JPH10117779A - Cancer cell having tumorigenicity and free from infiltrating and metastasizing ability, its preparation and screening of metastasis-related gene using the same cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish the subject cancer cell strain suitable for researching the difference between a signal transmission circuit for obtaining tumorigenicity and a signal transmission circuit for obtaining infiltrating and metastasizing ability by transducing a specific cancer gene into a recipient cell. SOLUTION: A cancer cell strain having tumorigenicity, but not having infiltrating and metastasizing ability is established by a method to transduce a cancer gene of ras family into a cell derived from BALB/C 3T3 A31, etc. A 1-1 ras 1000 cell (FERM BP-5406) is preferably used as the cancer cell. The resultant cancer cell strain is useful for screening of the resultant cancer metastasis-related gene and research of characteristics of infiltrating and metastasizing ability.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cancer cell having tumorigenic ability but not invasive / metastatic ability, a method for producing the same, and a method for screening metastasis-related genes using the same.

[0002]

2. Description of the Related Art Cancer cell invasion and metastasis occur at the final stage of cancer progression and are the greatest cause of death in cancer patients.
However, there are many unclear points about the molecular mechanism for developing the malignant trait of cancer cells such as invasion and metastasis, which makes treatment difficult. Although it has long been recognized that pathologically, it is a biological phenomenon that distinguishes tumor formation from the development of malignant traits such as invasion and metastasis during the carcinogenesis, its biochemical basis The information on is poor, and the identification of therapeutic target molecules has not been advanced.

[0003] Generally, it is thought that cells accumulate and become invasive / metastatic due to accumulation of changes in genetic information of oncogenes and tumor suppressor genes. That is,
A mutation occurs in a gene responsible for transmitting a signal from the outside to the nucleus of a cell, and the gene product always sets the downstream information transmission pathway to “ON”. This causes the expression of cancer cells. In addition, in the progression of cancer, it is thought that, in addition to the genetic changes required for tumorigenicity, further changes occur, resulting in the invasion and metastasis of cancer cells (Evans CW. The Metastatic Cell). : Behavior and Bio
chemistry, Chapman & Hall, London, 1991). Therefore, metastasis of cancer cells is induced by abnormalities in the intracellular signal transduction system, and it is considered that the signal transduction system is different from the signal transduction system necessary for obtaining tumorigenicity. Therefore, it is extremely important to elucidate the cancer-related signal transduction circuit specific to metastasis that causes cancer cells to acquire the invasion / metastasis ability in studying the mechanism of invasion / metastasis.

[0004] For such research, if cells are obtained which introduce oncogenes into cells and obtain tumorigenicity and invasive / metastatic potential separately, the above-mentioned circuits and molecules involved in the circuits will be identified. Useful for

[0005] While both cells originate from a common parent strain, some cells have tumorigenic potential but no invasion / metastasis, while one cell has both. If such a set of two types of cells is obtained, it is useful for studying a signal transmission circuit related to metastasis. Several cancer cell lines have been obtained that meet this criteria. For example, in a rodent-derived cell line, K1735, B16, T-lymphoma L5
178Y, Lewislung carcinoma,
rat assists hepatoma AH797
4, and a derivative strain obtained by recovering cells from the in vivo metastasized portion and culturing and selecting in vitro (Fidler IJ et al., J. Natl. Cancer Inst.
67: 947-956, 1981; Talmadge JE et al., J. Natl. Ca
ncer Inst. 69: 975-980, 1982; Fidler IJ, Nature 24
2: 148-149, 1973; Fidler IJ, Cancer Res. 35: 218-22
4, 1975; Fidler IJ et al., Am. J. Pathol. 97: 633-6
48, 1979; Ota et al., Clin. Exp.Metastasis 10: 297
-308, 1992; Olsson Let al., Cancer Res. 41: 4706-47
09, 1981; Brodt P, Cancer Res. 46: 2442-2448, 1986;
Pal K et al., Invasion Metastasis 5: 159-169, 198
5; Young MR etal., Cancer Res. 45: 3918-3923, 1985;
Kawaguchi T et al., Clin. Exp.Metastasis 10: 225-
238, 1992). However, the traits of these cell lines may be unstable during the experiment (Poste G et al., Proc. Nat.
l. Acad. Sci. USA 78: 6226-6230,1981)
Many properties of cells that are not involved in gaining metastasis change with in vivo selection.

It would be useful and valuable if a stable set of the above cell lines could be obtained by oncogene transfer. However, whether or not a tumorigenic and invasive / metastatic potential is obtained by oncogene transfer depends on the cell line used (Muschel RJ et a
l., Am. J. Pthol. 121: 1-8, 1985). Furthermore, the introduction of oncogenes often induces invasive / metastatic potential in recipient cells to a greater or lesser degree, together with the acquisition of tumorigenic potential (Thorgeirsson
UP, Mol. Cell. Biol. 5: 259-262, 1985; EganSE et a
l., Science 238: 202-205, 1987). To date, no report has been obtained that strictly established cells having tumorigenic ability but not invasion / metastasis and cells having both of them. Therefore, in an experimental system, it was not easy to separate the characteristics of the tumorigenicity of cancer cells and the characteristics of invasion / metastasis for use in research.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a tumorigenic, invasive / invasive tumor suitable for studying the difference between a signal transmitting circuit for acquiring tumorigenic ability and a signal transmitting circuit for acquiring invasive / metastatic ability. The purpose is to establish a cancer cell line that has no metastatic potential. Such cancer cell lines can be used to study the characteristics of invasive / metastatic potential by using them in combination with cell lines that develop from a common parental line and have both tumorigenic and invasive / metastatic properties. it can. It is also an object of the present invention to develop a method for screening for a metastasis-related gene using the cancer cell line of the present invention.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has shown that by introducing a specific oncogene into a recipient cell, it has tumorigenic potential,
A cancer cell line without metastatic potential could be established.

