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J Biol Chem, Vol. 274, Issue 33, 22941-22948, August 13, 1999
,From the Department of Biochemistry, Osaka University Medical School, Room B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Although the precise role of oligosaccharides in
metastasis is presently unknown, numerous studies suggest that the
The glycosylation of cell surface glycoproteins is thought to play
a critical role in a variety of specific biological interactions (1).
Numerous studies have concluded that alterations of cell surface
glycoprotein oligosaccharides cause significant changes in the adhesive
or migratory behavior of a cell (2). The high degree of branching of
N-glycans, in particular, the When the GnT-V gene was transfected into a lung epithelioid
cell, the transfectant showed an increased tumorigenicity, as evidenced
by an assay involving the subcutaneous injection of the cells into nude
mice (10). Interestingly, this cell showed an altered transformed cell
morphology, as is often observed for oncogenically transformed cells.
In addition, the overexpression of GnT-V resulted in a decrease in
serum growth requirements of the contact-inhibited parental cells, but
the migration rates of controls increased by 3- to 10-fold, and they
adhered less well to fibronectin or collagen type IV. In contrast, when
the overexpression of other glycosyltransferases, such as GnT-III, inhibited the action of GnT-V through substrate availability, lung
metastasis of melanoma cells was dramatically suppressed (11). These
data suggest that GnT-V is able to affect the phenotypes of cells
directly, that a high level of GnT-V expression in tumor cells with a
greater potential for malignancy may not be the result of malignant
transformation by other factors, and that GnT-V itself may well be the
key biological factor. For these reasons, it is important to elucidate
the mechanism by which GnT-V is expressed.
We recently isolated human GnT-V genomic DNA clones and showed that the
gene consists of 17 exons and spans 155 kilobases (12). The sequence
analysis of the 5' flanking region of GnT-V revealed that some putative
consensus sequences for tissue-specific transcription factors, such as
AP-1 and ets, are present in the promoter regions (Fig. 1). The AP-1
site is known to form complexes with the proto-oncogenes c-Jun and
c-Fos in response to a wide variety of growth factors (13). The Ets
family is a novel class of trans-acting phosphoproteins that
play important roles in the control of growth and development (14). The
family is defined by a highly conserved Ets domain (15), which encodes
a winged helix-turn-helix DNA-binding motif (16). ets binding sites, which contain a common core trinucleotide sequence, GGA, have been
identified in the regulatory regions of human T-cell receptor Whereas the stromal expression of c-Ets-1 is correlated with tumor
metastasis (20), the overexpression of Ets-1 in colon cancer cells has
been reported to result in the suppression of tumorigenesis (21). These
findings suggest that Ets-1 is a multifunctional transcription factor.
To demonstrate an involvement of the Ets family in GnT-V
gene regulation, we showed that the GnT-V gene is regulated
by Ets-1 protein in a human bile duct carcinoma cell line using a gel
mobility assay (22). Buckhaults et al. also reported that
the regulation of the GnT-V gene is mediated by the
src oncogene in the BHK (baby hamster kidney) fibroblast
cell line via the involvement of ets transcription factor (23). These results suggest that regulation of the GnT-V gene is
mediated by ets transcription factors in a cell-specific manner. Ets is also known to cooperate with AP-1 in the transcriptional regulation of
genes such as interleukin 2 (19), collagenase (24), and human tumor
necrosis factor (25). To provide more broad evidence concerning the
role of ets proteins in enhancing GnT-V gene expression, a
more general relationship between GnT-V and ets expression in cancer
cells would be desirable.
This study was undertaken to investigate the issue of whether the
expression of GnT-V is correlated with that of ets family mRNAs in
16 human and murine cancer cells and to better understand the effects
of overexpression of ets-1 and ets-2 and dominant negative ets-1 and
ets-2 on GnT-V expression as well as coordination of AP-1 and
GnT-V genes.
Cell Lines--
Human cancer cell lines MKN45 and Kato-III
(gastric cancer), A549, Lu99B and Lu65A (lung cancer), PacaII
(pancreatic cancer), MB231 (mammary cancer), Huh6, HepG2
(hepatoblastoma), Huh7 (hepatocellular carcinoma), A172 (glioblastoma),
mouse melanoma cell line B16-F1, and rat hepatoma cell line AH66tc were
obtained from the Japanese Cancer Research Resource (Tokyo, Japan), and
Hep3B (hepatocellular carcinoma) and Colo201 and Colo205 (colon cancer)
were obtained from American Type Culture Collection (Manassas, VA).
