WO2005007849A1 - Method of enhancing alkylating drug sensitivity - Google Patents

Abstract

A method of enhancing the sensitivity to alkylating drug through inhibition of the expression or activity of AlkB-homologue of mammal; an alkylating drug concomitant agent comprising a compound capable of inhibiting the expression or activity of the AlkB-homologue; and a method of screening a compound capable of enhancing the sensitivity to alkylating drug through measuring of the expression or activity of the AlkB-homologue. These enable enhancing of the effect of alkylating drug on cancer treatment, etc.

Description

 Methods for increasing sensitivity to alkylating drugs

 The present invention relates to methods and agents for increasing sensitivity to alkylating drugs. Background art

 Alkylating drugs are abundant in the environment and are frequently used in general cancer treatment. Alkyl glazes are known to act directly on intracellular DNA and RNA and modify the base to cause genomic mutations and translational abnormalities. As a result, in mammals, canceration, neurodegeneration, aging. Also, as a completely different action, alkylating drugs kill cancer cells rapidly. For this reason, alkylating drugs have severe side effects that are widely used as anticancer agents, and alkylic glaze resistant cancers are also known.

Genomic DNA damaged by alkylic drugs is repaired by DNA glycosylase or alkyl transferase. In humans, MYH and 0GG1 genes are known as DNA glycosylase and MGMT gene is known as alkyltransferase [Nakabeppu, Y. (2001) Prog Nucleic Acid Res Mol Biol. 68: 75 -94; Margison, GP and Santibanez-Koref, MF (20 02) Bioessays. 24: 255-66; Gerson, S. (2002) J Clin Oncol. 20: 2388-99]. In cells in which these genes are knocked out, it is known that sensitivity to alkylating drugs is increased. In particular, the expression of MGMT gene is high in cancer cells, and high expression in alkylating drug resistant cancer cells has been reported [Margison, GP and Santib anez-Koref, MF (2002) Bioessays. 24: 255-66 ; Gerson, SL (2002) J Clin Oncol. 20: 2388-99]. Therefore, the combined use of alkylating drugs and MGMT inhibitors showed the effects of alkylating drugs at lower concentrations, and the effects of alkylating drugs have been confirmed even in the case of alkyl-resistant drug-resistant cancer [ Margison, GP and Santibanez-Kor ef, MF (2002) Bioessays. 24: 255—66; Gerson, SL (2002) J Clin Oncol. 2 0: 2388-99] o In fact, it is a specific inhibitor of MGMT. 0 ⁶ -benzylguanine is a concomitant drug with BCNU (N, N, -bis (2-chloroethyl) -N-nitrosourea) and temozolomide, which are alkyl glazes. Four cases have been developed in Phase II and one in Phase III [Gerson, SL (2002) J Clin Oncol. 20: 2388-99]. Disclosure of the invention

 The present invention provides methods and agents for increasing sensitivity to alkylating drugs. The present invention also provides a screening method for a scavenger compound that increases sensitivity to an alkylating agent.

Most recently, the alkB gene of E. coli has been reported to be a new type of alkylated DNA repair enzyme [Begley, TJ and Samson, LD (2003) Trends Biochem S ci. 28: 2-5; Falnes, PO et al. (2002) Nature. 419: 178-82; Trewick, SC et al. (2002) Nature. 419: 174-8]. AlkB removes the methyl group from 1-methyladenine and 3-methylcytosine in DNA and RNA in the presence of 2-oxoglutarate and divalent iron ions. It is an enzyme that removes and restores normal bases. AlkB homologues are widely distributed in many organisms from mammals to bacteria. Human homologues are known to contain ABH, ABH2, and ABH3 genes, and among these three genes, ABH2 and ABH3 have been reported to exhibit enzyme activity similar to alkB [Duncan, T et al. (2002) Proc Nat 1 Acad Sci US A. 99: 16660-5]. ABH2 also efficiently removes methyl groups in double-stranded DNA, and ABH3 makes RNA a good substrate [Aas, PA et al. (2003) Nature. 421: 859-63]. Intracellular localization is that ABH2 exists only in the nucleus, and ABH3 exists in both the nucleus and the cytoplasm. It exists in a diffuse manner [Duncan, T. et al. (2002) Proc Natl Acad Sci US A. 99: 1 6660-5]. Both ABH2 and ABH3 are expressed in cancer cells, and overexpression is thought to indicate resistance to alkylic drugs.

 From this point of view, the present inventors considered that mammalian alkB homologues containing ABH2 and ABH3 are always promising molecular targets for alkylating drug combinations. Therefore, it was analyzed whether or not the alkB homologue was actually inhibited in mammalian cells, thereby showing sensitivity to the alkylic drug. As a result, it was found that by suppressing the expression of the alkB homolog, the sensitivity of the cells to the alkylating drug was significantly increased. That is, compounds that suppress or inhibit the expression or activity of mammalian alkB homologues are useful for increasing sensitivity to alkylating drugs, and such compounds can be used for cancer treatment, for example. It can be used in order to enhance the therapeutic effect by using in combination with an alkyl glaze. It is also possible to develop new alkylating drug combinations by screening for compounds that suppress or inhibit the expression or activity of alkB homologues.

 That is, the present invention relates to a method for increasing sensitivity to an alkylating agent, an alkylating agent combination agent that increases sensitivity to an alkylating agent, a screening method for a scavenger compound that increases sensitivity to an alkylating agent, and the like. More specifically, the present invention relates to the invention described in each claim. It should be noted that inventions consisting of one or more combinations of the inventions recited in the claims that already cite the same claim are already intended for the manifestation described in those claims. That is, the present invention

 (1) a method for increasing the sensitivity of a mammalian cell to an alkylich drug, comprising a step of inhibiting expression of a mammalian AlkB homolog gene or activity of a protein encoded by the gene,

 (2) The method according to (1), wherein the mammalian AlkB homolog gene is ABH2 or ABH3,

(3) RNAi inhibits mammalian AlkB homolog gene expression, (1) and Is the method described in (2),

 (4) The method according to any one of (1) Force et al. (3), wherein the mammalian cell is a cancer cell.

(5) an alkylating agent combination agent comprising, as an active ingredient, a compound that inhibits the expression of a mammalian AlkB homolog gene or the activity of a protein encoded by the gene,

 (6) The alkylating agent combination agent according to (5), wherein the mammalian AlkB homolog gene is ABH2 or ABH3,

 (7) The alkylating agent combination agent according to (5) or (6), wherein the compound is siRNA or a vector that expresses siRNA,

 (8) A method for screening candidate compounds that increase the sensitivity of mammalian cells to alkylating drugs,

 (a) measuring the expression of a mammalian AlkB homolog gene or the activity of a protein encoded by the gene in the presence of a test compound;

 (b) selecting a compound that inhibits the expression or activity relative to a control,

(9) A method for producing a concomitant alkylating drug comprising a step of mixing a compound selected by the method according to (8) with a pharmaceutically acceptable carrier. The present invention relates to a method for increasing the sensitivity of mammalian cells to an alkylating agent, which comprises the step of inhibiting the expression of a mammalian AlkB homologue gene or the activity of a protein encoded by the gene. The present inventors have succeeded for the first time in increasing the sensitivity to an alkyl glaze by targeting the mammalian AlkB homolog gene. Alkylating drugs generally have a strong anti-tumor effect and kill DNA by alkylating DNA and / or RNA. At this time, it is possible to enhance the effect of this alkylating agent by inhibiting the expression or activity of the AlkB homolog according to this effort. In the present invention, mammalian AlkB homolog gene (hereinafter also simply referred to as “AlkB homolog gene”) means 2-oxodaltalic acid (α-ketoglutaric acid) and divalent iron ion [Fe (II)] A nucleic acid encoding a protein having an activity to remove a methyl group, which is a dependent oxygenase and has the ability to remove 1-methyladenine and / or 3-methylcytosine in DNA and / or RNA Say. The mammalian AlkB homolog gene preferably complements the E. coli AlkB mutant (Chen BJ et al., J Bacteriol. 1994. 176 (20): 6255-61). That is, when the gene is introduced into an E. coli A1 kB mutant, the sensitivity to alkylating drugs is reduced. The capture should be significant when compared to the Escherichia coli AlkB mutant, and may be partially complemented without being completely recovered (to the same extent as the wild type).