That is, the present invention provides a cancer cell having a tumorigenic ability and no invasive / metastatic ability. A preferred cancer cell of the present invention having such properties is accession number FERM BP
This is 1-1ras1000 cells deposited at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology at -5406.

The present invention also relates to BALB / c 3T3 A
The present invention provides a method for producing a cancer cell having the above-mentioned tumorigenic ability and no invasion / metastasis by introducing a ras family oncogene into a 31-derived cell.

[0011] The present invention further comprises introducing DNA derived from a cancer tissue or a cancer cell line obtained from the surface of a mammal or from the body into a cancer cell having a tumorigenic ability and no invasion / metastasis, thereby obtaining invasion / metastasis. Provided is a method for screening a gene having a property of imparting invasion / metastasis, comprising isolating a substance having acquired the ability and extracting the DNA.

When selecting recipient cells that can be used to prepare the cell line of the present invention that has a tumorigenic ability and has no invasive / metastatic ability, the cells set as described above after the preparation of such a cell line are used. Strains, that is, those capable of producing cell lines having both tumorigenic ability and invasive / metastatic ability are preferred. In view of this, the recipient cell into which the oncogene is to be introduced has the following properties: 1) has a non-metastatic trait and is stable; ) Invasion / metastasis-capable cells can be obtained by the introduction of oncogenes. 3) Cells that have acquired invasion / metastasis by the introduction of oncogenes can be selected.

Recipient cells satisfying such properties include, for example, BALB / c 3T3A31 cells (Kaku
naga T, et al. Editing, Transformation Assay of Estab
lished Cell Lines: Mechanisms and Application, Oxf
ord University Press, New York, 1985, p55-73).
It can be used without limitation. BALB / c
Examples of 3T3 A31 cells include, for example, BALB / c established by Kakunaga used in Examples of the present invention.
3T3 A31-1-1, BALB / c 3T3 A3
1-1-8 and BALB / c 3T3 A31-1-
13 (Kakunaga et al., Sci.
ence 209: 505-507, 1980).

An oncogene to be introduced into a recipient cell is an active cancer that causes the recipient cell to form a focus (the formation of a focus, that is, the swelling of the cell means canceration of the cell) and does not impart invasive / metastatic ability. Gene, eg r
As family genes can be used. In the present invention, cancer cells having tumorigenic ability but no invasive / metastatic ability could be established using activated c-Ha-ras, but the present invention is not limited thereto.

To prepare the cancer cell of the present invention, a plasmid having the above-mentioned oncogene incorporated therein is prepared, and this is introduced into a recipient cell. Methods for introducing oncogenes into recipient cells include electroporation (Tatsuka et al., Exp.
Cell Res. 178: 154-162, 1988) is convenient, but other methods such as a calcium phosphate method and a microinjection method can also be used.

After transformation, the cells are seeded on a dish and subjected to a focus formation assay, and the transformed foci that have emerged are isolated and expanded as independent cell lines or pooled.

To confirm that the oncogene has been integrated into the transformed cells, Southern blot analysis is performed, and to determine whether the transgene has been transcribed into mRNA, the transcribed mRNA must be analyzed. Northern blot analysis can be performed. Furthermore, to confirm whether mRNA was translated into a protein, for example, anti-p21
Immunoblot analysis using ^ras antibodies can be performed.

The tumorigenicity and invasive / metastatic potential of the cell line thus obtained are confirmed using animals. In the present invention, BALB / c nude mice are used, and for tumorigenicity assay, transformed cells are injected subcutaneously into nude mice.
The presence or absence of tumor formation, the growth rate, and the incubation period (the period from injection to the recognition of cancer bumps) were observed. In the experimental metastasis assay, the transformed cells were injected into the tail vein of nude mice. Five weeks later, the mice were sacrificed, and the presence or absence of metastasis was confirmed by necropsy.

In the present invention, as described above, a cell line having tumorigenic ability but not invasive / metastatic ability could be established. Among the cell lines established in the present invention, a particularly preferred cell line is named 1-1ras1000 cell, and dated February 20, 1996, the Institute of Biotechnology and Industrial Science and Technology (1-Higashi, Tsukuba, Ibaraki, Japan). No. 1 (zip code 305) was deposited internationally under accession number FERM BP-5406. Further, 1-13ras1000 cells are also preferred cancer cells having the same properties as 1-1ras1000 cells.