These cell lines were cultured under standard conditions (RPMI 1640 medium or Dulbecco's modified Eagle's medium (Nikken Kagaku, Kyoto,
Japan) containing 10% fetal calf serum and antibiotics). The
antibodies to Ets-1 and Ets-2 were purchased from Santa Cruz Biotechnology.
Plasmids--
Human c-ets-1 cDNA was cloned into the pSVK3
vector, as described previously (22). To express the human ets-1
cDNA, its blunt-ended full-length fragment was inserted into the
gap-filled EcoRI site of pCAGGS (kindly provided by Dr. J. Miyazaki, Osaka University Medical School, Osaka, Japan), which was
controlled by a human Northern Blot Analysis--
Total cellular RNAs were extracted
from cancer cell lines according to the method reported by Chomczynski
and Sacchi (28). Total RNA (20 µg) was electrophoresed on a 1%
agarose gel containing 2.2 M formaldehyde. After blotting
onto a Zeta probe (Bio-Rad) nylon membrane, the filter was hybridized
with 32P-labeled human ets-1 (22) and ets-2 cDNAs (29)
or GnT-V cDNA (30), other ets family cDNAs (31) at 42 °C in
a hybridization buffer. The membrane filter was washed with 2×
standard saline-citrate, pH 7.4, and 0.1% SDS twice for 10 min each at
55 °C and then washed with 2× standard saline citrate, pH 7.4, and
0.1% SDS for 30 min. The filter was then exposed to x-ray films
(Kodak, Kyoto, Japan) with an intensified screen at Transient Expression of ets-1 and ets-2--
Various cancer
cells were plated at 1.7 × 105 cells/60-mm dish 1 day
before transfection. 10 µg each of ets-1 and the ets-2 expression
vectors were introduced into the cells using a LipofectAMINE reagent
(Takara, Shiga, Japan). To determine the efficiency of transfection,
pEGFP-N1 (CLONTECH), which expresses the bacterial green fluorescence protein, was used as a control plasmid. Briefly, 10 µg of DNA and 20 µl of LipofectAMINE reagent were mixed with serum-free medium to form a DNA-liposome complex. After incubation for
20 min, 1 ml of media was overlaid on the pre-rinsed cells. The cells
were incubated with the complex for 4 h, and the complex removed
by washing with ice-cold phosphate-buffered saline. After incubation
for 48 h, the cells were used for further analysis.
Western Blotting--
Approximately 1 × 107
cells were washed with ice-cold phosphate-buffered saline, followed by
lysis with Nonidet P-40 buffer (10 mM HEPES (pH 7.8)
containing 10 mM KCl, 2 mM MgCl2,
0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol
(8 µg/ml), 2 µg/ml leupeptin, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.2% (v/v) Nonidet P-40). After
centrifugation at 3,000 × g for 5 min at 4 °C,
nuclear protein was extracted from the pellet with 50 mM
HEPES buffer, pH 7.8, containing 420 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and 25% glycerol. 10 µg of the nuclear proteins were
boiled for 3 min in a sample buffer containing 0.125 M
Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol
and subjected to 10% SDS-polyacrylamide gel electrophoresis. The
separated proteins were electrophoretically transferred onto a
nitrocellulose membrane (Schleicher & Schüll). Nonspecific
binding sites on the filter were blocked by incubating the membrane in
TBS-T supplemented with 5% skim milk. The filters were then probed
with a primary rabbit antibody to Ets-1 (N-276; Santa Cruz
Biotechnology) and Ets-2 (C-20) for 2 h at room temperature. The
blots were washed with TBS-T for 30 min and then incubated with
anti-rabbit horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. After washing for 30 min, the membranes were
developed using ECL (Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclei were
isolated from cultured cells as described above. Extracts were cleared
by centrifugation at 14,000 × g for 15 min at 4 °C.
Aliquots of the resulting supernatants were frozen at Relationships between GnT-V and ets-1 mRNA Expression in
Various Cancer Cell Lines--
According to the diagram of the GnT-V
promoter region (Fig. 1) and the results
of our previous study (22), Ets-1 might be expected to have a high
potential for influencing GnT-V expression. To examine the
relationships between ets-1 and GnT-V expression at mRNA levels,
Northern blot analysis was performed on various cancer cell lines using
ets-1, GnT-V, ets-2, erg, and fli-1 cDNAs as probes (Fig.