 ABH gene family has been identified as a mammalian AlkB homolog gene. ABH gene family includes ABH (Wei YF et al., Nucl. Acids Res. 1996. 24: 931-37; Genbank accession X91992 (protein: CAA63047), AC008044 (protein: AAF01478, Q13686), BC025787 (protein: AAH25787) , ABH2 (Genbank accession XM— 058581 (protein: XP— 058581)), and ABH3 (Genbank accession 匪 —13917

8 (protein: NP_631917)) is known. Those skilled in the art can easily obtain information on the base sequences of these genes from public gene databases such as GenBank. ABH mouse homologs include Genbank accession XM-12704

9 (protein: XP-127049), ABH2 rat homologues are Genbank accession XM-222273 (protein: XP—222273), mouse homologues are Genbank accession XM_132383 (protein: XP—132383), and AK079195 (protein: BAC37576). Examples of ABH3 mouse homologues include Genbank accession XM_130317 (protein: XP_130317). Preferred mammalian AlkB homolog genes include the ABH2 and ABH3 genes and their counterparts.

More specifically, the mammalian AlkB homolog gene includes human ABH (SEQ ID NO: 1 ), Human ABH2 (SEQ ID NO: 3), human ABH3 (SEQ ID NO: 5), and their counterparts (including polymorphic opi variants) in other organisms. In general, mammalian genes may have multiple types of sequences even if they are the same gene due to polymorphisms in the base sequence. This polymorphism is not limited to single nucleotide polymorphisms (SNPs) consisting of single nucleotide substitutions, deletions, and insertion mutations, but also includes several consecutive nucleotide substitutions, deletions, and insertion mutations. Therefore, the base sequences of ABH, ABH2, and ABH3 shown above are merely examples, and are not necessarily limited to these sequences. Specific examples of mammalian AlkB homologues include the following proteins: 1_methyladenine in DNA and / or RNA in the presence of 2_oxodaltalic acid and divalent iron ions. And / or a gene encoding a protein having an activity of removing a methyl group from 3-methylcytosine.

 (a) a protein having 70% or more identity to a contiguous partial amino acid sequence of 100 or more residues of SEQ ID NO: 2, 4, or 6;

 (b) a protein comprising an amino acid sequence in which no more than 30% of amino acids have been substituted, deleted, and / or added in a sequence of 100 or more consecutive partial amino acids of SEQ ID NO: 2, 4, or 6;

 (c) A protein encoded by a nucleic acid that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 1, 3, or 5 or its complementary strand.

 These proteins may include naturally occurring AlkB homologous proteins (including polymorphisms and variants that maintain function) of each mammal.

The amino acid sequence identity can be calculated as the percentage of amino acids that match by aligning the full length amino acid sequences of the two proteins to be compared with appropriate gaps so that the amino acids match. For alignment of amino acids, a desired computer program can be used. For example, CLUSTAL W (Higgins, D. et al. (1994) Nucleic Acids Res. 22: 4673-4680), BLAST (National Center f) or Biothchnology Information). The gap is MISUMA The identity is determined as the ratio of the number of amino acids that matched in both sequences out of the total number of amino acids (including gaps) in the alignment. However, if there is a gap at the same position in both sequences, the gap is excluded from the calculation. The amino acid sequence identity is preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more. Further, the continuous partial amino acid sequence of 100 residues or more in (a) and (b) is preferably 120 residues or more, more preferably 150 residues or more, more preferably 200 residues or more, more preferably 250 residues. Group or more, more preferably SEQ ID NO:

: Total length of 2, 4, or 6.

 For example, when amino acid sequence identity is determined by BLAST, it can be carried out according to the method described in the literature “Altschul, SF. Et al., 1990, J. Mol. Biol. 215: 403-410”. Specifically, the blastn program is used to determine the identity of the base sequence, and the blastp program is used to determine the identity of the amino acid sequence. For example, on the BLAST web page of NCB I (National Center for Biothchnology Information) In this case, calculation is performed with all filter settings such as "Low complexity" turned OFF (A1 tschul, SF et al. (1993) Nature Genet. 3: 266-272; Madden, TL et al. (1996) Meth. Enzyraol 266: 131—141; Altschul, SF et al. (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. & Madden, TL (1997) Genome Res. 7: 649-656). For example, the cost of open gap is 5 for nucleotides and 11 for protein, the cost of extend gap is 2 for nucleotides and 1 for protein, the penalty for nucleotide mismat ch is 1 and the reward for nucleotide match is 1 and expect value 10, words i ze has 11 nucleotides, protein 2, Dropoff (X) for blast exten sions in bits is 20 for blastn and 7 for other programs, X dropoff value for gapped alignment (in bits) is 15 for non-blastn, final X dropoff value for gapped alignment

(in bits) should be 50 for blastn and 25 for other programs. BL0SUM62 can be used as a matrix for scoring in amino acid sequence comparisons . Two sequence alignments were created using the blast2sequences program (Tatiana A et al. (1999) FEMS Microbiol Lett. 174: 247-250), which compares two sequences.

The identity of the sequence can be determined.

 In the protein (b), the total number of amino acids replaced, deleted, and / or added in the continuous partial amino acid sequence is preferably within 27%, more preferably within 25%, more preferably Is within 20%, more preferably within 18%, more preferably within 15%, more preferably within 12%, more preferably within 10%, more preferably within 5% (however, converted into the number of amino acids). Rounded down to the nearest 1). Substitutions, deletions, and additions may be combined arbitrarily.

 In amino acid substitution, it is highly probable that a protein substituted with an amino acid having similar properties maintains the activity of the original protein. Such substitution is called conservative substitution. Conservative substitutions of amino acids are well known to those skilled in the art. Examples of amino acid groups corresponding to conservative substitutions include basic amino acids

(Eg lysine, arginine, histidine), acidic amino acids (eg aspartic acid, glutamic acid), uncharged polar amino acids (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar amino acids (eg alanine, valine) , Leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) / 3-branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine, tryptophan, histidine) Can be mentioned. The target protein in the present invention may be a protein comprising an amino acid sequence in which the amino acids of SEQ ID NO: 2, 4, or 6 are conservatively substituted.

In hybridization with the base sequence of SEQ ID NO: 1, 3, or 5, the base sequence or its complementary strand, or a part thereof is used as a probe to hybridize to the target sequence. Is the probe significantly hybridized to the target sequence after washing and washing under stringent conditions? Check. As a probe, SEQ ID NO: 1, 3, or 5 consecutive 15 bases or more, preferably 18 bases or more, more preferably 20 bases or more, more preferably 30 bases or more, more preferably 50 bases or more, more preferably Use at least 100 bases. If the probe contains sequences other than SEQ ID NO: 1, 3, or 5, or their phase capture strands, use only that sequence as a negative control and perform hyperpredation in the same way. Confirm that the probe does not significantly hybridize to the target sequence after washing under these conditions. Hybridization can be performed by a conventional method using a nitrocellulose membrane or a nylon membrane. For example, 6XSSC, 0.5% (W / V) SDS, lOO ^ g / ml denatured salmon sperm DNA, 5 X Denhardt solution (0.2 ° / 1 for 1 X Denhardt solution). In a solution containing polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% ficoll) at 42 ° C, preferably 50 ° C, more preferably 60 ° C, more preferably 65 ° C. Daisysi

3 XS, 4 XSSC, 0.5 ° / at the same temperature as the high pridization. SDS is a condition of washing 3 times for 20 minutes. More preferably, washing after 3 minutes of hybridization, 4XSSC, 0.5% SDS, 20 minutes twice, 2XSSC, 0.5% SDS, 20 minutes once at the same temperature as the hybridization. It is a condition to do. More preferably, the conditions are such that washing after 3 hours of high lysis is performed twice at 4 XSSC, 0.5% SDS, 20 minutes, followed by 1 XSSC, 0.5% SDS, 20 minutes at the same temperature as the high lysis. . More preferably, the post-hybridization washing is 2XSSC, 0.5 ° / at the same temperature as the hybridization. SDS, 20 minutes once, then 1XSSC, 0.5% SDS, 20 minutes once, then 0.5XSSC, 0.5% SDS 20 minutes once.

