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WO1997024439A1 - Genes permettant a des cellules cancereuses de redevenir normales - Google Patents

Genes permettant a des cellules cancereuses de redevenir normales Download PDF

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Publication number
WO1997024439A1
WO1997024439A1 PCT/US1996/020812 US9620812W WO9724439A1 WO 1997024439 A1 WO1997024439 A1 WO 1997024439A1 US 9620812 W US9620812 W US 9620812W WO 9724439 A1 WO9724439 A1 WO 9724439A1
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Prior art keywords
dna
cells
polypeptide
gene
expression
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Chiaki Takahashi
Hitoshi Kitayama
Makoto Noda
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Amgen Inc
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Amgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention pertains to a newly identified cancer-inhibiting gene which encodes a protein capable of transforming malignant cancer cells into normal cells, and to a plasmid vector for the expression of the DNA of this gene. It is possible to screen for and identify substances that are effective for cancer treatment by using the DNA of this gene as a probe to detect the expression level of the cancer- inhibiting gene itself. It is also possible to carry out a genetic treatment of cancer using an expression plasmid prepared by incorporating the DNA of this invention downstream from an expression promoter which effects expression in a mammalian host cell.
  • a cancer cell When a cancer cell is cultured in vi tro, it is known to exhibit various functional properties that are different from those of a normal cell originating from the same tissue. Many cancer genes have been isolated by screening cultured cells for morphological changes, and the like. On the other hand, cultured cancer cells may begin to manifest properties very close to normal properties, which is a phenomenon known as reversion. Any drug or gene which induces reversion is considered to provide an effective means for treating cancer. Such a drug or gene can be identified, for example, by using as a test specimen or target for research a cancer cell having a morphology significantly different from that of the normal cell. The proto-oncogene "ras" is known to be present in mutable form in various forms of human cancer.
  • rspl which encodes a protein having a domain structure in common with the Ras target protein (adenyl cyclase) ; see Cutler, M.L., et al . , Molecular Cell Biology, Volume 112, pages 3750-3756 (1982) .
  • An object of the present invention is to provide a method for isolating a novel cancer-inhibiting gene having an activity which allows cells transformed with an activated ras gene to return to normal (called "reversion activity") .
  • Another object of the present invention is to provide a method for identifying effective cancer- fighting substances through the use of the nucleotide sequence of this cancer-inhibiting gene as a probe.
  • Still another object of this invention is to provide a means for the effective treatment of cancer through gene therapy, in particular by preparing a recombinant DNA vector which enables the gene to be expressed in a mammalian host cell.
  • the present invention comprises a novel cancer-inhibiting gene having a nucleic acid sequence differing from those of Krev-1 and rspl .
  • the gene of this invention encodes a polypeptide that causes mouse fibroblast cells transformed by an activated ras gene to return to normal, as demonstrated further below.
  • this gene is cloned into a plasmid-type expression vector and expressed in various cancer cells, the malignant properties (such as reduced bonding, increased proliferation, and increased infiltration) disappear. Because of the activity and structure of its expression product, this gene is referred to herein as the "RECK" gene, which is short for "reversion-inducing cysteine-rich protein with Kazal- motif"
  • the present invention encompasses the following aspects :
  • an isolated DNA molecule comprising a nucleotide sequence (i) encoding a polypeptide having the amino acid sequence of SEQ. ID NO: 2 (i.e., RECK protein) and which has a biological activity that enables cancer cells to return to normal, or (ii) encoding a polypeptide having the amino acid sequence of SEQ. ID NO: 2 except that one or more amino acid residues have been deleted, added or substituted but which retains the same biological activity of enabling cancer cells to return to normal (i.e., reversion activity);
  • DNA molecules capable of hybridizing to the DNA molecules of (2) (4) DNA molecules of (3), above, which are capable of hybridization with the DNA molecule of (2) under at a temperature of 60-70 'C and in the presence of 6X SSC standard buffer solution (see further below for details) ;
  • DNA molecules comprising a nucleotide sequence having a homology of seventy percent or higher with the nucleotide sequence of SEQ. ID NO: 1 and encoding a polypeptide which has a biological activity that enables cancer cells to return to normal;
  • DNA molecules comprising a nucleotide sequence having a homology of fifty-five percent or higher with the nucleotide sequence SEQ. ID NO: 1 and encoding a polypeptide which has a biological activity that enables cancer cells to return to normal;
  • a method of screening for a substance effective for the treatment of cancer comprising detecting a messenger RNA hybridized to any of the DNA molecules described in (l)-(6) so as to measure any enhancement in the expression levels of the DNA molecule in response to treatment of the host cell with that substance;
  • an expression vector preferably plasmid-type, containing any of the nucleic acid molecules described under items (l)-(6), above, which enables the DNA molecules to be expressed in a mammalian cell; and (10) the recombinant plasmid-type expression vector pSR ⁇ neoRECK.
  • isolated refers to the original derivation of the DNA molecule by cloning. It is to be understood, however, that this term is not intended to be so limiting and, in fact, the present invention relates to both naturally occurring and synthetically prepared sequences, as will be clear from the following description.
  • the DNA molecules of this invention may be used in forms of gene therapy involving the use of an expression plasmid prepared by incorporating the DNA of this invention downstream from an expression promoter that effects expression in a mammalian host cell (e.g., cancel cell) .
  • a mammalian host cell e.g., cancel cell
  • this invention encompasses the polypeptides encoded by the above mentioned nucleic acid molecules, including the polypeptide of SEQ. ID NO: 2, as well as polypeptides of variant sequence in which one or more amino acid residues have been deleted, inserted or substituted while still being characterized by a biological activity that enables cancer cells to return to normal.
  • polypeptides are useful in methods for the treatment of cancer by contacting the affected cells with an effective amount which allows the cells to return to normal.
  • FIGURE 1 shows the structure of lambda phage expression vector ⁇ HK-1 which can be used to transform cells with DNA molecules in accordance with this invention.
  • the DNA molecules of this invention are preferably isolated from cDNA libraries obtained by preparing mRNA from normal cells of mammalian origin, including human, and converting them to double- stranded cDNA by the method described below.
  • the normal human fibroblast cell line, MRC-5 American Type Culture Collection, Rockville, Maryland, Accession No. 171
  • MRC-5 American Type Culture Collection, Rockville, Maryland, Accession No. 171
  • SDS or acid phenol methods can be employed.
