WO2004100990A1 - 癌細胞に対するアポトーシス誘導剤 - Google Patents
癌細胞に対するアポトーシス誘導剤 Download PDFInfo
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- WO2004100990A1 WO2004100990A1 PCT/JP2004/007145 JP2004007145W WO2004100990A1 WO 2004100990 A1 WO2004100990 A1 WO 2004100990A1 JP 2004007145 W JP2004007145 W JP 2004007145W WO 2004100990 A1 WO2004100990 A1 WO 2004100990A1
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/52—Isomerases (5)
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- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/99—Isomerases (5.)
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2510/00—Detection of programmed cell death, i.e. apoptosis
Definitions
- the present invention provides an apoptosis-inducing agent for cancer cells, comprising as an active ingredient a compound that suppresses the expression of a RecQ DNA helicase family gene, or a compound that suppresses the function of a protein encoded by the gene. And a method for screening a candidate compound for the apoptotic inducer.
- RecQ MA helicase family gene is a gene that exists widely from prokaryotes such as Escherichia coli to higher eukaryotes including humans, and is conserved in the process of evolution and diversified with multicellularization.
- the RecQ gene in Escherichia coli was the first discovery of a RecQ family member gene, and was identified as a gene that works in the conjugative recombination and UV damage repair RecF pathway (see Non-Patent Document 1) and has a function to suppress erroneous recombination. (See Non-Patent Document 2).
- Saccharomyces cerevisiae the SGS1 gene is known, and in fission yeast, the RQhl gene is known as the only RecQ homolog.
- Non-Patent Documents 3 and 4 there are multiple RecQ homologs in higher eukaryotes, and in humans, the RTS gene identified by the present inventors (see Non-patent Document 5, Patent Documents 1 and 2) and the RecQL5 gene (Non-patent Document 5, Patent Document 3). 5), including RecQL family genes, RecQLl (see Non-Patent Document 6), BLM, WRN, RTS, and RecQL5 gene.
- the BLM, WRN, and RTS genes are the causative genes of Bloom syndrome (see Non-Patent Document 7), Werner Syndrome (see Non-Patent Document 8), and Rossmond-Thomson Syndrome (see Non-Patent Document 9), respectively. These all play important roles in cell genome stability. You are.
- Non-Patent Document 10 Chromosome breakage and sister chromatid exchange (SCE) are frequently observed in cells derived from patients with Bloom syndrome (see Non-Patent Document 11). In lymphocytes derived from patients with Rossmond-Thomson syndrome, trisomy of chromosome 2 and chromosome 8 is frequently observed (see Non-Patent Document 12).
- Lymphocyte cell lines derived from patients with Werner syndrome have abnormal telomere length compared to cell lines derived from healthy persons (see Non-Patent Document 13).
- continuous passage of lymphoid cell lines derived from healthy individuals resulted in immortalization at a rate of about 15%, whereas cell lines derived from patients with Erna-1 syndrome did not show immortalization.
- WRN helicase works to maintain telomere structure and is essential for immortalization (carcinogenesis) of lymphocyte cell lines.
- WRN helicase forms foci in the nucleus in response to DNA damaging agents, and focuses and localizes RPA, a single-stranded DNA binding protein, one of the WRN binding proteins, and the recombinant repair factor RAD51.
- RPA DNA-dependent protein kinase complex
- FEN-1 flap endonuclease 1
- WRN helicase binds to FEN-1 and activates it, providing a place for accurate reconstruction of the replication fork by homologous recombination while processing the Okazaki fragment.
- BLM helicase is localized to a specific structure found in the nucleus, PML body, and binds to topoisomerase II (see Non-Patent Document 18). It is also thought to work for telomere maintenance and has the activity of unwinding the G-Qiiadniplex structure (see Non-Patent Document 19). In addition, it has been reported that Holliday junction is unfolded and interacts with Rad51 protein (see Non-Patent Document 20). These findings suggest that BLM helicopter also works in concert with other DNA metabolizing enzymes and plays an important role in DNA recombination repair and telomere maintenance.
- RecQl, BLM, WRN, RTS, and RecQ5 hardly express in cells in arrest, while they are transformed with viruses. And is highly expressed in cell lines in which proliferation has been promoted (see Non-Patent Document 21).
- TPA a carcinogenic promoter
- RecQl, BLM, WRN, and RTS is induced with the induction of cell division.
- the RecQ DNA helicopter family repairs cell genomes (BLM, WRN, RTS), while also being involved in telomere structure maintenance (BLM, WRN), and in some cells It plays an important role in the immortalization of tumors (WRN), and its expression is induced by cell division (RecQl, BLM, WRN, RTS), making it a promising molecule as an anticancer target .
- the expression of the RecQ DNA helicase family was found to be extremely high in tumor cells, and a method of screening for a compound that suppresses tumor growth using the suppression of the expression of the RecQ DNA helicase family gene as an index was known. (See Patent Document 4). In addition, it has been suggested that compounds that suppress the expression of the RecQ helicase gene may suppress the growth of cancer cells (see Patent Document 4).
- Patent Document 1 Japanese Patent Application No. 9-200387
- Patent Document 2 Japanese Patent Application No. 11-11218
- Patent Document 3 Japanese Patent Application No. 10-81492
- Patent Document 4 JP-A-2000-166600
- Non-Patent Document 1 Nakayama H, Nakayama, Nakayama R, Irino N, Nakayama Y, Ha Thigh alt PC, ⁇ Isolation and genetic characterization of a thymine less death-resistant mutant oi Escherichia coli K12: identification of a new mutation ( recQl) that blocks the RecF recombination pathway. ", Mol Gen Genet;., Ranji, Vol.195, p.474-480.
- Non-Patent Document 2 Hanada K, Ukita ⁇ , Kohno ⁇ , Saito ⁇ , Kato J, Ikeda H, ⁇ RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia col i. '', Proc Natl Acad SciUSA., 1997 Vol.94, p.3860-3865
- Non-Patent Document 3 Myung K, Datta A, Chen C, Kolodner RD, ⁇ SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and omeologous recombination. Genet., 2001, Vol.27, p.113-116
- Non-Patent Document 4 Doe CL, Dixon J, Osman F, WhitbyMC, rpartial suppression oi the fission yeast rahl (-) phenotype by expression of a bacterial Holliday junction resolvase.J, EMBO J., 2000, Vol. 19 , P.2751-2762
- Non-Patent Document 5 Kit ao S, Ohsugi I, IchikawaK, GotoM, Furuichi Y, Shimamoto A, ⁇ CIoning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes. '' ics-, 1998, Vol.54, p.443-452
- Non-Patent Document 6 Seki, M., Miyazawa, H., Tada, S., Yanagisawa, J., Yamaoka, T., Hos ino, S., Ozawa, K., Eki, T., Nogami, ⁇ .,, Okumura ⁇ ., Et al., “Molecular cloning of cDNA encoding human DNA hel icase Ql which has homology to Escherichia col i Rec Q hel icase and localization of the gene at chromosome 12P12.J, Nucleic Acids Res., 1994, Vol.22, No.22, p.4566-4573
- Non-Patent Document 7 Ellis NA, Groden J, Ye TZ, Straughen J, Lennon DJ, Ciocci S, Proytc eva M, German J, ⁇
- the Bloom's syndrome gene product is homologous to RecQ hel icases.J, Cell, 1995 Year, Vol.83, p.655-666
- Non-Patent Document 8 Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, Martin GM, Mulligan J, Scellenberg GD, ⁇ Positive cloning ⁇ the Werner's syndrome gene., ”Science, 1996, Vol.272, p.258-262.
- Non-Patent Document 9 KitaoS, ShinamotoA, GotoM, MillerRW, SmithsonWA, Lindor Marauder, Furuichi Y, ⁇ Mutations in RECQL4 cause a subset of cases of Rotkmnd- Thomson syndrome.J, Nat Genet., 1999, Vol.22 , P.82-84
- Non-Patent Document 10 GotoM, ⁇ HierarcMcal deterioration of body systems in Werner's syndrome: implications for concealed mal ageing. '', Mech.Aging Dev., 1997, Vol. 98, p. 239-254-
- Non-Patent Document 11 Ellis NA, German J, ⁇ Molecular genetics of Bloom's syndrome, j, Hum Mo 1 Gene, 1996, Vol. 5, p. 1457-1463.