2). Kato-III, HepG2, Huh7, Colo201,
Colo205, and B16-F1 cells showed higher levels of ets-1 expression than
the other cells. The expression of GnT-V mRNA showed a pattern
similar to that for ets-1 expression. In contrast, A549, Lu65A, Lu99B, MKN45, PacaII, MB231, Huh6, Hep3B, and AH66tc cells showed lower levels
of both GnT-V and ets-1 mRNA expression. The expression levels of
GnT-V and ets-1 mRNAs, as quantitated by densitometry, were plotted
(Fig. 2). A positive correlation was found between these mRNAs
expressed in cancer cell lines (r = 0.97;
p < 0.0001), suggesting that the control of
GnT-V gene expression by Ets-1 is widely distributed in a
variety of cell lines. Expression of ets-2 was positively correlated
with GnT-V expression in some cells such as HepG2, Huh7, Colo205, and
Kato-III cells. However, in the case of A172, Colo201, B16-F1, and
MB231 cells, no correlations were observed between ets-2 and GnT-V
expression. Another protein of the ets family, erg showed a low level
of expression in various cell lines and did not correlate with GnT-V
expression. The expression of fli-1, which is known to be 98%
homologous to erg (32), showed a pattern that was very similar to erg
(data not shown). Whereas the cDNA sequence homology of ets-1 among
human, mouse, and rat is in excess of 90%, cDNA of the ets-2
coding region between human and mouse was less than 70% homologous. In
the B16-F1 cell line, low levels of ets-2 could be due to its low
sequence homology. A high level of expression of ets-2, as well as
GnT-V and ets-1, was observed in Kato-III, HepG2, Huh7, and Colo205
cells, suggesting that Ets-2 could also regulate gene expression of
GnT-V in these cells. Whereas MKN45 cells expressed low levels of GnT-V
and ets-1, the expression of ets-2 was quite high.
To investigate the expression of Ets-1 and Ets-2 at the protein level,
Western blot analysis was performed using A549, B16-F1, HepG2, Huh7,
Kato-III, and MKN45 cells (Fig. 3). A
high level of expression of Ets-1 and Ets-2 was observed in HepG2,
Huh7, and Kato-III cells. In contrast, their expression was quite low in A549 cells, but a high expression of Ets-2 was observed in MKN45
cells. These results were consistent with the mRNA expression levels.
To determine whether the expression of GnT-V is dependent on Ets-1
protein, nuclear extracts from the MKN45, HepG2, and Colo201 cell lines
were subjected to an EMSA analysis. EMSA was performed using the
24-base pair GnT-V promoter-derived oligonucleotide E728 ( AP-1 Does Not Cooperate with Ets-1 in GnT-V Expression--
To
investigate the cooperative trans-activation of c-Jun on the
GnT-V promoter through the c-Jun binding element (AP-1) and the ets
binding element, we first performed a Northern blot analysis in various
cancer cell lines. The expression of c-jun mRNA was observed in
nearly all cells (Fig. 5) and was not
correlated with GnT-V expression.
To directly investigate the cooperation of AP-1 and ets binding
elements in GnT-V expression, an EMSA was performed using the 58-base
pair GnT-V promoter-derived oligonucleotide EJ565 (
To further confirm that AP-1 and the Ets-1 site do not cooperate,
antisense c-jun was transfected to A549 and PacaII cells. Although
these cells expressed low levels of both ets-1 and GnT-V (Fig. 2), the
transfection of a vector alone (mock transfectant) to these cells
brought a slight enhancement of GnT-V mRNA expression (Fig.
7). This result was reproduced in three
separate experiments. When antisense c-jun was transfected into cells,
GnT-V mRNA expression was unchanged (data not shown), suggesting
that c-Jun could not be linked to GnT-V expression.
The Effects of ets-1 Transfection on GnT-V Expression--
To
determine whether GnT-V mRNA expression is controlled by the
overexpression of ets-1, two types of Ets-1-expressing vectors were
transiently transfected into A549, MKN45, and PacaII cells, which were
shown to express low levels of both ets-1 and GnT-V (Fig. 2). The
transfection efficiency was about 15-20% of the total cell numbers,
as judged by fluorescence microscopy (data not shown). Although
intrinsic GnT-V expression was quite low, as shown in Fig. 2, the
transfection of a vector alone (mock transfectant) slightly enhanced
the expression of GnT-V mRNA (Fig. 7). This enhancement was very
reproducible, but the details of its mechanism are presently unknown.