  • the use of the guanidine hydrochloride method is preferred herein; for a description see Stroh an et al. , Cell, Volume 10, pages 265-273 (1977) .
  • mRNA molecules in the cytoplasm of eukaryotic cells have been known to have a poly A sequence at the 3' terminal. This characteristic may be utilized to adsorb the mRNA on an oligo(dT)cellulose column, which is subsequently eluted for purification.
  • the mRNA may be fractionated further by use of the well-known sucrose density gradient centrifugation method.
  • RNA prepared by the above method is used as a template to synthesize a single-stranded cDNA using reverse transcriptase, and this single-stranded cDNA is used to synthesize a double-stranded cDNA.
  • SI nuclease method as described by Efstratiadis et al. in Cell, Volume 7, pages 279-288 (1976), the method described by Land et al. in Nucleic Acids Research, Volume 9, pages 2251-2266 (1981), or the method described by Yoo et al. in the Proceedings of the National Academy of Science, USA Volume 79, pages 1049-1053 (1983) is utilizable.
  • the Okayama-Berg method is most preferred; see Okayama and Berg, Molecular Cell Biology, Volume 2, pages 161-170 (1982) .
  • DNA molecules of this invention may be prepared by the following method:
  • the vector is transfected into a cancerous cell that manifests a different morphology from a normal cell
  • ⁇ HK-1 (see Figure 1) , which is a lambda phage expression vector.
  • Suitable cancer cells for use with this vector are DT cells.
  • the latter are derived from a cloned cell line obtained by twice transfecting NIH 3T3 cells, lacking phosphoribosyl transferase, with the Kirsten mouse sarcoma virus; see Noda et al . , Proceedings of the National Academy of Science, USA, Volume 80, pages 5602-5606 (1983) .
  • Suitable restriction sites for use with such transfected cancer cells include, but are not limited to, Xbal or SpII sites.
  • Escherichia coli pSR neoRECK containing plasmid pSR neoRECK
  • a DNA molecule having the nucleotide sequence of SEQ. ID NO: 1 can be obtained by culturing this E. coli cell line, isolating and purifying pSR ⁇ neoRECK from the biomass using methods well-known to those skilled in the art, digesting with restriction enzymes Kpnl and NotI, and then isolating a DNA fragment of about 3600 base pairs (bp) .
  • the sequence of the DNA prepared as described above can be determined using, for example, the chemical denaturation method described by Maxam and Gilbert in Methods in Enzymology, Volume 65, pages 499-559 (1980) , or by the dideoxynucleotide chain termination method using M13 phage as described by Messing and Vieira in Gene, Volume 19, pages 269-276 (1982) .
  • a popular method for determining DNA base sequences involves the use of a fluorescent pigment instead of a radioisotope. This method allows the use of a computer controlled robot to carry out the dideoxynucleotide chain termination method and a computer to carry out base sequencing after electrophoresis.
  • An example is the Seekgen II system manufactured by the Biorad Company. Such a system can be used to determine the DNA base sequence both efficiently and safely.
  • the cloned fragment containing a DNA molecule encoding a polypeptide that induces reversion in cancerous cells, is incorporated into a suitable vector for transforming other prokaryotic host cells or eukaryotic host cells.
  • the DNA can be expressed in the host by including a suitable expression promoter.
  • Suitable prokaryotic host cells can include, for instance, Escherichia coli , Bacillas subtilis, and the like. By transfecting host cells with replicons originating from species adaptable to the hosts, that is, plasmid vectors containing replication starting point and regulator sequences, these host cells can be transfected with the desired gene (or cDNA) .
  • Such vectors are preferably those having a sequence that provides the transfected cells with a property (phenotype) by which they can be selected.
  • E. coli hosts the strain E. coli K12 is typically used, and for the vector either pBR322 or pUC plasmids are generally employed, although other known strains and vectors are also utilizable.
  • Suitable promoters for E. coli hosts include the tryptophan (trp) promoter, the lactose (lac) promoter, the tryptophan-lactose (tac) promoter, the lipoprotein (lpp) promoter, the bacteriophage lambda ( ⁇ )PL promoter, and the polypeptide chain propagation factor Tu (tufb) promoter, all of which are well known to those skilled in the art.
  • trp tryptophan
  • lac lactose
  • tac tryptophan-lactose
  • lpp lipoprotein
  • ⁇ bacteriophage lambda
  • Tu polypeptide chain propagation factor Tu
  • Bacillus subtilis hosts the 207-25 strain is preferred, together with the vector pTU228; see Oh ura et al., Journal of Biochemistry, Volume 95, pages 87-93 (1984) .
  • secretion of the expression product through the cell membrane can be effected by connecting a DNA sequence coding for the signal peptide sequence of ⁇ -amylase of B . subtilis at the 5' upstream side of the gene.
  • Eukaryotic hosts include cells derived from vertebrates, insects, yeast, etc. As a vertebrate host cell, one can employ monkey COS cells such as those described by Gluzman in Cell, Volume 23, pages 175-182 (1981); or Chinese hamster ovarian (CHO) cells taken from a dihydrofolic acid reductase dependent line such as those described by Urlaub and Chasin in the Proceedings of the National Academy of Science, USA, Volume 77, pages 4216-4220 (1980) .
  • monkey COS cells such as those described by Gluzman in Cell, Volume 23, pages 175-182 (1981)
  • Chinese hamster ovarian (CHO) cells taken from a dihydrofolic acid reductase dependent line such as those described by Urlaub and Chasin in the Proceedings of the National Academy of Science, USA, Volume 77, pages 4216-4220 (1980) .
  • promoters positioned on the 5' upstream side of the gene to be expressed, and having RNA splicing positions, polyadenylation, and transcription termination sequences, etc.
  • the vector may also contain an origin of replication site.
  • An example of such a vector is pSR ⁇ neo, containing an SR ⁇ promoter which is a combination of a promoter of SV40 virus origin and the U5 segment from the human leukemia virus (HTLV-1) long terminal repeat (LTR); see Takebe et al. , Molecular Cell Biology, Volume 8, pages 466-472 (1988).
  • pSR ⁇ neoRECK containing a DNA of this invention incorporated into pSR ⁇ neo
  • pSR ⁇ neoRECK can be isolated and purified from the deposited pSR ⁇ neoRECK-containing E. coli cell line (FERM BP-5324) using well known methods.
