JP2007000002A - Method for enhancing sensitivity to alkylating drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for enhancing sensitivity to alkylating drugs by which sensitivity of mammal cells to alkylating drugs is enhanced. <P>SOLUTION: The method for enhancing sensitivity to alkylating drugs comprises inhibiting the expression or activity of AlkB-homologue of mammals. An alkylating-drug concomitant agent comprises a compound inhibiting the expression or activity of the AlkB-homologue. A method for screening a compound enhancing the sensitivity to alkylating drugs comprises measuring the expression or activity of the AlkB-homologue. These enable enhancement of the effect of alkylating drugs on cancer treatment and the like. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

【００７１】[結果]
ABH2およびABH3遺伝子発現を抑制したときに、癌細胞がアルキル化薬に感受性を示すかどうか、ABH2およびABH3遺伝子に対するsiRNAを用いて実験を行った。まず、ヒト子宮頚部癌細胞であるHeLa細胞を用いてABH2、ABH3のsiRNAによりABH2とABH3の遺伝子発現抑制効果を調べた。その結果、上記に記載したABH2およびABH3に対する全てのsiRNAで発現が抑制された。以下、ABH2およびABH3において、それぞれ配列番号：7、8、および配列番号：13、14で示したRNAからなるsiRNAを用いて実験を行った。このsiRNAにより、ABH2は4%、ABH3は2%にまで遺伝子発現が抑制された (図1)。ヒト肺癌細胞であるA549細胞においても、同様にsiRNAを処理したところABH2は14%、ABH3は8%にまで遺伝子発現を抑制した (図2)。ABH2、ABH3遺伝子の発現を抑制しても、HeLa細胞およびA549細胞の生育増殖には影響はなかった。
【００７２】
ABH2、ABH3の siRNA処理したHeLa細胞およびA549細胞におけるアルキル化薬感受性を調べた。アルキル化薬として、DNAおよびRNAを効率的にメチル化する化合物として知られているメタンスルホン酸メチル (MMS)を用いて、生細胞数を測定する事により感受性を調べた。その結果、図3で示すようにABH2、ABH3の発現をそれぞれ抑制したときに、HeLa細胞およびA549細胞いずれにおいてもNS (コントロールとして用いた2本鎖RNAで発現抑制を示さない) と比較して、MMSに対して感受性が増す事が確認できた。
【００７３】
MMS以外に臨床で抗癌剤として使用されているアルキル化薬に対する影響を調べた。抗癌剤としてはシクロフォスファミド、カルムシチンを使用した。IC₅₀値で比較した結果を表1に示した。HeLa細胞において、MMSのIC₅₀値はNSと比較して、1/3程度の値を示し、シクロフォスファミドやカルムシチンにおいてもNSより低いIC₅₀値を示す事が確かめられた。また、A549細胞においても同様の結果を得た。
以上の結果より、ABH2、ABH3はアルキル化薬との併用剤としての全く新しいターゲットであり、ABH2、ABH3のsiRNAはアルキル化薬との有用な併用剤となる事が証明された。
【００７４】
【表１】 種々のアルキル化薬に対する感受性

【００７５】
【発明の効果】
本発明により、細胞のアルキル化薬に対する感受性を上昇させることが可能になった。アルキル化薬は癌治療等において広く用いられており、本発明の方法により、癌治療におけるアルキル化薬の効果を高めることが可能である。哺乳動物のAlkBホモログの発現または活性を阻害する化合物は、アルキル化薬併用剤として極めて有用である。
【００７６】
[参考文献]
1. Dinglay S, Trewick SC, Lindahl T, Sedgwick B.
Defective processing of methylated single-stranded DNA by E. coli AlkB mutants.
Genes Dev. 2000. 14(16):2097-105.
2. Dinglay S, Gold B, Sedgwick B.
Repair in Escherichia coli alkB mutants of abasic sites and 3-methyladenine residues in DNA.
Mutat Res. 1998. 407(2):109-16.
3. Wei YF, Carter KC, Wang RP, Shell BK.
Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.
Nucleic Acids Res. 1996. 24(5):931-37.
4. Wang G, Palejwala VA, Dunman PM, Aviv DH, Murphy HS, Rahman MS, Humayun MZ.
Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli.
Genetics. 1995. 41(3):813-23.
5. Wei YF, Chen BJ, Samson L.
Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes.
J Bacteriol. 1995. 177(17):5009-15.
6. Chen BJ, Carroll P, Samson L.
The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity.
J Bacteriol. 1994. 176(20):6255-61.
7. Volkert MR, Gately FH, Hajec LI.
Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents.
Mutat Res. 1989. 217(2):109-15.
8. Rebeck GW, Coons S, Carroll P, Samson L.
A second DNA methyltransferase repair enzyme in Escherichia coli.
Proc Natl Acad Sci U S A. 1988. 85(9):3039-43.
9. Volkert MR.
Adaptive response of Escherichia coli to alkylation damage.
Environ Mol Mutagen. 1988. 11(2):241-55.