When ets-1 was transfected into A549 and PacaII cells that expressed
low levels of Ets-1, the expression of GnT-V was increased in
comparison with control or mock-transfected cells (Fig. 7). In the case
of MKN45, however, the level of GnT-V expression was not increased by
ets-1 transfection, suggesting that Ets-1 is not sufficient for
GnT-V gene expression in MKN45 cells.
The Effects of Dominant Negative ets-1 Transfection on GnT-V
Expression--
To demonstrate the enhancement of GnT-V by Ets-1
proteins by a different approach, the action of Ets-1 was inhibited by
the transfection of dominant negative ets-1 in Kato-III and B16-F1 cells, which showed high levels of intrinsic ets-1 and GnT-V
expression. It has been reported that the dominant negative mutant
containing the DNA-binding domain of ets-1 (N70) specifically inhibits
both ras stimulation and the constitutive The Effects of ets-2 and Dominant Negative ets-2 Transfection on
GnT-V Expression--
To determine whether GnT-V mRNA expression
is controlled by Ets-2, ets-2- or dominant negative ets-2-expressing
vector was transiently transfected into A549, MKN45, PacaII, Kato-III,
and B16-F1 cells, respectively. The transfection efficiency was about 15-20% of the total cell numbers, as judged by cotransfection of a
GFP-producing vector, followed by fluorescence microscopy (data not
shown). When ets-2 was transfected into A549 and PacaII cells that
expressed low levels of ets-2, the expression of GnT-V was not changed
in comparison with control or mock-transfected cells (Fig.
9). In contrast, whereas the level of
GnT-V expression in MKN45 cells was not increased by ets-2
transfection, transfection of a dominant negative ets-2 suppressed
GnT-V mRNA at the 40% levels of GnT-V expression in mock
transfectants. This suggests that Ets-2 regulates gene expression of
GnT-V in MKN45 cells. When the dominant negative mutant of ets-2 was
transfected into Kato-III and B16-F1 cells, the expression of GnT-V was
not changed, suggesting that ets-1 is a key factor for the control of
GnT-V expression in these cells (Fig. 9). Transfection of ets-2 into Kato-III cells induced increases in the expression of urokinase plasminogen activator mRNA (data not shown).
The present study demonstrated that GnT-V expression is regulated
by Ets-1 in many different cancer cells. This evidence provides a
possible new pathway of tumor metastasis via the up-regulation of GnT-V
by Ets-1.
As shown in Fig. 1, three ets binding sites and one AP-1 site are
located in the 5' flanking region of the GnT-V gene. It is
already known that ets cooperates with AP-1 in the transcriptional regulation of genes such as interleukin 2, collagenase, and tumor necrosis factor. However, this was not observed in the regulation of
the GnT-V gene by Ets-1 (Figs. 5 and 6). Expression of c-jun was not correlated with GnT-V levels, and antisense c-jun did not
change the level of GnT-V expression.
Although other ets family proteins are able to bind to these ets
binding sites, GnT-V expression was correlated most with ets-1
expression. Correlations between GnT-V and Ets-2 were not so high
compared with Ets-1. Fig. 9 showed the noninvolvement of Ets-2 in GnT-V
expression in Kato-III and B16-F1 cells. However, in MKN45 cells, Ets-2
protein is a key factor in gene regulation of GnT-V because a dominant
negative of ets-2 suppressed GnT-V expression (Fig. 9), and
overexpression of ets-1 showed no effects on GnT-V expression (Fig. 7).