  • yeasts can also be employed as hosts, including Saccharomyces yeasts such as S. cerevisiae, which is preferred.
  • Suitable promoters for use with yeast hosts in the practice of this invention include the alcohol dehydrogenase gene promoter described by Bennetzen and Hall in the Journal of Biological Chemistry, Volume 257, pages 3018-3025 (1982), and the acidic phosphatase gene promoter described by Miyanohara et al. in the Proceedings of the National Academy of Science, USA, Volume 80, pages 1-5 (1983).
  • COS cells The use of COS cells as hosts is illustrated as follows.
  • the expression vector is constructed to include the SV40 replication starting point, a transcription promoter, transcription termination signal and RNA splicing sites, and has the ability to cause self- proliferation of COS cells.
  • the expression vector is introduced into COS cells by the DEAE-dextran method described by Luthman and Magnusson in Nucleic Acids Research, Volume 11, pages 1295-1308 (1983) ; by the calcium phosphate-DNA co-precipitation method described by Graham and van der Eb in Virology, Volume 52, pages 456-457 (1973); or by the electroporation method described by Neumann et al. in EMBO Journal, Volume 1, pages 841-845 (1982).
  • a vector containing a neo gene functioning as a G418 resistance marker can be effected.
  • a vector containing a neo gene functioning as a G418 resistance marker examples include pRSVneo as described by Sambrook et al., "Molecular Cloning, A Laboratory Manual, “ Cold Spring Harbor Laboratory, New York (1989) , and pSV2neo as described by Southern and Berg, Journal of Applied Molecular Genetics, Volume 1, pages 327-341 (1982) .
  • Clones characterized by G418 resistance are then selected to obtain stably transfected cells which produce the polypeptide encoded by the DNA of this invention.
  • the desired transfectant prepared as described above can be cultured by conventional means, and the polypeptide encoded by the DNA is produced either inside or outside of the cell, depending on the means employed for expression.
  • a culture medium various kinds of conventionally used media depending on the employed host are possible.
  • a medium such as RPMI-1640, Dulbecco's denatured Eagle minimal medium (DMEM) , with serum components such as fetal bovine serum (FBS), etc., added, if necessary.
  • this invention also encompasses nucleic acid molecules with partial nucleic acid sequences, so long as the partial sequence results in reversion activity.
  • a DNA molecule having the nucleic acid sequence represented by SEQ. ID NO: 1 is the most preferred.
  • eukaryotic genes are considered to show polymorphism, as is known in the case of the gene which encodes interferon; see, for example, Nishi et al., Journal of Biochemistry, Volume 97, pages 152-159 (1985).
  • One or more amino acids may be substituted because of this polymorphism, or alternatively, the amino acid sequence and corresponding nucleotide sequence can be left unchanged.
  • Reversion activity may also be observed in polypeptides with one or more amino acid residues deleted, inserted or substituted at one or more sites in the amino acid sequence of the polypeptide of SEQ. ID NO: 2.
  • polypeptides can be referred to as equivalent polypeptides.
  • a polypeptide prepared by converting the nucleotide encoding for cysteine in the interleukin 2 (IL-2) gene to a nucleotide encoding for serine is known to retain IL-2 activity. Therefore, the present invention embraces DNA molecules which encode such equivalent polypeptides.
  • DNA molecules of this invention can also be prepared by nucleic acid chemical syntheses using conventional methods, such as the phosphite triester method; see Hunkapiller et al. , Nature, Volume 310, pages 105-111 (1984) .
  • codons for the desired amino acid residues in the sequence to be expressed may be selected and can be determined, for example, by taking into account the codon usage frequency of the host used; see Grantham et al. , Nucleic Acids Research, Volume 9, pages 143-174 (1981) .
  • partial codon denaturation may be carried out in these nucleotide sequences according to conventional methods, such as the site-specific partial mutagenesis method; see Mark et al.. Proceedings of the National Academy of Science, USA, Volume 81, pages 5662-5666 (1884).
  • Whether any DNA will hybridize with the DNA of SEQ. ID NO: 1 can be determined by using the following Southern hybridization procedures. Specifically, a probe with ⁇ - [ 32 p]dCTP-labeled DNA containing the nucleotide sequence of SEQ. ID NO: 1 is prepared by the random primer method described in Analytical Biochemistry, Volume 132, pages 6-13 (1983), or by the thick translation method described by Maniatis et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, New York (1982) . Further, the DNA in the sample is permitted to become adsorbed on a nitrocellulose or nylon membrane and is immobilized by heating or UV irradiation.
  • the membrane is dipped in a pre- hybridization solution containing 6X SSC (or SSPE), 5% Denhardt's solution and 0.1% sodium dodecyl sulfate (SDS), and then maintained at 55'C for four hours or longer.
  • the labeled probe is also added to the pre-hybridization solution so that the final relative activity is 1 x IO 6 cpm/mL, and the solution is then maintained at 60'C overnight.
  • the membrane is washed five times with 6X SSC, for five minutes each time, then washed at 57'C for twenty minutes, and autoradiography of the membrane is carried out to determine if the probe and the DNA in the sample are hybridized or not.
  • the above hybridization method with a labeled probe is also utilizable in the case of cultured cells or mRNA extracted from animal tissues (Northern hybridization) .
  • the amount of the probe hybridized with a constant amount of mRNA can be shown as a numerical value, by measuring the residual radioactivity after washing or by carrying out a densitometer measurement of the X-ray film after autoradiography.
  • the numerical results may be compared among multiple mRNA samples for a quantitative evaluation of the amounts of the corresponding mRNAs. Using this method, it is possible to search for a substance which promotes or inhibits the expression of a specific mRNA.
  • mRNAs are extracted from cells cultured for a fixed period of time in media with and without "substance A”, constant amounts are then immobilized by the above method, and hybridization is carried out. The amount of labeled probe hybridized is measured for each sample. If the result of the measurement of the sample originating from cells cultured in the medium containing substance A is found to be larger than that of the sample originating from the cells cultured in the medium with no substance A present, the possibility that substance A promotes the expression of DNA according to this invention is high. Moreover, that substance is expected to have a pharmacological effect that causes reversion of cancerous cells to normal. Thus, this method is useful for the evaluation or screening (i.e., identification) of a substances that are effective for treating cancer.