10. Kondo H, Nakabeppu Y, Kataoka H, Kuhara S, Kawabata S, Sekiguchi M. Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA.
J Biol Chem. 1986. 261(33):15772-7.
11. Samson L, Derfler B, Waldstein EA.
Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.
Proc Natl Acad Sci U S A. 1986. 83(15):5607-10.
12. Teo I, Sedgwick B, Kilpatrick MW, McCarthy TV, Lindahl T.
The intracellular signal for induction of resistance to alkylating agents in E. coli.
Cell. 1986. 45(2):315-24.
13. Kataoka H, Sekiguchi M.
Molecular cloning and characterization of the alkB gene of Escherichia coli.
Mol Gen Genet. 1985. 198(2):263-9.
14. Kataoka H, Yamamoto Y, Sekiguchi M.
A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.
J Bacteriol. 1983. 153(3):1301-7.
【００７７】
【配列表】

【図面の簡単な説明】
【図１】 HeLa細胞におけるABH2およびABH3のsiRNAによる遺伝子発現抑制を示す図である。siRNAのトランスフェクション後48時間の細胞から全RNAを抽出し、半定量的RT-PCRでABH2およびABH3遺伝子の発現を測定した。NSはコントロールRNAである。ABH2およびABH3遺伝子の発現をNSに対する相対値で示した。
【図２】 A549細胞におけるABH2およびABH3のsiRNAによる遺伝子発現抑制を示す図である。siRNAのトランスフェクション後48時間の細胞から全RNAを抽出し、半定量的RT-PCRでABH2およびABH3遺伝子の発現を測定した。NSはコントロールRNAである。ABH2およびABH3遺伝子の発現をNSに対する相対値で示した。
【図３】 ABH2およびABH3のsiRNA処理によるアルキル化薬に対する感受性への影響を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to methods and agents for increasing sensitivity to alkylating drugs.
[0002]
[Prior art]
Alkylating drugs are abundantly present in the environment and are frequently used in general cancer treatment. Alkylating drugs are known to act directly on intracellular DNA and RNA, modify the base portion, and cause genomic mutations and translational abnormalities. As a result, it causes canceration, neurodegeneration, and aging in mammals. Also, as a completely different action, alkylating drugs kill cancer cells rapidly. For this reason, alkylating drugs are widely used as anticancer agents, but they have serious side effects, and alkylating drug resistant cancers are also known.
[0003]
Genomic DNA damaged by alkylating drugs is repaired by DNA glycosylase or alkyltransferase. In humans, MYH and OGG1 genes are known as DNA glycosylases and MGMT genes are known as alkyltransferases [Nakabeppu, Y. (2001) Prog Nucleic Acid Res Mol Biol. 68: 75- 94; Margison, GP and Santibanez-Koref, MF (2002) Bioessays. 24: 255-66; Gerson, SL (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 MGMT gene is highly expressed in cancer cells, and high expression in alkylating drug resistant cancer cells has been reported [Margison, GP and Santibanez-Koref, MF (2002) Bioessays. 24: 255-66; Gerson , SL (2002) J Clin Oncol. 20: 2388-99]. Thus, 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 in alkylating drug-resistant cancers [Margison, GP and Santibanez-Koref, MF (2002) Bioessays. 24: 255-66; Gerson, SL (2002) J Clin Oncol. 20: 2388-99]. In fact, O is a specific inhibitor of MGMT ⁶ -Benzylguanine is a concomitant drug with the alkylating drugs BCNU (N, N'-bis (2-chloroethyl) -N-nitrosourea) and temozolomide, 7 in Phase I clinical trials, 4 in Phase II, One case has been developed in Phase III [Gerson, SL (2002) J Clin Oncol. 20: 2388-99].
[0004]
[Non-Patent Document 1]
Nakabeppu Y. (2001) Regulation of intracellular localization of human MTH1, OGG1, and MYH proteins for repair of oxidative DNA damage.Prog Nucleic Acid Res Mol Biol. 68: 75-94.
[Non-Patent Document 2]
Margison GP, Santibanez-Koref MF. (2002) O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. Bioessays. 24: 255-66.
[Non-Patent Document 3]
Gerson SL. (2002) Clinical relevance of MGMT in the treatment of cancer.J Clin Oncol. 20: 2388-99.
[0005]
[Problems to be solved by the invention]
The present invention provides methods and agents for increasing sensitivity to alkylating drugs. The present invention also provides a method for screening candidate compounds that increase sensitivity to alkylating drugs.
[0006]
[Means for Solving the Problems]
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 Sci. 28: 2-5; Falnes, PO et al (2002) Nature. 419: 178-82; Trewick, SC et al. (2002) Nature. 419: 174-8]. AlkB removes methyl groups from 1-methyladenine and 3-methylcytosine in DNA and RNA in the presence of 2-oxoglutarate and divalent iron ions, An enzyme that repairs normal bases. AlkB homologues are widely distributed in many organisms from mammals to bacteria. Human homologues are known to have 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 Natl 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]. In the intracellular localization, ABH2 exists only in the nucleus, and ABH3 exists in both the nucleus and cytoplasm. [Duncan, T. et al. (2002) Proc Natl Acad Sci US A. 99: 16660-5 ]. Both ABH2 and ABH3 are expressed in cancer cells, and overexpression is considered to indicate resistance to alkylating drugs.