In the case of isolating a gene from a desired mammal by hybridization, for example, the base sequence of the mammalian AlkB homolog gene exemplified in the present specification (eg, SEQ ID NO: 1, 3, or 5) or its phase sequence is used. Tang Dynasty or part of it as probe Then, a cDNA library prepared from the organism to be isolated is screened. In the present invention, the AlkB homolog gene encoded by the natural gene thus obtained can be targeted. The material for identifying the AlkB homolog is not particularly limited. Mammalian genomic DNA or cDNA is preferred. Specific examples include desired mammals such as mice, rats, and goats, primates such as monkeys, and humans.

 The target AlkB homologous protein in the present invention is DNA from 1-methyladenine and / or 3-methylcytosine to methyl in DNA and / or RNA in the presence of 2-oxodaltalic acid and divalent iron ions. Has activity to remove groups. The activity of removing a methyl group can be assessed by the method described below.

In cells expressing the above AlkB homolog gene, inhibition of the expression of this gene can increase the sensitivity of the cell to alkylic drugs. The alkylating agent used at this time is a compound that alkylates intracellular DNA or RNA to produce 1-methyl adenine and / or 3-methyl cytosine. There are many known alkylating agents such as SN ₂ type alkylating agents such as methyl methanesulfonate (MMS). In addition to MMS, cyclophosphamide and camlucitin can be exemplified. In addition, clinical examples of menorephalan, chlorambucinole, ifosfamide, busunolevane, semustine, dacarpazine, nitromine, dimustine, and ranimustine are not limited thereto. The order of the alkylating agent administration step and the suppression of AlkB homolog expression or activity is not particularly limited, and the AlkB homolog expression or activity is suppressed before, simultaneously with, or after the addition of the alkyl glaze to the cell. It's okay. The sensitivity of cells to alkylich drugs can be determined, for example, by measuring cell survival. For measurement of cell viability, for example, the MTT Atsey method or the living cell count reagent SF (Nacalai Tester) can be used.

Suppression of the expression of the AlkB homologue gene is due to the raRNA level of the gene and / or the protein. There may be a suppression of white matter levels. Preferred examples of the compound that suppresses the expression of the AlkB homolog gene include RNA having an RNAi (RNA interferance) effect on the gene. In general, RNAi refers to target RNA by introducing or expressing into the cell RNA that includes a sense RNA consisting of a sequence homologous to part of the mRNA sequence of the target gene and an antisense RNA consisting of a complementary sequence. This refers to a phenomenon in which the expression of the target gene is inhibited by inducing the destruction of the gene mRNA (Genes Dev. 2001,

15: 188-200; Elbashir, SM et al. (2001) Nature 411: 494-498). When double-stranded RNA with the thigh i effect is introduced into the cell, an enzyme called DICER (a member of the RNase III nucleic acid-degrading enzyme family) force ¾ contacts with double stranding, and double-stranded RA becomes small interfering R It is broken down into small pieces called NA (siRNA). In the present invention, siRNA is collectively referred to as siRNA as well as RNA generated by such intracellular processing, and RNA molecules artificially synthesized or expressed to inhibit target gene expression by RNAi. The RNA having an RNAi effect in the present invention includes these siRNAs. siRNA can suppress the expression of target genes in vivo (Anton P. et al., Nature Vol. 418 38-39 2002; David L. et al., ature genetics Vol. 3 2 107-108, 2002).

The siRNA for a target gene is usually a sequence of 15 bases or more in the transcription sequence (mRNA sequence) of this gene (more preferably a sequence of 16 bases or more, 17 bases or more, 18 bases or more, or 19 bases or more) , And its complementary sequence, and these sequences are RNA that is hybridized to form a double strand. Preferably, a sequence of 19 to 30 bases, more preferably 20 to 25 bases, more preferably a sequence of 21 to 23 bases or a complementary sequence thereof is included in one strand so as to form a complementary pair with this. RNA containing the other strand. However, even if the RNA contains a longer sequence, the length of the RNA is not particularly limited because it is expected to be degraded into siRNA having an RNAi effect in the cell. In addition, a long double-stranded RA corresponding to the full length or almost the full length region of the target gene mRNA can be decomposed with DICER in advance and the degradation product can be used. Is possible. This degradation product is expected to contain an RNA molecule (siRA) having the RNAi effect. According to this method, it is not necessary to select a region on raRNA that is expected to have an RNAi effect. That is, the sequence having the RNAi effect of the target gene may not necessarily be precisely defined.

Usually, double-stranded RNA having an overhang of several bases at the end is known to have a high RNAi effect. The siRNA used in the present invention is not essential, but preferably has an overhang of several bases at the end (preferably the 3 ′ end). The length of the base forming this overhang is not particularly limited, but is preferably a 2-base overhang. In the present invention, for example, TT (2 thymines), UU ( ² uracils), double-stranded RNA having an overhang of other bases (most preferably, a double-stranded RNA part of an I ⁹ base pair and Molecules having an overhang of 2 bases (TT) can be preferably used. The siRNA of the present invention includes molecules in which the base that forms the overhang is DNA.

 In addition, two strands forming a base pair in siRNA may be linked via a spacer. That is, RNA in which the spacer forms a loop and the RNA sequences before and after the spacer anneal to form a double strand can also be suitably used. The length of the spacer is not limited, but may be 3 to 23 bases, for example.

In addition, vectors capable of expressing the above siRNA can also be used in the present invention. That is, the present invention relates to the use of a vector capable of expressing a thigh having an RNAi effect. The vector capable of expressing the RNA may be, for example, a nucleic acid linked to a separate promoter so that one strand of a double-stranded siRNA and the other strand are separately expressed. Alternatively, two types of RA may be transcribed from one promoter by alternative splicing or the like. Alternatively, it may be a vector that expresses a single-stranded RNA in which a sense strand and an antisense strand are linked via a spacer (forms a loop). RNA expressed from this vector forms an RNA stem with RNAi effect and suppresses expression of the target gene. The stem length is the same as the above siRNA For example, it may be 19 to 29 bases. There is no limit to the length of the spacer, but for example 3 to 23 bases. It may or may not have an overhang of several bases at 5 'and / or 3'. Those skilled in the art can easily produce these vectors by general genetic engineering techniques (Bru thigh elkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001 ) Nature Biotechnology 19: 500-5 05; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddis on PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et al. (2002) Proc Nat 1 Acad Sci USA 99 (8): 5515-5520; Proc Natl Acad Sci USA 99: 14943-14945, 2002; Paddison, PJ et al. (2002) Genes Dev. 16: 948-958). More specifically, it can be constructed by appropriately inserting DNA encoding a target RNA sequence into various known expression vectors. As the promoter, RA polymerase III promoter and the like can be preferably used. Specifically, for example, U6 Pol I II promoter and HI RNA promoter (HI RNA is a component constituting RaseP) can be used.