  • a plasmid vector containing a DNA molecule of this invention causes cancerous cells to revert to normal when introduced into such cells to effect expression therein of the DNA.
  • the DNA molecules of this invention are directly useful as chemotherapeutics for genetic therapy by introducing them into the cancerous foci of patients and expressing them in situ.
  • An example of a suitable method for genetic therapy is the one described by Anderson et al. in U.S. Patent No. 5,399,346.
  • the normal human fibroblast cell line, MRC-5 (ATCC, deposit no. CCL-171) , was cultured in a 150-mm plastic dish (Falcon Company) with a denatured Eagle minimum essential medium ( ⁇ -MEM, below) containing 10% fetal bovine serum (FBS, below) , and the cells were allowed to proliferate until reaching confluency. Subsequently, the culture medium was discarded, the cells were dissolved in a guanidine thiocyanate solution (4M guanidine thiocyanate, O.lM Tris hydrochloride at pH 7.5, and 1% 2-mercaptoethanol) , and the solution was recovered.
  • a guanidine thiocyanate solution 4M guanidine thiocyanate, O.lM Tris hydrochloride at pH 7.5, and 1% 2-mercaptoethanol
  • a 10-mL injection cylinder with a 23-G injection needle was used to suck up and discharge the cell solution repeatedly for several times. Subsequently, sodium N-lauroylsarcosinate was added so that the final concentration was 0.5%, the solution was centrifuged at room temperature and 5000 rpm for ten minutes, and the supernatant was recovered.
  • a polyallomer centrifuge tube for a Beckman SW41 rotor bucket was filled first with 3 mL 5.7M cesium chloride-0.01M ethylenediamine tetraacetate (EDTA, pH 7.5) and then layered over the cell solution. Ultracentrifugation was carried out at 20°C and 32,000 rpm for twenty-four hours.
  • RNA was then dissolved in 400 ⁇ L TE buffer (10 mM Tris hydrochloride, pH 7.6, and 1 mM EDTA) containing 0.1% sodium dodecyl sulfate (SDS), and treated with ethanol to obtain precipitated nucleic acid.
  • the precipitate was re-dissolved in 400 ⁇ L TE buffer containing 0.1% SDS.
  • An equal amount of chloroform:1-butanol (4:1, volume/volume) was added, the mixture was stirred, then centrifuged at 4°C and 11,000 rpm for five minutes, and the aqueous layer was recovered. Ethanol treatment of the aqueous layer was carried out to obtain a precipitate which was dissolved in 150 ⁇ L TE buffer. About 0.1 mg of total RNA was thus prepared.
  • the total RNA prepared as described above was applied to an oligo(dT) cellulose chromatographic column to purify poly(A) + RNA.
  • the total RNA was dissolved in an adsorption buffer (0.5M sodium chloride, 20 mM Tris hydrochloride, pH 7.6, 1 mM EDTA and 0.1% sodium N-lauroylsarcosinate) , and the solution was heated at 65°C for five minutes and applied to an oligo(dT)cellulose column (type 7, manufactured by Pharmacia) that was saturated with the adsorption buffer.
  • an adsorption buffer 0.5M sodium chloride, 20 mM Tris hydrochloride, pH 7.6, 1 mM EDTA and 0.1% sodium N-lauroylsarcosinate
  • Poly(A) + RNA was then eluted with an elution buffer (composed of 10 mM Tris hydrochloride, pH 7.5, 1 mM EDTA and 0.05% SDS) and recovered. Twenty micrograms of poly(A) + RNA was thus obtained.
  • an elution buffer composed of 10 mM Tris hydrochloride, pH 7.5, 1 mM EDTA and 0.05% SDS
  • the preparation of a cDNA library was carried out using the method of Okayama-Berg. More specifically, 5 ⁇ g of poly(A)+RNA prepared as described above were added to 50 ⁇ L of a solution containing 10 Units of Superscript reverse transcription enzyme (manufactured by Stratagene Company) , reaction buffer, 10 mM of dithiothreitol (DTT,), 0.6 mM of deoxyadenosine triphosphate (dATP), 0.6 mM of deoxyguanosine triphosphate (DGTP), 0.6 mM of thymidine deoxythymidine triphosphate (dTTP) , 0.6 mM of 5-methyldeoxythymidine triphosphate, and 0.05 ⁇ g/mL of linker primer DNA (the latter having a Sail restriction enzyme recognition sequence) .
  • DTT dithiothreitol
  • dATP deoxyadenosine triphosphate
  • DGTP deoxyguanosine triphosphate
  • the mixture was incubated at 37°C for one hour to synthesize single-stranded cDNA.
  • the reaction tube was placed in ice to terminate the reaction, then 40 ⁇ L of 10X concentrated second strand buffer (Stratagene) , 15 ⁇ L of O.lM DTT, 6 ⁇ L of 10 mM second strand nucleotide mixture (Stratagene), 285.6 ⁇ L of sterile distilled water and 2 ⁇ L of ⁇ - [ 32 p] -dCTP were added.
  • ribonuclease H (1 U/ ⁇ L)
  • 6.9 ⁇ L DNA polymerase I (14.5 U/ ⁇ L) were added, and the mixture was incubated at 16°C for two and one-half hours to synthesize double-stranded cDNA.
  • the reaction tube was cooled with ice, the reaction mixture was extracted with phenol-chloroform, and the aqueous phase was treated with ethanol to obtain a precipitate.
  • the precipitate obtained was dissolved in 50 ⁇ L of sterile distilled water to a volume of 39 ⁇ L of the solution.
  • the amount corresponding to 1 ⁇ g of the lambda phage expression vector XHK-1 of Figure 1 (1 ⁇ L of 1 ⁇ g/mL) digested with restriction enzymes NotI and Sail were joined with the cDNA digested with Sail by co-reacting in the presence of T4 DNA ligase (at 12° for twelve hours) .
  • 1 ⁇ L of the reaction mixture was removed, 15 ⁇ L of Gigapack II packaging extract (Stratagene) were added, and the mixture was incubated at 22°C for two hours.
  • E. coli cell line LE392 was transfected with the whole amount of the reaction mixture, and as a result 5 x 10 clones of independent cDNAs were found to be contained in the reaction mixture.