The 1-1 ras1000 cell is one of the clones obtained by performing an amplification operation in order to increase the expression level of the introduced oncogene, and is about 16 times as large as the parent strain when isolated from the foci. Is amplified and it can be said that the clone is overexpressed. 1-1ras100
The non-metastatic trait of 0 cells is extremely stable, and conversion to metastatic does not occur by in vitro passage or treatment of an initiator such as ultraviolet light or methylcholanthrene and TPA. Further, even if selection is performed by passing through a micropore filter coated with a basement membrane (Matrigel), it is very difficult to obtain a clone having invasion / metastasis ability.

Therefore, 1-1ras1000 cells do not express a metastatic trait, and their non-metastatic traits are very stable. However, this property is 1-
It cannot be denied that 1ras1000 cells may have no invasion / metastasis potential. Therefore,
When these cells were transfected with the oncogene src, numerous metastatic foci were observed in the lungs. From this, 1-
It was found that 1ras1000 cells did not lack the step of acquiring invasion / metastasis. The motility of cells that had taken up src with invasive / metastatic ability was higher than that of cells that had taken up ras on the basement membrane (Matrigel). The src-transformed cells also showed invasiveness to the basement membrane in experiments using a micropore filter.

According to the present invention, a cancer cell line having tumorigenic ability but no invasive / metastatic ability and a cell line having both of the above-mentioned tumorigenic ability and invasive / metastatic ability were obtained as a set. As a result, it became possible to separate the characteristics of invasion / metastasis from those of tumorigenicity for use in research.

Using the cell line of the present invention which has tumorigenic ability but does not have invasive / metastatic ability, screening for a gene imparting invasive / metastatic ability can be carried out. In order to carry out the screening method of the present invention, a cell line of the present invention is obtained by introducing DNA derived from a cancer tissue or a cancer cell line obtained from the surface of a mammal or from the body into a cell line of the present invention, and acquiring invasion / metastasis ability. By separating and extracting the DNA, a gene having a property of imparting invasion / transfer ability can be screened.

A specific screening method for a gene having the property of imparting invasion / metastasis using the 1-1ras1000 cell line of the present invention can be performed by the following procedure. 1) Genomic gene of metastatic cancer cells (derived from cancer tissues or cancer cell lines obtained from or on the body of mammals) or cDN prepared using mammalian cell expression vectors
An A library (derived from a cancer tissue or cancer cell line obtained from the body surface or body of a mammal) is prepared. 2) The genomic gene or cDNA library obtained in 1) and a selection marker (eg, a hygromycin resistance gene) are introduced into 1-1ras1000 cells. It can be introduced using a calcium phosphate method, an electrical method, a liposome method, or the like. As a selection method, a neo resistance gene, gpt selection, or the like can be used. 3) When the selectable marker is a hygromycin resistance gene, the cells are cultured in a medium containing hygromycin to obtain hygromycin resistant cells. 4) Hygromycin-resistant cells are selected based on the invasive / metastatic ability. 5) c-Ha containing Alu sequence specific to human gene
Recover gene fragments that are not ras. Plaque hybridization can be used for recovery of the gene fragment, for example.

When a cDNA library prepared using a mammalian cell expression vector is introduced, it may be recovered as an episome (Tatsuka M et al., Nature
359: 333-336, 1992), plasmid rescue and PC
Methods such as R and library production can be used. 6) Each of the collected gene fragments is introduced alone or in a mixture into 1-1ras1000 cells.
Check to obtain metastatic ability. Specifically, for example,
Phage DNA obtained by plaque hybridization
Is introduced into 1-1ras1000 cells individually or mixed, and the transfected cells are injected subcutaneously or into the tail vein of a nude mouse to obtain a phage clone in which cells that have become metastatic appear. 7) Using a gene conferring invasion / metastasis as a probe,
Near-full-length cDNA is isolated from the NA library and the gene sequence is determined. The amino acid sequence is determined based on this. 8) Using the cDNA isolated in 7) as a probe, a human genomic gene library is screened, and a human genomic gene containing all exons is isolated. 9) Incorporate the cDNA separated in 7) into a mammalian cell expression vector, and introduce it into 1-1ras1000 cells to confirm whether or not to acquire invasive / metastatic ability.

The gene thus obtained can be said to be a gene that imparts invasive / metastatic ability to cancer cells.

Conventionally, various studies have been made to screen for metastasis-related genes. For example, Weiberg
The group attempted to clone the transgene using NIH3T3 cells, but failed to do so (Bernstein SC and Weiberg R, Proc. Nat.
l. Acad. Sci. USA 82: 1726-1730, 1985). In addition, by extracting mRNA from highly metastatic cancer cells, separately extracting mRNA from low metastatic cancer cells, and comparing these genes to identify mRNA that is more abundant in highly metastatic cancer cells,
nm23 (Leone A et al., Cell 65: 25-35, 1991),
CD44 (Gunthert U et al., Cell 65: 13-24, 199
1), Kai-1 (Dong JT et al., Science 268: 884-88)
6, 1995). Alternatively, an endogenous transgene (Tiam-1) by the virus-insertion mutagenesis method has been cloned (Habets GGM et al., Cell 77: 537-549, 1994). However, the genes isolated by such a method are not necessarily transfer-related genes in many cases, and the efficiency of the transfer-related gene screening is low.

