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Department of Veterinary Physiology and Pharmacology, Texas A&M University (C.Q., S.S.), College Station, Texas 77843-4466; and the Department of Anatomy and Neurosciences (P.S.), University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: Dr. Stephen 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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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-4 has also been widely detected in breast tumors and cells in
culture, and it has been reported that IGFBP-4 expression positively
correlated with ER status in mammary tumors (12, 33).
E2 induces IGFBP-4 messenger RNA (mRNA) and
protein levels in ER-positive breast cancer cells, and antiestrogens
inhibited these responses (34, 35, 36, 37); however, the molecular mechanisms
of ER action have not been determined. Structural and functional
analyses of the IGFBP-4 gene promoter have recently been reported
(38, 39), and the 5'-promoter region contains multiple
cis-elements including cAMP response elements, activated
protein-1 (AP-1)/AP-2 sites, Egr-1 sites, and GC-rich sequences. This
study shows that E2 induces IGFBP-4 expression,
and analysis of the 5'-promoter region has identified GC-rich sites at
-559 to -553 and -72 to -64 that bind Sp1 protein and are important
for E2 responsiveness. ER/Sp1 action at GC-rich
sites has previously been reported for cathepsin D, retinoic acid
receptor 
1, and c-fos genes and does not require direct
interaction of the ER with genomic DNA (40, 41, 42, 43, 44). The results obtained
in this study for IGFBP-4 further extend the number of
E2-responsive genes regulated by this
transcription factor complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP (3000 Ci/mmol) and
[-32P]CTP (3000 Ci/mmol) were purchased from NEN Research Products (Boston, MA). Horseradish peroxidase substrate
for Western blot analysis was purchased from DuPont NEN
(Boston, MA). Hybond-N nylon membrane for Northern blot analysis and
Hybond enhanced chemiluminescence nitrocellulose membrane for
Western blot analysis were purchased from Amersham International (Aylesbury, UK). IGFBP-4 antibodies were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Polydeoxy-(inosinic-cytidylic) acid [polyd(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 provided
by Dr. Ming-jer Tsai (Baylor College of Medicine, Houston, TX).
Recombinant human Sp1 protein, reporter lysis buffer, and
luciferase reagent for luciferase studies were purchased from
Promega Corp. (Madison, WI), and baculovirus-expressed hER
proteins were obtained from Panvera (Madison, WI). ß-Galactosidase
(ß-Gal) reagent was purchased from Tropix (Bedford, MA). The plasmid
preparation kit was purchased from Qiagen (Chatsworth,
CA). All other chemicals and biochemicals were the highest quality
available from commercial sources. All primers and oligonucleotides
used in this study were synthesized and/or sequenced by Texas
Agricultural Experiment Station, Department of Veterinary Pathobiology,
Texas A&M University (College Station, TX). InstantImager and LumiCount
were purchased from Packard (Meriden, CT).
Northern blot analysis
MCF-7 cells were seeded in 5% charcoal-stripped FBS/DMEM/Ham’s
F-12 medium for 24 h and in serum-free DMEM/Ham’s F-12 medium for
another 24 h. Fresh serum-free medium was then used, and cells
were treated with E2, antiestrogens, or
dimethylsulfoxide (DMSO) for different times before harvesting. Total
RNA was isolated using the STAT-60 Kit (Tel-Test). Twenty
micrograms of total RNA were diluted in 2 x FPF [20%
formaldehyde, 1.65% Na2HPO4 (pH 6.8), 63.5%
formamide, and 1 x loading buffer] and separated on 1.2%
agarose gel with 1 M formaldehyde in 1 x SPC buffer.
After transfer onto Hybond-N nylon membrane (Amersham),
the blots were prehybridized and hybridized in NENhybe solution (5
x SSPE, 10% dextran sulfate, 0.1% polyvinylpyrolidine, 0.1% Ficoll,
0.1% BSA, and 1% SDS) at 65 C without or with
[32P]CTP-labeled IGFBP-4 complementary DNA probe for
16 h. Blots were visualized by autoradiography and quantitated on
InstantImage (Packard). The membrane was then stripped and rehybridized
with ß-tubulin probe as a control. The IGFBP-4 c-DNA probe was
determined by RT-PCR using the following primers: IGFBP-4 forward,
5'-TGC AGA AGC ACT TCGCCA AA-3' (+702/+721); and IGFBP-4 reverse,
5'-ACA GGA CTC AGA CTC AGA CT-3' (+1141/+1160).