  • a shaker culture of the library mixed with E. coli line LE392 in a proportion of 0.05 M.O.I, (multiplicity of infection) was carried out for about eight hours at 37°C in 500 mL of an LB medium (composed of 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 1% sodium chloride) .
  • LB medium composed of 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 1% sodium chloride
  • Sodium chloride was added to the treated culture so that the final concentration was IM.
  • the mixture was centrifuged at 11,000 rpm and 4°C for thirty minutes, and the resulting supernatant was recovered.
  • Polyethylene glycol 8000 was added to the supernatant so that the final concentration was ten percent.
  • the solution was centrifuged at 11,000 rpm and 4°C for ten minutes, and the resulting precipitate was dissolved in 5 mL of SM buffer (composed of 0.05M Tris hydrochloride, pH 7.5, O.lM of sodium chloride, 2 g/L of magnesium sulfate heptahydrate and 0.01% gelatin) .
  • SM buffer composed of 0.05M Tris hydrochloride, pH 7.5, O.lM of sodium chloride, 2 g/L of magnesium sulfate heptahydrate and 0.01% gelatin
  • a Scotch tape was pasted to the side of the centrifuge tube after ultracentrifugation, and a 21-G injection needle was gently inserted at the tape, which was pasted below a phage layer visible at the boundary between the 1.45 and 1.50 g/mL layers under visible light. The tip of the needle was adjusted until it was positioned immediately below the phage layer, and the phage layer was recovered through the injection needle. Ultracentri ⁇ fugation at 38,000 rpm and 4°C using a Beckman Ti50 rotor was carried out for twenty-four hours in a 1.5 g/mL cesium chloride solution, and the precipitates were dissolved in 3 mL of SM buffer to obtain a phage solution.
  • the solution was dialyzed for twenty-four hours against an aqueous solution containing 10 mM sodium chloride, 50 mM Tris hydrochloride (pH 8.0) and 10 mM magnesium chloride. After SDS was added in a final concentration of 0.5%, proteinase (50 mg/mL) was added, and the mixture was incubated at 56°C for one hour. The reaction mixture was extracted with phenol-chloroform, and the aqueous phase was treated with ethanol to obtain a DNA precipitate. As a result, 0.22 mg of lambda phage DNA was recovered.
  • the DT cell line utilized in the following procedures was established by the method of Noda et al. described in the Proceedings of the National Academy of Science, USA, Volume 80, pages 5602-5606 (1983) .
  • the cell was a virus non-producer prepared by twice-infecting NIH 3T3 cells lacking hypoxanthine-guanine phosphoribosyltransferase with Kirsten mouse sarcoma virus, then cloning the cells by the colony formation method using agar medium.
  • the DT cells were inoculated onto a 60-mm diameter collagen-coated dish (Corning) at a density of 1.0 x IO 5 cells/dish and cultured overnight at 37°C in a DMEM medium (containing 4 mM of L-glutamine, 100 U/mL of penicillin G and 100 ⁇ g/mL of streptomycin sulfate) under an atmosphere containing 5% C0 2 .
  • DMEM medium containing 4 mM of L-glutamine, 100 U/mL of penicillin G and 100 ⁇ g/mL of streptomycin sulfate
  • 120 ⁇ g of the cDNA expression library prepared as described above were dissolved in 180 ⁇ L of TE buffer. To 10 ⁇ L of the resulting solution were added
  • Transfection was carried out by using a mammalian transfection kit II (Stratagene) and the manufacturer's recommended procedure. In particular, to 135 ⁇ L of the above DNA solution, there were added
  • the same procedures were used for eighteen dishes to introduce the cDNA library into a total of 1.8 x 10 6 DT cells.
  • the cells were cultured for twenty-four hours, the medium was exchanged with fresh medium, and the cells were removed with trypsin and cultured further in a medium containing 1 g/L of the antibiotic, geniticin G418 (Gibco) .
  • After three days, 6.9 x IO 4 cells were counted.
  • the cells were treated with trypsin, and 3 x 10 cells were inoculated into three 150-mm dishes.
  • the side of each dish was tapped manually, a phosphate buffered physiological saline solution (PBS) was added, and the cells that floated in the PBS were removed.
  • PBS phosphate buffered physiological saline solution
  • Each of the reverted cell lines was cultured at 37°C under an atmosphere containing 5% of C0 2 until reaching confluency.
  • the cultured cells were washed twice with PBS, 5 mL of PBS were added, and the cells were removed using a rubber policeman and centrifuged at 1000 rpm for ten minutes.
  • the resulting pellet was suspended in a solution containing 10 mM Tris hydrochloride (pH 8.0), 10 mM of EDTA, 20 mg/mL of ribonuclease (bovine spleen origin) , 100 mg/mL of proteinase K, and 0.5% SDS, and the mixture was incubated at 37°C for six hours. After incubation, the mixture was extracted with phenol- chloroform, and the aqueous layer was recovered and treated with ethanol to obtain precipitated genomic DNA.
  • the DNA prepared as described above was dissolved in TE buffer at a concentration of 1 ⁇ g/mL, and 5 ⁇ L of the solution were digested with restriction enzyme Xbal or SpII.
  • the reaction mixture was treated with phenol-chloroform and the aqueous phase was recovered.
  • the aqueous layer was treated with ethanol to obtain precipitated DNA, and the precipitate was washed with 75% ethanol and dried under a vacuum.
  • the DNA was dissolved in TE buffer to a concentration of 240 ⁇ g/mL.
  • One ⁇ L of the cyclized DNA was introduced into 40 ⁇ L of transformation E. coli DH5 ⁇ electrocompetent cells (Stratagene) by using an electroporation gene introduction device Genepulser (Biorad) . More specifically, a cuvette from the gene introduction device (Biorad, electrode spacing 1 mm) was cooled with ice, a mixture of the above DNA solution and transformation-competent E. coli was loaded into it, and the loaded cuvette was installed on the gene introduction device. Subsequently, 2.5 volts of current were supplied with a capacitance of 25 microFarads and resistance of 200 ohms.
  • the mixture was then immediately transferred to 1 mL of SOC culture medium composed of 20 g/L of Bactotryptone (Difco) , 5 g/L of Bacto-yeast extract (Difco), 0.5 g/L of sodium chloride and 20 mM of glucose) .
  • Shaker culture (using 200 rpm) was carried out at 37°C for one hour.