Since the 1-1ras1000 cells of the present invention have tumorigenicity and no invasive / metastatic ability, a gene derived from a cancer cell is introduced into these cells. Genes are thought to be deeply involved in the acquisition of invasive and metastatic potential. By using such a screening method of the present invention, a gene that imparts invasion / metastasis to cancer cells can be efficiently identified, which is extremely useful in studying the mechanism of metastasis.

In the present invention, it was confirmed that when the v-src oncogene was actually introduced into 1-1ras1000 cells, the cells acquired invasive / metastatic ability. Also, v-src
Also invaded the parent strain, BALB / c 3T3 A31.
Transferability was imparted. These facts suggest that the src gene is a gene involved in imparting invasive / metastatic ability.

Further, in the present invention, DNA is extracted from cancer cell lines having various metastatic potentials, and the cells are used to transfect 1-1ras1000 cells of the present invention having no metastatic potential, and the transformed cells are transferred to nude mice. The injection was tested for metastatic potential. As a result, it was confirmed that the transformed cells had acquired the metastatic ability, and the metastatic ability was stable even after subculture, and no change was observed in the metastatic ability. Therefore, 1-1ras1000 cells have D
It proved to be a useful tool for screening NA.

Further, according to the present invention, a set of cancer cells having tumorigenic ability but not invasive / metastatic ability and cancer cells derived from the same parent strain and having both tumorigenic ability and invasive / metastatic ability were obtained. These are extremely useful for studying the difference between the signal transmission circuit for acquiring tumorigenic ability and the signal transmission circuit for acquiring invasive / metastatic ability.

By using the screening method of the present invention, it is possible to discriminate a benign tumor from a malignant tumor, to predict whether the metastasis is high or low, or to predict an organ-specific metastasis. Furthermore, a metastasis-related protein can be obtained from a gene identified by the screening method of the present invention as a gene that imparts invasion / metastasis to cancer cells, and new diagnostic and therapeutic agents for cancer can be developed. Can be expected. Industrial applicability of the present invention is extremely large.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.

[0034]

EXAMPLES The cell lines and plasmids used in the examples of the present invention are briefly described below. Cell line BALB / c 3T3 used as a recipient cell in the present invention
Clones 1-1 and 1-13 from A31 have been established by Kakunaga (Kakunaga T et al., Science 209: 505-50).
7, 1980), all of which are cancerous by radiation and chemicals, but their susceptibility is different. In this example, the original cells used in the initial stage of the culture were used. UV and M
Mutant cells induced by CA (3-methylcholanthorene) (T
atsuka M, Nature 359: 333-336, 1992), and Ki-
Cells mutated with MSV (Kirsten strain mouse sarcoma virus) (Kakunaga T, in Omen GS et al., (Eds), Genetic
Variability in Responses to Chemical Exposure, Co
ld Spring Harbor Laboratory, New York, 1984, p257-
274) was also used. All these cell lines were injected subcutaneously into nude mice and tested for tumorigenicity. Negative control cells used for experiments to confirm invasion / metastasis were 3Y
1. Positive control cells were fos-SR-3Y1-2
02 (Taniguchi S et al., Cancer Res. 49: 6738-674
4, 1989).

All cell lines are E-cells containing 10% FCS.
The cells were cultured in a MEM medium under 5% CO ₂ at 37 ° C. Plasmid pSV2neo-ras is the BamHI of pSV2neo.
Activated c-Ha-ras is inserted at the site. p
cDsrc has a v-src oncogene inserted at the EcoRI site of pcD (Yagi T et al., Mol. Carcin.
og. 1: 222-228,1989; Kizaka et al., Mol. Cell. Bio
l. 9: 5669-5675, 1989). The pHyg plasmid carrying the hygromycin resistance gene was also used. Example 1: Transformation of BALB / c 3T3 A31-derived cells Clone 1-1 (hereinafter sometimes referred to as A31-1-1) and 1-13 derived from BALB / c 3T3 A31 established by Kakunaga (A31-1-13
Was used to perform a susceptibility test for in vitro transformation. For the transformation experiment, the above-mentioned UV irradiation, MCA exposure, and the virus by Ki-MSV having the v-Ki-ras oncogene were used. The focus forming ability A31-1-13 of the transformed cells , as compared to A31-1-1, was more effective in transforming chemically and physically induced cancers as reported previously (Tatsuka M et al., Supra, 1992). For the transformation with Ki-MSV, the sensitivity of both clones was as reported previously (Ka
kunaga T, supra, 1984) There were morphological differences in the foci formed between the two clones. However, there was no difference in the form of the focus between the carcinogens used for the induction of transformation. A3 than A31-1-1
The focus derived from 1-1-13 was larger and faster in growth rate. Tumor-forming potential and invasive / metastatic potential of transformed cells All transformed cells were either nude mice or neonatal BA.
LB / c mice were injected subcutaneously and tested for tumor formation. On the other hand, injection of the parent strain and MOCK cells (eg, transformed cells without MCA alone) did not produce cancer. There was no significant difference in growth rate and incubation period between the two clones.

It is known that active ras oncogene often induces invasion / metastasis in transformed cells.
Therefore, the transformed cells into which the active ras oncogene was introduced were injected into the tail vein of nude mice, and it was examined whether a metastatic focus was formed in the lung.