- Non-Patent Document 12 Lindor Thigh, Devries EM, Michels VV, Schad CR, Jalal SM, Donovan KM, Siithson WA, Kvols LK, Thibodeau SN, Dewald GW,
- Non-Patent Document 13 Tahara H, Tokutake Y, Maeda S, Kataoka H, Watanabe T, Satoh M, Mat sumo to T, Sugawara M, Ide T, GotoM, Furuichi Y, Sugimoto M, ⁇ Atmormal telomere dynamics of B -lymphoblastoid cell strains from Werner 's syndrome patients transformed by Epstein-Barr virus., Oncogene, 1997, Vol. 15, p. 1911-1920.
- Non-Patent Document 14 Sugimoto M, Furuichi Y, Ide T, Goto M, ⁇ Incorrect us of "immortalization" for B-lympho lastoid cell lines transformed by Epstein-Barr virus.J, Virol., 1999, Vol. 73, p. 9690-9691
- Non-Patent Document 15 Sakamoto S, Nishikawa K, Heo SJ, Goto M, Furuichi Y, Shimamoto A, ⁇ Werner helicase relocates into nuclear foci in response to MA damaging agents and co-localizes with RPA and Rad51.J, Genes Cells., 2001, Vol.6, p.421-430
- Non-Patent Document 16 Yaniione SM, Roy S, Chan D, Murphy MB, Huang S, Campisi J, Chen DJ, ⁇ Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase.J, J Biol Chem., 2001, Vol.276, p.38242-38248
- Non-Patent Document 17 Brosh RMJr, von Kobbe C, Sommers JA, Karmakar P, Opresko PL, Piotrowski J, Dia ⁇ a I, Dianov GL, Bohr VA, ⁇ Werner syndrome protein interacts with human flap endonuc lease 1 and stimulates its cleavage activity. J, EMB0 J., 2001, Vol. 20, p.5791-5801
- Non-Patent Document 18 Johnson FB, Lombard DB, Neff NF, Mastrangelo MA, Dewolf W, Ellis NA, Marciniak RA, Yin Y, Jaenisch R, Guarente L, ⁇ Association of the Bloom syndrome protein with topoisomerase Illalpha in somatic and meiotic cells. J, Cancer Res., 2000, Vol.60, p.1162-1167
- Non-Patent Document 19 Mohaghegh P, Karow JK, Brosh Jr RM Jr, Bohr VA, Hickson ID, ⁇
- the Bloom's and Werner's syndrome proteins are DNA
- Non-Patent Document 20 Wu L, Davies SL, Levitt NC, Hickson ID, rpotential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51. J, J Biol Chem., 2001, Vol. 276, p.19375-19381
- Non-Patent Document 21 awabe, T., Tsuyama, ⁇ ., Kitao, S., Nishikawa, ⁇ ., Shimamoto, A., Shiratori, M., Matsumoto, T., Anno, ⁇ ., Sato, ⁇ . , Mitsui, ⁇ ., Seki, ⁇ ., Enomoto, ⁇ ., Goto, ⁇ ., Ellis, ⁇ .
- the present invention has been made in view of such circumstances, and it is an object of the present invention to provide a compound that suppresses the expression of a RecQ DNA helicase family gene or a compound that suppresses the function of a protein encoded by the gene as an active ingredient.
- An object of the present invention is to provide an apoptosis inducer for cancer cells. It is another object of the present invention to provide a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells.
- the present inventors have intensively studied to solve the above-mentioned problems. It is known that the expression of the RecQ DNA helicase family gene is increased in tumor cell lines (for example, cancer cells). The present inventors have studied the effect of the suppression of the expression of each RecQ helicase family gene on the growth of cancer cells using siRNA having the effect of suppressing the expression of the human RecQ helicase family gene. investigated. As a result, the present inventors found that apoptosis is induced in various cancer cells by suppressing the expression of WRN, BLM, and RecQl genes, which are RecQ helicity family genes, thereby suppressing cell proliferation. I found it for the first time.
- WRN-siRNA and RecQ siRNA inhibit tumor growth in tumor-bearing nude mice in vivo without significant adverse effects. Therefore, WRN, BLM and RecQl genes, which are RecQ helicopter family genes, are considered to be very excellent target molecules for anticancer drugs with few side effects. That is, a compound that suppresses the expression of the RecQ helicity family gene or the function of the RecQ helicase protein is considered to have an apoptosis-inducing effect, and the compound is highly expected to be an anticancer agent with few side effects. Is done. For example, compounds that suppress the expression or activity of RecQ helicase family 1 protein such as sIRA based on the mRNA sequences of WRN, BLM and RecQl can be expected as a repressed cancer drug with few side effects.
- the present invention provides an apoptosis inducing agent for cancer cells, comprising as an active ingredient a compound that suppresses the expression of a RecQ MA helicopterase family gene or a compound that suppresses the function of a protein encoded by the gene. And a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells, more specifically,
- RecQ DNA-inducing apoptosis inducer for cancer cells which contains as an active ingredient a compound that suppresses the expression of ligase family 1 gene
- Double-stranded RNA is a sense RNA consisting of a sequence homologous to any continuous 20 to 30 nucleotides in the mRNA of RecQ helicase family gene and an antisense RNA consisting of a sequence complementary to the sense RNA
- the apoptosis inducing agent according to [1], wherein the compound that suppresses the expression of the RecQ DMA kinase family gene is the following (a) or (b):
- an apoptosis inducer for cancer cells comprising as an active ingredient a compound that suppresses the function of a protein encoded by the RecQ MA helicopterase family gene;
- RecQ DM helicase family one protein variant with dominant negative properties for the protein encoded by the RecQ DNA helicase family gene
- the apoptosis inducing agent according to any one of (1) to (7), wherein the RecQ DNA helicase family gene is any of a WRN gene, a BLM gene, or a RecQl gene;
- the apoptosis-inducing agent according to any one of [1] to [7], wherein the nucleotide sequence of the RecQ DNA helicase famili gene is the nucleotide sequence according to any one of SEQ ID NOs: 6 to 30; [Cl 0] One of the strands of the double-stranded RNA having the RNAi effect has the nucleotide sequence of any one of SEQ ID NOS: 1 to 5, 31 to 40, and 43 to 69, [2] to [4].
- the apoptosis inducing agent according to any of the above,
- an anticancer agent comprising an apoptosis-inducing agent listed in any one of (1) to (10) as an active ingredient;
- a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells comprising the following steps (a) to (c) (a method for screening an apoptosis-inducing agent for cancer cells):
- (c) a step of selecting a protein encoded by the RecQ DNA helicase family gene or a compound that binds to a partial peptide thereof [13] Inducing apoptosis in cancer cells, including the following steps (a) to (c): Screening method of candidate compounds for induction agents (Screening method of apoptosis inducing agent for cancer cells),-
- a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells comprising the following steps (a) to (c):
- a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells comprising the following steps (a) to (c). (Screening of an apoptosis-inducing agent for cancer cells) Method) ,
- the RecQ DNA helicase family gene is preferably a human RecQ DNA helicase family gene.
- the present inventors have found that suppression of the expression of the RecQ DNA helicase family 1 gene induces apoptosis in cancer cells (tumor cells).
- the present invention first provides an apoptosis-inducing agent comprising, as an active ingredient, a compound that suppresses the expression of a RecQ DNA helicase family gene.
- the apoptosis-inducing agent of the present invention particularly has an apoptosis-inducing action selectively on cancer cells.
- a cancer cell-selective (specific) apoptosis-inducing effect comprising, as an active ingredient, a compound that suppresses the expression of the RecQ DNA helicase zefamiri gene or the function of the protein encoded by the gene.
- a drug having the following is provided.
- Apoptosis generally refers to cell death aggressively caused by cells themselves under physiological conditions.