Thus, gene expression of GnT-V is regulated in a cell type-specific
manner, although Ets-1 contributes to GnT-V gene regulation
in a variety of cancer cell lines. Although an ets-2 dominant negative
mutant contains a sequence that is similar to the DNA-binding domain of
ets-1, it was not able to inhibit GnT-V expression, as did the ets-1
dominant negative mutant (Fig. 8). A similar phenomenon was reported in
which the dominant negative mutant of ets-2 was unable to suppress ras
stimulation and the constitutive The mechanism of tumor metastasis is complicated and is not fully
understood at present. The ets family has been reported to be involved
in tumor metastasis through angiogenesis and the enhancement of
expression of metalloproteinase or collagenase (33, 34). In addition,
it has been suggested that the increased expression of GnT-V is
associated with metastasis and the invasion of tumor cells. Therefore,
Ets-1 would be expected to promote tumor metastasis not only via
angiogenesis and the expression of metalloproteinase, but also through
enhancing the expression of GnT-V. Moreover, because changes of
N-linked oligosaccharide structures in tumor cells are
associated with the expression of tissue inhibitors of
metalloproteinase I (35), the enhancement of GnT-V expression by ets-1
would be expected to participate in the regulation of metalloproteinase
activity. These considerations are consistent with the suggestion that
Ets-1 contributes to the malignancy potential of tumor cells via an
increase in the expression of GnT-V, which, in turn, leads to
alteration of N-linked oligosaccharides in tumor cells.
It has been reported that stromal fibroblasts adjacent to tumor cells
may express Ets-1 in response to tumor invasion (20); thus, it is
uncertain whether the high expression levels of ets-1 and GnT-V
detected in human hepatoma tissues were derived from hepatoma cells.
However, our analyses of a variety of cancer cells, including hepatoma
cells, indicate that many of the cancer cells investigated in the
present study expressed Ets-1.
It was recently reported that the use of antisense oligonucleotide for
ets-1 is one of the strategies by which tumor growth is suppressed
through the inhibition of angiogenesis (36). This latter report also
indicated that Ets-1 controls not only tumor angiogenesis but also
normal vascular development. The issue of whether GnT-V plays a role in
these vascular developments involving Ets-1 is not known and remains to
be examined. We also indicated that the impairment of the action of
GnT-V by transfection of GnT-III, the product of which prevents the
addition of a 
1-6 branching structure of N-linked oligosaccharides
plays a role in tumor metastasis. N-Acetylglucosaminyltransferase V (GnT-V), which catalyzes
the formation of the 1-6 branch, therefore appears to play a
crucial role in tumor metastasis. Recently, we demonstrated that the
expression of the GnT-V gene is regulated by a
transcriptional factor, Ets-1 (Kang, R., Saito, H., Ihara, Y., Miyoshi,
E., Koyama, N., Sheng, Y., and Taniguchi, N. (1996) J. Biol.
Chem. 271, 26706-26712). In this study, we report an
investigation of the general requirement for Ets-1 in the expression of
GnT-V in cancer cell lines. In 16 cancer cell lines, the levels of
GnT-V mRNA were closely correlated with ets-1 expression
(r = 0.97; p < 0.0001). An increase
in ets-1 levels by transfection of its cDNA led to an enhancement
in GnT-V expression in cells that normally expressed low levels of
ets-1. In contrast, the transfection of dominant negative ets-1 into cells that express high levels of ets-1 resulted in a decrease in GnT-V
expression. Although Ets-1 cooperates with c-Jun in certain gene
expressions, this was not the case in the regulation of the GnT-V gene. These results suggest that Ets-1 plays a
significant role in regulating the expression of GnT-V in a variety of
cancers and might be involved in the potential for malignancy via the action of GnT-V.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-6 branching type,
appears to be related to a potential for the development of malignancy
(3). UDP-GlcNAc, 
-mannoside
-1,6-N-acetylglucosaminyltransferase V
(GnT-V),1 represents an
enzyme that catalyzes such branching. To understand the molecular basis
of this oligosaccharide structure in terms of tumor metastasis, we and
other groups have purified GnT-V from rat kidney (4) and from a human
lung cancer cell line QG (5) and cloned the gene. The expression of
GnT-V is enhanced by malignant transformation through the
ras oncogene (6), by cell proliferation (7), and by
hepatocarcinogenesis (8). However, GnT-V is also highly expressed in
normal tissues, suggesting that the synthesis of oligosaccharides by
GnT-V might be different in normal cells versus cancer cells
(9). The most important issue, however, is how GnT-V modifies the
metastatic potential of tumor cells.
