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Tumor Biology |
Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences [H. I., K. N., M. O., E. J. C., Y. N., K. Su.], and Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry [H. I., K. Sh.], Kyushu University, Fukuoka 812-0054, and Division of Gene Therapy Science, School of Medicine, Osaka University, Osaka [Y. K.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-). Oligodeoxynucleotides (ODNs) were synthesized with
either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a
mutated form of this sequence (mt-Sp1 decoy ODNs). Using the
hemagglutinating virus of Japan (HVJ)-liposome method, we transferred
these ODNs into cultured cancer cells (A549 and U251 cells). The
TNF--mediated expression of both VEGF and transforming growth factor
ß1 and tissue factor (TF) by the cancer cells could be
simultaneously suppressed to less than 30% by transfection of Sp1
decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in
vitro invasiveness, synthesis of mRNA for urokinase-type
plasminogen activator, and cell proliferation of both cell lines were
also inhibited to 40% by the transfection of only Sp1 decoy ODNs.
These results suggested that the Sp1 decoy strategy would be effective
for regulating tumor growth by simultaneously reducing cancer cell
(a) angiogenic growth factor expression,
(b) proliferation, and (c) invasiveness. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the VEGF promoter region, there are several consensus sequences of
the binding sites for transcription factors AP-1, AP-2, HIF-1, and Sp1
(22)
. Expression of the VEGF gene in
tumor cells is thought to be induced by hypoxia. Recently, expression
of the VEGF gene was reported to be stimulated by TNF-,
produced by tumor-associated macrophages, through the action of
transcription factor Sp1 (23)
.
In this study, we examined the inhibitory effects of a
cis-trans element decoy containing Sp1 binding sites on the
TNF--induced synthesis of VEGF by tumor cells.
TGF-ß1 is multipotent with marked effects on
cell proliferation, differentiation, adhesion, and migration, as well
as other activities (24)
. It is also reported to be
regulated by Sp1 activation (25)
. Although the mechanism
of the angiogenic effects of TGF-ß1 is not
completely clear, TGF-ß1 stimulates
angiogenesis in vivo by local injection (26)
and in vitro via the stimulation of tube formation by
cultured capillary endothelial cells (27)
. TF is a
transmembrane glycoprotein that can be a potent cofactor of FVIIa. The
TF/FVIIa complex initiates the coagulation cascade leading to thrombin
formation. TF is thought to be a genuine receptor inducing an
intracellular signaling response on binding to its specific ligand,
FVII. TF is reported to be highly expressed in several cancer cells and
enhances the metastatic potential of these cells (28
, 29)
.
In addition, TF is reported to affect tumor growth (30)
.
Furthermore, a recent report indicated that VEGF synthesis is dependent
on TF in response to FVIIa (31)
. This suggests that TF may
influence tumor angiogenesis through regulation of VEGF synthesis. TF
transcription is reported to be transactivated by NF-
B, AP-1,
and Sp1 under several stimuli. The TNF--induced transcription of the
TF gene is thought to be regulated by Sp1 (32)
.
Simultaneous inhibition of the synthesis of these angiogenic factors and other proteins related to cell migration and proliferation, such as uPA, by altering Sp1 action via Sp1 decoy strategy, should dramatically suppress tumor growth. This study examined the inhibitory effects of a cis-trans element decoy against Sp1 binding site on the in vitro expression of angiogenesis-related factors and the growth and invasiveness of cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis of Sp1 Decoy ODNs and Selection of Target Sequences.
ODNs included the binding sequence (underlined) for transcriptional
factor Sp1 centrally and dummy sequences upstream and downstream from
the Sp1 binding sequence (33)
. Double-stranded ODNs were
prepared by incubation at 90°C for 5 min and were designated "Sp1
decoy ODNs." Mutated Sp1 decoy ODNs (designated "mt-Sp1 decoy
ODNs") were mutated at two positions (shown as lowercase letters) of
the Sp1 binding sequence found in the Sp1 decoy ODNs. Inspection of the
resulting nucleotide sequences showed no sequence homologous to other
known transcription factors using databases on transcriptional
regulation (34)
: Sp1 decoy ODNs,
5'-ATTACCGGGCGGGCGGGCTAC-3' and
3'-GTAGCCCGCCCGCCCGGTAAT-5'; mt-Sp1 decoy ODNs, 5'-ATTACCGG t a
GGTAGGGCTAC-3' and 3'-GTAGCCCT a t CTACCGGTAAT-5'.