- Examples of the form of cis include chromosome aggregation of cell nuclei, fragmentation of cell nuclei, disappearance of cell surface microvilli, aggregation of cytoplasm, and the like. Therefore, the apoptosis-inducing action in the present invention refers to, for example, an action of inducing the above-mentioned form of apoptosis in cells, but is not necessarily limited to only the above-mentioned form of induction. Those skilled in the art can appropriately determine whether or not cell apoptosis is induced.
- the apoptosis-inducing agent for cancer cells of the present invention is, for example, an anticancer agent (cancer inhibitor) having an apoptosis-inducing action as a mechanism.
- RecQ DNA helicopterase family gene includes RecQl gene, WRN gene, BLM gene, RTS gene, RecQ5 gene, etc. Can be mentioned. Information on the base sequence of each of these genes can be easily obtained by those skilled in the art from a public gene database (for example, GenBank or the like). An example of the GenBank accession number of the above gene is shown below.
- RecQl gene NM_002907 (SEQ ID NO: 6), ⁇ —032941 (SEQ ID NO: 7), BC001052 (SEQ ID NO: 8), D37984 (SEQ ID NO: 9), L36140 (SEQ ID NO: 10) WRN gene: NMJ00553 ( SEQ ID NO: 11), AF091Z14 (SEQ ID NO: 12), L76937 (SEQ ID NO: 13), AL833572 (SEQ ID NO: 14) BLM gene: U39817 (SEQ ID NO: 15), Bandai 000057 (SEQ ID NO: 16), BC034480 (SEQ ID NO: 17)
- RTS gene hired—004260 (SEQ ID NO: 18), AB006532 (SEQ ID NO: 19), BC020496 (SEQ ID NO: 20), BC011602 (SEQ ID NO: 21), BC013277 (SEQ ID NO: 22) RecQ5 gene: NMJ04259 (SEQ ID NO: 22) No .: 23), AK075084 (SEQ ID NO: 24), AB006533 (SEQ ID NO: 25), AB042825 (SEQ ID NO: 26), AB042824 (SEQ ID NO: 27),
- the amino acid sequence of the protein encoded by the RecQ enzyme gene of the present invention for example, based on the accession number described above.
- amino acids of protein encoded by each gene of RecQl gene, W gene, BLM gene, RTS gene and RecQ5 gene The sequences are shown in the sequence listing (RecQl protein is SEQ ID NO: 70, WRN protein is SEQ ID NO: 71, BLM protein is SEQ ID NO: 72, RTS protein is SEQ ID NO: 73, RecQ5 protein is SEQ ID NO: 73 : 74).
- the RecQ helicase gene may be assigned a plurality of accession numbers even for the same gene depending on the presence or absence of a polymorphism in the nucleotide sequence.
- This “polymorphism” is not limited to single nucleotide polymorphisms (SNPs) consisting of single nucleotide substitutions, deletions and insertion mutations, but also includes substitutions, deletions and insertion mutations of several consecutive nucleotides. Therefore, the nucleotide sequence of the RecQ helicase gene is not necessarily limited to the sequence obtained by the above accession number.
- the amino acid sequence of the protein encoded by the RecQ helicase gene is not particularly limited to the amino acid sequences described in SEQ ID NOs: 70 to 74. That is, the protein of the present invention is not limited to the amino acid sequences described in SEQ ID NOs: 70 to 74. In the amino acid sequence, one or more amino acid residues are added, deleted, substituted, or inserted. Consisting of the amino acid sequence Proteins that are functionally equivalent to the proteins described in any of SEQ ID NOs: 70 to 74.
- RecQ helicase gene of the present invention preferably, a WRN gene, a BLM gene, or a RecQl gene can be suitably shown.
- the RecQ helicopter gene of the present invention is not particularly limited, it is usually derived from an animal, more preferably from a mammal, and most preferably from a human.
- RNAi refers to a target gene by introducing into a cell a double-stranded RNA consisting of sense RNA consisting of a sequence homologous to the mRNA sequence of the target gene and antisense RNA consisting of a sequence complementary thereto. A phenomenon that induces mRNA destruction and inhibits target gene expression.
- DICER a type of RNase III nuclease family
- the double-stranded RNA having the RNAi effect in the present invention also includes this siRNA.
- a DNA capable of expressing the double-stranded RNA of the present invention is also included in the present invention. That is, the present invention provides a DNA (vector) capable of expressing the double-stranded RNA of the present invention.
- the DNA (vector 1) capable of expressing the double-stranded RNA of the present invention usually encodes DM that encodes one strand of the double-stranded RNA, and encodes the other strand of the double-stranded RNA.
- the above-mentioned MA of the present invention can be easily prepared by those skilled in the art by general genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting a DNA encoding the RNA of the present invention into various known expression vectors.
- RNA used for RNAi need not be completely identical (homologous) to the RecQ helicase gene or a partial region of the gene, but has complete identity (homologous) Is preferred.
- the double-stranded RNA having an RNAi effect of the present invention is usually a sense RNA consisting of a sequence homologous to any continuous RNA region in the mRNA of the RecQ helicase gene, and an antisense RNA consisting of a sequence complementary to the sense RNA.
- a double-stranded RNA consisting of The length of the “arbitrary continuous RNA region” is usually 20 to 30 bases, and preferably 21 to 23 bases. However, even if the RNA is long A that has no fruit at the same length, it is expected to be degraded into sRNA having RNAi effect in the cell.
- the length of the strand RNA is not particularly limited.
- long double-stranded RNA corresponding to the full-length or almost full-length region of the mRNA of the RecQ helicity gene is, for example, previously degraded with DICER, and the degradation product is used as the apoptosis-inducing agent of the present invention. It is possible to use.
- This degradation product is expected to include a double-stranded RNA molecule (siRNA) having an RNAi effect.
- siRNA double-stranded RNA molecule
- the region on the mRNA expected to have the RNAi effect does not need to be particularly selected. That is, the region on the RecQ helicopter mRNA having the RNAi effect does not necessarily need to be precisely defined.
- double-stranded RNA having an overhang of several bases at the end is generally known to have a high RNAi effect, and the double-stranded RNA of the present invention may have an overhang of several bases at the end. desirable.
- the length of the base forming the overhang is not particularly limited, but is preferably a two-base overhang.
- TT two thymines
- UU two peracil
- a double-stranded RNA having an overhang of other bases most preferably a double-stranded RNA of 19 bases and two bases ( A molecule having an overhang of TT) can be suitably used.
- the double-stranded RNA of the present invention also includes a molecule whose base forming the overhang is DNA.
- double-stranded RNA having an RNAi effect on the Rec Q helicity gene is known to those skilled in the art as the target of the double-stranded RNA RecQ helicity gene. It can be appropriately prepared based on the base sequence.
- the nucleotide sequence of the RecQ helicase gene is as described above. It can be easily obtained from public gene databases overnight.
- the double-stranded RNA of the present invention can be prepared based on the nucleotide sequence of any one of SEQ ID NOs: 6 to 30.
- RNA region of mRNA which is a transcript of the sequence is selected, and a double-stranded RNA corresponding to this region is selected. It is easy for a person skilled in the art to carry out.
- the sequence of the gene (target gene) serving as the base of the double-stranded RNA of the present invention does not necessarily need to have a known full-length nucleotide sequence. It is only necessary to know any contiguous RNA region to be selected (for example, 20 to 30 bases). Therefore, the double-stranded NA of the present invention can be obtained from gene fragments, such as ESTs (Expressed Sequence Tags), for which a part of the niRNA is known, but whose full length is not known, based on the base sequence of the fragment. It can be manufactured.
- ESTs Expressed Sequence Tags
- accession numbers of EST sequences that show high homology with the human RecQ DNA helicopter and zefamily gene in the GenBank database are listed below for each gene name. However, these are only examples of a large number of EST sequences, and those skilled in the art can easily obtain information on an appropriate EST fragment from a public database.