Western blot analysis
MCF-7 cells were seeded in 5% charcoal-stripped FBS/DMEM/Ham’s
F-12 medium in six-well plates. At 85% cell confluence, cells were
washed twice with sterile PBS buffer and incubated in serum-free
DMEM/Ham’s F-12 medium for 24 h. Cells were then changed to fresh
serum-free DMEM/Ham’s F-12 medium and treated with
E2 or DMSO (solvent control). After incubation
for 24 h, cells were changed to 0.75 ml fresh serum-free medium
and treated again. After 16–24 h, conditioned medium was collected,
and cells in each well were counted to normalize results. The
conditioned medium was concentrated using microconcentrators
(12,000 x g, 60 min) to less than 100 µl and
transferred to fresh 1.5-ml tubes. Samples were boiled for 2 min,
separated on 12% SDS-PAGE at 180 V for 4 h, and transferred to
Hybond enhanced chemiluminescence nitrocellulose membrane
(Amersham) at 14 V overnight at 4 C. Nitrocellulose
membranes were then soaked in 5% milk/Tris-buffered saline with
gentle shaking for 15 min and incubated in fresh 5% milk-TBS with
1:500–1000 primary antibody (Santa Cruz Biotechnology, Inc.) for 1–2 h with gentle shaking. After washing with TBS for
15 min (once) and 5 min (twice), the membrane was incubated in 5%
milk-TBS with 1:1000–2000 secondary antibody for 1–2 h with gentle
shaking. The membrane was washed with TBS for 15 min (once) and 5 min
(twice); 10 ml horseradish peroxidase substrate (DuPont NEN) were then added and incubated for 1.0 min. The membrane was
exposed to Kodak X-Omat film (Eastman Kodak Co.,
Rochester, NY), visualized by autoradiography, and quantitated by
densitometry using the Molecular Dynamics, Inc. Zero-D
software package (Sharp Corp., Mahwah, NJ).
Cloning
pXP2 luciferase reporter plasmid (American Type Culture Collection) was modified with the insertion of TATA sequence
into its polylinker site immediately upstream of the luciferase
expression gene. IGFBP-4 promoter fragments (-1214 to -379, -575 to
-379, -569/-569m to -540, and -83/-83m to -54) were
amplified/enzyme cut or synthesized as double stranded DNA with
5'-overhangs and inserted into the vector between HindIII
and BamHI polylinker sites; fragments (-373 to +18 and
-125 to +18) that contain TATA sequences were inserted into pGL2
luciferase reporter plasmid (Promega Corp.) at
KpnI and XhoI sites. All plasmids are designated
with a p, followed by the size of the promoter insert. All ligation
products were transformed into competent Escherichia coli
cells. Plasmids were isolated and clones were confirmed by restriction
enzyme mapping and DNA sequencing. High quality plasmids for
transfection were prepared using QIAGEN Plasmid Mega Kit.
The sequences of primers and oligonucleotides are listed below. GC-rich
elements are capitalized, and mutations in the oligonucleotides are
underlined; (s)1 means sense; (m)** means mutant: -1214
forward primer, cca agc ttc tcg tga tct gcc; -575 forward primer, cca
agc ttc cct ggg gag a; -354 reverse primer, aga aag gga ctt cct a;
-373 forward primer, gcg gta ccc aga gcc ggg agt cc; -125 forward
primer, gcg gta ccg cga ctc agg aca gc; +18 reverse primer, cga gct cgg
cag ggg gct gag; -569 oligonucleotide(s)1(-569 to -540), agc ttg gga
gat tgc gGG GGC GGG aga ggt tgc aag; -569 oligonucleotide(s)1(m)**,
agc ttg gga gat tgc gGA ATC TTG aga ggt tgc
aag; -83 oligonucleotide(s)1(-83 to -54), agc ttt ctc ccc ctc gCC
CGC CCC ggc tcc ccc acg; -83 oligonucleotide (s)1(m)**, agc ttt ctc
ccc ctc gCA AGA TCC ggc tcc ccc acg; and
consensus Sp1 oligonucleotide(s)1, agc tta ttc gat cgg ggc ggg gcg agc
g.
Transient transfection and luciferase activity assay
Cultured MCF-7 cells were seeded in charcoal-stripped
FBS/DMEM/Ham’s F-12 medium in 60-mm plates 1 day before transfection.