Examples of preferable siRNA are illustrated below, but the siRNA used in the present invention is not limited thereto. First, a transcription sequence region downstream of 50 bases or more, preferably 60 bases or more, more preferably 70 bases or more from the start codon of the target gene is selected. From this region, preferably the M sequence is found and the 17-20 nucleotides following the AA (eg 19 nucleotides following M) are selected. Although the next base of AA is not particularly limited, a sequence that is G or C is preferably selected. Here, the GC content of the sequence to be selected is preferably 20 to 80%, more preferably 30 to 70%, more preferably 35 to 65%. In addition, the selected sequence is preferably a sequence specific to the target gene among genes expressed in the tissue to which siRNA is administered. For example, it is possible to search a public gene sequence database by selecting a selected sequence as a query, and to confirm that there is no gene having the same sequence as the transcription sequence other than the target gene in the gene of the administered individual. preferable. The sequence is preferably selected from the protein coding sequence (CDS) of the target gene. A sequence containing the sequence excluding the first AA of the sequence thus selected

(Preferably, UU or TT is added to 3 ′), and its phase-trapping sequence (preferably having UU or TT in 3 ′) is a suitable siRNA. Alternatively, instead of searching for the sequence following M, you may search for the sequence following CA as above. Alternatively, it may be a sequence other than M or CA. It is also possible to appropriately select an RNA having an optimal RNAi effect from a plurality of types of siRNAs. Specific examples of suitable siRNAs include the RNAs shown in the examples. That is, siRNA containing the 1st to 19th sequences of SEQ ID NOs: 7 and 8; siRNA containing the 1st to 19th sequences of SEQ ID NOs: 9 and 10; SEQ ID NO:

: SiRNAs containing the 1st to 19th sequences of 11 and 12 (bases such as TT or UU may be added 3 ′ of each RNA) are preferably used. Similarly, siRNA containing sequences 1 to 19 in each pair of SEQ ID NOs: 13 and 14, 15 and 16, 17 and 18 (bases such as TT or UU are added to the 3 ′ of each RNA) May be preferably used.

 The RA having the RNAi effect can be appropriately prepared by those skilled in the art based on the base sequence of the target gene. The base sequence of the target AlkB homolog gene can be easily obtained from the public gene database as described above, and the base sequence of the human AlkB homolog gene (SEQ ID NO: 1, 3, or 5) is probed. In addition, it can be obtained by screening a cDNA library of mammals, or by RT-PCR based on a primer prepared based on the base sequence of the human AlkB homolog gene. Based on the base sequence of the obtained gene, siRNA can be designed according to the above explanation.

It is not always necessary to know the base sequence of the full length of the target gene. Any continuous RNA region that can be selected as siRNA (for example, 20 to 30 bases) may be known. Therefore, the siRNA of the present invention is prepared from a gene fragment whose partial length is known, such as EST (Expressed Sequence Tag), but whose full length is not known, based on the base sequence of the fragment. It is possible. Below, GenBank In the database, an EST sequence accession number showing high homology with human ABH2 and ABH3 was described. However, these are merely examples of a large number of EST sequences, and those skilled in the art can easily obtain information on appropriate EST fragments from public databases. In the mRNA corresponding to each EST, AlkB homolog gene expression may be inhibited by using RNA having an RNAi effect with a part of a continuous RNA region as one strand.

 (a) ABH2

 BU857344, BM713799, BU621636, CB306784, BU535633, BU858046, CA425782, BU6 21592, AL528750, BU539036, BF812735, AW245078, AW246209, BI822466, BM7909 74, BM852409, AI201794, BE222844, AI078333, AI374763, 382, AI374763, BI BQ920902, AI885651, AW206726, AW005870, AA782579, B G831183, BU542852, BG057635, AW971683, BE253917, AW952528, AI040632, H097 45, AI 124006, BM701520, AI138654, BQ013942, BE742854, 283153, 59483, AI253156, W73383, 28383 BG116821, BE328465, AW731884, CB115208, BQ22 7530, AI278638, BF683378, BF591258, AI421482, AI417338, AI073924, BF18320 8, BM799140, AI675781, BU623121, BQ014087, AW071763, BF, 115, 785 BI861887, M256089, T7963 1, T79716, BM785502, BI666861, BI861994, BM546765, AW952001 BI767047, BF 766222, BG751427, AI269764, BG284978, BE255140, CB11 8993, BI045022, AW005 663, AI589576, AI284851, CB115220, AA333965, AI784306, M911501, N92173, AI183736, AA865002, AW169802, BE857214

 (b) ABH3

BI860137, BX406644, AL570007, AL570007, BE250119, BE249876, AL571973, AL5 51946, BG283085, BF724816, BM824362, BM792655, BE293567, BG282971, BE7890 24, BE293665, BM545966, BF037645, B4272207, AL 545207, AL AL546752, BM981193, BM985113, AL554302, BX406645, B M999346, CB048424, BI918447, BM694348, BU602469, AL577048, BQ939674, BM70 4858, BE408144, BU179848, BI258292, BQ233246 _S BG772137, BM548608, BE27441 9, BU553708, BI823026, BU838356, M172190, BM98454, BM9845 BQ017150, BM971930, BM681387, BE891036, BE892721, BFO 62547, BE791860, BQ181579, BM011189, BU788104, BI222582, BF026256, BI9089 85, BE887345, AI636076, BU784435, BI561482, BG831972, BF933573, BE260 229, BF933573, BE260 229 BX361668, BQ375283 _S BX089006, B F823012, BE937862, BG823846, BQ375279, BI833740, AI916587, AI656885, BQ57 6341, BG187465, AW001947, BG191648, BG825625, BG370227, AA932508, AW45147 2, 495370BM, 388147

 When the synthesized siRNA is used clinically, siRA can be modified. For example, phosphorothioate type RNA can be used to reduce susceptibility to nuclease degradation and maintain the effect. It has been reported that substitution of phosphoric acid ester of siRNA with phosphoroate does not change the RNAi effect and increases stability. In addition, siRNA uridine and cytidine can be converted to 2 '_fluoro-2, -deoxyuridin e and 2, -fluoro-2, -deoxycytidi'n, respectively, and the RNAi effect will not change, and it will become resistant to nucleases. [Harborth J. et al. (2003) Nucleic Acid Drug Dev. 13: 83-105]. Therefore, siRNA containing such an RNA analog is suitable for clinical use. The siRNA of the present invention includes such thigh analogs. In order to suppress the expression of the AlkB homolog gene, for example, the following nucleic acid (a) or (b) can be used.

 (a) Antisense nucleic acid for transcript of AlkB homolog gene or part of it

 (b) Ribozyme that specifically cleaves the transcript of the AlkB homolog gene

As a method for inhibiting (suppressing) the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. Antisense nucleic acid is the target gene There are several factors as the action of inhibiting the expression of: Ie

Inhibition of transcription initiation by triplex formation, inhibition of transcription by formation of a hybrid with a site where an open loop structure is locally formed by RNA polymerase, inhibition of transcription by formation of a hybrid with RNA, which is being synthesized, intron and exon Splicing inhibition by hyperid formation at the junction with, splicing inhibition by hyperid formation with spliceosome formation site, inhibition of transition from nucleus to cytoplasm by hyperid formation with mRNA, capping site poly (A) addition site Inhibition of splicing due to the formation of a hybrid with the translation initiation factor, inhibition of translation initiation due to the formation of a hybrid with the translation initiation factor binding site, inhibition of translation due to the formation of a hybrid with the ribosome binding site near the initiation codon, translation region of the mRNA with the polysome binding site By hybrid formation Elongation inhibition of peptide chain, and gene expression inhibition by high Puriddo forming the interaction site between a nucleic acid and protein, and the like. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (by Hirashima and Inoue, Shinsei Chemistry Laboratory 2 Nucleic acid IV gene replication and expression) , Japan Biochemical Society, Tokyo Chemical Doujin, 1993, p. 319-347).