- RecQl gene BQ436743, AU130503, BI756143, BQ962215. AW149458.BG113470, BG177944, AV718094, BQ952333, BQ647346, AU127897, AA830035, BQ215072, BE708578, BM467595, AL042375, AA298927, BU858680, CB158017, AA298835, BG536173, AU280724, BU194296, BI090858, BG118 673131, BE888299, BE794392, CA394258, Z33439> BX093234, AA29895K AA459772, BG542269, BF344325, BQ359629, CA396832.
- BLM gene AL556823, AL556853, BM451903 BI091601, BG199179, BI091772, BQ230262, BG772975, BG875917.BM54246L BM040993, BE618504, BM04166 KBG574669,
- BE889560 BQ316432, BE963549, AI39460KBG397477, BG756262, BX283839, AI097184, AW503829, AW404657, BE535950, AA747832, 590, 599 BQ359304, AA974756, BE778486, BM151892, AA769336, BG192554, BG187329 3 ⁇ 4 AW575595, AA214549, AA480209, AI423875, AW173139, AI63052K CA488994, AA862803, BE940055, BU431321, AA249737, BM150628, AW138812, AA903504, BE245666, EB906666, EB906666, BE806666, BE245666,
- RTS gene AL561020, BU90297L CA454998, BM557643, BU944576, BQ065027, BQ649577, BG824628, BQ072016, AL582326, BU173357, BU902969, BE560845, BG388102, BG337750, CA489272, BM763376, BQ646647, BU171, BU957 , BE513519, BE379488, BE513709, BQ66835L BG338114, BQ883533, BM724503, BM849415, BE295951, BI457058, BG398209, BM679729, BM794167, BM853252, BM461584, BE466402, BE281293 E378846, BQ215879, BM84778KBA90637, BK, 316, BK, , BM854499, AI858255,
- a potosis inducer is provided.
- the RecQ helicopterase gene may contain various polymorphisms even for the same gene.
- the RNAi effect of the sequence described in any one of SEQ ID NOs: 6 to 30 or the above-mentioned EST sequence is considered by adding information from the public polymorphism database regarding the RecQ helicase gene, for example.
- the sequence of the RNA expected to have can be appropriately designed.
- An apoptosis inducer containing such RNA is also included in the present invention. Further, those skilled in the art can appropriately select an RNA having an optimal RNAi effect from a plurality of double-stranded RNAs of the present invention prepared as an apoptosis inducer.
- the double-stranded RNA described in FIG. 1 or FIG. 2 can be suitably shown.
- an s iRNA molecule in which the base sequence described in FIG. 3 or the sequence listing is one strand of double-stranded RNA (SEQ ID NO: 1 to 5, 31 to 40, 43 to 69 (SiRNA molecule consisting of a base sequence and its complementary strand). That is, in one embodiment of the present invention, one of the double-stranded RNAs having an RNAi effect has any one of SEQ ID NOs: 1 to 5, 31 to 40, and 43 to 69.
- a cancer cell-specific apoptosis-inducing agent comprising:
- an apoptosis-inducing agent wherein the compound that suppresses the expression of the RecQ DNA helicase family 1 gene is the following (a) or (b):
- Nucleic acid in the present invention means RNA or DNA.
- a method for inhibiting (suppressing) the expression of a specific endogenous gene a method utilizing antisense technology is well known to those skilled in the art.
- the effect of the antisense nucleic acid on inhibiting the expression of the target gene is caused by the following factors.
- inhibition of transcription initiation by triplex formation inhibition of transcription by hybridization with a site where an open loop structure was locally formed by RNA polymerase, inhibition of transcription by hybridization with RNA that is undergoing synthesis, intron and exon Inhibition of splicing by hybridization at the junction with the protein, inhibition of splicing by hybridization with the spliceosome-forming site, inhibition of translocation from the nucleus to the cytoplasm by hybridization with mRNA, and the addition of the capping site to the poly (A) addition site Inhibition of translation initiation by hybridization with the translation initiation factor binding site, translation inhibition by hybridization with the ribosome binding site near the initiation codon, hybridization with mRNA translation region and polysome binding site Outgrowth inhibitory downy peptide chain by, and the like by that gene expression inhibition Haipuriddo formation with the site of interaction between nucleic acids and proteins.
- antisense nucleic acids inhibit target gene expression by inhibiting various processes such as transcription, splicing, or translation.
- the antisense nucleic acid used in the present invention may inhibit the expression of the RecQ helicase gene by any of the above actions.
- designing an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the RecQ helicopter gene would be effective in inhibiting gene translation.
- a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
- the nucleic acid containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the RecQ helicity gene is also included in the antisense nucleic acid used in the present invention.
- the antisense nucleic acid to be used is ligated downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is ligated on the 3 ′ side.
- the antisense nucleic acid used is usually a synthetic oligomer, which is used to reduce the susceptibility to nuclease degradation and maintain the activity as an antisense nucleic acid. It is generally known to use S-oligos (phosphorothioate-type oligonucleotides) in which 0 (oxygen) is replaced with S (sulfur). This S-oligo is currently undergoing clinical trials as an antisense drug injected directly into the affected area. In the present invention, the S oligo can be suitably used.
- the sequence of the antisense nucleic acid is preferably a sequence complementary to the target gene or a part thereof, but may not be completely complementary as long as gene expression can be effectively suppressed.
- the transcribed RNA has preferably 90% or more, and most preferably 95% or more complementarity to the transcript of the target gene.
- the length of the antisense nucleic acid is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more.
- Liposomes refer to RNA molecules that have catalytic activity. There are various liposomes with various activities, It is also possible to design ribozymes that cleave site-specifically. Lipozymes include those with a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, but those with an active domain of about 40 nucleotides called hammerhead type and hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191).
- the self-cleaving domain of the hammerhead lipozyme cleaves the 3 'side of C15 in the sequence G13U14C15, but its activity depends on the base pairing of U14 and A9. It has been shown that A15 or U15 can also be cleaved (Koizumi, ⁇ ⁇ et al., FEBS Lett, 19S8, 228, 228.).
- a lipozyme in which the substrate binding site is complementary to the RNA sequence near the target site a restriction enzyme-like RNA cleavage lipozyme that recognizes the sequence UC, UU or UA in the target RNA can be created (Koizumi, M. et al.).
- Hairpin-type liposomes are also useful for the purpose of the present invention.
- This lipozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin-type liposomes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology , 1992, 30, 112.). As described above, expression of the gene can be inhibited by specifically cleaving the transcript of the RecQ helicase gene of the present invention using the lipozyme.
- the present invention contains, as an active ingredient, a compound that suppresses the function (activity) of a protein encoded by the RecQ DNA helicase family gene (hereinafter, sometimes abbreviated as “RecQ helicase protein”). Apoptosis inducer.
- the RecQ helicase protein of the present invention has an amino acid sequence in the protein.
- a mutant or homologous protein of the RecQ helicase protein containing an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and a protein functionally equivalent to the RecQ helicase protein.
- a “functionally equivalent protein” is a protein having the same activity as that of the RecQ helicase protein.
- a protein having 90% or more, preferably 95% or more, more preferably 99% or more homology to the amino acid sequence of RecQ helicity protein is transferred to RecQ. It can be shown as a protein that is functionally equivalent to the zeoprotein.
- the compound that suppresses the function (activity) of the protein encoded by the DNA virulence gene is a compound described in any one of the following (a) to (c):
- An apoptotic inducer is provided. These compounds are considered to have an apoptosis-inducing effect on cells, particularly on cancer cells, by inhibiting (reducing) the function or activity of the RecQ helicase protein.
- a RecQ DNA helicase protein mutant having the property of being dominant negative with respect to the protein encoded by the RecQ DNA helicase family gene means that the activity of the endogenous wild-type protein is lost or Refers to a mutant RecQ helicase protein that has the function of decreasing. For example, it is particularly well conserved in the helicase domain of RecQ helicase family proteins.
- mutants in which the lysine residue found in motif I (ATP-binding mot if) is substituted with alanine or methionine are known to have no ATP-degrading activity. I have.
- This mutant is thought to function as a dominant negative mutant. Therefore, a specific example of the dominant negative mutant of the present invention is a mutant in which the lysine residue in motif I is substituted.
