/RXR-Sp1 Interaction: Characterization of Binding Between Sp1 and GC Box Motif Mol. Endocrinol., October 1, 2001; 15(10): 1677 - 1692. [Abstract] [Full Text] [PDF]
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I. Samudio, C. Vyhlidal, F. Wang, M. Stoner, I. Chen, M. Kladde, R. Barhoumi, R. Burghardt, and S. Safe Transcriptional Activation of Deoxyribonucleic Acid Polymerase {{alpha}} Gene Expression in MCF-7 Cells by 17{{beta}}-Estradiol Endocrinology, March 1, 2001; 142(3): 1000 - 1008. [Abstract] [Full Text] [PDF]
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W. Xie, R. Duan, I. Chen, I. Samudio, and S. Safe Transcriptional Activation of Thymidylate Synthase by 17{beta}-Estradiol in MCF-7 Human Breast Cancer Cells Endocrinology, July 1, 2000; 141(7): 2439 - 2449. [Abstract] [Full Text] [PDF]
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L. Salvatori, L. Ravenna, M. P. Felli, M. R. Cardillo, M. A. Russo, L. Frati, A. Gulino, and E. Petrangeli Identification of an Estrogen-Mediated Deoxyribonucleic Acid-Binding Independent Transactivation Pathway on the Epidermal Growth Factor Receptor Gene Promoter Endocrinology, June 1, 2000; 141(6): 2266 - 2274. [Abstract] [Full Text] [PDF]
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A. van den Wijngaard, W. R. Mulder, R. Dijkema, C. J .C. Boersma, S. Mosselman, E. J. J. van Zoelen, and W. Olijve Antiestrogens Specifically Up-Regulate Bone Morphogenetic Protein-4 Promoter Activity in Human Osteoblastic Cells Mol. Endocrinol., May 1, 2000; 14(5): 623 - 633. [Abstract] [Full Text]
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Y. Liu, X. Zhong, W. Li, M. G. Brattain, and S. S. Banerji The Role of Sp1 in the Differential Expression of Transforming Growth Factor-beta Receptor Type II in Human Breast Adenocarcinoma MCF-7 Cells J. Biol. Chem., April 14, 2000; 275(16): 12231 - 12236. [Abstract] [Full Text] [PDF]
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 G. Liu, J. A. Schwartz, and S. C. Brooks Estrogen Receptor Protects p53 from Deactivation by Human Double Minute-2 Cancer Res., April 1, 2000; 60(7): 1810 - 1814. [Abstract] [Full Text]
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B. Saville, M. Wormke, F. Wang, T. Nguyen, E. Enmark, G. Kuiper, J.-A. Gustafsson, and S. Safe Ligand-, Cell-, and Estrogen Receptor Subtype (alpha /beta )-dependent Activation at GC-rich (Sp1) Promoter Elements J. Biol. Chem., February 25, 2000; 275(8): 5379 - 5387. [Abstract] [Full Text] [PDF]
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H. Inadera, T. Sekiya, T. Yoshimura, and K. Matsushima Molecular Analysis of the Inhibition of Monocyte Chemoattractant Protein-1 Gene Expression by Estrogens and Xenoestrogens in MCF-7 Cells Endocrinology, January 1, 2000; 141(1): 50 - 59. [Abstract] [Full Text] [PDF]
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L. Dong, W. Wang, F. Wang, M. Stoner, J. C. Reed, M. Harigai, I. Samudio, M. P. Kladde, C. Vyhlidal, and S. Safe Mechanisms of Transcriptional Activation of bcl-2 Gene Expression by 17beta -Estradiol in Breast Cancer Cells J. Biol. Chem., November 5, 1999; 274(45): 32099 - 32107. [Abstract] [Full Text] [PDF]
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R. Duan, W. Porter, I. Samudio, C. Vyhlidal, M. Kladde, and S. Safe Transcriptional Activation of c-fos Protooncogene by 17{beta}-Estradiol: Mechanism of Aryl Hydrocarbon Receptor-Mediated Inhibition Mol. Endocrinol., September 1, 1999; 13(9): 1511 - 1521. [Abstract] [Full Text]
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W. Wang, L. Dong, B. Saville, and S. Safe Transcriptional Activation of E2F1 Gene Expression by 17{beta}-Estradiol in MCF-7 Cells Is Regulated by NF-Y-Sp1/Estrogen Receptor Interactions Mol. Endocrinol., August 1, 1999; 13(8): 1373 - 1387. [Abstract] [Full Text]
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C. Qin, P. Singh, and S. Safe Transcriptional Activation of Insulin-Like Growth Factor-Binding Protein-4 by 17{beta}-Estradiol in MCF-7 Cells: Role of Estrogen Receptor-Sp1 Complexes Endocrinology, June 1, 1999; 140(6): 2501 - 2508. [Abstract] [Full Text]
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N. Tanaka, H. Yonekura, S.-i. Yamagishi, H. Fujimori, Y. Yamamoto, and H. Yamamoto The Receptor for Advanced Glycation End Products Is Induced by the Glycation Products Themselves and Tumor Necrosis Factor-alpha through Nuclear Factor-kappa B, and by 17beta -Estradiol through Sp-1 in Human Vascular Endothelial Cells J. Biol. Chem., August 11, 2000; 275(33): 25781 - 25790. [Abstract] [Full Text] [PDF]
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M. Stoner, F. Wang, M. Wormke, T. Nguyen, I. Samudio, C. Vyhlidal, D. Marme, G. Finkenzeller, and S. Safe Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor alpha and Sp3 Proteins J. Biol. Chem., July 21, 2000; 275(30): 22769 - 22779. [Abstract] [Full Text] [PDF]
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K. J. Scheidegger, B. Cenni, D. Picard, and P. Delafontaine Estradiol Decreases IGF-1 and IGF-1 Receptor Expression in Rat Aortic Smooth Muscle Cells. MECHANISMS FOR ITS ATHEROPROTECTIVE EFFECTS J. Biol. Chem., December 1, 2000; 275(49): 38921 - 38928. [Abstract] [Full Text] [PDF]
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E. J. Cram, B. D. Liu, L. F. Bjeldanes, and G. L. Firestone Indole-3-carbinol Inhibits CDK6 Expression in Human MCF-7 Breast Cancer Cells by Disrupting Sp1 Transcription Factor Interactions with a Composite Element in the CDK6 Gene Promoter J. Biol. Chem., June 15, 2001; 276(25): 22332 - 22340. [Abstract] [Full Text] [PDF]
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E. Castro-Rivera, I. Samudio, and S. Safe Estrogen Regulation of Cyclin D1 Gene Expression in ZR-75 Breast Cancer Cells Involves Multiple Enhancer Elements J. Biol. Chem., August 10, 2001; 276(33): 30853 - 30861. [Abstract] [Full Text] [PDF]
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B. Saville, H. Poukka, M. Wormke, O. A. Janne, J. J. Palvimo, M. Stoner, I. Samudio, and S. Safe Cooperative Coactivation of Estrogen Receptor alpha in ZR-75 Human Breast Cancer Cells by SNURF and TATA-binding Protein J. Biol. Chem., January 18, 2002; 277(4): 2485 - 2497. [Abstract] [Full Text] [PDF]
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