To enhance the transport of the transfected decoy ODNs to the nucleus, protein-DNA complexes with high mobility group proteins HMG-1 and -2 were prepared before transfection by incubating the ODNs at 20°C for 20 min with high mobility group proteins HMG-1 and -2.
HVJ-Liposome Preparation.
In this study, we transferred ODNs into cancer cells using the
HVJ-liposome method (35)
. Briefly, phosphatidylserine
(sodium salt), phosphatidylcholine, and cholesterol (all purchased from
Sigma Chemical Company, St. Louis, MO) were mixed in a weight ratio of
1:4.8:2 in 3.9 ml of tetrahydrofuran. The lipid mixture (10 mg) was
deposited on the sides of the flask by removing the organic solvent in
a rotary evaporator. Dried lipid was hydrated in 200 µl of Sp1 or
mt-Sp1 decoy ODNs, and the mixture was vigorously shaken in a Vortex
mixer for 30 s. After the addition of 300 µl of BSS [140
nmol/liter NaCl, 5.4 nmol/liter KCl, 10 mmol/liter Tris-HCl (pH 7.6)],
the sample was placed on a mechanical reciprocal shaker (120
strokes/min) at 37°C for 30 min. Purified HVJ (Z strain) was
inactivated by fragmentation of genomic RNA by UV irradiation (11
J/m2
/s) for 120 s, and 30,000 HAU of the HVJ
solution were added to the liposome suspension. The total volume was
brought to 4 ml through the addition of BSS. The mixture was incubated
at 4°C for 10 min to adhere HVJ to liposomes. The mixture was
incubated with gentle shaking at 37°C for 60 min to fuse liposome
membrane and HVJ. Free HVJ was removed from HVJ-liposomes by sucrose
density gradient centrifugation at 60,000 x g at 4°C for 3 h.
The HVJ-liposomes-containing Sp1 or mt-Sp1 decoy ODNs were stored at 4°C and used for the following transfection study. HVJ-liposomes prepared without ODNs (empty HVJ vehicles) were used as a control.
Gene Transfer.
A549 and U251 cells were grown to 50% confluency. The 10%
FBS-supplemented RPMI 1640 was replaced with FBS-free RPMI 1640 and was
harvested 12 h later. The serum-free medium was exchanged for
HVJ-liposome solution in balanced salt solution (10 mM
Tris-HCl, 137 mM NaCl, 5.4 mM KCl; 100
HVJ-liposome particles/cell), containing either decoy ODNs or no ODNs.
Transfer of the ODNs was allowed to occur by incubation at 37°C for
12 h. Cells were then washed five times with PBS to completely
remove any HVJ-liposome that had not been transferred. Transfected
cells were immediately used for the following experiments.
Flow Cytometry.
To detect ODNs located within cultured cells after transfection, Sp1
and mt-Sp1 decoy ODNs were labeled at the 5' end with FITC (Wako Pure
Chemical Industries Co. Ltd., Osaka, Japan) and T4 polynucleotide
kinase (Toyobo Co. Ltd., Osaka, Japan), respectively. Flow cytometry
was used to observe A549 and U251 cells 3 h after FITC-labeled Sp1
decoy ODNs’ transfection. Cells were trypsinized, washed three times
with PBS, and analyzed on a FACScan II (Becton Dickinson, Mountain
View, CA), gating on live cells. Fluorescence was monitored at 488 nm
with a 525-nm band pass filter. A549 and U251 cells without
transfection of FITC-labeled decoy ODNs were used as a control.
Fluorescence Microscopic Examination.