- the antibody that binds to the RecQ helicase protein (b) can be prepared by a method known to those skilled in the art.
- a polyclonal antibody can be obtained, for example, as follows. Immunize small animals such as rabbits with RecQ helicase protein, which is a recombinant or recombinant RecQ helicase protein or a recombinant protein expressed in microorganisms such as Escherichia coli as a fusion protein with GST, or a partial peptide thereof. Obtain serum. This is prepared by, for example, purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column to which RecQ helicase protein or synthetic peptide is coupled.
- a monoclonal antibody for example, a small animal such as a mouse is immunized with RecQ helicity-zenoprotein or its partial peptide, a spleen is excised from the mouse, and cells are separated by grinding the spleen. The cells and mouse myeloma cells are fused using a reagent such as polyethylene glycol, and a clone that produces an antibody that binds to the RecQ helicase protein is selected from the resulting fused cells (eight hybridomas). I do.
- a reagent such as polyethylene glycol
- the obtained eight hybridomas are transplanted into a mouse intraperitoneal cavity, ascites is collected from the mouse, and the obtained monoclonal antibody is transferred to, for example, ammonium sulfate precipitation, protein and protein G columns, DEAE ion exchange chromatography, and RecQ. It can be prepared by purifying zeoproteins or synthetic peptides by affinity coupling with coupled rams or the like.
- the antibody of the present invention is not particularly limited as long as it can bind to the RecQ helicase protein of the present invention.
- Antibodies, humanized antibodies obtained by genetic recombination, and antibody fragments and modified antibodies thereof are also provided.
- the RecQ helicase protein of the present invention used as a sensitizing antigen for obtaining antibodies is not limited to the animal species from which it is derived, but is preferably a protein derived from mammals, such as mice and humans, and particularly preferably a protein derived from humans. .
- the protein used as the sensitizing antigen may be a partial peptide of the protein in addition to the intact protein.
- the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
- the term “antibody” generally refers to an antibody that reacts with the full length or fragment of a protein.
- human lymphocytes for example, human lymphocytes infected with EB virus
- the lymphocytes can be fused with human-derived myeloma cells having permanent cleaving ability, for example, U266, to obtain a hybridoma producing a desired human antibody having protein binding activity.
- a human antibody or a human antibody is preferable in order to reduce immunogenicity.
- RecQ family proteins to RecQ include, for example, monoclonal and polyclonal antibodies to RecQl helicase, WRN helicase, BLM helicase, RTS helicase, RecQ5 helicase. Can be listed.
- the compound that suppresses the expression of the RecQ hepatase gene of the present invention or the function (activity) of the protein encoded by the gene may be any of natural or artificial compounds. Usually, it is a compound which can be produced or obtained / isolated by using methods known to those skilled in the art. For example, single compounds such as organic compounds, inorganic compounds, nucleic acids, proteins, peptides, sugars, etc., or compound libraries, genetics Examples include expression products of a sub-library, cell extracts, cell culture supernatants, fermentation microorganism products, marine biological extracts, plant extracts, and the like, or compounds isolated and purified from the extracts.
- RecQ helicin family proteins include, for example, distamycin A and netropsin, which have an inhibitory effect on WRN and BLM helicases.
- distamycin A and netropsin which have an inhibitory effect on WRN and BLM helicases.
- the present invention provides a method for screening a candidate compound for an apoptosis-inducing agent for cancer cells (a method for screening an apoptosis-inducing agent for cancer cells).
- Compounds screened by the method are expected to have an apoptosis-inducing action.
- One embodiment thereof is a method using the binding between a test compound and a RecQ helicase protein or a partial peptide thereof as an index.
- a compound that binds to the RecQ helicase protein or its partial peptide is expected to have an effect of inhibiting the function of the RecQ helicase protein.
- a protein encoded by the RecQ helicase gene or a partial peptide thereof is brought into contact with a test compound.
- RecQ helicopter Depending on the indicator for detecting the binding to the test compound, for example, the purified form of RecQ helicase protein or its partial peptide, the form expressed in or outside the cell, Alternatively, it may be in a form bound to an affinity column.
- the test compound used in this method can be appropriately labeled and used as necessary. Examples of the label include a radiolabel, a fluorescent label and the like.
- the binding between the protein encoded by the RecQ helicase gene or its partial peptide and the test compound is detected.
- the binding of the test compound to the RecQ helicase protein or its partial peptide can be detected, for example, by the label attached to the test compound bound to the RecQ helicase protein or its partial peptide.
- a change in the activity of the protein caused by the binding of the test compound to the RecQ helicase protein or a partial peptide thereof expressed inside or outside the cell can be detected as an index.
- a test compound that binds to the protein encoded by the RecQ helicase gene or a partial peptide thereof is selected.
- test compound used in the present method is not particularly limited.
- single compounds such as natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins, peptides, and sugars, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, and fermentation microorganisms Examples include, but are not limited to, organisms, marine organism extracts, plant extracts, man-made compounds and the like.
- a test compound is contacted with a cell or a cell extract expressing the RecQ DNA helicase family 1 gene.
- the “cells expressing the RecQ DNA helicase family gene” the cells expressing the endogenous RecQ lectase gene or the exogenous RecQ into which the lipase gene is introduced, Can be used.
- Cells in which an exogenous RecQ helicase gene has been expressed usually contain an expression vector containing the gene. It can be prepared by introducing the evening into a host cell. This expression vector can be produced by those skilled in the art by general genetic engineering techniques.
- cells expressing RecQ helicity gene include, for example, MCF7 (breast cancer), A549 (lung cancer), U20S (osteosarcoma), C33A (cervical cancer), HT1080 (fibrosarcoma) , PA-1 (ovarian teratocarcinoma), Tera2 (embryonic carcinoma), T24 (bladder cancer), K562 (chronic myelogenous leukemia), Mol t4 (acute lymphoblastic leukemia), A172 (glioblastoma) ), HeLa (cervical cancer), HepG2 (liver cancer), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (kidney cancer), KP4 (knee cancer), MKN45 (gastric cancer), LNCaP (prostate)
- Various types of tumor cells such as cancer can be suitably used.
- a test compound with cells expressing the RecQ helicase gene is usually performed by adding the test compound to a culture of cells expressing the RecQ helicase gene, but is not limited to this method.
- the test compound is a protein or the like
- “contact” can be performed by introducing a DNA vector expressing the protein into the cell.
- the expression level of the RecQ helicopterase gene is measured.
- “gene expression” includes both transcription and translation.
- the expression level of a gene can be measured by a method known to those skilled in the art. For example, extracting nRNA from cells that express the gene for RecQ enzyme in accordance with a standard method, and performing a Northern High 'predication method or an RT-PCR method using the nRNA as a type II. Thus, the transcription level of the gene can be measured.
- the protein fraction was collected from cells expressing the RecQ helicase family gene, and the expression of RecQ helicity protein was detected by electrophoresis such as SDS-PAGE to measure the translation level of the gene. You can do it too.
- RecQ As an antibody used for detection of lipase protein, if it is a detectable antibody, both monoclonal antibodies and polyclonal antibodies can be used. For example, the above-described antibody of the present invention can be used.
- a compound that reduces the expression level is selected as compared to the case where the measurement is performed in the absence of the test compound (control).
- the compound selected in this manner is expected to have an action of inducing apoptosis in cancer cells.
- the compound is expected to be an anticancer drug (anticancer drug) having an apoptosis induction as a mechanism of action.
- Yet another embodiment of the method of the present invention is a method for screening a compound of the present invention that reduces the expression level of the RecQ DNA helicopter zefamilli gene using a reporter gene.
- a test compound is brought into contact with a cell or cell extract containing DNA having a structure in which the transcription regulatory region of the RecQ helicity gene and the repo overnight gene are functionally linked.
