JPH10137000A - Screening of metastasis suppressor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a metastasis suppressor by utilizing that a GnT-V enzyme participates in metastasis and Etx-1 protein participates in transcriptional control of gene, and a kit for screening the suppressor. SOLUTION: DNA comprising the following base sequences: 5'-GGGAGTGA- GGATGATGTAGGGAAG-3' (sequence No.1), 5'- ATGGGGCAGAGGAACTTACGTTAT-3' (sequence No.2) or at least either one thereof, (b) Ets-1 protein or its fragment and (c) a sample are together incubated and binding of the DNA (a) to Ets-1 protein or its fragment (b) is measured. The method is useful for primary screening of metastasis suppressor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for screening for a metastasis inhibitor and a kit therefor.

[0002]

BACKGROUND OF THE INVENTION Cell surface oligosaccharides that bind to asparagine residues of membrane glycoproteins appear to be involved in a variety of specific biological interactions (Hakomori, SI (19).
89) Adv. Cancer Res. 52,257-
331). Increased branching of N-glycans appears to be related to cancer potential, and in particular, increased β1-6 branching of N-glycans is directly related to enhanced tumor metastasis (De.
nnis JW et al., (1987) Science 236,
(582-585). This structure, as shown in FIG. 1, comprises UDP-N-acetylglucosamine: α-
6-D-mannoside β1-6-N-acetylglucosaminyltransferase V (GnT-V) [EC2.
4.1.155].

[0003] GnT-V is the β-form of N-glycan oligosaccharides.
1-6 UDP-N- to generate linked branches
The transfer of N-acetylglucosamine from acetylglucosamine to α-6-D-mannoside is catalyzed to control the polylactosamine content. A GnT-V gene containing 17 exons and spanning 155 kb has been cloned by the present inventors and other groups (Shoreibah, M. et al. (1992) J. Biol. Ch.
em. 267, 2920-2927; Gu, J. et al., (19.
93) J. Amer. Biochem. 113,111-11
6)). This gene is expressed in a tissue- and cell type-specific manner and is regulated at the level of transcription by multiple promoters (Saito, H. et al., N. (199).
5) Eur. J. Biochem. 233, 18-2
6).

The expression of the GnT-V gene is induced by viral and oncogene transfection, TGFβ and phorbol ester (Yamashita K. et al., JB).
iol. Chem. 260 3963-3969), suggesting that a complex mechanism exists for its induction. The Ets transcription factor family shares a common core-trinucleotide sequence, a common DNA-binding domain that specifically interacts with GGA-containing sequences (Wasylyk.
B. et al., (1993) Eur. J. Biochem. 2
11, 7-18).

The Ets-binding site (EBS) is a human TCR
-Α (Ho. IC et al., (1990) Science 2
50, 814-818) and -β (Prosser, HM et al.
(1992) Proc. Natl. Acad. Sci.
USA 89, 9934-9938), and IL-2β
R (Thompson CB et al., (1992) Mol. Cel.
l. Biol. 12, 1043-1053), and the regulatory regions of other cellular and viral enhancers, and their Ets-binding sites regulate their transcriptional activity (Wu. H. et al., (1994) Mol. C
ell. Biol. 14, 2129-2139). Et
s-1 is the ve in replication defective retrovirus E26
It was first described as a cell homolog of ts and is thought to be involved in tumorigenesis and embryogenesis (Klaes.
A. et al., (1994) Cell 78, 147-16.
0).

[0006]

The present invention utilizes the fact that the GnT-V enzyme is involved in cancer metastasis and that the Ets-1 protein is involved in the regulation of gene transcription, as described above. Accordingly, it is intended to provide a method for screening for a metastasis inhibitor and a kit therefor.

[0007]

As described above, GnT-
The V gene is involved in promoting cancer metastasis, and Ets-1
Proteins are involved in the regulation of gene transcription and are known to be involved in tumorigenesis and the like. We have developed Gn
As a result of examining the relationship between the transcription and expression of the TV gene and the Ets-1 protein, the Ets-1 protein binds to a specific site in the promoter region upstream of the GnT-V gene, and
The present inventors have found that the transcription of the TV gene is promoted, and solved the above-mentioned problems.