The intracellular location of decoy ODNs in the cultured cells
transfected with HVJ-liposome was determined by fluorescent microscopy
of cells transfected with FITC-labeled decoy ODNs. A549 and U251 cells
were seeded on chamber slides and incubated with 10% FBS-supplemented
medium for 12 h to allow the cells to adhere. Three and 6 h
after initiation of the transfection protocol described above, cells
were washed with PBS and subjected to microscopic examination. A549 and
U251 cells without transfection of FITC-labeled decoy ODNs were the
controls.
EMSAs.
After replacing conditioned media, supplemented with serum, with
serum-free media containing TNF- (100 ng/ml; R&D Systems,
Minneapolis, MN), nuclear proteins were isolated from cancer cells
before and 15, 30, 60, and 120 min after TNF- treatment
(36)
. Twenty µg of nuclear extracts were subjected to
EMSA as reported previously (37)
. Sp1 decoy ODNs or mt-Sp1
decoy ODNs (0.35 pmol/ml) were end-radiolabeled using T4 polynucleotide
kinase, [
-32P]dATP, and commercially
available buffer (Life Technologies, Inc., Rockville, MD) for a 30-min
incubation at 37°C in a total volume of 10 µl. The reaction was
stopped with EDTA. Ten µg of nuclear extract and 2.0 µg of
poly(deoxyinosinic-deoxycytidylic acid) were incubated with the
radiolabeled Sp1 decoy ODNs or mt-Sp1 decoy ODNs in a total volume of
15 µl. ODN-protein complexes were analyzed by electrophoresis through
5% polyacrylamide gels in 22.3 mM Tris, 22.3
mM boric acid, and 0.5 mM EDTA. Visualization
was performed using a BAS 2000 Bioimage Analyzer (Fuji Photo Film Co.,
Tokyo, Japan).
Northern Blot Analysis.
Radiolabeled cDNA probes (VEGF165,
TGF-ß1, TF, bFGF, and uPA) were prepared
according to the manufacturer’s instructions with
[-32P]dCTP (Amersham) and a DNA Labeling Kit
purchased from Pharmacia Biotech (-dCTP).
Total RNA was examined to determine the effects of ODNs on
TNF--mediated expression of the VEGF,
TGF-ß1, TF,
bFGF, and uPA genes. A549 and U251 cells with or
without transfection of ODNs (Sp1 decoy ODNs or mt-Sp1 decoy ODNs) were
treated with TNF- (100 ng/ml) for 12 h. Total RNA was isolated
from the cells by the guanidine isothiocyanate extraction method
described previously (38)
. Twenty µg of total RNA were
electrophoresed on 1.2% agarose/3% formaldehyde RNA gels and
transferred onto N nylon membranes (Amersham Life Sciences,
Amersham, United Kingdom). The membranes were prehybridized in 1
mM EDTA, 0.5 M NaHPO4 (pH
7.2), and 7% SDS at 52°C for 2 h. Hybridization was performed
with purified, radiolabeled VEGF, TGF-ß1, TF,
bFGF, or uPA cDNA in the above solution at 52°C for 16 h. The
membrane was initially washed in 0.1x SSC/0.1% SDS at 52°C.
Visualization was performed using the BAS 2000. The mRNA expression was
quantitatively evaluated using a densitometer. Applied total RNA was
routinely standardized with either human GAPDH mRNA expression or
ethidium bromide-stained rRNA.
ELISA.
The presence of VEGF and TGF-ß1 proteins
secreted by A549 and U251 cells into conditioned medium was determined
with a human Quantikine Kit from R&D Systems. VEGF or
TGF-ß1 concentrations were quantitatively
determined according to the manufacturer’s instructions with minor
modifications relative to standard curves derived from diluted
recombinant human VEGF165 or
TGF-ß1 proteins. ELISA values were read using a
microplate reader (Immuno-mini, NJ-2300; Inter Med, Tokyo, Japan).
Cell Growth.