Five micrograms of test plasmid, 2.5 µg wild-type hER, and
ß-Gal-lacZ plasmid (1.0 µg) obtained from
Invitrogen (Carlsbad, CA) were cotransfected into MCF-7
cells using the calcium phosphate-DNA coprecipitation method. After
incubation for 16–20 h, cells were washed with PBS and treated with 10
nM E2 or DMSO (as control)
in fresh medium for 40 h. Cells were then washed with PBS and
lysed with 400 µl 1 x reporter lysis buffer
(Promega Corp.). Cell lysate was frozen in liquid nitrogen
and thawed at room temperature; 20 µl cell extract were assayed with
luciferase (Promega Corp.) and ß-Gal reagents (Tropix).
LumiCount (Packard) was used to quantitate luciferase and ß-Gal
activities. The luciferase/ß-Gal ratio was used to represent
normalized luciferase activity for each treatment group.
Gel electrophoretic mobility shift assay (GEMSA)
Oligonucleotides were synthesized, purified, and annealed, and
10 pmol of specific oligonucleotides were 32P-labeled at
the 5'-end using T4 polynucleotide kinase and
[-32P]ATP. GEMSAs were performed by incubating varying
amounts of recombinant human Sp1 protein (Promega Corp.)
in 25 µl 1 x binding buffer (5% glycerol, 0.477% HEPES,
0.546% KCl, 1 mM EDTA, and 0.4 mM
dithiothreitol, pH 8.0) and 0.16 mg/ml BSA. After incubation for 10 min
at 4 C, 32P-labeled oligonucleotides (100,000 cpm) were
added to the reaction mixture in the presence of 1 µl polyd(I-C) and
0.33% Ficoll and incubated for an additional 15 min at 25 C.
Competition studies were carried out with excess unlabeled DNA before
the addition of 32P-labeled oligonucleotides. The following
procedures were used for ER-enhanced Sp1 binding studies: 1) 0–400
fmol pure hER protein (Panvera) in 1 x binding buffer containing
40 mM E2 and BSA were incubated for
10 min at 4 C; 2) different amounts of Sp1 protein were added to the
mixture and incubated on ice for 5 min; 3) 32P-labeled
oligonucleotides (100,000 cpm) were added to the reaction mixture in
the presence of 1 µl polyd(I-C), and the mixture was incubated for
another 15 min at 25 C. Five percent polyacrylamide gel
(acrylamide-bisacrylamide, 30:0.8) was used to separate the reaction
mixture. Electrophoresis was carried out at 110 V in 1 x TBE (0.9
M Tris-borate and 2 mM EDTA, pH 8.3). Gels were
dried, and protein-DNA interactions were determined by scanning on an
InstantImage (Packard) and visualized by autoradiography.
Statistical analysis
Statistical differences between treatment groups were determined
by ANOVA and Scheffe’s test for significance. The data are presented
as the mean ± SD, and at least three determinations
were carried out for each treatment group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Northern analysis showed that
E2 significantly induced IGFBP-4 mRNA levels
within 2 h after treatment, and a more than 3-fold increase was
observed after 6 h; moreover, 10-6 M
4'-hydroxytamoxifen and 10-6 M ICI 182,780
inhibited E2-induced mRNA levels. A more than
6-fold increase in the 28-kDa glycosylated form of IGFBP-4 was detected
in conditioned medium by IGFBP-4 antibodies 6 h after treatment
with E2.
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). The loss of activity for
p-125/+18 indicated that elements within the -373 to -125 region of
the IGFBP-4 gene promoter were essential for maximal basal activity in
MCF-7 cells. E2 caused an approximately 3-fold
increase in luciferase activity in MCF-7 cells transfected with the
full-length construct (-1214 to +18) and induction was more than
10-fold using the 3'-deletion construct p-1214/-379 (Fig. 2B). The
E2 responsiveness of this upstream region of the
IGFBP-4 gene promoter was further investigated by 5'- and 3'-deletion
analysis (p-575/-379, p-569/-540, and p-569/-540m), and the
GC-rich site at -559 to -553 was identified as the important upstream
cis-element required for ER action. The downstream -373 to
+18 (p-373/+18) region of the promoter was also
E2 responsive and contained two GC-rich sites at
-313 to -305 and -72 to -64. Transient transfection of MCF-7 cells
with p-373/+18, p-125/+18, or p-83/-54 showed that only the -72 to
-64 GC-rich site was required for hormone responsiveness in MCF-7
cells (Fig. 2B). Mutation of the -72 to -64 GC-rich site
(p-83/-54m) resulted in the loss of E2
responsiveness.