[0037] Throughout the present invention, tumorigenicity and an experimental metastasis assay were performed by the following methods.

BALB / c nude mice 6 to 7 weeks old were prepared. Cells were collected after trypsinization and washed with PBS. In the tumorigenic assay, 10 ⁶ cells were injected subcutaneously in nude mice. In an experimental metastasis assay, 5x
10 ⁵ cells were injected into the tail vein of 7-8 week old nude mice. The mice injected with the cells transformed with ras were sacrificed 5 weeks later, and the presence or absence of metastasis was confirmed by autopsy.
On the other hand, in a preliminary experiment, it was found that injection of mice with src-transformed cells metastasized to the lungs and proliferated very quickly, resulting in death in 18 to 24 days. To avoid this phenomenon, when performing metastatic experiments using mice injected with src transformed cells, they were sacrificed at 2 weeks and necropsied. Metastatic nodules in the lung are 15% Indian, Inc. (Wexler H, J. Natl. Cancer Inst. 36: 641-64).
5, 1966) into the lungs.

In some experiments, BALB / c mice and natural killer (NK) cells were
B / c mice were used. In order to remove the NK activity, in injection three days before the cancer cells, the anti-asialo GM ₁ serum of 200μl,
BALB / c mice, 7-8 weeks old, were injected into the tail vein.
The anti-asialo GM ₁ serum specifically binds to NK cells,
It is known to suppress its function (Kasai M et a
l., Eur. J. Immunol. 10: 175-180, 1980).

A31-1-1 cells and A31-1-1
The results obtained for the tumorigenicity and invasive / metastatic potential of the three cells are shown in Table 1 below.

[0041]

Table 1 A31-1-1 and A transformed with UV, MCA and Ki-MSVMetastasis test in 31-1-13 cells Number of mice with nodules injectedCell line No. of mice Nodules (nodules / mouse)  Cells transformed with UV * 1-1UV 5 † 0 1-13 UV 5 † 0 0 Cells transformed with MCA * 1-1MCA 5 † 0 1-13MCA 5 † 0 0 Cells transformed with Ki-MSV * 1-1ki-msv1 5 † 0 0 1-1ki-msv2 5 † 0 0 1-1ki-msv3 5 † 0 0 1-1ki-msv4 5 † 0 0 1-1ki-msv5 5 † 0 0 1-13ki- msv1 5 † 0 0 1-13ki-msv2 5 † 0 0 1-13ki-msv3 5 † 0 0 1-13ki-msv4 5 † 0 0 1-13ki-msv5 5 † 0 0 Positive control fos-SR-3Y1-202 3 ‡ 324 3 (149,103,72) Negative control3Y1 5 † 0 0 * 10⁶When transformed cells were injected subcutaneously into BALB / c nude mice, all transformed cells were tumorigenic. ５ 5x10 in tail vein of BALB / c nude mouse^FiveCells were injected and animals were sacrificed 5 weeks later and necropsied for metastasis to the lungs. ‡ Animals were necropsied two weeks later.

As is evident from Table 1, the transformed cells did not show any ability to infiltrate and metastasize into the lung. As a positive control, fos-SR-3Y1-20 obtained by transforming rat 3Y1 cells with v-src and v-fos.
Injection of No. 2 showed numerous metastatic foci in the lungs.

Therefore, BALB / c 3T3 A3
A31-1-1 and A31-1-1 which are 1-derived cells
3 was found to be prone to cancer by different carcinogens such as UV irradiation, chemicals and the ras oncogene.
However, although the transformed cells had tumorigenicity, the invasion / metastasis was negligible when the invasion / metastasis was observed experimentally. Example 2: Introduction of activated c-Ha-ras oncogene into BALB / c 3T3 A31-derived cells To isolate cells expressing a large amount of activated Ras oncoprotein (oncoprotein), A31 was used.
-1-1 and A31-1-13 have activated c-Ha-
ras (derived from bladder cancer cells T24) and a plasmid (pSV2neo-r) carrying a neo gene as a selection marker.
as) was introduced for transformation. Transformation was performed by electroporation (Tatsuka M, et al., Exp. Ce.
ll Res. 178: 154-162, 1988). 400 transformed cells
And in the presence of 1000 μg / ml G418. In the presence of 400 μg / ml G418, 1-1ra
s and 1-13ras were isolated and 100 clones were isolated.
1-1ras100 in the presence of 0 μg / ml G418
Two clones, 0 and 1-13ras1000, were isolated. All of the isolated clones had properties similar to those exhibited by cells transformed with v-Ki-ras. These cells were used for the next experiment. Southern blot analysis of the incorporated gene The genomic DNA of each cell transformed with ras was digested with the restriction enzyme SacI, and adsorbed on a nitrocellulose membrane using a BIO-DOT SF device (Bio-Rad). The membrane was hybridized with a ras probe labeled with ³² P and detected with X-ray film. The copy number of the introduced gene was determined with a densitometer. As a result, a 3.0 kb SacI band was detected in the ras gene introduced into the cells. Northern blot analysis of transcribed mRNA Cytoplasmic RNA was prepared by guanidinium thiocyanate-chloroform extraction method (Chomczynski P et al., An
al. Biochem. 162: 648-657, 1977). Blotting and hybridization were performed by the method of Maniatis (Maniat
is T et al., Molecular Cloning: A Laboratory Mannu
al, Cold Spring Harbor Laboratory, New York, 198
2) was used. Introduced c-Ha-ras and vs
The mRNA of the rc gene was detected with a specific probe labeled with ³² P, respectively. As a result, ras mRNA was detected.