A549 and U251 cells were plated onto tissue culture wells at
1 x 104 cells/2.1
cm2
, cultured with RPMI 1640 containing 10% FBS
for 12 h to allow adherence. This was followed by incubation with
serum-free medium for 12 h. After transfection by the HVJ-liposome
method, the culture medium was replaced with RPMI 1640 containing 10%
FBS, renewed every 2 days. Cell cultures were washed twice with PBS and
removed from the tissue culture wells by trypsinization (0.25% in PBS)
at 3, 6, and 9 days. Cell numbers were determined using a
hemocytometer. Both of the cell lines with or without transfection of
empty HVJ-liposome were used as controls. Each experiment was performed
in triplicate. The results are given as mean cell numbers ± SD/dish.
In Vitro Migration Assay.
The Boyden chamber procedure was used as described elsewhere
(39)
to evaluate cell migration activity in a
24-microwell chemotaxis chamber (Falcon). The upper and lower wells
were separated by a polyvinyl-pyrolidone-free polycarbonate filter (8
µm pore size), coated with type I collagen and fibronectin. The lower
wells contained RPMI 1640 with 10% FBS. Cell suspension (1.2 x 104 cells in 50 µl of serum-free
medium) of A549 and U251 cells, transfected with Sp1 decoy ODNs or
mt-Sp1 decoy ODNs, or treated with empty HVJ-liposomes, was added to
the upper wells. The chamber was incubated at 37°C for 6 h. The
filters were then removed and fixed in methanol overnight. Nonmigrating
cells on the upper surface of the filters were removed with a cotton
swab. Cells were stained with Giemsa and counted using a microscope at
x40 in 10 random fields per each well. Migration was assayed by
measuring the number of cells moving across the filter. Each experiment
was performed in triplicate. The number of cells that migrated is
given as the mean ± SD. The number of cells that
migrated without any treatment was also determined.
Statistical Analysis.
Results are expressed as the mean ± SD. Statistical
comparison of several means was carried out by an ANOVA, and comparison
of two means was done with Student’s t test. All of the
Ps were analyzed on two sides.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment on VEGF, TGF-ß1, and
TF Expression. treatment on the expression of VEGF,
TGF-ß1, and TF was examined in A549 and U251
cells. All of these mRNA species were increased in a dose-dependent
manner by TNF- treatment (0, 10, 50, and 100 ng/ml) for 12 h.
The amounts of VEGF, TGF-ß1, and TF mRNA were
most prominent in both of the cell lines after stimulation by 100 ng/ml
of TNF-. The increases were about 4- (VEGF and
TGF-ß1) and 2-fold (TF; Figs. 1
and 2
). No cytotoxic effect on cultured cells was observed with this
TNF- dose. Subsequent experiments used 100 ng/ml TNF-. Treatment
with TNF- (100 ng/ml) had no effect on bFGF mRNA synthesis in A549
and U251 cells compared with treatment with serum-free medium
alone (Fig. 2)
.
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. treatment, EMSA was performed using radiolabeled Sp1 decoy ODNs
or mt-Sp1 decoy ODNs as a probe. The binding of Sp1 decoy ODNs to
nuclear protein extracted from either A549 or U251 cells was observed
within 15 min after stimulation with TNF- (100 ng/ml) and continued
for at least 120 min (Fig. 3)
. No complex formation with radiolabeled mt-Sp1 decoy ODNs was
detected. These results indicate not only that TNF- treatment
induces the transactivation of Sp1 in the cancer cells examined, but
also that the sequences of the Sp1 decoy ODNs bind the activated Sp1
protein, whereas those of the mt-Sp1 decoy ODNs do not.
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.
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. Six h after transfection, FITC-labeled decoy ODNs were
localized in the nucleus (Fig. 5b)
.
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(100 ng/ml) for
12 h was examined for the amount of various mRNA species.
Cells treated with empty HVJ-liposomes were used for the mock treatment
control. As shown in Fig. 2
, the treatment of both cell lines with Sp1
decoy ODNs reduced the TNF--induced mRNAs expression,
i.e., 9% (A549) and 30% (U251) for VEGF mRNA, 23% (A549)
and 8% (U251) for TGF-ß1 mRNA, and 14% (A549)
and under constitutive level (U251) in TF mRNA. In contrast,
transfection of mt-Sp1 decoy ODNs or treatment with empty HVJ-liposomes
had no effect on the TNF--induced synthesis of VEGF,
TGF-ß1, or TF mRNA in either A549 or U251
cells.