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compare the
binding of human recombinant Sp1 protein to consensus
[32P]Sp1 oligonucleotide (lane 2),
[32P]-569 (lanes 3–6), and mutant
[32P]-569m probes (lanes 7 and 8). A retarded band with
similar mobility (see arrow) was observed only for the
consensus [32P]Sp1 oligonucleotide and
[32P]-569 probe. The specificity for
[32P]-569-Sp1 binding was confirmed in competitive
binding studies (Fig. 3B), showing that the intensity of the retarded
band was decreased after competition with excess unlabeled -569 probe
(lanes 3 and 4) and consensus Sp1 oligonucleotide (lanes 6 and 7), but
not mutant -569m probe (lane 5) or mutant Sp1m oligonucleotide (lane
8). The effects of recombinant hER protein on formation of the
[32P]-569-Sp1 retarded band were also investigated by
GEMSA (Fig. 3C). The intensity of the retarded band (lane 2) was
incrementally increased after the addition of 0.1, 0.2, and 0.4 pmol
recombinant human ER protein (lanes 3–5); the retarded band intensity
was decreased after incubation with excess unlabeled -569 probe (lane
6), but was unaffected by coincubation with mutant -569m probe (lane
7). ER protein alone did not form a retarded band with
[32P]-569 (lane 8). Recombinant human Sp1 protein also
formed a retarded band with [32P]Sp1 oligonucleotide
(Fig. 4A, lanes 1
and 2) and [32P]-83 probe (lanes 3 and 4), but not with
mutant [32P]-83m (lanes 5 and 6). The specifically bound
[32P]-83-Sp1 band (Fig. 4B, lane 2) was competitively
decreased by competition with excess unlabeled -83 probe (lanes 3 and
4) and Sp1 oligonucleotide (lanes 6 and 7), but not by mutant -83
probe (lane 5) or Sp1m (lane 8). Recombinant human ER protein alone did
not bind [32P]-83 probe (Fig. 4C, lane 8); however, ER
enhanced retarded band intensity after coincubation with Sp1
protein/[32P]-83 (lanes 3–5) compared with the band
formed with Sp1 protein alone (lane 2). The intensity of the
specifically bound retarded band was decreased after competition with
unlabeled wild-type -83 probe (lane 6), and band intensity only
slightly decreased after competition with -83m probe. The latter
result could be attributed to upstream (CCCCCTCG) and downstream
(CCCCCACG) motifs that could also bind Sp1 protein. These results are
consistent with previous studies using 32P-labeled
consensus Sp1 oligonucleotide and GC-rich motifs in the cathepsin D,
c-fos, and retinoic acid receptor 1 gene promoters
(40, 41, 42, 43, 44).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) confirm that IGFBP-4 is E2
responsive. Qin and co-workers (39) previously analyzed the promoter
activity of the IGFBP-4 gene 5'-flanking region in COS-7 monkey kidney
cells and three human osteoblast-like osteosarcoma (SaOS-2, TE-85, and
MG-63) cell lines. Deletion analysis showed that maximal basal activity
in all four cell lines required the -836 to -54 regions of the
IGFBP-4 gene promoter, and important elements that may contribute to
this activity include putative Sp1, AP-2, and EGR-1 sites (39). Results
obtained in MCF-7 cells (Fig. 2) demonstrate that high basal activity
is primarily associated with the -373 to -125 region of the IGFBP-4
gene promoter containing a GC-rich Sp1-binding site at -313 to -305
and an AP-2 site centered at -247.
Multiple cis-elements have been identified within the
5'-promoter region of the IGFBP-4 gene; these include a TATAA element
(-18 to -14), binding sites for ATF/CREB (-1169 to -1161), AP-1
(-855 to -859), early growth response factor-1 (-562 to -554), an
Alu repetitive element (-1150 to -848), and numerous GC-rich
Sp1-binding sites (-1204 to -1199, -904 to -899, -559 to -553,
-313 to -305, and -72 too -64) (38, 39). Classical palindromic
estrogen-responsive elements were not detected in the IGFBP-4 gene
promoter; however, recent studies show that
E2-induced trans-activation can be
mediated through ER interactions with DNA-bound AP-1 or Sp1 proteins
(40, 41, 42, 43, 44, 50). Deletion analysis of the IGFBP-4 gene promoter showed
that constructs containing both upstream (p-1214/-379) and downstream
(p-373/+18) sequences were E2 responsive in
transient transfection studies in MCF-7 cells (Fig. 2B). Subsequent
deletion analysis showed that E2 responsiveness
was primarily associated with the upstream GC-rich sequence at -559 to
-553 (p-569/-540). In addition, E2 induced
luciferase activity (2-fold) in MCF-7 cells transfected with
p-83/-54, but not -85/-54m (GC mutations), suggesting that the
downstream Sp1-binding site at -72 to -64 was also weakly
E2 responsive.