- “functionally linked” refers to the transcription regulatory region of the RecQ helicase gene such that the transcription factor binds to the transcription regulatory region of the RecQ helicase gene, thereby inducing expression of the repo overnight gene. And the repo overnight gene are linked. Therefore, even when the reporter gene is linked to another gene and forms a fusion protein with another gene product, the transcription factor binds to the transcription regulatory region of the RecQ helicase gene. If the expression of the fusion protein is induced by this, it is included in the meaning of the above “functionally linked”. Based on the cDNA base sequence of the RecQ helicase gene, those skilled in the art can obtain the transcription regulatory region of the RecQ helicase gene present in the genome by a well-known method.
- the repo overnight gene used in the present method is not particularly limited as long as its expression can be detected, and examples thereof include a CAT gene, a lacZ gene, a luciferase gene, and a GFP gene.
- the transcriptional regulatory region of the RecQ DNA helicase family 1 gene For example, as a ⁇ cell containing a DNA having a structure in which a reporter gene is functionally linked to a reporter gene, '' for example, a vector having a structure in which a transcription regulatory region of a RecQ DNA helicase family gene is functionally linked to a reporter gene has been introduced. Cells and the like. The vector can be prepared by those skilled in the art using general genetic engineering techniques.
- the vector can be introduced into cells by a general method, for example, calcium phosphate precipitation, electric pulse perforation, ribophenamine, microinjection, and the like.
- Cells containing DNA having a structure in which a transcription regulatory region of a RecQ DNA helicase family gene and a reporter gene are functionally linked also include cells having the structure inserted into a chromosome. Insertion of a DNA structure into a chromosome can be performed by a method generally used by those skilled in the art, for example, a gene transfer method utilizing homologous recombination.
- ⁇ Cell extract containing DNA having a structure in which the transcription regulatory region of the RecQ DNA helicase family gene and the repo allele gene are functionally linked '' is included, for example, in a commercially available in vitro transcription / translation kit.
- a cell extract obtained by adding a DNA having a structure in which a transcription regulatory region of a RecQ DNA helicase family 1 gene and a reporter gene are functionally linked to each other can be mentioned.
- ⁇ contacting '' refers to adding a test compound to the culture medium of ⁇ cells containing DNA having a structure in which the transcription regulatory region of the RecQ DNA helicase family gene and the reporter gene are functionally linked ''.
- the method can be performed by adding a test compound to the above-mentioned commercially available cell extract containing the DNA, but is not limited to these methods.
- the test compound is a protein
- the contact can be performed by introducing a DNA vector expressing the protein into the cell.
- the expression level of the reporter gene is measured.
- the expression level of the reporter gene can be measured by a method known to those skilled in the art according to the type of the reporter gene.
- the repo overnight gene is a CAT gene
- acetylation of chloramphenicol by the gene product is detected.
- the expression level of the reporter gene can be measured.
- the repo one gene is a 1 acZ gene
- the coloration of a pigment compound by the catalytic action of the gene expression product is detected, and when the gene is a luciferase gene, the gene expression product is catalyzed.
- the expression level of the reporter gene can be measured. .
- a compound that decreases the measured expression level of the repo overnight gene is compared with that measured in the absence of the test compound.
- the compound selected in this way is a candidate compound for an apoptosis inducer for cancer cells.
- Another embodiment of the method of the present invention is a method of screening a compound using the activity of the protein encoded by the RecQ DNA helicase family gene of the present invention as an index.
- a test compound is brought into contact with a protein encoded by the RecQ helicase gene, or a cell or cell extract expressing the protein.
- the activity of the protein is measured.
- the activity of the protein include DNA helicase activity and DNA-dependent ATP degradation activity.
- Helicity activity generally refers to the activity of unraveling double-stranded DNA into single-stranded DNA.
- the DNA helicase activity can be measured by those skilled in the art by a known method. For example, M13 phage single-stranded circular DNA is annealed with radiolabeled complementary oligo DNA to form a partially double-stranded structure as a substrate, which is reacted with an enzyme in the presence of ATP and magnesium ions.
- the reaction product can be separated into single-stranded DNA and double-stranded DNA by polyacrylamide gel electrophoresis or agarose gel electrophoresis, and detected by radioautography.
- Another method is to anneal mutually complementary single-stranded DNAs labeled with europium and Qcy7 to form a double-stranded MA as a substrate, And the method of reacting with an enzyme in the presence of magnesium and then measuring the fluorescence of europium-labeled single-stranded DNA released from double-stranded DNA by the use of helicase activity.
- Electrochemi luminescence-based hel icase assay Scintillation ion proximity assay.Homogeneous time-reso lved florescence quenching assay, DELFIA hel icase assay (Zhang, L., Schwartz, G., 0 'Do Haku 11, 11, M. & Harrison, RK Development of a novel l he icase assay using elec trochemi luminescence. Anal. Biochem. 293, 31-37. 2001).
- the DNA-dependent ATP degradation activity can be measured by those skilled in the art by a known method.
- plasmid DNA or salmon sperm DNA is reacted with an enzyme in the presence of magnesium ion using ATP as a substrate, and the inorganic phosphate generated when ATP is decomposed into ADP is colorimetrically determined by the malachite green method.
- the RecQ helicase protein used in the above method is preferably a full-length protein containing no mutation, but if it has the same activity as the protein, a part of the amino acid sequence may be replaced and / or deleted. Protein.
- the RecQ MA helicase family gene in the above screening method is preferably a WRN gene, a BLM gene, or a RecQl gene.
- the present invention also provides an anticancer drug (pharmaceutical composition for treating cancer), which comprises, as an active ingredient, a (cancer cell-specific) apoptosis-inducing agent for cancer cells of the present invention. Further, the present invention provides a method for producing an apoptosis-inducing agent as a pharmaceutical composition. In this method, first, a candidate compound for an apoptosis-inducing agent for cancer cells is selected by the screening method of the present invention. The selected compound is then mixed with a pharmaceutically acceptable carrier.
- These pharmaceutically acceptable carriers include, for example, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, Examples include a suspending agent, a tonicity agent, a binder, a disintegrant, a lubricant, a fluidity promoter, a flavoring agent, and the like, but are not limited thereto, and other conventional carriers can be appropriately used. .
- the above carrier can be added as necessary according to a conventional method.
- examples thereof include pyrrolidone, gelatin, medium-chain fatty acid tridalicelide, polyoxyethylene hydrogenated castor oil 60, sucrose, potassium oxymethylcellulose, corn starch, and inorganic salts.
- Examples of the dosage form of the above drugs include oral tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film coatings, pellets, sublinguals, and pastes.
- Parenteral preparations include injections, suppositories, transdermals, ointments, plasters, and solutions for external use.A person skilled in the art can select the optimal dosage form according to the administration route and administration target. it can.
- DNA expressing the protein encoded by the RecQ DNA helicase family member MA expressing the antisense RNA, ribozyme, or siRNA that suppresses the RecQ DNA helicity family member gene to the living body
- Viral vectors such as retrovirus, adenovirus and Sendai virus, and non-viral vectors such as ribosome can be used.
- non-viral vectors such as ribosomes, polymer micelles, and cationic carriers can be used.
- the administration method include the /// vivo method and the r / 'ra method.
- the present invention also includes the above-mentioned pharmaceutical composition having an apoptosis-inducing action.
- the dose of the drug or pharmaceutical composition of the present invention can be finally determined as appropriate by the judgment of a physician in consideration of the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, and the like. it can.
- FIG. 1 is a diagram showing the nucleotide sequence of the siRNA used in the examples and the nucleotide sequence of si RNA in which extremely strong suppression of gene expression was observed.
- FIG. 2 is a continuation of FIG.
- FIG. 3 is a diagram showing the nucleotide sequences of siRNAs showing high gene expression suppressing ability of RecQl and WR BLM used in Examples.
- TT indicates an overhanging region in DNA, and other siRNAs form a duplex with a complementary strand in RNA.
- Fig. 4 shows that 311 « ⁇ for 5 human pheasants (3 ⁇ 81 ⁇ , 1 ⁇ 5, 1 ⁇ 0 ( ⁇ 1, 1 ⁇ 1 ⁇ 2) were introduced into HeLa cells, respectively, 72 hours later.
- 2 is a graph showing the results of quantifying the expression of each mRNA by Tatiman PCR NS is control RNA.