Therefore, it was confirmed that the ras gene was integrated into the gene of the cell and that the gene was transcribed into mRNA. Confirmation of ras copy number by slot blot analysis 5, 10 or 15 μg of genomic D digested with SacI
NA was hybridized with a ³² P-labeled SacI / SacI fragment of the c-Ha-ras gene from T25 human bladder cancer by slot blotting. Figure 1 shows the results obtained.
Shown in The copy number of ras was 400 μg / ml G4
18-1 ras100 than cells selected in the presence of 18
0 and 1-13ras1000 were found to be higher (16-fold and 4-fold, respectively). Immunoblot analysis of p21 ^ras The extent of expression of Ras oncoprotein was confirmed by immunoblot analysis using an anti-p21 ^ras monoclonal antibody.

The cells collected by centrifugation were placed in a Laemmli sample solution (Laemmli UK, Nature 227: 680-685, 1979).
It was dissolved by applying a slight ultrasonic wave. 2mg protein concentration
/ Ml. The cell lysate was treated at 100 ° C. for 3 minutes and run on a 15% polyacrylamide gel. The proteins in the gel were electrically transferred to a Durapore filter (Towbin H et al., Proc. Nalt. Acad. Sci. USA 7
6: 4350-4354, 1979), an 18 hour anti-p21 ^ras monoclonal antibody, NCC-RAS-004 (Kanai T et.
al., Jpn. Cancer Res. 78: 1314-1318, 1987). This antibody is c-Ha-ras 1
Protein p derived from a gene having a mutation in the second codon
21 ^ras can be detected. At this time, nonspecific binding on the membrane was suppressed with skim milk. The protein transcribed on the membrane was further purified by ¹²⁵ I-labeled protein G (Akerstrom Be
tal., J. Immonol. 135: 2589-2592, 1984) and autoradiographed with Fuji RX-X-ray film.

FIG. 2 shows the obtained results. The weaker band migrating earlier corresponds to endogenous p21 ^ras .
The band of the p21 ^ras oncoprotein that migrates slowly is indicated by the mutated c-Ha-ras gene (Taparowski E e).
t al., Nature 300: 762-765, 1982).
As was specifically detected in cells transformed with as. The slowly migrating p21 ^ras oncoprotein band was not detectable by gel electrophoresis with a 10-fold excess (50 μl) of cell lysate of the parent strain (see FIG. 2). Cells transformed with c-Ha-ras and v-src
Examination of tumorigenicity and invasive / metastatic ability of p- ^{ras In} order to examine whether an increase in the expression level of the p21 ^ras oncoprotein affects the invasive / metastatic ability of transformed cells, c-H
The tumorigenicity and invasion / metastasis of the cells transformed with a-ras and v-src were tested. The results obtained are shown in Table 2 below.

[0047]

[Table 2] As is clear from Table 2, even if the expression level of the p21 ^ras oncoprotein was increased, it did not affect the invasion / metastasis ability of the transformed cells.

From the above, the traits that do not have the ability to invade and metastasize cells transformed with ras are extremely stable,
It was also found that there was almost no change during in vitro culture. Example 3: BALB with viral oncogene v-src
/ C Acquisition of invasive / metastatic ability of 3T3 A31-derived cells The viral oncogene v-src has strong transforming ability and can also induce invasive / metastatic ability at the same time depending on the type of recipient cell. Therefore, this gene was transferred to the parent strain BA
The cells were introduced into LB / c 3T3 A31-derived cells and cells transformed with ras, and the effect of v-Src oncoprotein was examined.

The v-src was replaced with the parent strain (BALB / c 3T3).
A31 cells), the cells formed foci in vitro. This suggests that the v-src gene itself has a transforming ability for cells. The shape of the cells forming the foci of the transformed parent strain was not significantly different from that transformed with the mutated ras gene.

Next, by the Southern blot analysis, the transformed cells were transformed with the introduced v-src gene by 2.9 kb of E.
It was confirmed that it had a coRI fragment. Also, v-S
Whether the rc oncoprotein was functionally expressed was confirmed as follows. Immunoblot analysis and tyrosine kinase of p60 ^src
The expression level of the v-src gene product in cells mutated by the zassay src was confirmed by immunoblot analysis of p60 ^src . This experiment demonstrates that anti-p specific for the Src protein
This was performed using 60 ^src serum (Cooper JA et al., J. Vi.
rol. 48: 752-764, 1983). Phosphorylation of the Src protein in cells mutated with src was determined by the Kation assay by Jove et al. (Joce R et al., J. Virol. 60: 849-85).
7, 1986).