That is, (a) the following base sequence: 5'-GGGAGTGAGGATGATGTGTAGGA
AG-3 '(SEQ ID NO: 1) 5'-ATGGGGCAGAGGAACTTACGTT
A DNA containing AT-3 '(SEQ ID NO: 2) or at least one thereof;
The ts-1 protein or fragment thereof and (c) the sample are incubated together and the DNA (a) and Ets
A method for screening for a cancer metastasis inhibitor by measuring the binding to a -1 protein or a fragment thereof.

The present invention further provides (a) the following base sequence: 5'-GGGAGTGAGGATGATGTAGGGGA
AG-3 '(SEQ ID NO: 1) 5'-ATGGGGCAGAGGAACTTACGTT
A DNA containing AT-3 '(SEQ ID NO: 2) or at least one thereof;
a DNA comprising a ts-1 protein or a fragment thereof;
A kit for measuring the binding between (a) and an Ets-1 protein or a fragment thereof is provided.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION First, the present inventors constructed an expression vector by ligating various sites of a promoter region upstream of a GnT-V gene to a luciferase gene (with a promoter removed) as a reporter gene. And
While the Ets-1 protein was acting, the expression of the luciferase gene was measured. In addition, the binding between the Ets-1 protein and various sites in the promoter region of the GnT-V gene was measured by a gel migration shift assay and a supershift assay.

From these measurements, the Ets-1 protein binding site in the promoter region of the GnT-V gene was changed to the following site: E266 5'-GGGAGTGA GGA TGATGT
AGGGAAG-3'-278 to -255 (SEQ ID NO:
1) E728 5'-ATGGGGCAGA GGA ACTT
ACGTTTAT-3'-741 to -718 (SEQ ID NO:
2).

Therefore, if a substance that inhibits the binding between the DNA having the above-described sequence or a sequence containing the same and the Ets-1 protein is found, it is highly possible that it is a substance that suppresses cancer metastasis. Accordingly, in the present invention, a DNA having the above-mentioned sequence or a sequence containing the above-mentioned sequence is used as a DNA having E.
The ts-1 protein or a fragment thereof, which binds to the DNA, is incubated with the test sample,
If the binding between NA and protein is inhibited, it is estimated to be a cancer metastasis inhibitor. Therefore, the present invention is useful as a primary screening method for selecting a cancer metastasis inhibitor.

[0013]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG. 5'-untranslated region of human GnT-V gene
Of functional regulatory elements within the human cholangiocarcinoma HuCC-T1 cells
Obtained from urces Bank. 10% fetal bovine serum (Li
fe Technologies, Inc.), RPMI 1640 medium supplemented with 50 mg / ml streptomycin sulfate and 50 units / ml penicillin (Nakarai Tesque,
Japan).

The 5'-upstream region of the GnT-V gene is defined as Hu
Cloned from the genome of CC-T1 cells (Saito,
H. et al., (1995), Eur. J. Biochem.
233, 18-26). A 5'-upstream region of various lengths was inserted at the XhoI site (Klenow-filled in) of the pGV-enhancer vector (Toyo Ink Co., Ltd., Japan) in front of the luciferase gene lacking the promoter.

[0015] This structure corresponds to each of exon 1 and 2.
It contains 2 kb and 0.6 kb upstream regions and 1.0 kb intron 1 and contains pGV-2760 / -440 (S
alI / XbaI, Klenow-filled in), p
GV-642 / + 23 (XbaI / PuvII, Klenow-filled in) and pGV-23 / + 1123
(FIG. 2). 2 μg of SV40 promoter p
GV-control vector (lane 1), pGV-enhancer vector lacking promoter (lane 2), -276
0 / -440 pGV-construct (lane 3), -642
/ + 23 pGV-construct (lane 4) and + 23 / +
The 1123 pGV-constructs (lane 5) were each Hu
Transiently transfected into CC-T1 cells.