  • the culture mixture was centrifuged at 3,000 rpm for ten minutes, the resulting pellet was suspended in 100 ⁇ L of SCO medium, and the whole amount was inoculated onto a solid LB medium plate (90-mm dish) containing 75 ⁇ g/mL of ampicillin and cultured at
  • the plasmid DNA was adjusted to a concentration of 0.5 ⁇ g/mL, introduced into DT cells by the same method as employed above, and cultured in DMEM culture medium containing 1 g/L of G418. Both the number of G418-resistant cells which appeared and the number of cells with a flat morphology were counted, and the latter was divided by the former to calculate the reversion induction rate.
  • the plasmid DNA of ctl92 prepared above (see 1.4), in an amount corresponding to 10 ⁇ g (20 ⁇ L of a 0.5 ⁇ g/ ⁇ L DNA solution), was digested with NotI and Sail at 37°C for six hours. Electro ⁇ phoresis of the reaction mixture (using 100 volts applied for two hours) was carried out in 1% agarose gel, using a submarine-type electrophoresis tank containing IX concentrated TAE buffer (0.04M Tris acetate, pH 7.6, and 1 mM EDTA, pH 8.0) . Then the gel was dipped into a 1 ⁇ g/mL aqueous solution of ethidium bromide to stain the DNA.
  • DNA template 3 ⁇ L
  • 5 ⁇ L of ⁇ -[ 32 p]dCTP relative activity of 3000 Ci/mmol
  • E. coli Yl090r (Clonetech) was cultured overnight in LB culture medium containing 0.2% maltose. To 600 ⁇ L of the culture there were added 5 x IO 4 clones from a ⁇ gtll human fibroblast cell cDNA library
  • a 10 x 15 cm nitrocellulose membrane was placed over the culture and allowed to stand for one minute. Then the membrane was peeled off, placed on a filter dipped in a denaturation solution (composed of 1.5M sodium chloride and 0.5M sodium hydroxide) for one minute, and subsequently dipped into a neutralization buffer (composed of 1.5M sodium chloride and 0.5M Tris hydrochloride, pH 8.0) for one minute. After washing in 3X SSC, the membrane was first air-dried and then further dried by heating at 65°C under vacuum for two hours. In this manner a total of 5 x IO 5 plaques were immobilized on the nitrocellulose membrane.
  • a denaturation solution composed of 1.5M sodium chloride and 0.5M sodium hydroxide
  • a neutralization buffer composed of 1.5M sodium chloride and 0.5M Tris hydrochloride, pH 8.0
  • the membrane was incubated at 42°C in 15 mL of hybridization solution (composed of 50% formaldehyde, 5X SSPE, 5X concentrated Denhardt's solution, 0.1% SDS, and 100 ⁇ g/mL of denatured salmon DNA) for three hours .
  • the membrane was incubated overnight at 42°C in 10 mL of the hybridization solution with the whole amount of the thermally denatured labeled probe prepared as described above (1.5.1) . (Denaturing of the labeled probe was effected by treatment in a boiling water bath for three minutes followed by quenching) .
  • the membrane was washed twice at room temperature for thirty minutes in 2X SSC containing 0.1% SDS, then twice under the same conditions in 0.5X SCC containing 0.1% SDS. Autoradiography was then carried out, and thirty positive plaques that had hybridized with the probe were identified from the sensitized spots on the X-ray film.
  • plaques determined to be positive were isolated using the tip of a sterile Pasteur pipette and then recovered in SM buffer. To obtain a complete length of a single cDNA clone from the positive phages, the plaque hybridization procedures described above were repeated. To avoid overlapping of plaques during culturing, the concentration was adjusted to about 100 plaques/90-mm dish, and as a hybridization probe template, a 5' terminal fragment of ctl92 containing about one hundred base pairs was used. As a result, five individual positive plaques were identified, isolated and recovered in SM buffer. To obtain DNA for amplifying the phage clones in these plaques, E.
  • coli Yl090r infected with a highly concentrated phage solution was cultured at 37°C until the plaques covered the solid culture medium almost completely (5 x 10 cm square dish) . Subsequently, SM buffer was poured over the culture medium and incubated at 4°C for eight hours while shaking gently. The SM buffer was recovered as a phage solution, and DNA was prepared from the phage solution by the method described above (see 1.2) . The phage DNA prepared in this manner was digested with EcoRI, and agarose electrophoresis was carried out. A clone having the longest insert (approximately 3.6 kbp) was selected, and the band of the inserted DNA fragment of about 3.6 kbp of the clone was cut out.
  • the 3.6-kbp DNA fragment was ligated with a plasmid vector pBlueScriptSK (+) (Stratagene) in which the terminal had been dephosphorylated previously by digestion with EcoRI. Thereafter, E. coli line XL-lBlue (Stratagene) was transfected to select for an ampicillin-resistant colony. The colony selected was inoculated on 50 mL of TB culture medium composed of 12 g/L of Bacto-tryptone (Difco), 24 g/L of Bacto-yeast extract (Difco) , 0.4% of glycerol, 0.017M monobasic potassium phosphate and 0.072M dibasic potassium phosphate. Culturing was carried out at 37°C for eight hours. The culture mixture was subsequently centrifuged to collect the biomass, and the same alkali method described above was used to extract the plasmid DNA.
  • pBlueScriptSK (+) (Stratagene)
  • E. coli line XL-lBlue
  • the plasmid DNA isolated referred to herein as pBSK(+)RECK, was digested with restriction enzymes Kpnl and Xhol.
  • a nested deletion kit (Pharmacia) was used to prepare twenty DNA molecules of varying nucleotide chain lengths from the insert. The ends of the DNA molecules were "blunted” using SI nuclease, and the blunt-ended molecules were cyclized by using T4 DNA ligase.
  • the treated DNA molecules were used to transform XL-lBlue using the same procedures described above. An ampicillin-resistant colony was selected, - 27 -
  • the gene was found to contain an open reading frame of 2196 base pairs (SEQ. ID NO: 1) . Since there was no previously known sequence corresponding to the predicted amino acid sequence (SEQ. ID NO: 2) encoded by the nucleotide sequence of the open reading frame, the gene was determined to be novel. An analysis of the predicted amino acid sequence showed that it was rich in cysteine, and the segment corresponding to amino acid numbers 396-415 had a sequence of "C-X 8 -C-X 6 -Y-X 3 -C" (where C is cysteine, Y is tyrosine and X fertiliz shows the presence of n residues of unspecified amino acids) .