The antisense nucleic acid used in the present invention may inhibit the expression of the AlkB homolog gene by any of the actions described above. The antisense nucleic acid may be a nucleic acid containing an antisense sequence for a sequence of 13 nucleotides or more, preferably 14 nucleotides or more, more preferably 15 nucleotides or more of the sequence to be transcribed of the AlkB homolog gene. For example, an exon-intron boundary in the initial transcription sequence, an intron-exon boundary, a region including the translation initiation codon, an untranslated region near the 5 'end, or a sequence of protein coding sequences (CDS) in the mature mRNA 13 A nucleic acid containing an antisense sequence for nucleotides or more, preferably 14 nucleotides or more, more preferably 15 nucleotides or more is preferred. Moreover, when considering clinical application, the antisense nucleic acid used is usually a synthetic oligomer. The antisense nucleic acid may be DNA and may be further modified. For example, against nuclease degradation In order to reduce the sensitivity and maintain the activity as an antisense nucleic acid, the use of S oligos (phosphorothioate oligonucleotides) in which 0 (oxygen) in the phosphate ester bond is replaced with S (sulfur) Generally known. This S oligo is now in clinical trials as an antisense drug that is injected directly into the affected area. In the present invention, this S oligo can be preferably 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 the gene expression can be effectively suppressed. Transcribed R

NA preferably has 90% or more complementarity, and most preferably 95% or more complementarity to the target gene transcript. In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is preferably 17 bases or more, more preferably 20 bases or more, more preferably 25 bases or more, Preferably 30 bases or more

More preferably, it is 40 bases or more, more preferably 50 bases or more, and even more preferably 100 bases or more. Antisense RNA can also be expressed intracellularly. A vector in which a nucleic acid encoding the target RNA is linked downstream of a promoter active in the target cell may be prepared and introduced into the cell. As a vector, a retrovirus vector, an adenovirus vector, a virus vector such as an adeno-associated virus vector, or a non-viral vector such as a ribosome can be used. High-molecular carriers are known as non-viral vectors other than ribosomes. Polyethylene Daricol-Polycation Block as a delivery system for nucleic acid drugs that stably exist in the body and can circulate stably in the bloodstream. Polymer nanomicelles formed by self-association of copolymers and nucleic acid drugs are listed [Harada A. & Kataoka K. Science, 283, 65-67 (1999); Katayose S. & Kataoka K. Bioconjugate Chemistry, 8 (5), 702-707 (1997)]. This improved type, calcium phosphate micelles, an inorganic-organic composite nanostructure formed from polyethylene glycol-polyaspartate (PEG-PAsp) block copolymer and calcium phosphate [Kakizawa Y. & Kataoka K. Langmuir, 18 (2), 4539-4543 (20 02)], and polymer nano micelles with improved blood stability by introducing disulfide bridges into hemorrhoids [Kakizawa Y., Harada A. & Kataoka KJ Amer. Chem. Soc., 121 (48), 11247- 11248 (1999)], and these derivatives are considered to be effective as siRNA and nucleic acid carriers. Another polymer carrier is bioconjugate-type carrier strength S [Asayama S. et al. Bioconjugate Chemistry, 9 (4), 476-481 (1998); Park JU. Et al. Prep. Biochem & Biotechnol., 29 (4), 353-370 (1999)]. Using these vector systems or carriers for gene transfer, gene therapy can be performed by administering to a patient by ex vivo method or in vivo method. In addition, inhibition of the expression of the AlkB homolog gene can be carried out using a ribozyme or a vector encoding a ribozyme. Ribozyme refers to an RNA molecule that has a catalytic activity. Some ribozymes have various activities, and it is possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as the Ml RNA contained in the dalup I intron type Rase P, but the hammerhead type (Rossi et al. (1991) Pharmac. Ther. 50: 245-254) and hairpin type (Hampel et al. (1990) Nucl. Acids Res. 18: 299-304, and US Pat. No. 5, 254, 678) Some have (Makoto Koizumi, Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 219 1).

For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a liposome that has a substrate binding site that is complementary to the RNA sequence near the target site, a restriction enzyme-like RNA cleavage liposome that recognizes the UC, UU, or UA sequence in the target RNA can be created (Koizumi, M. et al. , FEB S Lett, 1988, 239, 285., Makoto Koizumi, Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059.) Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of the satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin-type liposomes can also produce target-specific RA-cleaved ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the transcription product of the target gene using a ribozyme.

 Furthermore, in the present invention, it is allowed to use a compound that suppresses the function of a protein encoded by an AlkB homolog gene (also referred to as “AlkB homolog protein” in the present specification). In a preferred embodiment, the compound that suppresses the function of the protein encoded by the AlkB homolog gene is a compound described in the following (a) or (b). These compounds increase the cell's sensitivity to alkyl glazes by P and harming (decreasing) the function or activity of the AlkB homolog protein.

 (a) Antibody that binds to AlkB homolog protein

 (b) Low molecular weight compound that binds to AlkB homolog protein

Antibodies that bind to the AlkB homolog protein can be prepared by methods known to those skilled in the art. For example, a polyclonal antibody can be obtained as follows. Serum is obtained by immunizing small animals such as Usagi with natural or recombinant AlkB homolog protein, or recombinant AlkB homolog protein expressed in microorganisms such as Escherichia coli as a fusion protein with GST, or a partial peptide thereof. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, AlkB homolog protein or affinity column coupled with synthetic peptide. In the case of a monoclonal antibody, for example, AlkB homolog protein or a partial peptide thereof is immunized to a small animal such as a mouse, and the spleen is excised from the mouse, and this is ground and finely divided. Antibodies that bind to the AlkB homolog protein from the intermediate force of the fusion cells (hypridoma) formed by fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol. A clone that produces is selected. Next, the obtained hyperidoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is used for, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography. It can be prepared by purifying with an affinity column coupled with AlkB homolog protein or its partial peptide. The AlkB homolog protein used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or human, and particularly preferably a protein derived from a human.

 The antibody of the present invention is not particularly limited as long as it can bind to the AlkB homolog protein. In addition to the polyclonal antibody and the monoclonal antibody, a human antibody, a humanized antibody by genetic recombination, and a fragment containing an antibody variable region (Including Fab, Fc, F (ab ′) 2, scFv, etc.), and modified antibodies.

 In the present invention, the protein used as the sensitizing antigen may be a partial peptide of the protein in addition to the complete protein. Examples of the partial peptide of protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of protein. In the present specification, the “antibody” usually means an antibody that reacts with the full length or fragment of a protein.

In addition to immunizing non-human animals with antigens to obtain the above-mentioned hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Then, sensitized lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain a hybridoma that produces a desired human antibody having protein binding activity. When the antibody of the present invention is used for the purpose of administering to the human body (antibody therapy), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity. The compound that suppresses the expression of the AlkB homolog gene or the function (activity) of the protein encoded by the gene may be either a natural or artificial compound. For example, compounds that are 1-methyladeyun analog and 3-methylcytosine analog that interact with AlkB homolog protein but are not degraded by the protein can be preferably used. 2-Oxodaltaric acid analogs Ν-oxalylg ycine N-oxalyl-4S-alanine N-oxalyl_4R-anine 2-thiono (N-oxalylglycine) (Welford RW et al. (2003) J Biol Chem. 278: 10157-61). These compounds and other 2-oxoglutaric acid analogs may be used to inhibit the activity of the AlkB homolog protein. Other than that, for example, organic compounds, inorganic compounds, nucleic acids, proteins, peptides, single compounds such as sugars, or compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermenting microorganisms It may be a marine organism extract, a plant extract or the like, or a compound isolated and purified from the extract.

 The present invention also relates to the use of a compound that suppresses the expression of the AlkB homolog gene or the activity of the protein deduced by the gene in combination with an alkylating agent. The present invention also relates to the use of the compounds to enhance the action of alkyl glazes. The present invention also relates to the use of the compound for the production of an alkylating agent combination agent or an alkylating agent sensitizer. Furthermore, the present invention relates to the use of the compound for enhancing the anticancer effect of the alkyly glaze and the use in combination with an anticancer drug containing the alkyl glaze.