As a result, v-Src and intrinsic c-Sr
c was difficult to distinguish by immunoblot analysis (FIG. 3A). However, from phosphorylation assays and subsequent immunoprecipitation experiments (using anti-p60 ^src serum), enhanced autophosphorylation of p60 ^src was observed in transformed cells (FIG. 3B). Acquisition of tumorigenicity and invasion / metastasis of src transformed cells
Injection of pooled portions of src transformed cells from experiments A31-1-1 and A31-1-13 resulted in 1
Tumors formed within ~ 2 weeks. The incubation period was the same as that of the ras transformed cells. Pooled transformed cells and each transformed clone (1-1src clones 1-3, 1-13src clones 1-3, and 1-1
ras1000src) formed a number of metastatic foci in the lung in an assay for experimental invasion / metastasis (see Table 2).

Therefore, in an assay for experimentally confirming the invasive / metastatic ability, a clear difference in the invasive / metastatic ability was observed between cells transformed with active ras and v-src. The difference is that the ras transformed cells
In order to confirm whether the sensitivity was to be indicative of sensitivity to nude mice, 1-1ras1000 cells transformed with ras using wild-type and BALB / c mice in which NK activity was suppressed, instead of nude mice, and src The transferability of 1-1src cells transformed with the above was compared. as a result,
The same results were obtained in both wild type and mice in which NK activity was suppressed (see FIG. 4).
This is not due to differences in sensitivity to K activity. Example 4 In Vitro Invasiveness and Cell Motility Test of ras and src Transformed Cells The in vitro cell motility and invasiveness were
It is known to be closely related to metastatic ability. Boyden with Matrigel-coated filter
Using a modified Boyden chamber method, the method of Albini et al. (AlbiniA et al., Cancer Res. 47: 3239-3245,
1987) analyzed the invasiveness of the cells. As a result, v-sr
c-transformed A31-1-1 and A31-1-1
The three cells had a 4- to 5-fold higher invasion index than the cells transformed with ras (FIG. 5A). Its invasiveness did not change with increased expression of mutant Ras oncoprotein.

Cell motility was determined by digital image analysis using a computer on a matrix-coated dish (Tatsuka M et al., Exp. Cell Res. 185: 342).
-352, 1989) and the motility index (motility ind
ex). As a result, the motility index of the cells transformed with src was 6 to 7 times higher than that of the cells transformed with ras (FIG. 5B). Therefore, it was suggested that the v-Src oncoprotein induces various cell traits not induced by the active Ras protein, and as a result, the src-transformed cells acquire the invasion / metastasis ability. Example 5: Acquisition test of metastatic ability using DNA derived from human cancer cells DNA was extracted from various human cancer cells having metastatic ability,
It was tested whether they were introduced into 1-1ras1000 cells without metastatic ability to acquire metastatic ability. A. Experimental method (1) DNA extraction Using the human cancer cell lines described below as experimental materials, the method of Maniatis et al. (Maniatis, T. et al., Molecular Cloning: AL)
aboratory Manual, Cold Spring Harbor Laboratory, N
ew York, 1982): A204 (ATCC HTB-82): Rhabdomyosarcoma (rh)
Malme-3M (ATCC HTB-64): Malignant melanoma metastasized to the lung; Mewo (JCRB0066): Malignant melanoma; SW480 (ATCC CCL-228): Colon adenocarcinoma.

The above cells were obtained from American Type Culture Coll
Section (ATCC) or Japanese Collectio
n of Research Biosources (JCRB).

The size of each DNA extracted from the cell line was confirmed by pulse field electrophoresis.
It was over -90 kb. (2) DNA transfection DNA transfection was performed by the calcium phosphate method (Graham, FL and Van Der EB, AJ, Transform
ation of rat cells by DNA of human adenoma5, Virol
ogy, 96: 64-79, 1973). Prior to transfection, 5 × 10 ⁵ 1-1ras1000 cells were grown to a diameter of 10
Soaked in a 0 mm dish. 100 mg of pSV2gpt DNA (dominant selacta) is added to 1 mg of genomic DNA.
ble marker) was mixed to prepare each genomic DNA sample, which was precipitated and added to 20 dishes. 4
After time, cells were shocked with 15% glycerol for 60 seconds. Cells were cultured for 16 hours in Eagle's MEM medium containing 10% fetal bovine serum. Next, xanthine (250
μg / ml), hypoxanthine (15 μg / ml), thymidine (10 μg / ml), aminopterin (2 μg / ml)
ml) and mycophenolic acid (MPA: 25 μg / m
Selection medium containing 1) was added to each dish. MPA resistant colonies that appeared on the dish were pooled and used for further experiments. (3) Assay for metastatic potential 6-7 week old Balb / c nude mice (male) were obtained. MPA-resistant cells were collected by trypsinization. Pooled cells from each transfection were washed with Hank's solution and 5 × 10 ⁵ cells were injected into the tail vein of nude mice (7-8 weeks old). The mice are sacrificed after 5 weeks,
Necropsy was performed. Cells were obtained by mincing the lungs and culturing them. If metastatic foci were formed in the lung, colonies appeared when cultured in MPA selective medium. Usually, each colony evolved from each foci that metastasized to the lungs of one mouse. The number of colonies was counted, and all colonies from each mouse were used for the second transfection. B. Result A204, Malme-3M, Mewo or SW48
Colonies that incorporated DNA from cell line 0 and showed MPA resistance metastasized to the lung (Table 3). MPA-resistant colonies collected from the lungs with metastatic foci were pooled, cultured, and injected again into mice. As a result, metastasis to the lung was observed in all cases (Table 3).