HuCC-T1 cells were plated at 1.7 × 10 ⁵ cells per 60 mm dish for one day prior to transfection. They were combined with 2 μg of reporter plasmid, various expression vectors and pCH110 (N
ovagen) were transfected with Lipofectamine reagent (Gibco BRL) containing 2-5 μg of various expression vectors, where the β-galactosidase expression vector was used to measure transfection efficiency. Briefly, diluted DNA2
~ 5 µg and 5 µl of Lipofectamine reagent into 200 µl
mixed with 1 serum-free RPMI 1640, DNA
-Formed a liposome complex. After a 30 minute incubation, 800 μl of serum-free medium was coated on previously rinsed cells.

The cells are incubated with the complex for 6 hours, followed by addition of 1 ml of 20% fetal calf serum medium,
Incubation was continued for another 18 hours. After removing the complexes by washing, the cells were washed with RP containing 10% FCS.
Cultured in MI1640 for 24 hours. Luciferase activity in cell lysates, Pica Gene ^TM Lumines
cence Kit (Toyo Ink Co., Ltd., Japan). Β-galactosidase activity in cell lysates was determined as reported (Herbomel, P. et al.
(1984) Cell 39, 653-662),
And used as an internal control for variation on transfection efficiency. The results are shown in FIG. 3 as the mean ± SEM of 3-5 independent experiments. D. Indicated by

To prevent the possibility that the genomic insert in the pGV-2760 / + 23 construct contains negative elements,
Two additional pGVs containing small genomic fragments were analyzed (pGV-2760 / -440 and pGV-6
42 / + 23). Both constructs expressed luciferase activity, while pGV-2760 / -440 did not
It showed lower promoter activity compared to the V40 promoter vector. However, the corresponding deleted construct, pGV-1460 / -440, has a strong promoter activity (3.
13 times) (FIG. 4).

Among these constructs, pGV-642 /
+23 shows 182 fold luciferase activity when compared to the vector lacking the promoter,
V-2760 / -440 and pGV + 23 / + 1123
Shows 124- and 169-fold luciferase activities, respectively, and was thus found to be the most active. The results show that the two fragments acted as promoters and the negative element was pGV-2760 /
It strongly suggests that it is present in the -1460 structure.

Embodiment 2 FIG . 5 'of human GnT-V gene
-Deletion analysis of untranslated region To further characterize the potential regulatory domains containing an Ets binding site in the 5'-untranslated region of exon 1, plasmids pGV-2760 / -440 and pGV-64
A series of 5 'deletion constructs of the GnT-V 5'-untranslated region were prepared using 2 / + 23, and then the deleted construct was inserted into HuCC-T1 cells for determination of luciferase activity. The body was transferred (FIG. 2). pGV-
A series of 5'-deletions of 2760 / -440 and pGV-642 / + 23 were generated by the nested deletion method as previously described (Henikoff, S. (1987).
Methods Enzymal. 155.156-1
65). The deleted nucleotide site was confirmed by general sequence analysis.

Plasmids pGV-243 / + 23 and pGV-112 / + 23 upstream of exon 1 of 200 and 100 bp in length, respectively, were ligated to a set of oligonucleotide primers (sense, 5'-ggacgcgtTCT).
TACCATATAGAAC-3 ′ (SEQ ID NO: 3),
5'-ggacgcgtGCCTAGATGATCAG
TC-3 '(SEQ ID NO: 4) and antisense, 5'-
ggctcgagGCCTCTTACTGTTTTC-
3 '(SEQ ID NO: 5)). The PCR products were subcloned into the pT7 Blue-T vector (Novagen), sequenced, and then their sequences were compared to the sequence of the 2.76 kb fragment. Individual fragments were converted to MluI
And XhoI, followed by subcloning into a pGV-enhancer vector containing only the SV40 enhancer element (MluI / XhoI sites).