Treatment of A549 and U251 cells with TNF- (100 ng/ml) for 12 h
had no effect on the synthesis of bFGF mRNA relative to FBS-free medium
alone. Treatment of A549 and U251 cells with either ODNs or with empty
HVJ-liposomes had no effect on bFGF mRNA synthesis regardless of
treatment with TNF- (100 ng/ml).
The Effects of Transfection with Sp1 Decoy ODNs on TNF-induced VEGF
and TGF-ß1 Protein Production.
To confirm that the effects of Sp1 decoy ODNs transfection on the
synthesis of mRNA, described above, resulted in an effect on secretion
of VEGF and TGF-ß1 proteins by A549 and U251
cells into the conditioned media, the concentrations of VEGF and
TGF-ß1 proteins in conditioned media were
determined with ELISA assays. As shown in Fig. 6, A and B
, the secretion of these proteins was
enhanced 3-fold with TNF- stimulation (100 ng/ml). This
TNF--induced secretion of both proteins was reduced to the
respective constitutive level by transfection with Sp1 decoy ODNs.
Neither transfection with mt-Sp1 decoy ODNs nor treatment with empty
HVJ-liposomes had any effect on the secretion of VEGF and
TGF-ß1 proteins by A549 and U251 cells.
|
. In contrast, transfection of Sp1 decoy
ODNs reduced the growth of A549 and U251 cells on day 9, to 40 and
46%, respectively, relative to cells without any treatment. This
inhibitory effect of Sp1 decoy ODNs on the growth of A549 and U251
cells is probably a consequence of reduction of Sp1 protein binding to
the Sp1 consensus sequence in the promoter regions of the genes related
to serum-dependent cancer cell growth. This interpretation is
buttressed by the observation that neither mt-Sp1 decoy ODNs
transfection nor empty HVJ-liposome treatment affected proliferation of
either cell type.
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. The invasiveness of A549 and U251 cells was reduced to about one-half
by treatment with Sp1 decoy ODNs, as compared with no treatment. In
contrast, either A549 and U251 cells transfection with mt-Sp1 decoy
ODNs or treatment with empty HVJ-liposome had no effect on the
invasiveness of the cells. The effect of Sp1 decoy ODNs transfection on
TNF--induced uPA synthesis in A549 and U251 cells was evaluated by
analysis of mRNA expression. Fig. 9
shows that treatment of A549 and U251 cells with Sp1 decoy ODNs reduced
uPA mRNA expression. In contrast, transfection with mt-Sp1 decoy ODNs
or treatment with empty HVJ-liposome had no effect on uPA mRNA
synthesis in either A549 or U251 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced expression of the
VEGF, TGF-ß1, and
TF genes. A comparable reduction in secreted VEGF and
TGF-ß1 protein by these cells in response to
stimulation with TNF- is presented. Transfection of both A549 and
U251 cells by the Sp1 decoy ODNs also suppresses the invasion
activities of these cells. Therefore, a possible advantage of the Sp1
decoy strategy is the simultaneous suppression of the transcription of
several factors, including angiogenic molecules, through modification
of Sp1 function.
Although there are many angiogenic factors related to tumor
angiogenesis, VEGF is thought to be the most important growth factor in
the vascularization of the adjacent stromal tissue, especially for
solid tumors. Previous in vitro and in vivo
studies of the inhibition of VEGF function used VEGF antisense mRNA
(12, 13, 14)
, neutralizing antibodies against VEGF (15
, 16)
, a soluble isoform of the VEGF receptor (17)
,
and a dominant-negative isoform of the VEGF receptor (18)
.
In this study, we tested a new "decoy" strategy as a specific gene
therapy for cancers involving angiogenic suppression. The decoy
strategy was originally reported by Bielinska et al.