One of the pooled cells, SW480
1-1ras1000 cells that had taken up DNA were used for further transfection experiments. When DNA was extracted from the cells and re-introduced into 1-1ras1000 (second transfection), the cells metastasized to the lung. When the same operation was repeated (third transfection), lung metastasis was confirmed. As the number of transfections increased, the number of colonies that had metastasized to the lung increased (Table 3). The transformed cells were stable even after culturing them for at least 5 generations in a normal medium, and did not change their metastatic potential.

[0057]

[Table 3]

According to the present invention, a cancer cell leaf having a tumorigenic ability and no invasive / metastatic ability, a gene derived from a cancer cell is introduced into these cells, and if the invasive / metastatic ability is imparted to the cells, the introduced gene Is thought to be deeply involved in the acquisition of invasive and metastatic potential. By using the screening method of the present invention, a gene that imparts invasion / metastasis to cancer cells can be identified. Therefore, the screening method of the present invention is extremely useful for studying the mechanism of metastasis.

Further, according to the present invention, a set of cancer cells having tumorigenic ability but not invasive / metastatic ability and cancer cells derived from the same parent strain and having both tumorigenic ability and invasive / metastatic ability were obtained. Using these, it is extremely useful for studying the difference between a signal transmission circuit for acquiring tumorigenic ability and a signal transmission circuit for acquiring invasive / metastatic ability. In addition, the development of new diagnostic and therapeutic agents for cancer can be expected using the present invention.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the introduction of a mutant c-Ha-ras gene,
The copy number by slot blot hybridization of A31-derived cells transformed with as is shown (photograph of electrophoresis).

FIG. 2: Anti-p21 ^ras monoclonal antibody NCC-R
Figure 5 shows an immunoblot analysis of ras-transformed A31-derived cells using AS-004 (electrophoresis picture). Lane 1: 5 ng + of standard protein p21Gly-12 corresponding to the product of normal human c-Ha-ras1
5 μl of cell lysate of T24 bladder cancer cells having the mutant c-Ha-ras1 gene; lane 2: 5 μl of cell lysate of A31-1-1 cells; lane 3: 50 μl of cell lysate of A31-1-13 cells ( Lane 10: A3
5 μl of cell lysate of 1-1-1ras cells; lane 5:
Lane 6: 5 μl of cell lysate of A31-1-13 cells; Lane 7: 5 μl of cell lysate of 1-13 ras1000 cells.

FIG. 3. BALB / c 3T transformed with v-src
3 shows an immunoblot analysis (A) and a phosphorylation assay (B) in A31-derived cells (photograph of electrophoresis). Lane 1: 1-1; Lane 2: 1-13; src transformants from Lanes 3-6: 1-1; Lane 7
Src transformants derived from -10 to 1-13.

FIG. 4: BALB / c with wild-type or NK activity suppressed
1-1ras1 transformed with ras using mouse
1 shows the invasion and metastasis ability of 000 cells and 1-1src cells transformed with src (photograph showing the form of an organism).

FIG. 5: In vitro invasiveness assay (A) in which the number of cells infiltrating a Matrigel-coated filter was counted under a microscope at 400 ×, and cell motility as indicated by a motility index measured by digital image analysis using a computer. (B) is shown. Each column represents the mean and standard deviation of three tests. Column 1: 1-ras; Column 2: 1-1 ras 1000; Column 3: 1-13 ras
s; column 4: 1-13 ras1000; column 5: 1
-1 src; column 6: 1-13 src.

Claims (11)

[Claims] [Claim 1] A cancer cell having a tumorigenic ability and no invasive / metastatic ability. 2. The cancer cell according to claim 1, wherein the tumorigenicity is provided by an oncogene of the ras family. 3. The ras family of oncogenes is c-Ha
The cancer cell according to claim 2, which is a -ras gene. 4. A cell containing 1-1ras1000 cells (accession number F)
The cancer cell according to any one of claims 1 to 3, which is ERM BP-5406). 5. The method for producing a cancer cell according to claim 1, wherein a ras family oncogene is introduced into a BALB / c 3T3 A31-derived cell. 6. The BALB / c 3T3 A31-1-1 cell or the BALB / c 3T3 A31-1-1 cell is a BALB / c 3T3 A31-derived cell.
The method according to claim 5, which is ALB / c 3T3 A31-1-13 cells. 7. The ras family of oncogenes is c-Ha
The method according to claim 5, which is a -ras gene. 8. The cancer cell according to claim 5, wherein the cancer cell is 1-1ras1000 cell (accession number: FERM BP-5406).
The production method according to any one of the above. 9. A cancer cell or a cancer cell line-derived DNA obtained from the body surface or body of a mammal is introduced into the cancer cells according to claim 1, and those having acquired invasion / metastasis are separated. A method for screening a gene having a property of imparting invasion / metastasis, comprising extracting the DNA. 10. The screening method according to claim 9, wherein the DNA is a genomic gene of a metastatic cancer cell or a cDNA library prepared using a mammalian cell expression vector. 11. The method according to claim 11, wherein the cancer cell into which the DNA is introduced is 1-1ra.
s1000 cells (Accession number FERM BP-5406)
The screening method according to claim 9 or 10, wherein