PGV-2664 / -440 (FIG. 4) or pGV-642 / + 23 (FIG. 5) were transfected into HuCC-T1 cells and the relative luciferase activity of each deletion mutant was determined 3-5 times. About the independent experiment of
% Of the activity of the pGV-enhancer vector in HuCC-T1 cells, i.e. mean ± S. D. Expressed as

HuCC-T1 cells express endogenous GnT-V activity, suggesting that the cells contain the nuclear factors required for GnT-V promoter activity. The results are shown in FIGS. 4 and 5 where pGV
The -1460 / -440 construct is pGV-EA-2760
It shows a 2.5-fold increase in luciferase activity as compared to / -440, indicating that the adjacent region is more active than the full length of the distant region. An additional deletion for nucleotide -710 bp removes a potential Ets-1 binding site (position -728) and results in a 3.4-fold reduction in luciferase activity.

Compared to the pGV-642 / + 23 construct, pGV-362 / + 23 showed more promoter activity, indicating that this region functions as the core promoter of the GnT-V gene. Nucleotide-2
Deletion in this region, extending to 43 bp, removes another Ets-binding site (position -266), resulting in a 13.2-fold reduction in luciferase activity. Thus, the most proximal region (-362 / -243) is about 3.9-fold more active than the distal region (-1460 / -710), which is a very significant increase in the upstream region of the GnT-V gene. Shows high luciferase activity.

The results show that the region in its proximal region (-36
2 / -243) and the distal region (-1460 / -71)
0) strongly acts as a positive regulatory element that regulates the promoter activity of the GnT-V gene in HuCC-T1 cells. However, an additional deletion of about 100 bp (pGV-112 / + 23) resulted in an 8.3-fold increase in activity in HuCC-T1 cells, which was-
-243 / -1 can regulate GnT-V transcription in HuCC-T1 cells like the 2760/1460 element
12 shows the presence of a negative regulatory element in 12 regions.

Embodiment 3 FIG . Regulatory region of GnT-V gene
Analysis of the binding of the Ets-1 protein to the putative sequence of E. coli.
The 710 and -362/243 regions are HuCC-T1-
It has been shown to act as a cis-acting element for restriction transcription. LBP-1 (position-1334, -1038,
-935 and -932), AP-2 (pos. -1264 and -227), NF-IL6 (pos. -1385, 100
5, -965 and -255), c-myb (position -28
7), and several putative binding sites for Ets-1 (positions 728 and 266) have been shown to be included in those two elements.

We analyzed three putative binding sites for the transcription factor Ets-1, a product of the proto-oncogene associated with malignant transformation and metastasis of tumors. Ets
To determine if the -1 protein can bind to their putative binding sites in the GnT-V gene regulatory region, gel mobility shift experiments were performed on the 5 'of the GnT-V gene.
-24-bp GnT-V promoter-derived oligonucleotides, E266, E565 and E728, corresponding to the three putative Ets-binding sites in the untranslated region
This was performed using First, we confirmed that the in vitro transcribed / translated truncated Ets-1 protein and nuclear extracts of MOLT4 cells did indeed bind to the Ets-1 consensus sequence (FIG. 6).

A fragment encoding the full-length sequence of Ets-1 was obtained by polymerase chain reaction (PCR), and then the pSVK3 vector (Pharma
cia). The nucleotide sequence was confirmed by general sequence analysis. This plasmid is called X
digested with hoI / SacI and treated with Ets-1cD
NA was converted to Xh of pET19b vector (Novagen).
subcloned into the oI-SacI site, where the protein is E. coli. It was expressed in E. coli BL21 (DE3) under the control of the T7 promoter.