The present invention also provides a method for screening a candidate compound that increases the sensitivity of mammalian cells to alkylich drugs. One embodiment thereof is a method using as an index the binding between an AlkB homolog protein or a partial peptide thereof and a test compound. Normally, a compound that binds to AlkB homolog protein or a partial peptide thereof is expected to have an effect of inhibiting the function of AlkB homolog protein. In this method, first, a test compound is contacted with a protein encoded by an AlkB homolog gene (for example, ABH2 or ABH3) or a partial peptide thereof. A IkB homolog protein or a partial peptide thereof can be used, for example, in a purified form, cell line or cell of the AlkB homolog protein or a partial peptide thereof, depending on the indicator for detecting binding to the test compound. It can be in an externally expressed form or a form bound to an affinity column or other carrier. The test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label. Subsequently, the binding between the test compound and the protein encoded by the AlkB homolog gene or a partial peptide thereof is detected. The binding between the A IkB homolog protein or its partial peptide and the test compound can be detected by, for example, a label added to the test compound bound to the A1 kB homolog protein or its partial peptide. It is also possible to detect the change in the activity of the protein caused by the binding of the test compound to the Alk B homolog protein or its partial peptide expressed intracellularly or extracellularly as an indicator. Next, a test compound that binds to the protein encoded by the AlkB homologue gene or a partial peptide thereof is selected.

 The test compound used for screening is not particularly limited. For example, natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins, peptides, single compounds such as sugars, compound libraries, gene library expression products, cell extracts, cell culture supernatants, Examples include, but are not limited to, fermented microbial products, marine organism extracts, plant extracts, and man-made products.

Another embodiment of the screening method of the present invention is a method of screening for a compound that inhibits the binding between an AlkB homolog protein and a substrate. AlkB homolog protein uses DNA and / or RNA as a substrate to remove 1-methyladenine and / or 3-methylcytosine contained in the substrate. Therefore, the AlkB homolog protein (eg, ABH2 or ABH3) is contacted with the substrate in the presence of the test compound, and the binding between the two is detected. Covered Select compounds that inhibit the binding of the AlkB homolog protein to the substrate compared to the absence of a test compound or the presence of a low dose (control). Such a compound becomes a compound that inhibits the activity of the AlkB homologue protein, and can be used as a drug that increases the sensitivity to an alkylating drug.

 In another embodiment of the screening method of the present invention, compounds that suppress the expression of the AlkB homolog gene are screened. In this method, first, a test compound is brought into contact with a cell or cell extract expressing an AlkB homolog gene (for example, ABH2 or ABH3). As such a cell, a cell endogenously expressing an AlkB homolog gene can be preferably used. Examples of cells used for screening include MCF7 (breast cancer), A549 (lung cancer), U20S (osteosarcoma), C33A (cervical cancer), HT1080 (fibrosarcoma), PA-1 (ovarian teratocarcinoma), Tera2 (embryonic cancer), T24 (bladder cancer), K562 (chronic myeloid leukemia), Molt4 (acute lymphoblastic leukemia), A172 (glioblastoma), HeLa (cervical cancer), etc. Tumor cells, and desired normal cells.

Contact of the test compound with the cell expressing the AlkB homolog gene is usually not limited to the force S obtained by adding the test compound to the culture medium of the cell expressing the AlkB homolog gene. When the test compound is a protein or the like, it can be brought into contact with the cell by introducing a vector expressing the protein into the cell. Next, in this method, the expression of the AlkB homolog gene is measured. Here, gene expression includes both transcription and translation. That is, mRNA may be detected or protein may be detected. The gene expression level can be measured by methods known to those skilled in the art. For example, mRNA can be extracted from cells expressing the AlkB homolog gene according to a standard method, and the transcription level of the gene can be measured by performing the Northern hyperpridization method or the RT-PCR method. When measuring the level of gene translation by protein detection, the protein fraction is collected from cells expressing the AlkB homologue gene, and the expression of the AlkB homologue protein is expressed in S. It can be detected by electrophoresis such as DS-PAGE. Furthermore, by performing Western blotting using an antibody against the AlkB homolog protein, the expression of the protein can be detected and the translation level of the gene can be measured. The antibody used for detecting the AlkB homolog protein is not particularly limited as long as it is a detectable antibody. For example, either a monoclonal antibody or a polyclonal antibody can be used.

 In this method, a compound that decreases the expression level is selected as compared with the case where the measurement is performed in the absence of the test compound or in the presence of a low dose (control). Compounds selected in this way are expected to have the effect of increasing sensitivity to alkylic drugs. The compound is useful as a concomitant agent for alkylating drugs, and is expected to be a concomitant agent that enhances the effect of, for example, an alkylich anticancer agent (anticancer agent).

In yet another embodiment of the method of the present invention, the compound that decreases the expression level of the AlkB homolog gene of the present invention can be screened using a reporter gene. Specifically, such screening methods include: (a) measuring the activity of the endogenous promoter of the mammalian AlkB homolog gene in the presence of the test compound; (b) comparing the activity to that of the control. Selecting a compound to be reduced. For this purpose, the test compound is first contacted with a cell or cell extract containing DNA in which the transcriptional regulatory region of the AlkB homolog gene and the reporter gene are functionally bound. Here, “functionally linked” means that the transcription factor binds to the transcriptional regulatory region of the AlkB homolog gene, so that the expression of the reporter gene is induced, and the transcriptional regulatory region of the AlkB homolog gene and the reporter gene. And are connected. For example, the reporter gene may be linked in frame with the coding sequence downstream of the translation initiation sequence of the AlkB homolog gene. Based on the cDNA base sequence of the AlkB homologue gene, those skilled in the art can obtain the transcriptional regulatory region of the AlkB homologue gene present in the genome by a well-known method. A By creating a knock-in animal or knock-in cell in which the code region of the lkB homologue gene is replaced with that of the reporter gene, a reporter gene can be expressed according to the promoter activity of the AlkB homolog gene. It is also possible to use it for screening. A compound that decreases the expression level compared to the control level when the expression level of the reporter gene is measured in the presence of the test compound and measured in the absence of the test compound or in the presence of a low dose (control) Select. The compound selected in this way becomes a candidate compound for an alkylating drug combination. The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, and GFP gene. As a cell containing a nucleic acid functionally linked to the transcriptional regulatory region of the AlkB homolog gene and the reporter gene, for example, a vector containing a nucleic acid functionally linked to the transcriptional regulatory region of the AlkB homologous gene and the reporter gene is introduced. Cells and the like. Those skilled in the art can produce the above-described vectors by general genetic engineering techniques. Introduction of the vector into the cells can be carried out by a general method such as calcium phosphate precipitation, electric pulse perforation, ribophetamine, microinjection and the like. A cell containing a nucleic acid in which a transcriptional regulatory region of an AlkB homolog gene and a reporter gene are operably linked includes a cell in which the nucleic acid is integrated into a chromosome. The integration of DNA into the chromosome can be performed by a method generally used by those skilled in the art, for example, a gene introduction method using homologous recombination.

 In addition, when screening is performed using a cell extract containing a nucleic acid having a structure in which a transcriptional regulatory region of an AlkB homolog gene and a reporter gene are functionally linked, for example, a commercially available in vitro transcription translation kit is included. A cell extract obtained by adding a nucleic acid having a structure in which a transcriptional regulatory region of an AlkB homolog gene and a reporter gene are functionally linked can be used.

In screening, test compounds should be added to cells or cell extracts. However, it is not limited to these methods. When the test compound is a protein, for example, contact can be performed by introducing a vector expressing the protein into the cell. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting the acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when it is a luciferase gene, the fluorescent compound catalyzed by the gene expression product Further, in the case of the GFP gene, the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.