  • This subsequence is referred to as a "Kazal domain” or “Kazal motif” and is commonly found in some serine protease inhibitor proteins; for reference, see Laskowski et al. , Annual Review of Biochemistry, Volume 49, pages 593-626 (1980) . Accordingly, this gene was termed “RECK” (an abbreviation for "reversion-indueing cysteine-rich protein with Kazal motif”) .
  • the plasmid pBSK(+)RECK prepared as described in Example 1 was digested with Kpnl and NotI, then agarose electrophoresis of the reaction mixture was carried out, and the band containing the insert was cut out. Using a ligation kit (Takara Shuzo K.K.), ligation was applied to 200 ng of the DNA and 100 ng of expression vector pSR ⁇ neo
  • pSR ⁇ neoRECK One colony harboring pSR ⁇ neoRECK was inoculated into 50 mL of TB culture medium, and shaker culture was carried out at 37°C for twelve hours. Subsequently, the biomass was collected by centrifugation of the culture. About 100 mg of plasmid DNA were extracted and purified from the biomass using a Qiagen column (Qiagen) in accordance with the manufacturer's recommendations.
  • Vector pSR ⁇ neoRECK prepared as described in Example 2, was introduced into DT cells using the same method described in Example 1, and the cells were cultured in DMEM medium containing 1 g/L of
  • the pSROtneo vector containing no insert was also introduced into separate DT cells as a control.
  • the reversion induction rate was calculated for these two cases. A significant degree of reversion was found to have been induced in the case of pSR ⁇ neoRECK, compared to pSR ⁇ neo, indicating that the RECK expression product induced reversion activity.
  • the cells used in this Example were all cultured in 15-cm dishes until confluency was achieved.
  • the cultured cells were washed twice with PBS, removed with 5 mL of PBS using a rubber policeman, and centrifuged at 1000 rpm for ten minutes to obtain pellets.
  • the pellets were dissolved in a solution containing 2% of SDS, 0.2M of Tris hydrochloride (pH 8.0), 25 mM of EDTA, 0.2M of sodium chloride and 50 mg/mL of proteinase K.
  • a 10-mL injection cylinder with a 23 G injection needle was used to carry out suction and discharge several times, and then the cell solution was incubated at 45°C for thirty minutes.
  • the sodium chloride concentration of the cell solution was adjusted to 0.5M, 50 mg of oligo-dT-cellulose bead powder were added, and the mixture was incubated at room temperature for twenty minutes.
  • washing buffer composed of 20 mM of Tris hydrochloride, pH 7.6, 0.5M of sodium chloride, 1 mM of EDTA and 0.1% of sodium N-lauroylsarcosine
  • the suspension was centrifuged at 3000 rpm for one minute, and the supernatant was removed.
  • the washing procedures were repeated twice.
  • the precipitates were suspended again in the washing buffer, and the suspension was transferred to a C3 Ultrafree Filter (Millipore) and then was washed three times.
  • 200 ⁇ L of elution buffer (composed of 10 mM of
  • Tris hydrochloride, pH 7.6, 1 mM of EDTA and 0.05% of SDS) were added to the precipitates, which were kept in suspension using a pipette. The suspension was centrifuged at 3,000 rpm for one minute, and the supernatant was recovered. The supernatant was treated with ethanol to obtain a poly(A) + RNA precipitate. Each poly(A) + RNA was dissolved in the elution buffer at a concentration of 0.5 mg/mL. To 5 ⁇ L of the solution there was added 2.5 ⁇ L of 10X MOPS buffer, composed of
  • MOPS 3- (N-morpholino)propanesulfonic acid
  • the nucleic acids in the gel were transferred to a nylon membrane (Biodyne A membrane, Paul Co.) using the capillary method. After the transfer, the membrane was dried in air, and 1200 Joules of ultraviolet light was applied in the presence of a UV crosslinker (Stratagene) . As a result, the nucleic acids were immobilized on the membrane. Determination of the expression pattern in various human organs was carried out using human MTN (multiple tissue Northern blot) purchased from Clonetech.
  • MTN multiple tissue Northern blot
  • the membrane was incubated at 42°C for twelve hours in a hybridization solution composed of six-fold concentrate of SSPE, 0.15M of sodium chloride, 0.01M of monobasic sodium phosphate and 1 mM of EDTA, 5X Denhardt's solution, 0.1% of SDS, 100 ⁇ g/mL of salmon testis DNA and 50% formaldehyde. Then, 50 ng of labeled probe prepared by the same method as used in 1.5.1, above, were thermally denatured by heating in a boiling water bath for five minutes . The denatured probe material was added to the hybridization solution, and the resulting mixture was incubated at 42°C for sixteen hours.
  • the membrane was subsequently washed at room temperature for one hour with a two-fold concentrate of SSC solution containing 0.1% SDS, then at 65°C for thirty minutes with 0.5X SSC solution containing 0.1% SDS, after which autoradiography was carried out. All of the lanes of the samples thus examined displayed bands at a position corresponding to about 4.6 kb for all normal organ samples, with the expression of the RECK gene being confirmed.
  • the results obtained for cultured cell line samples are shown in Table 1.
  • Example 1 The procedures described in Example 1 were used to introduce pSR ⁇ neoRECK or expression vector pSR ⁇ neo into cell lines NIH 3T3,
  • the resulting transfected cell lines were cultured in media containing 1 g/L of G418 (see Table 1 for specific details of the media) .
  • the number of G418-resistant colonies developed for each cell line was counted, and the G418- resistant cells were removed with trypsin and recovered.
  • 3 x 10 cells from each cell line were cultured for two weeks at 37°C under an atmosphere of 5% C0 2 in a soft agar medium composed of minimum essential medium (MEM), 0.33% Bacto-agar (Difco) , nonessential amino acid solution (Gibco) , 100
  • Example 2 The same method used in Example 1 was also used to introduce pSR ⁇ neoRECK or pSR ⁇ neo into readily metastasizing mouse melanoma cell line B16-F10; see Fidler, Nature, Volume 242, pages 148-149 (1973) .
  • the transfected cell line was cultured in a medium containing 1 g/L of G418, and the invasiveness of the G418-resistant cells were examined by the following method.