- FIG. 5 is a graph showing the results of introducing WRN, BLM, and RecQl s iRNA into TIG3 cells and semi-quantifying the expression of WRN, BLM, and RecQl mRNA 72 hours later by TaQman PCR.
- NS is control RNA.
- FIG. 6 is a graph showing the results of semi-quantitative RT-PCR quantifying the expression level of each gene 48 hours after introduction of sRNA against RecQl, ⁇ , and BLM into HeLa cells.
- NS indicates non-silencing s iRNA treatment.
- -FIG. 7 is a photograph showing the results of Western blot analysis of WRN, BLM, RTS, RecQK and RecQ5 protein expression in HeLa cells in which mRNA expression suppression was observed.
- FIG. 8 The upper part of FIG. 8 is a graph showing the results obtained by introducing five human RecMA helicase siMAs into HeLa cells, and examining the cell viability 4 days later by MTT assay.
- NS is a control RNA.
- the lower part is a graph showing the results of introducing the WRN, BLM, and RecQl s iRNA into TIG3 cells, respectively, and examining the cell viability 4 days later by MTT assay.
- NS is a control RNA.
- FIG. 9 shows the results of transfection of RecQl, WRN, and BLM sRNA to HeLa cells.
- FIG. 3 is a diagram showing the survival rate of HeLa cells detected at the time point. Non-silencing s Shows the number of cells when the number of cells transfected with iRNA is 100%.
- FIG. 10 is a graph showing the results obtained by introducing WRN, BLM and RecQl s iRNA into A549 cells, and quantifying the expression of WRN, BLM and RecQl mRNA 72 hours later by Taciman PCR.
- NS is control RNA.
- FIG. 11 is a photograph showing the results of Western blot analysis of WRN, BLM, and RecQl protein expression in A549 cells in which mRNA expression suppression was observed.
- FIG. 12 is a graph showing the results obtained by introducing WRN, BLM, and RecQl s iRNA into A549 cells, respectively, and examining cell viability 4 days later by MTT assay.
- NS is control RNA.
- FIG. 13 is a photograph showing the results of the TUNEL method for examining whether apoptosis was induced 48 hours after introduction of the recitation, BLM, and RecQlsRNA into HeLa cells.
- the green on the left of each figure shows the nucleus in which apoptosis was induced, and the right of each figure shows the nucleus of the cells present in the visual field.
- FIG. 14 is a photograph showing the inhibition of tumor growth in nude mice by administration of the silencing RecQl-s iRNA.
- FIG. 11 is a photograph showing the overall tumor size of a tumor injected with RecQl-s iRNA (upper left photograph) and a tumor injected with NS-s iRNA (upper right photograph) 32 days after inoculation. The photo below shows an A549 tumor removed from a mouse. Tumor (NS) (upper) into which NS-s iRNA was injected, and tumor (RecQl) (lower) into which RecQ s iRNA was
- FIG. 15 is a graph showing the time course of the inhibitory effect of WRN-s iRNA and RecQl-s iRNA on the growth of A549 tumor in nude mice.
- FIG. 16 is a photograph in which a tumor section was prepared after administration of sRNA to a nude mouse transplanted with a tumor (A549), and whether or not apoptosis was induced was detected by the TUNEL method.
- Control, RecQl and WRN are images to which NS s iRNA, RecQl s iRNA and WRN s iRNA were administered, respectively. Red-brown indicates nuclei in which apoptosis has been induced, and blue-violet indicates nuclei in which apoptosis has not been induced.
- HeLa human cervical cancer cells
- A549 human lung cancer cells
- MCF 7 human breast cancer cells
- TIG3 normal diploid fibroblasts
- U20S osteosarcoma
- HepG2 liver cancer cells
- U251 glioblastoma
- UACC62 melanoma
- Caki-1 kidney cancer
- KP4 ⁇ cancer
- MKN45 stomach cancer
- LNCaP prostate cancer.
- All human cells are 10% fetal calf serum, 37 degrees Dulbecco 's modif ied Eagle' s medium containing 50 g / ml Gen evening puromycin, were cultured in 5% C0 2 conditions.
- the selected sRNA and the synthetic sequence that forms the basis of the sRNA (the lowercase region represents the homologous part to the mRNA of the underlying RecQ DNA family gene), and a very strong suppression of gene expression was observed.
- the siRNA sequences are shown in FIG. 1 and FIG. In the sequence listing, only the sense strand was described, and the corresponding antisense strand was omitted from the sequence listing.
- sRNAs were derived from human cervical carcinoma-derived HeLa cells and normal cells, fetal embryos. Fibroblasts were introduced into TIG3 cells. When introducing siRNA,
- the cells were seeded at a density of 1 to 3 ⁇ 10 4 cells in a 24-well plate, and transfection was performed at 20-50% confluence.
- Transfection of siRNA into each cell was performed 24 hours after plating using 01 igofectoamine (Invitrogen) and Lipofectoamine2000 (Invitrogen), and the method was performed according to the manufacturer's protocol.
- total RNA was extracted from the cells 48 hours and 72 hours using the RNeasy Mini Kit (Qiagen), respectively. Quantitative PCR was performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosysteins).
- the RecQ helicase gene group, the primer for RT-PCR of the j8-actin gene, and the TaciMan probe were purchased from Applied Biosystems.
- the RT-PCR reaction was performed using a TaQMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems) according to the manual. Quantitative comparison was performed using ⁇ -actin as a standard.
- siRNAs for three human RecQ helicases, RecQl, WRN, and BLM, and selected 14 types for RecQl, 11 types for WRN, and 7 types for BLM. Selected sequences include those already described above. You. These selected siRNA sequences are shown in FIG. Only the sense strand is described in the sequence listing, and the corresponding antisense strand is omitted from the sequence listing.
- siRNAs were introduced into HeLa cells. 48 hours after transfection, the expression of RecQl, WRN and BLM genes was quantified by semi-quantitative RT-PCR. Assuming that the gene expression when the non-silencing siRNA was treated (NS) was 100%, the expression levels when each siRNA was introduced were compared.
- the expression of WRN, BLM, RTS, RecQK and RecQ5 proteins in HeLa cells in which suppression of mA expression was observed was analyzed by Western plot.
- the cells 48 hours and 72 hours after the siRNA transfection were collected, respectively, and collected in a RIPA solution (50 mM Tris-HCl pH 8.0, 0.1% SDS, 1% triton X100, 1% sodium deoxycholate). The cells were disrupted by).
- the extracted proteins were separated by SDS-PAGE using a 4-20 gradient polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (0PVDF membrane).
- the primary antibodies used were RecQl, polyclonal antibodies for BLM, monoclonal antibodies for W, RTS, RecQ5, and antibodies against ⁇ -actin (ICN).
- a secondary antibody a peroxidase-labeled antibody was used, and detection was carried out by means of ECL Plus Western Blotting Detection Ion Reagents (Amersham Biosciences) using luminescence.
- Example 2 Five human RecQ helicinase siRNAs selected in Example 2 were used for HeLa cells And 96 hours later, the viable cell count reagent SF (Nacalai) was measured by colorimetric assay of cell viability based on cleavage of the tetrazolium salt WST-8 by mitochondrial dehydrogenase. Tesque). Three hours after the addition of the reagent, the absorbance of the formazan dye was measured at 450 nm.
- the present inventors introduced newly designed sRNAs for RecQl, WRN, and BLM into HeLa cells, respectively, and examined the cell viability 96 hours later by MTT assay.
- the siRNA shown in FIG. 3 was used.
- Apoptosis induction of WRN-siA, BLM-s iRNA and RecQl-s iRNA on HeLa cells To clarify whether the effect is tumor cell-specific or occurs in other tumor cell lines, we studied 11 tumor cell lines derived from various tumors (HeLa, A549 ( Lung cancer), MCF7 (breast cancer), HepG2 (liver cancer), U20S osteosarcoma), U251 (glioblastoma), UACC62 (melanoma), Caki-1 (kidney cancer), KP4 ( ⁇ cancer), MKN45 (stomach cancer) And LNCaP (prostate cancer)) to examine the apoptosis-inducing effect of sRNA in the same manner as in Example 6. As a result, it was confirmed that apoptosis was induced in some cancer cells but not in normal cells.