(20)
and by Morishita et al.
(21)
who applied NF-B decoy ODNs to suppress the acute
inflammatory process in myocardial ischemia and reperfusion injury.
VEGF synthesis is regulated by the transactivation of transcription
factors such as AP-1, hypoxia-inducible factor-1 (HIF-1), and Sp1
(22)
. To regulate TNF--induced synthesis of VEGF, we
selected a decoy system targeted to inhibit Sp1 function for several
reasons: (a) inflammatory cytokines may play important roles
in tumor progression via transcription factors including Sp1;
(b) TNF- can participate in tumor angiogenesis
(23)
; and (c) synthesis of TNF- itself is
reported to depend on Sp1 activation (41)
.
Sp1 decoy ODNs, but not mt-Sp1 decoy ODNs, showed a mobility shift in
the presence of nuclear extract prepared from TNF--treated cells.
This indicates that the Sp1 decoy ODNs used in this study bound
activated Sp1, and mt-Sp1 decoy ODNs could be used as a negative
control. Transfection of Sp1 decoy ODNs reduces the synthesis of
TNF--induced VEGF, TGF-ß1, and TF mRNA in
both A549 and U251 cells by 10–30%, thereby reducing the levels of
these mRNAs to approximately the constitutive levels of expression.
These effects of Sp1 decoy ODNs are considered specific for
Sp1-mediated transcription for three reasons. First, treatment with
either mt-Sp1 decoy ODNs or empty HVJ-liposome had no effect. Second,
transcription of the bFGF gene, thought to be independent of
Sp1 function, was not influenced by either TNF- treatment or
transfection with either Sp1 decoy ODNs or mt-Sp1 decoy ODNs. Third,
VEGF is believed to be up-regulated by hypoxia, via activation of
transcription factors HIF-1 and AP-1. We confirmed induction of
VEGF gene expression in both A549 and U251 cells under
hypoxic conditions (data not shown). Both cell lines showed no
alteration of hypoxia-induced VEGF synthesis with Sp1 decoy ODNs
transfection (data not shown).
Both cell proliferation and the invasiveness of A549 and U251
cells were apparently reduced by transfection with Sp1 decoy ODNs.
Neither transfection with mt-Sp1 decoy ODNs nor empty HVJ-liposome
treatment showed any effect. Cancer cell invasiveness is thought to be
regulated by many factors, such as matrix metalloproteases, uPA,
cell-matrix interaction, and other factors (38)
. To
clarify inhibitory mechanism of FBS-induced invasion, which the Sp1
decoy ODNs influences, we examined the TNF--induced synthesis of uPA
mRNA. Transfection with Sp1 decoy ODNs inhibited TNF--induced
synthesis of uPA mRNA in both cell lines. This is consistent with a
previous report showing Sp1 binding site(s) in the promoter regions of
the uPA gene (42)
. Although FBS contains many
factors that stimulate cancer cell invasiveness, the inhibition of uPA
synthesis by transfection with Sp1 decoy ODNs may be effective as a
suppressor of invasiveness.
As described above, the decoy strategy against Sp1 could
theoretically provide a therapeutic method for inducing "tumor
dormancy," as proposed by Holmgren et al.
(43)
, through the inhibition of both tumor angiogenesis
and tumor cell growth and invasiveness. However, the strategy of
treatment using decoy ODNs may possess both favorable and unfavorable
effects in vivo. Therefore, a number of issues concerning
the in vivo therapeutic potential of Sp1 decoy ODNs on tumor
growth, such as transfer efficiency, duration of intranuclear retention
of decoy ODNs in tumor tissue and side effects (both local and
systemic), remain to be examined. In addition, Sp1 is considered a
representative transcription factor, comparable with AP-1, NF-B, and
Egr-1 and -2, which participate in the induction and regulation of the
inflammation-repair process. Therefore, the Sp1 decoy strategy may also
be clinically pertinent in the treatment of inflammatory angiogenic
diseases, including rheumatoid arthritis, atherosclerosis, and
proliferative retinopathies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Pathophysiological and Experimental Pathology, Department
of Pathology, Graduate School of Medical Science, Kyushu University,
3-1-1 Maidashi, Fukuoka 812-0054, Japan. 