  • a total of 2 x 10 of G418-resistant cells were suspended in DMEM medium containing 0.1% BSA and then placed in the upper plate of a Matrigel Invasion Chamber (Becton-Dickinson) ; see Kobayashi et al., Cancer Research, Volume 52, pages 3610-3614 (1992) .
  • a cytotoxic factor 25 ⁇ g/mL of culture medium containing fibronectin were placed in the lower plate, and cell culture was carried out in a C0 2 incubator with 5% C0 2 at 37°C for seven or ten hours, respectively.
  • the bottom of the upper plate had a structure of gel laminate comprising an extracellular matrix constituent on a membrane having many pores of 8 ⁇ m in diameter.
  • Invading cells were induced by the fibronectin in the bottom plate to move through the gel and pores to reach the back side of the membrane.
  • the cells which reached the back side of the membrane were fixed and stained using crystal violet in methanol, and then counted under a microscope. It was found that the invasiveness of the B16-F10 cells containing pSR ⁇ neoRECK was inhibited at both the seven-hour and ten-hour incubation times, compared with the results obtained for the cells containing pSR ⁇ neo.
  • the survival of the transformed cells in a serum-free medium was good even after ten hours, and no correlation was observed between the degree of invasiveness and the proliferation rate. It was concluded that the reduced invasiveness due to the presence of the RECK DNA was not caused by reduced cell vitality, but rather was due to the effects of the expressed RECK protein. Additionally, the
  • B16-F10 cells containing pSR ⁇ neoRECK had a flat morphology.
  • transfected B16-F10 cells described in Example 6 were suspended by trypsinization and collected by centrifugation at 1,000 rpm for five minutes, then re-suspended (lxlO 7 cells/ml) in PBS containing 500 ⁇ /ml of penicillin-G and 500 ⁇ g/ml of streptomycin sulfate, and subsequently injected subcutaneously into six-week-old BALB/c athymic mice (nine mice, using a volume of 0.2 ml per site of injection) .
  • mice After injection, the viability of the cells left in the used syringes was examined with trypan blue-staining and was found to be more than ninety-eight percent in all the cases.
  • the injected mice were observed periodically and sacrificed on the twenty-first day following injection, before the largest tumor reached one-tenth of the body weight.
  • the original tumors and lungs were dissected and stored in Bouin's solution. The occurrence of metastasis in other visceral organs was also evaluated.
  • the tumors forming after the three-week period were excised and weighed.
  • the average weight of the tumors that had formed in the mice injected with RECK-transfected cells i.e., pSR ⁇ neoRECK
  • pSR ⁇ neoRECK RECK-transfected cells
  • Lung Liver Lymph Node Lung Colonies mean ⁇ standard error pSR ⁇ neo 1 . 73 ⁇ 0 . 55 5 / 5 2 / 5 3 / 5 4 . 67 ⁇ 0 . 33 pSR ⁇ neoRECK 0 . 91 ⁇ 0 . 24 5 /5 0 / 5 0 / 5 4 . 50 ⁇ 2 . 10
  • Transfected B16-F10 cells prepared as described in Example 6 were suspended at a concentration of 2.5xl0 5 cells/ml and injected into the lateral tail vein of six-week-old C57BL mice (injection volume of 0.2 ml per animal) . Cell viability following injection was confirmed as described in Example 7. The injected mice were observed periodically and sacrificed on the twenty-first day. The mice were dissected and inspected for metastasis in visceral organs, with etastasized organs being stored in Bouin's solution.
  • Example 6 To evaluate whether the polypeptide expression product of the RECK gene is secreted from host cells, the invasion assay described in Example 6 was carried out using untransfected B16-F10 melanoma cells in the presence of various concentrations of conditioned media harvested from cells that had been transfected with either the "control" vector (pSR ⁇ neo) or the RECK expression vector
  • Cys Lys Ser Asp Cys Val Glu lie Leu Lys Lys Cys Gly Asp Gin Asn 195 200 205
  • Cys lie Pro Lys Pro Gin Val Cys Leu Thr Thr Phe Asp Lys Phe Gly 450 455 460

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Abstract

Un gène d'inhibition du cancer a été identifié. Ce gène code une protéine permettant de transformer des cellules cancéreuses malignes en cellules normales. En outre, il permet de détecter et d'identifier des composés ou d'autres substances qui sont efficaces pour traiter le cancer à l'aide de séquences de nucléotides provenant de ce gène, sous forme de sondes. Cela permet de contrôler les niveaux d'expression du gène qui sont élevés en réponse au contact avec ces composés ou ces autres substances. La thérapie génique du cancer peut être réalisée à l'aide d'un plasmide d'expression préparé par incorporation de l'ADN selon l'invention, en aval d'un promoteur d'expression qui assure l'expression dans une cellule hôte d'un mammifère. Le polypeptide codé par ce gène peut être utilisé pour traiter les cellules cancéreuses et leur permettre de redevenir normales.
PCT/US1996/020812 1995-12-27 1996-12-24 Genes permettant a des cellules cancereuses de redevenir normales Ceased WO1997024439A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9018438B2 (en) 2010-07-29 2015-04-28 Kyoto University Screening method for anticancer drugs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028009A1 (fr) * 1993-06-01 1994-12-08 Ludwig Institute For Cancer Research FRAGMENTS DE NEUROFIBROMINE (NF1) ET PROCEDE POUR INVERSER, DANS DES CELLULES MAMMIFERES, L'ACTIVATION DE LA TRANSFORMATION MALIGNE INDUITE PAR LES Ras
WO1995017885A1 (fr) * 1993-12-27 1995-07-06 Rutgers, The State University Of New Jersey Inhibiteurs de l'urokinase activateur du plasminogene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028009A1 (fr) * 1993-06-01 1994-12-08 Ludwig Institute For Cancer Research FRAGMENTS DE NEUROFIBROMINE (NF1) ET PROCEDE POUR INVERSER, DANS DES CELLULES MAMMIFERES, L'ACTIVATION DE LA TRANSFORMATION MALIGNE INDUITE PAR LES Ras
WO1995017885A1 (fr) * 1993-12-27 1995-07-06 Rutgers, The State University Of New Jersey Inhibiteurs de l'urokinase activateur du plasminogene

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9018438B2 (en) 2010-07-29 2015-04-28 Kyoto University Screening method for anticancer drugs

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