(17)
and (18) and the interleukin 2 receptor (19), as well as other
cellular and viral enhancers.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin promoter and the KpnI and
BamHI site of pHook-2 (Invitrogen, NVLeek, Netherlands)
controlled by a cytomegalovirus promoter. The plasmids that encoded the
ets-1 (15, 26) and ets-2 (
1-328) (23; Ref. 27) dominant negative
mutants were constructed with polymerase chain reaction fragments that
were designed to code the DNA binding domain. To appropriately
translate the ets-1 dominant negative mutant, the sense primer
contained a consensus AUG initiation codon (Table
I; ND-ets-1-N), and the antisense primer
was designed to code for the final 441 (corresponding to 485 in p68
ets-1) amino acids of Ets-1 (ND-ets-1-C). The dominant negative ets-1
and ets-2 were also cloned into the EcoRI site of plasmid
pCAGGS. The probe of ets-2, erg-1, erg/fli-1 fragment, and full-length
cDNA of c-jun (sense, jun-N; antisense, jun-C) were also prepared
from a human fetal liver cDNA library
(CLONTECH) by the polymerase chain reaction using
oligonucleotide primers (Table I). The polymerase chain reaction
products were cloned into the T vector (Novagen, Madison, WI) and
confirmed with DNA sequencing (Applied Biosystem, Chiba, Japan).
Antisense c-jun was prepared from a clone in which the PstI
fragment (0.6 kilobase) had been cloned in a reverse manner into the
EcoRI site of plasmid pCAGGS. Various DNA sequence analyses
were performed with DNAsis (Version 3.6; Hitachi).
Oligonucleotides used in cDNA cloning
80 °C for 1 day.
70 °C. The
protein concentration of the nuclear extract was measured using a BCA
kit (Pierce) with bovine serum albumin as the standard. These nuclear
proteins were used for the electrophoretic mobility shift assay. The
DNA fragment containing the putative ets binding sequences of the
upstream regulation regions of GnT-V was synthesized as described in
Table II. Complementary oligonucleotides were annealed and used as probes or competitors. The probes were end-labeled with [
-32P]ATP (Amersham Pharmacia
Biotech) using T4 polynucleotide kinase (Takara, Osaka, Japan). For the
gel mobility shift assay, DNA (10,000 cpm; labeled with
32P) and 5 µg of nuclear proteins were preincubated for
10 min at room temperature with 400 ng of poly(dI-dC) (Sigma) in 20 µl of binding buffer (25 mM Tris-HCl, pH 7.9, 65 mM KCl, 6 mM MgCl2, 0.25 mM EDTA, and 10% glycerol). For the competition assay, the unlabeled competitor oligonucleotides were preincubated with nuclear proteins 10 min before the addition of labeled oligonucleotide. For the
supershift assays, anti-Ets-1 (N-276, Santa Cruz Biotechnology) and
anti-Ets-2 (C-20; Santa Cruz Biotechnology) antibodies were added to
the reaction mixture, which was then incubated for 1 h at room
temperature. Samples were loaded onto 5% nondenaturing polyacrylamide
gels, 0.25× TAE (1× TAE = 40 mM Tris-HCl (pH 7.8), 1.1 mM EDTA, and 37 mM sodium acetate)
containing 2.5% glycerol, and then electrophoresis was then carried
out at 4 °C at 100 V for 1 h. After electrophoresis, the gels
were dried with a gel dryer and exposed to x-ray films (Kodak).
Oligonucleotides used in electrophoretic mobility shift assay
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of transcription
factor binding sites in the GnT-V promoter region. The location of
both the ets and AP-1 sites are described. Numbers represent
nucleotides that are upstream of exon 1 (12).
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Fig. 2.
Expression of ets-1, ets-2, and GnT-V
mRNA in various cancer cells. Expression of ets-1, GnT-V, and
ets-2 was investigated by Northern blot analysis using 20 µg of total
RNA. Hybridization was performed with each probe as described under
"Materials and Methods." Ethidium bromide staining showed
comparable amounts of RNA in each lane. A and B
represent different agarose gels. C, correlation of GnT-V
and ets-1 mRNA levels.
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Fig. 3.
Immunoblot analyses of nuclear extracts from
A549, B16-F1, HepG2, Huh7, Kato-III, and MKN45 cells. Nuclear
extracts from A549, B16-F1, HepG2, Huh7, Kato-III, and MKN45 cells were
subjected to 10% SDS-polyacrylamide gel electrophoresis, followed by
immunoblot analyses with anti-rabbit Ets-1 and Ets-2 antibodies.
Procedural details are described under "Materials and
Methods."