- Table 1 summarizes the results of examining the apoptosis-inducing effect of siRM in Examples 6 and 7.
- RecQl ++ f ++++++++++ H In the above table, +++ indicates 70% or more of apoptosis-inducing cells, ++ indicates 70-40%, and + indicates 40-20%.
- Example 8 Inhibition of tumor cell growth by s iRNA in a tumor-bearing animal model
- mice Male BALBZcA nude mice were purchased from CLEA Japan, Inc. A549 cells (5 ⁇ 10 6 cells / nil) were injected subcutaneously into the back of nude mice (6-7 weeks old), and administration of siRNA was started 8 days after subcutaneous injection of tumor cells.
- the tumor suppression effect of RecQl-s iRNA was tested ( Figures 14 and 15). 5'-phosphorylated 25 gs iRNA was mixed with 5 g of polyethyleneimine (MW 10,000, Wako) in 50 d of saline, and the solid formed 8 days after inoculation of A549 cells top to 3 days per tumor (volume about 40 thigh 3) were injected subcutaneously (8, 11, 14, 17, 20, 23, 27 and 32 days). Tumor volume was measured with calipers. The formula for estimating the tumor volume of an ellipsoid is set to LXW '2 Z2. Where L is the major axis of the tumor and W is the minor axis. Tumor volumes were prayed for statistical significance by using the multiple comparison method (Holm-Sidak method).
- RecQl-siRNA significantly inhibited the development of A549 tumors for 32 days after inoculation, but NS-s iRNA mixed with polyethyleneimine had no effect, and the volume of A549 tumors increased (Fig. 1).
- Mice receiving the mixture of RecQl-s iRNA and polyethyleneimine did not lose weight compared to untreated tumor-bearing mice, indicating that this treatment had no significant side effects.
- Fig. 15 shows that RecQl-s iRNA swelled on day 50 compared to NS-s iRNA (PEI / control). This clearly indicates that WRN-s iRNA inhibited the increase in tumor volume by approximately 80% (P ⁇ 0.01), and WRN-s iRNA approximately 50% (P ⁇ 0.05).
- the compound that inhibits the expression of the RecQ DNA helicase family gene of the present invention or the function of the protein encoded by the gene has a cancer cell-selective apoptosis-inducing action.
- Pharmaceuticals containing the compound are considered to be anticancer agents with few side effects, which are induced by apoptosis.
- an anticancer agent having a mechanism of inducing apoptosis targeting RecQ helicase was provided for the first time.
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EP04733937.9A EP1625853B1 (en) | 2003-05-19 | 2004-05-19 | Apoptosis inducer for cancer cell |
US10/556,968 US20070243570A1 (en) | 2003-05-19 | 2004-05-19 | Apoptosis Inducer for Cancer Cell |
JP2005506299A JP4299299B2 (ja) | 2003-05-19 | 2004-05-19 | 癌細胞に対するアポトーシス誘導剤 |
US12/643,938 US8299044B2 (en) | 2003-05-19 | 2009-12-21 | Apoptosis inducer for cancer cell |
US13/629,226 US8809296B2 (en) | 2003-05-19 | 2012-09-27 | Apoptosis inducer for cancer cell |
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US12/643,938 Continuation US8299044B2 (en) | 2003-05-19 | 2009-12-21 | Apoptosis inducer for cancer cell |
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Cited By (5)
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WO2006054625A1 (ja) * | 2004-11-19 | 2006-05-26 | Genecare Research Institute Co., Ltd. | 癌細胞特異的細胞増殖抑制剤 |
US8193332B2 (en) | 2004-04-09 | 2012-06-05 | Genecare Research Institute Co., Ltd. | Cancer cell-specific apoptosis-inducing agents that target chromosome stabilization-associated genes |
WO2017022650A1 (ja) * | 2015-07-31 | 2017-02-09 | 株式会社ジーンケア研究所 | 修飾siRNA及びそれを含む医薬組成物 |
WO2021132648A1 (ja) * | 2019-12-27 | 2021-07-01 | ルクサナバイオテク株式会社 | Recqlの発現を阻害するアンチセンスオリゴヌクレオチド及びその用途 |
WO2024004779A1 (ja) * | 2022-06-30 | 2024-01-04 | 株式会社ジーンケア研究所 | RecQL1ヘリカーゼ遺伝子を標的とするsiRNA |
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US20070243570A1 (en) | 2003-05-19 | 2007-10-18 | Genecare Research Institute Co., Ltd | Apoptosis Inducer for Cancer Cell |
IT1398977B1 (it) | 2009-06-25 | 2013-03-28 | Icgeb | Agente anticancro specifico per i tumori cerebrali con meccanismo di soppressione di recq1 |
JP2019505562A (ja) | 2016-02-22 | 2019-02-28 | ニューヨーク インスティチュート オブ テクノロジーNew York Institute Of Technology | Brca1/fancm相互作用を無効化することによってがんを治療する方法 |
CN113797338B (zh) * | 2021-09-10 | 2022-07-15 | 中国医学科学院基础医学研究所 | Dbc1调控细胞衰老及其应用 |
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US5726195A (en) | 1995-07-28 | 1998-03-10 | Cubist Pharmaceuticals, Inc. | Aminoacyl adenylate mimics as novel antimicrobial and antiparasitic agents |
US5877215A (en) | 1996-03-20 | 1999-03-02 | Medical University Of South Carolina | Method of treating neoplastic cells with prostaglandin and radiation treatment or prostaglandin and platinum-based anti-tumor agents |
JPH11276173A (ja) | 1998-03-27 | 1999-10-12 | Eijiin Kenkyusho:Kk | ヘリカーゼをコードする遺伝子、RecQ5 |
US6489344B1 (en) | 1998-06-19 | 2002-12-03 | Chiron Corporation | Inhibitors of glycogen synthase kinase 3 |
JP2000166600A (ja) | 1998-09-30 | 2000-06-20 | Eijiin Kenkyusho:Kk | 腫瘍の増殖を抑制する化合物をスクリ―ニングする方法 |
WO2000032179A2 (en) | 1998-12-01 | 2000-06-08 | University Of Kentucky Research Foundation | Use of nicotonic acid derivatives for the treatment of dna damage in skin cells |
CZ302719B6 (cs) * | 2000-12-01 | 2011-09-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Izolovaná molekula dvouretezcové RNA, zpusob její výroby a její použití |
US20030171310A1 (en) * | 2001-02-23 | 2003-09-11 | Isis Pharmaceuticals Inc. | Antisense modulation of RECQL expression |
US6448080B1 (en) * | 2001-02-23 | 2002-09-10 | Isis Pharmaceuticals, Inc. | Antisense modulation of WRN expression |
US20070243570A1 (en) | 2003-05-19 | 2007-10-18 | Genecare Research Institute Co., Ltd | Apoptosis Inducer for Cancer Cell |
EP2520652B1 (en) | 2004-04-09 | 2015-06-10 | Genecare Research Institute Co., Ltd | Cancer cell-specific apoptosis-inducing agents that target chromosome stabilization-associates genes |
JP4809240B2 (ja) | 2004-11-19 | 2011-11-09 | 株式会社ジーンケア研究所 | 癌細胞特異的細胞増殖抑制剤 |
-
2004
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- 2004-05-19 JP JP2005506299A patent/JP4299299B2/ja not_active Expired - Fee Related
- 2004-05-19 EP EP04733937.9A patent/EP1625853B1/en not_active Expired - Lifetime
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EP1625853A1 (en) | 2006-02-15 |
JP4299299B2 (ja) | 2009-07-22 |
US8809296B2 (en) | 2014-08-19 |
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EP1625853B1 (en) | 2015-07-08 |
US8299044B2 (en) | 2012-10-30 |
US20070243570A1 (en) | 2007-10-18 |
US20100168209A1 (en) | 2010-07-01 |
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EP1625853A4 (en) | 2009-07-01 |
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