In another embodiment of the screening method of the present invention, compounds are screened using the activity of the protein encoded by the AlkB homolog gene as an index. First, a test compound is brought into contact with a protein encoded by an AlkB homolog gene (for example, ABH2 or ABH3), or a cell expressing the protein or a cell extract. Next, the activity of the protein is measured. Then, a compound that reduces the activity of the AlkB homolog protein is selected as compared with the case where it is measured in the absence of the test compound or in the presence of a low dose. The AlkB homolog protein may be a protein endogenously expressed by a cell, or a protein expressed from an exogenous AlkB homolog gene introduced therein. A cell that expresses an exogenous AlkB homolog gene can usually be prepared by introducing an expression vector containing the gene into a host cell. This expression vector can be prepared by a person skilled in the art by a general genetic engineering technique. The AlkB homolog protein used for this screening is preferably a full-length protein that does not contain mutations, but some amino acid sequences may be substituted and / or missing if they have the same activity as the protein. Losing protein It may be quality. Further, it may be a fusion protein with another protein.

 Examples of the activity of the AlkB homolog protein include an activity of removing a methyl group from 1-methyladenine and / or 3-methylcytosine in DNA and / or RNA. In order to measure this activity, DNA and / or RNA is modified with a radiolabeled methyl group, for example, with N- [¾] methyl-N-nitrosourea. AlkB homolog protein reacts with this substrate, DNA and / or RA is ethanol precipitated, and radioactivity is measured with a liquid scintillation counter [Aas PA et al. (2003) Natur e. 421: 859-63.] Alternatively, the activity can be quantified by analysis by high performance liquid chromatography using a reverse phase column [Falnes P. 0. et al. (2002) Nature. 419: 178-82., Trewick SC (2002) Nature. 419: 174-8.].

 Alternatively, AlkB homolog protein is reacted with DNA and / or RNA treated with an alkylating agent such as methyl methylsulfonate as a substrate. The reacted DNA and / or RNA is heat-treated at 90 ° C to 180 ° C under acidic conditions to release 1-methyladenine and 3-methylcytosine, and a high-speed liquid using a reverse phase column or ion exchange column You can assemble it by analyzing with kuguchimatography. [Falnes P. 0. et al. (2002) Nature. 419: 178—82 ·, Trewick S. C. (2002) Nature. 419: 1 74-8.]

Alternatively, a method in which AlkB homolog protein is reacted with DNA and / or RNA treated with an alkyl-rich agent such as methyl sulfonate and the oxygen consumed is measured by an oxygen electrode [Trewick SC (2002) Nature. 419: 174-8 ·], reacting AlkB homolog protein with DNA and / or RNA treated with an alkylating agent such as methyl methylsulfonate, and reacting formaldehyde with Nash reagent to produce fluorescence. Measurement method [Falnes P. 0. et al. (2002) Nature. 419: 178-82.], DNA and / or RNA treated with alkylating agents such as methyl methylsulfonate were radiolabeled on the substrate 2- Reaction of AlkB homologue protein with oxodaltalic acid and liquid scintillation of the radiolabeled diacid-carbon produced A method of measuring with a counter [Welford RW et al. (2003) J Biol Chem. 278: 10 157-61.] Can also be exemplified.

 Moreover, the activity of AlkB homolog protein can be used to complement E. coli AlkB mutants. The alkB mutant of Escherichia coli is sensitive to alkylating agents (methyl methanesulfonate) at low concentrations. By transforming this Escherichia coli with the AlkB homologous gene, resistance to the alkylating agent is restored. Complementary genes can be screened using resistance to methyl methanesulfonate as an index. [Wei Y. F et al. (1995) J Bacteriol. 177: 5009-15.]

 The present invention also relates to a kit containing a compound that inhibits AlkB homologue gene expression or AlkB homologue protein activity, and an alkylating agent. Such a kit is used when administering an alkylich drug to a cell or a living body. A compound that inhibits the expression or activity of an AlkB homolog can be used in combination with an alkyl glaze to increase sensitivity to alkylating drugs. Each drug can be appropriately combined with a pharmaceutically acceptable carrier.

The present invention also relates to an alkylating agent sensitizer and an alkylating agent combination agent comprising a compound that inhibits the expression or activity of an AlkB homolog. These agents can be formulated, for example, as a pharmaceutical composition. The alkylating agent combination agent of the present invention is useful for enhancing the effect of the alkylating agent by using it together with the alkylating agent. In particular, it can be used in cancer treatment to enhance the anticancer effect of alkylating drugs. When formulating as a pharmaceutical composition, first, a compound that inhibits the expression or activity of the AlkB homolog is selected by the screening method of the present invention. The selected compound is then mixed with a pharmaceutically acceptable carrier. Examples of these pharmaceutically acceptable carriers include surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrating agents, Examples include lubricants, fluidity promoters, and flavoring agents, but are not limited thereto, and other commonly used carriers can be used as appropriate. In formulating the drug of the present invention, the above-mentioned carrier can be added as necessary according to a conventional method. Specific examples include light anhydrous dianhydride, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polybucetal jetylaminoacetate, polybulu Examples include pyrrolidone, gelatin, medium-chain fatty acid tridalylide, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts. Examples of the dosage forms of the above drugs include tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film coatings, pellets, sublinguals, and pastes as oral preparations. Examples of parenteral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and liquids for external use. Those skilled in the art should select the optimal dosage form according to the route of administration and the subject of administration. Can do. In addition, when vectors that express proteins or nucleic acids (such as siRNA or antisense) that inhibit the expression or activity of AlkB homologs are administered in vivo, viral vectors such as retroviruses, adenoviruses, and Sendai viruses, and liposomes Non-viral vectors can be used. Polymer carriers are known as non-viral vectors other than ribosomes. Polyethylene Daricool-Poly Power is used as a delivery system for nucleic acid drugs that exist stably in the body and can circulate stably in the bloodstream. Examples include polymeric nanomicelles formed by self-association of thione block copolymers and nucleic acids [Harada A. & Kataoka K. Science, 283, 65-67 (1999); Katayose S. & Kataoka K. Bioconjuga te Chemistry, 8 (5), 702-707 (1997)]. And this improved type, calcium phosphate micelles, an inorganic-organic composite nanostructure formed from polyethylene glycol-polyaspartate (PEG-PAsp) block copolymer and calcium phosphate [Kakiza a Y. & Kataoka K. Langmuir, 18 (2), 4539-4543 (2002)] and polymer nano micelles with improved blood stability by introducing disulfide bridges into the inner core [Kakizawa Y., Harada A. & Kataoka J. Amer. Chem. Soc., 121 (48), 11247-11248 (1999)], Furthermore, these derivatives are considered to be effective as siRNA and nucleic acid carriers. Other polymer carriers include pyoconjugate carriers [Asayama S. et al. Bioconjugate Chemistry, 9 (4), 476-481 (1998); Park JU. Et al. Prep. Biochem. & Biotechnol., 29 (4), 353-370 (1999)]. Examples of the administration method include an in vivo method and an ex vivo method. The dosage of the drug or pharmaceutical composition of the present invention should be determined appropriately according to the judgment of the doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, etc. Is possible. Brief Description of Drawings

 FIG. 1 is a diagram showing suppression of gene expression by ABH2 and ABH3 siRNA in HeLa cells. Total RNA was extracted from cells 48 hours after siRNA transfection and the expression of ABH2 and ABH3 genes was measured by semiquantitative RT-PCR. NS is a control rod. The expression of ABH2 and ABH3 genes is shown relative to NS.

 FIG. 2 shows suppression of gene expression by siRNA of ABH2 and ABH3 in A549 cells. Total RNA was extracted from cells 48 hours after transfection of siRNA, and the expression of ABH2 and ABH3 genes was measured by semiquantitative RT-PCR. NS is a control RNA. The expression of ABH2 and ABH3 genes is shown relative to NS.