741/718)
(22), which has been shown to have moderate binding affinity among
three ets binding sites, which are located in the 5'-untranslated
regions of the GnT-V gene. As shown in Fig.
4, when the radiolabeled Et-B, E728
oligonucleotides were incubated with nuclear extracts prepared from
MKN45, HepG2, and Colo201, retarded protein-DNA complexes were detected
(lanes 1 and 2). The intensities of the complexes
of HepG2 and Colo201 were higher than that of MKN45. Specificity of
binding was identified with a mutant oligonucleotide (mE728; Fig. 4,
lane 3). To confirm ets antibody-specific binding, the
supershift assay was performed by means of the addition of anti-Ets-1
and anti-Ets-2. Whereas both antibodies were shown to supershift in the
HepG2 cells, a supershifted band by the Ets-2 antibody was clearer than
that of the Ets-1 antibody in MKN45 cells. The supershifted band by Ets-1 and Ets-2 was a competitive pattern in Colo201 cells (Fig. 4,
lanes 4 and 5). This is because Colo201 cells are
not adhering cells like HepG2 and MKN45 cells, and the amounts of
nuclear proteins in 5 µg of proteins applied on EMSA might be
relatively small as compared with the others. As a result, its
supershift might appear as a competitive pattern. Collectively, these
results strongly reflect the importance of Ets-1 and Ets-2 in GnT-V
expression in each cell.
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Fig. 4.
Electrophoretic mobility shift assay on the
ets site (E728) of the GnT-V promoter. 32P-labeled
oligonucleotide probes Et-B, E728, and mE728 were incubated with
nuclear extracts from MKN45, HepG2, and Colo201 cells (lanes
1-3). Supershift was performed using anti-Ets-1 and anti-Ets-2
antibodies on labeled probe E728 (lanes 4 and 5).
Procedural details are described under "Materials and
Methods."
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Fig. 5.
Expression of c-jun mRNA in various
cancer cells. Expression of c-jun was investigated by Northern
blot analysis using 20 µg of total RNA. Ethidium bromide staining
showed comparable amounts of RNA in each lane.
578/522), which
contains the ets and AP-1 sites (Table II). When the radiolabeled EJ565
oligonucleotide was incubated with nuclear extracts prepared from
HepG2, protein-DNA complexes were retarded (Fig.
6, lane 1). The radiolabeled
mJ565, which contained a mutant AP-1 site and was designed to detect
the specificity of the AP-1 site, was incubated with nuclear extracts.
The level of a retarded protein-DNA complex pattern was the same as
when EJ565 was used (Fig. 6, lane 2). ets-specific binding
was confirmed using competition analysis and the supershift assay.
Levels of retarded protein-DNA complex were decreased during an
incubation with unlabeled EJ565 (Fig. 6, lane 3), and the
bands were supershifted during incubation with anti-Ets-1 in the case
of both EJ565 and mJ565 (Fig. 6, lanes 4 and 5).
These results indicate that the DNA-protein complex with E565
nucleotide is dependent on ets binding, but not on AP-1.
View larger version (74K):
[in a new window]
Fig. 6.
Electrophoretic mobility shift assay on ets
and AP-1 site (E565) of the GnT-V promoter.
32P-labeled oligonucleotide probes E565 and mJ565 were
incubated with nuclear extracts from HepG2 cells (lanes 1 and 2). For competition analysis, a 100-fold amount of
unlabeled oligonucleotide (EJ565) was added before the incubation
(lane 3). Supershift assay was performed using 1 µg of
anti-Ets-1 antibody on the incubation with labeled probes EJ565 and
mJ565 (lanes 4 and 5).
View larger version (57K):
[in a new window]
Fig. 7.
Effects of ets-1 transfection on GnT-V
expression. Plasmids that contain ets-1 with a -actin promoter
(pCAGGS) and a cytomegalovirus promoter (pHook-2) were transiently
expressed in A549, MKN45, and PacaII cells, in which a very low level
of ets-1 and GnT-V mRNA was expressed. Total RNA (30 µg)
extracted from various cancer cells was electrophoresed on a 1%
agarose gel containing formaldehyde and analyzed by Northern blot.