GnT of Ets-1 consensus binding sequence
A DNA fragment containing the putative Ets-binding sequence of the regulatory region upstream of -V was synthesized as follows: E26
6,5'-GGGAGTGA GGA TGATGTAGG
GAAG-3 '(SEQ ID NO: 1); E565, 5'-C
TTGTTAAA GGA TAGGCTGTGGAC-
3 '(SEQ ID NO: 6); E728, 5'-ATGGGG
CAGA GGA ACTTACGTTTAT-3 '(SEQ ID NO: 2); EB, 5'-CGGCCAACCGGAAG
CAGTTGC-3 '(SEQ ID NO: 7), mE266
5'-GGGAGTGA GTA TGATGTAGGGGA
AG-3 '(SEQ ID NO: 8) and mE728,5'-
ATGGGGCAGA GTA ACTTACGTTTAT-
3 '(SEQ ID NO: 9). Complementary oligonucleotides were annealed and used as probes or competitors.
Probes were probed with [γ- ³² P] dATP (Amersham Corp.) and T4 polynucleotide kinase (Takara, Osaka, Jap.
an) labeled with their 5'-extended ends.

For the gel mobility shift assay, D
NA (10,000 cpm, ³² P-labeled) and protein prepared by nuclear extract or in vitro transcription / translation were combined with 65 mM KCl, 25 mM Tris-HCl (p
H7.9), 6 mM MgCl ₂ , 0.25 mM EDTA
And a buffer containing 10% glycerol in a total volume of 20 ml. 2 μg of poly (dI-dC) (Si
gma) was also added to the individual reaction mixtures. For competition analysis, proteins were incubated in the presence of excess unlabeled oligonucleotide.

In FIG. 7, γ- ³² P-labeled oligonucleotide probes E266 (lane 2), E7
28 (lane 7) and E565 (lane 12) were incubated with in vitro transcribed / translated Ets-1 protein. For competition analysis, the protein was added to excess unlabeled oligonucleotide E2.
Incubation was performed in the presence of E.66 (lanes 3-5), E728 (lanes 8-10) and E565 (lane 13). In FIG. 8, the γ- ³² P-labeled oligonucleotide probes E266 (lane 2) and E728
(Lane 6) was incubated with nuclear extracts of MOLT4 cells.

For competition and supershift assays, add 100-fold unlabeled oligonucleotide or 1 μg of anti-Ets-1 antiserum, respectively.
Subsequently, it was incubated at room temperature for 1 hour before electrophoresis (lanes 3 and 4, and 7 and 8 in FIG. 8). FIG. 9, m
The E266 and mE728 probes are E266 and E72
Eight probe point mutations, converting three GGA sequences in the GnT-V promoter to GTA. The unlabeled oligonucleotides were added in excess and incubated with the reaction mixture of MOLT4 nuclear extract (Figure 9, lanes 4 and 7).

For the supershift assay, Et
Anti-Ets-1 I which does not cross react with s-2 protein
gG (Cambridge Research Biochemicals) was added to the reaction mixture, followed by incubation at room temperature for 1 hour. Samples were run on a 6% non-denaturing polyacrylamide gel (acrylamide: bisacrylamide, 29: 1),
0.5 × TBE (1 × TBE = 89 mM Tris, 89 mM
Boric acid, 2 mM EDTA) and then electrophoresis was performed at 4 ° C. and 150 V for 1 hour. After electrophoresis, the gel was dried with a gel dryer and then exposed to X-ray film (Kodak, Japan).

As a result, radioactively labeled E266,
When E565 and E728 were incubated as described above with the in vitro transcribed / translated Ets-1 protein, the protein-DNA complex was converted to E26.
6 and E728 were delayed, but E
This was not the case with the 565 oligonucleotide (FIG. 7, lanes 2, 7 and 12). FIG. 7 shows the results of the competition analysis. Excessive unlabeled E266, E56
5 and E728 oligonucleotides were used.

Unlabelled E266 and E728
Oligonucleotides were labeled E266 and E72
8 was found to compete with the protein binding to the probe, and Et to the Ets-binding site of the GnT-V regulatory region.
Specific and high affinity binding of the s-1 protein was demonstrated. The protein-DNA complex effectively cross-competed with either an excess of E266 or E728, but did not compete with the oligonucleotides mE266 and mE728 containing the mutated Ets binding sequence (data shown). Absent).