2 The abbreviations used are: VEGF,
vascular endothelial growth factor; TNF-, tumor necrosis factor ;
ODN, oligodeoxynucleotide; TF, tissue factor; FVII, factor VII; uPA,
urokinase-type plasminogen activator; FBS, fetal bovine serum; HVJ,
hemagglutinating virus of Japan; EMSA, electrophoretic mobility shift
assay; bFGF, basic fibroblast growth factor; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Received 5/12/00. Accepted 9/20/00.
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 T. Sato and K. Furukawa Transcriptional Regulation of the Human {beta}-1,4-Galactosyltransferase V Gene in Cancer Cells: ESSENTIAL ROLE OF TRANSCRIPTION FACTOR Sp1 J. Biol. Chem., September 17, 2004; 279(38): 39574 - 39583. [Abstract] [Full Text] [PDF]
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D. Wei, L. Wang, Y. He, H. Q. Xiong, J. L. Abbruzzese, and K. Xie Celecoxib Inhibits Vascular Endothelial Growth Factor Expression in and Reduces Angiogenesis and Metastasis of Human Pancreatic Cancer via Suppression of Sp1 Transcription Factor Activity Cancer Res., March 15, 2004; 64(6): 2030 - 2038. [Abstract] [Full Text] [PDF]
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L. Wang, D. Wei, S. Huang, Z. Peng, X. Le, T. T. Wu, J. Yao, J. Ajani, and K. Xie Transcription Factor Sp1 Expression Is a Significant Predictor of Survival in Human Gastric Cancer Clin. Cancer Res., December 15, 2003; 9(17): 6371 - 6380. [Abstract] [Full Text] [PDF]
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 A. Tsuchiya, M. Sakamoto, J. Yasuda, M. Chuma, T. Ohta, M. Ohki, T. Yasugi, Y. Taketani, and S. Hirohashi Expression Profiling in Ovarian Clear Cell Carcinoma: Identification of Hepatocyte Nuclear Factor-1{beta} as a Molecular Marker and a Possible Molecular Target for Therapy of Ovarian Clear Cell Carcinoma Am. J. Pathol., December 1, 2003; 163(6): 2503 - 2512. [Abstract] [Full Text]
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 R L Stephen, J E Crabtree, T Yoshimura, C L Clayton, M F Dixon, and P A Robinson Increased zinc finger protein zFOC1 transcripts in gastric cancer compared with normal gastric tissue Mol. Pathol., June 1, 2003; 56(3): 167 - 171. [Abstract] [Full Text] [PDF]
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Y. Tang, D. Zhang, L. Fallavollita, and P. Brodt Vascular Endothelial Growth Factor C Expression and Lymph Node Metastasis Are Regulated by the Type I Insulin-like Growth Factor Receptor Cancer Res., March 15, 2003; 63(6): 1166 - 1171. [Abstract] [Full Text] [PDF]
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M. Borgatti, I. Lampronti, A. Romanelli, C. Pedone, M. Saviano, N. Bianchi, C. Mischiati, and R. Gambari Transcription Factor Decoy Molecules Based on a Peptide Nucleic Acid (PNA)-DNA Chimera Mimicking Sp1 Binding Sites J. Biol. Chem., February 21, 2003; 278(9): 7500 - 7509. [Abstract] [Full Text] [PDF]
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M. Abdelrahim, I. Samudio, R. Smith III, R. Burghardt, and S. Safe Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells J. Biol. Chem., August 2, 2002; 277(32): 28815 - 28822. [Abstract] [Full Text] [PDF]
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)
and U251 (
) cells—after transfection of Sp1 decoy ODNs
(Decoy) or mt-Sp1 decoy ODNs (mt-Decoy) or after
treatment with empty HVJ liposomes (Empty HVJ-lipo)—were
cultured in the presence of TNF-