Results of GEMSAs with [32P]-569 and
[32P]-83 (Figs. 3 and 4) demonstrated that both
oligonucleotides formed specifically bound Sp1-DNA complexes.
Coincubation with ER did not result in a supershifted ER/Sp1-DNA
ternary complex, but caused a 2-fold increased intensity of the binary
Sp1-DNA retarded band. Previous gel mobility shift studies using
32P-labeled consensus Sp1 oligonucleotide or similar
GC-rich sequences from the c-fos, retinoic acid receptor
1, adenosine deaminase, and cathepsin D gene promoters also showed
that wild-type ER enhanced the rate of Sp1-DNA complex formation
(40, 41, 42, 43, 44) and increased (2- to 3-fold) the binding capacity for the
retarded band; this was comparable to results obtained with
[32P]-569 and [32P]-83 (Figs. 3 and 4).
Coimmunoprecipitation and pulldown assays using chimeric
glutathione-S-transferase Sp1 and ER proteins confirmed that
ER and Sp1 physically interact, and ER preferentially bound to the
C-terminal regions of Sp1 protein (40). The failure of ER to form a
supershifted ternary ER/Sp1-DNA complex is not unprecedented, as 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 (51, 52, 53).
In summary, the results of this study have
identified GC-rich regions in the IGFBP-4 gene promoter that are
important for E2 responsiveness. ER/Sp1-mediated
trans-activation through GC-rich sites plays an important
role in the induction of several genes, including c-fos,
cathepsin D, and retinoic acid receptor 1 in MCF-7 cells, and this
complements ER/Sp1 action through Sp1(N)xERE half-sites
identified in c-myc, creatinine kinase B, cathepsin D, and
heat shock protein 27 genes (54, 55, 56, 57). The importance of the Sp1 protein
for transcriptional activation by members of the nuclear receptor
superfamily is not confined to the ER, as progesterone receptor-Sp1
complex interactions with GC-rich sites in the p21 gene promoter are
required for progesterone-mediated trans-activation (58).
Current studies in this laboratory are further investigating the
integrating role of Sp1 in the cell-specific regulation of several
E2-responsive genes and genes regulated via other
nuclear transcription factors.
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Footnotes |
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Received September 21, 1998.
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1 gene expression: role of estrogen
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D. Zhang, X.-L. Zhang, F. J. Michel, J. L. Blum, F. A. Simmen, and R. C. M. Simmen Direct Interaction of the Kruppel-like Family (KLF) Member, BTEB1, and PR Mediates Progesterone-Responsive Gene Expression in Endometrial Epithelial Cells Endocrinology, January 1, 2002; 143(1): 62 - 73. [Abstract] [Full Text] [PDF]
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S. A. Krieg, A. J. Krieg, and D. J. Shapiro A Unique Downstream Estrogen Responsive Unit Mediates Estrogen Induction of Proteinase Inhibitor-9, a Cellular Inhibitor of IL-1{beta}- Converting Enzyme (Caspase 1) Mol. Endocrinol., November 1, 2001; 15(11): 1971 - 1982. [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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 S. Nilsson, S. Makela, E. Treuter, M. Tujague, J. Thomsen, G. Andersson, E. Enmark, K. Pettersson, M. Warner, and J.-A. Gustafsson Mechanisms of Estrogen Action Physiol Rev, October 1, 2001; 81(4): 1535 - 1565. [Abstract] [Full Text] [PDF]
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P. G. V. Martini and B. S. Katzenellenbogen Regulation of Prothymosin {alpha} Gene Expression by Estrogen in Estrogen Receptor-Containing Breast Cancer Cells via Upstream Half-Palindromic Estrogen Response Element Motifs Endocrinology, August 1, 2001; 142(8): 3493 - 3501. [Abstract] [Full Text] [PDF]
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 C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [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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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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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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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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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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