These results indicate that in vitro transcribed / translated truncated Ets recognizing the Ets consensus sequence.
-1 protein at positions -266 and -728, Gn
TV promoter sequence, ie, 5'-GGGAGT
GA GGA TGATGTAGGGAAG-3 '(SEQ ID NO: 1) and 5'-ATGGGGCAGA GGA ACT
It suggests that it can bind to TAGTTTAT-3 '(SEQ ID NO: 2).

To determine whether the Ets-binding site from the GnT-V promoter is involved in nuclear factors in Ets-1 expressing cells, it was prepared from MOLT4 cells that highly express Ets-1 protein. The results of performing a gel mobility shift assay using the same Ets-binding sequence from the GnT-V promoter incubated with the extracted nuclear extract are shown in FIG. The radiolabeled E266 and E728 oligonucleotides are
When incubated with nuclear extracts prepared from LT4 cells, a delayed protein-DNA complex was detected (FIG. 8, lanes 2 and 6), and specific binding was identified on competition analysis (FIG. 8). , Lanes 3 and 7).

The binding of Ets was confirmed by an Ets-specific antiserum. That is, anti-Ets-1. Addition of IgG resulted in competition with the protein-DNA complex (FIG. 8, lanes 4 and 8). However, the mE266 and mE728 oligonucleotides containing the mutated Ets binding sequence did not compete (FIG. 9, lanes 4 and 7). These results indicate that the HuCC-GnT-V gene
T1-24 bp Ets- of restricted promoter region
The binding of the Ets-related factor to the binding site was shown.

Embodiment 4 FIG . Human GnT-V promoter
In regulating GnT-V transcription in transactivated viable cells
To confirm the importance of -1, the Ets-1 cDNA
Was subcloned into the pSVK3 vector. Where S
The V40 promoter is c-Ets- in mammalian cells.
1 Drive expression of cDNA. GnT-V gene is E
To determine if it could be activated by ts stimulation alone, 2 μg of the construct pGV-1460 / -440 was added to
Co-transfected into HuCC-T1 cells with 1 μg (FIG. 10, lane 4) or 2 μg (FIG. 10, lane 5) of Ets-1 expression plasmid SVETS-1.
As shown in FIG. 10, HuCC-T1 cells were SVE
After transient co-transfection with TS-1 and pGV-1460 / -440, luciferase activity is increased 2.1-fold or more in the presence of 1 μg of Ets-1 expression vector, while promoter activity is increased by Ets-1 expression vector. It decreased in the presence of 2 μg.

1 μg of pGV-362 / + 23 (FIG. 1)
1, lane 4) or 2 μg (FIG. 11, lane 5) SV
It was co-transfected into HuCC-T1 cells with ETS-1. Relative luciferase activity was expressed as mean ± SEM for 3-5 independent experiments. D. Expressed as S
VETS1 and pGV-362 / + 23 were converted to HuCC-T
When co-transfected into one cell, it was observed that the promoter activity increased in a dose-dependent manner (FIG. 1).
1).

The result of this trans-activation was Ets-1
To study its regulatory function in more detail, suggesting that it can activate the GnT-V promoter containing its binding site, the antisense Ets-1 mR
Using NA, human GnT- in HuCC-T1 cells
The formation of Ets-1 which increases the promoter activity of the V gene was inhibited.

The HuCC-T1 cells were transformed with 2 μg of pGV-
1460 / -440 (FIG. 12) or pGV-362 / +
23 (FIG. 13) construct and varying amounts of antisense E
Co-transfected with the ts-1 expression plasmid SVETSA. Relative luciferase activity is 3 ~
Mean ± SEM for 5 independent experiments. D. Expressed as As shown in FIG. 12, pGV-1460 / -440 causes a 60% reduction in luciferase activity when co-transfected with 1 μg of antisense expression plasmid (SVETSA) and is co-transfected with 10 μg of antisense expression plasmid. In that case, it caused a 100% decrease in luciferase activity. However, no change in the promoter activity of pGV-362 / + 23 was observed (FIG. 13). These findings suggest that the transcription factor Ets-1 protein positively regulates the complex transcription machinery mediated by GnT-V gene transcription.