 FIG. 3 is a graph showing the effect of ABH2 and ABH3 on the sensitivity to alkylating drugs by siRNA treatment. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention described in the present specification can easily be conceived by those skilled in the art, and these embodiments are included in the scope of the present invention. References cited in this specification are incorporated as part of this specification. EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. [Example 1]

 In this example, a method for increasing the sensitivity to an alkylating drug by suppressing the expression of the AlkB homologue gene in human cells is exemplified.

[Cell culture, transformation]

HeLa (human chick cervical cancer cells) and A549 (human lung cancer cells) were used as human cells. All human cells are 10% fetal bovine serum, and cultured at 37 ° C, 5% C0 ₂ under conditions D ulbecco 's modified Eagle' s medium containing 50 / zg / ml gentamicin. SiRNA was used to suppress the expression of the AlkB homologue gene. siRNA was synthesized at Fasmac Co., Ltd. Twenty-four hours prior to transfection of siRNA, cells were seeded on a 24-well plate and transfection was performed at 20-50% confluence. Transfection of siRNA (20 pmol each) into each cell was carried out according to the method using Oligofectoamine ™ (Invitrogen) and Lipofectoamine ™ 2000 (Invitrogen). As a control siRNA (NS), a double-stranded polynucleotide comprising uucu ccgaacgugucacgudTdT (ti column number: 19) and acgugacacguucggagaadTdT (laying [J 畨: 20) force was used.

[Quantification of mRNA]

 Total RNA was extracted from cells 48 hours after siRNA transfection using the R easy ™ Mini Kit (Qiagen). Quantitative PCR was performed using the ABI PRISM ™ 7000 Sequence Detection System (Applied Biosystems). ABH2 gene (SEQ ID NO: 3) (XM058581), ABH3 gene (SEQ ID NO: 5) (匪 _139178), β-actin gene RT-PCR primers and TaqMan ™ probe are from Applied Biosystems Purchased. The RT-PCR reaction was performed using TaqMan ™ One-Step RT-PCR Master Mix Reagents Kit (Applied Biosysytems) in accordance with the system. Quantitative comparison was performed using β-cutin as a standard.

[Alkylating agent sensitivity test] As alkylating agents, methyl methanesulfonate, cyclophosphamide, and carmuscithin were used. After 24 hours of siRNA transfection, each drug was added, and the cells after 24 hours were further measured with a living cell count reagent SF (Nacalai Tester).

The sequence of siRNA used in the experiment is shown below.

ABH2

 1. gage cagcuguguugggagdTdT (酉 己 歹! J 号 "^: 7)

 cucccaacacagcuggcucdTdT (酉 S column number "^: 8)

2. gaaagcacaaggaagaggcdTdT (酉 己 列 畨 "^: 9)

 gccucuuccuugugcuuucdTdT (SEQ ID NO: 10)

3. gauggcugugaccacaucgdTdT (G row number: 11)

 cgauguggucacagccaucdTdT (酉 己 歹! j number: 12)

ABH3

 1. gacuuacagcaugguauggdTdT (SEQ ID NO: 13)

 ccauaccaugcuguaagucdTdT (酉 己 歹 lj number: 14)

2. gagccaucuccaccagaagdTdT (Rating number: 15)

 cuucugguggagauggcucdTdT (酉 己 歹 幡 号 ： 16)

3. gaaccucagcagguaguacdTdT (SG column number: 17)

 guacuaccugcugagguucdTdT (酉 己 歹 U number: 18)

 [Result]

Cancer cells are sensitive to alkylating drugs when ABH2 and ABH3 gene expression is suppressed Experiments were conducted using siRNA against the ABH2 and ABH3 genes. First, ABH2 and ABH3 gene expression suppression effects were examined using ABH2 and ABH3 siRNAs using HeLa cells, which are human eclampsia cancer cells. As a result, the expression of all siRNAs against ABH2 and ABH3 described above was suppressed. Hereinafter, in ABH2 and ABH3, experiments were conducted using siRNAs consisting of thighs shown in SEQ ID NOs: 7, 8 and SEQ ID NOs: 13, 14, respectively. This siRNA suppressed gene expression to 4% for ABH2 and 2% for ABH3 (Figure 1). In the same way, A549 cells, human lung cancer cells, were treated with siRNA. ABH2 was 14 ° and ABH3 was 8 ° /. In addition, the gene expression was suppressed (Fig. 2). Suppression of ABH2 and ABH3 gene expression did not affect the growth and proliferation of HeLa and A549 cells.

We investigated the sensitivity of alkyl addictive drugs in ABLA2 and ABH3 siRNA-treated HeLa cells and A549 cells. Sensitivity was examined by measuring the number of living cells using methyl methanesulfonate (匪 S), which is known as a compound that efficiently methylates DNA and RNA. As a result, as shown in Fig. 3, when both ABH2 and ABH3 expression was suppressed, NS (double-stranded RNA used as control console showed inhibition of expression in both HeLa and A549 cells. It was confirmed that the sensitivity to MMS increased compared to

In addition to MMS, the effects on alkylating drugs used clinically as anticancer agents were investigated. Cyclophosphamide and carmustine were used as anticancer agents. IC ₅ . Table 1 shows the results of comparison by value. He S IC ₅ in HeLa cells. The value was about 1/3 of that of NS, and it was confirmed that cyclophosphamide carmuscitin also showed a lower IC ₅₀ value than NS. Similar results were obtained with A549 cells.

From the above results, it was proved that ABH2 and ABH3 are completely new targets for concomitant use with alkylich drugs, and siRNA of ABH2 and ABH3 are useful concomitant drugs with alkylating drugs. Sensitivity to various alkylating drugs

ABH2 ABH3 IC ₅₀ value (mM)

HeLa

 Methyl methanesulfonate 0.3 0.1 0.1

 Cyclophosphamide 17.2 14.3 10.8

 Cam Lecithin 0.5 0.3 0.4

A549

 Methyl methanesulfonate 0.8 0.4 0.3

 Camnolecithin 0.9 0.6 0.2

Industrial applicability

 The present invention makes it possible to increase the sensitivity of cells to alkylating drugs. Alkyl glaze is widely used in cancer treatment and the like, and the method of the present invention can enhance the effect of alkyl glaze in cancer treatment. Compounds that inhibit the expression or activity of mammalian AlkB homologues are extremely useful as alkylating agent combinations.

[References]

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Claims

The scope of the claims 1. A method for increasing the sensitivity of a mammalian cell to an alkylating agent, comprising a step of inhibiting expression of a mammalian AlkB homolog gene or activity of a protein encoded by the gene.  2. The method of claim 1, wherein the mammalian AlkB homologue gene is ABH2 or ABH3.  3. The method according to claim 1, wherein the expression of the AlkB homologue gene in mammals is damaged by RNAi.  4. The method of claim 1, wherein the mammalian cell is a cancer cell.  5. An alkylating agent combination agent comprising, as an active ingredient, a compound that inhibits the expression of a mammalian AlkB homologue gene or the activity of a protein encoded by the gene.  6. The alkylating agent combination agent according to claim 5, wherein the mammalian AlkB homolog gene is ABH2 or ABH3.  7. The alkylating drug combination agent according to claim 5, wherein the compound is siRNA or a vector that expresses siRNA.  8. A method of screening a candidate compound that increases the sensitivity of a mammalian cell to an alkylating agent comprising: (a) measuring the expression of a mammalian AlkB homolog gene or the activity of a protein encoded by the gene in the presence of a test compound;  (b) selecting a compound that inhibits the expression or activity relative to a control.  9. A method for producing an alkylating drug combination agent, comprising a step of mixing a compound selected by the method according to claim 8 with a pharmaceutically acceptable carrier.