Lanes 1, 5, and 9 contain the transfectant of the
control plasmid pEGFP-N1; lanes 2, 6, and 10 contain the mock transfectant (pCAGGS); lanes 3, 7, and
11 contain the transfectant of the ets-1-expressing plasmid
(pCAGGS), and lanes 4, 8, and 12 contain the
transfectant of the ets-1-expressing plasmid (pHook-2). GnT-V
expression levels were described according to their relative intensity
compared with the mock transfectant of each cell.
-domain activity of Ets-1 (26). When the dominant negative mutant of ets-1 was transfected into
Kato-III and B16-F1 cells, the expression of GnT-V was decreased in
comparison with a mock transfectant, suggesting that Ets-1 regulates GnT-V expression in these two cell lines (Fig.
8).
View larger version (32K):
[in a new window]
Fig. 8.
GnT-V expression by transfection of the
dominant negative ets-1 mutant. A dominant negative mutant of
ets-1 was transfected into Kato-III and B16-F1 cancer cells, which
showed high levels of expression of both ets-1 and GnT-V. Total RNAs
(30 µg) were extracted from these cells, electrophoresed on a 1%
agarose gel containing formaldehyde, and analyzed by Northern blot.
Lane 1 contains the transfectant of the control plasmid
pEGFP-N1; lanes 2 and 4 contain the mock
transfectant (pCAGGS), and lanes 3 and 5 contain
the transfectant of the dominant negative mutant of ets-1. The mRNA
of the dominant negative ets-1 is indicated by an arrowhead.
GnT-V expression levels are according to their relative intensity
compared with mock transfectant of each cell. Experimental details are
described under "Materials and Methods."
View larger version (34K):
[in a new window]
Fig. 9.
Effects of ets-2 or dominant negative ets-2
mutant transfection on GnT-V expression. The plasmids that contain
ets-2 or dominant negative ets-2 mutant were transiently expressed in
A549, MKN45, and PacaII and MKN45, Kato-III, and B16-F1 cells,
respectively. Total RNA (20 µg) extracted from each cell was
electrophoresed on a 1% agarose gel containing formaldehyde and
analyzed by Northern blot. A, effects of ets-2 transfection
on GnT-V expression, Lanes 1, 4, and 7 contain
the transfectant of the control plasmid pEGFP-N1; lanes 2, 5, and 8 contain the mock transfectant (pCAGGS);
lanes 3, 6, and 9 contain the transfectant of the
ets-2-expressing plasmid. B, effects of ets-2 dominant
negative transfection on GnT-V expression. Lane 1 contains
the transfectant of the control plasmid pEGFP-N1; lanes 2, 4, and 6 contain the mock transfectant (pCAGGS), and
lanes 3, 5, and 7 contain the transfectant of the
dominant negative mutant of ets-2. GnT-V expression levels are
described according to their relative intensity compared with the mock
transfectant of each cell. Experimental details are described under
"Materials and Methods."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-domain activity as Ets-1 did (26).
A similar correlation between the expression levels of ets-1 and GnT-V
was observed in 12 human hepatoma tissues (data not shown), suggesting
that Ets-1 regulates the expression of GnT-V in vivo.
1-6 GlcNAc residue, dramatically suppresses lung
metastasis of mouse melanoma cells that express high levels of GnT-V
(11). This clearly suggests that a 1-6 branch of an
N-glycan is involved in tumor metastasis. Therefore, it can
be concluded that the pathophysiological significance of Ets-1 in
malignancy potential such as tumor metastasis can be ascribed to the
increased expression of GnT-V. The prevention of the action of GnT-V as
well as Ets-1 would provide new insights into cancer therapy.
| |
ACKNOWLEDGEMENT |
|---|
We are grateful for the expert technical assistance and suggestion of Ayako Okado.
| |
FOOTNOTES |
|---|
* This work was supported by Grants-in-Aid No. 10178105 for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Tokyo Biochemical Research Foundation fellowship.
§ To whom correspondence should be addressed. Tel.: 81-6-6879-3420; Fax: 81-6-6879-3429; E-mail: proftani@biochem.med.osaka-u.ac.jp.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
GnT-V, UDP-GlcNAc,
-mannoside -1,6-N-acetylglucosaminyltransferase V;
GFP, green fluorescence protein;
AP-1 activator protein 1, TBS-T,
Tris-buffered saline containing 0.05% Tween-20;
EMSA, electrophoretic
mobility shift assay.
| |
REFERENCES |
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RESULTS
DISCUSSION
REFERENCES
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