[0043]

[Sequence list] SEQ ID NO: 1 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGGAGTGAGG ATGATGTAGG GAAG 24

SEQ ID NO: 2 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATGGGGCAGA GGAACTTACG TTAT 24

Sequence number: 3 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGACGCGTTC TTACCATATA GAAC 24

SEQ ID NO: 4 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGACGCGTGC CTAGATGATC AGTC 24

SEQ ID NO: 5 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGCTCGAGGC CTCTTACTGT TTTC 24

SEQ ID NO: 6 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CTTGTTAAAG GATAGGCTGT GGAC 24

SEQ ID NO: 7 Sequence length: 21 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: synthetic DNA sequence CGGCCAACCG GAAGCATGTG C 21

SEQ ID NO: 8 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGGAGTGAGT ATGATGTAGG GAAC 24

SEQ ID NO: 9 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATGGGGCAGA GTAACTTACG TTAT 24

[Brief description of the drawings]

FIG. 1 shows the formation of branched glucans that are expected to be involved in cancer metastasis.

FIG. 2 is a diagram showing various structures of the 5 ′ untranslated region of the GnT-V gene. Ets binding sites in the GnT-V regulatory region are at positions -266, -565 and -72.
8 exists. SalI, XbaI and PuvII indicate specific restriction sites. The numbers on the left indicate the origin of the individual deletion constructs associated with the genomic map shown above. Arrows indicate early transcription sites. The indicated restriction sites are: S, SalI; X, XhoI; H, Hinf.
I.

FIG. 3 shows the basic promoter activity in HuCC-T1 cells relative to luciferase activity.

FIG. 4 is a graph showing the transcriptional activities of various GnT-V promoters as luciferase relative activities based on transfection into HuCC-T1 cells.

FIG. 5 is a graph showing transcriptional activities of various GnT-V promoters as luciferase relative activities based on transfection into HuCC-T1 cells.

FIG. 6 shows G directly linked to the luciferase gene.
HuCC-T1 of nT-V promoter and deletion construct
It is a figure which shows activity in a cell. Consensus sequence probe used as probe.

FIG. 7 is the same as FIG. 6, but shows the results using E266, E728 and E565 as probes.

FIG. 8 is a view showing the results of a gel migration shift assay using the Ets-1 protein for the control region of the GnT-V gene. E266 and E728 used as probes.

FIG. 9 is a view similar to FIG. 8, but showing the results using E266, E728, mE266 and mE728 as probes.

FIG. 10 shows Ets for human GnT-V gene promoter activity in HuCC-T1 cells.
1 is a graph showing the effect of -1 protein.

FIG. 11 shows Ets for human GnT-V gene promoter activity in HuCC-T1 cells.
1 is a graph showing the effect of -1 protein.

FIG. 12 is a graph showing suppression of GnT-V promoter expression by an antisense Ets-1 gene.

FIG. 13 is a graph showing the activity of the GnT-V promoter by the antisense Ets-1 gene.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12N 15/09 C12N 15/00 A

Claims (2)

[Claims] 1. (a) the following base sequence: 5′-GGGAGTGAGGATGATGTAGGGGA
AG-3 '(SEQ ID NO: 1) 5'-ATGGGGCAGAGGAACTTACGTT
A DNA containing AT-3 '(SEQ ID NO: 2) or at least one thereof; (b) an Ets-1 protein or a fragment thereof; and (c) a sample, and the DNA (a) A method for screening for a metastasis-suppressing substance, which comprises measuring the binding of the compound to Ets-1 protein or a fragment thereof. 2. (a) the following nucleotide sequence: 5′-GGGAGTGAGGATGATGTAGGGGA
AG-3 '(SEQ ID NO: 1) 5'-ATGGGGCAGAGGAACTTACGTT
Binding of the DNA (a) comprising AT-3 '(SEQ ID NO: 2) or a DNA containing at least one thereof, and (b) an Ets-1 protein or a fragment thereof to the Ets-1 protein or a fragment thereof Measurement kit.