[0007]
From this point of view, the present inventors considered that mammalian alkB homologues containing ABH2 and ABH3 are very promising molecular targets as alkylating drug combinations. Therefore, we analyzed whether or not the alkB homolog is actually inhibited in mammalian cells to show sensitivity to alkylating drugs. As a result, it was found that by suppressing the expression of alkB homolog, the sensitivity of cells to alkylating drugs was significantly increased. That is, a compound that suppresses or inhibits the expression or activity of a mammalian alkB homolog is useful for increasing the sensitivity to an alkylating agent, and the compound can be treated by combining with an alkylating agent, for example, in the treatment of cancer. It can be used to increase the effect. 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.
[0008]
That is, the present invention relates to a method for increasing sensitivity to an alkylating drug, an alkylating drug concomitant drug that increases sensitivity to an alkylating drug, a screening method for candidate compounds that increase sensitivity to an alkylating drug, and the like. In terms of
(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 according to (1), wherein the mammalian AlkB homolog gene is ABH2 or ABH3,
(3) The method according to (1), wherein RNAi inhibits mammalian AlkB homolog gene expression,
(4) The method according to (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 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 drug combination agent according to (5), wherein the compound is siRNA or a vector that expresses siRNA,
(8) A method for screening a candidate compound that increases the sensitivity of a mammalian cell to an alkylating agent,
(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) The present invention relates to a method for producing an alkylating drug combination agent, which comprises a step of mixing a compound selected by the method described in (8) with a pharmaceutically acceptable carrier.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for increasing the sensitivity of a mammalian cell to an alkylating drug, which comprises a step of inhibiting the expression of a mammalian AlkB homolog gene or the activity of a protein encoded by the gene. The present inventors have succeeded for the first time in increasing sensitivity to alkylating drugs by targeting the mammalian AlkB homolog gene. Alkylating drugs generally have a strong anti-tumor effect and alkylate DNA and / or RNA to kill cancer cells. At this time, it is possible to enhance the effect of the alkylating agent by inhibiting the expression or activity of the AlkB homolog according to the present invention.
[0010]
In the present invention, the mammalian AlkB homolog gene (hereinafter also simply referred to as “AlkB homolog gene”) is 2-oxoglutarate (α-ketoglutarate) and a divalent iron ion [Fe (II)]-dependent oxygenase. A nucleic acid encoding a protein having an activity of removing a methyl group from 1-methyladenine and / or 3-methylcytosine in DNA and / or RNA. The mammalian AlkB homolog gene preferably complements the E. coli AlkB mutant. That is, when the gene is introduced into an Escherichia coli AlkB mutant, the sensitivity to alkylating drugs is reduced. Complementation is only required to be significant when compared with the E. coli AlkB mutant, and may be partial complementation even if it is not completely recovered (to the same as the wild type).
[0011]
The ABH gene family has been identified as a mammalian AlkB homolog gene. The 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 NM_139178 (protein: NP_631917)) are known to those skilled in the art for public information such as GenBank. Genbank accession XM_127049 (protein: XP_127049) for ABH mouse homologs, Genbank accession XM_222273 (protein: XP_222273) for rat homologues of ABH2, and Genbank accession for mouse homologues XM_132383 (protein: XP_132383) and AK079195 (protein: BAC37576), etc. ABH3 mouse homologs include Genbank accession XM_130317 (protein: XP_130317). As AlkB homolog gene, preferably, it includes ABH2 genes and ABH3 genes, and their counterparts.
[0012]
More specifically, mammalian AlkB homologous genes include human ABH (SEQ ID NO: 1), human ABH2 (SEQ ID NO: 3), and human ABH3 (SEQ ID NO: 5), as well as those in other organisms. Counterparts (including polymorphisms and variants) are included. In general, a mammalian gene may have a plurality of types of sequences even in 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. The mammalian AlkB homologue gene specifically includes the following proteins, which include 1-methyladenine and / or 3 in DNA and / or RNA in the presence of 2-oxoglutarate and divalent iron ions. -Genes encoding proteins with the activity of removing methyl groups from methylcytosine are included.
(A) a protein having 70% or more identity with a continuous 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 obtained by substituting, deleting, and / or adding 30% or less of amino acids in a continuous partial amino acid sequence of 100 or more residues 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 of mammals (including polymorphisms, variants that maintain function, etc.).
[0013]
The amino acid sequence identity can be calculated as the ratio of amino acids that match by aligning the entire 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 for Biothchnology). Information) can be used. The gap is handled in the same manner as the mismatched amino acid, and the identity is determined as the ratio of the number of amino acids that match in both sequences out of the total number of amino acids (including the gap) in the alignment. However, if a gap is inserted 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, 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. As described above, the full length of SEQ ID NO: 2, 4, or 6 is more preferable.
[0014]
For example, when amino acid sequence identity is determined by BLAST, it can be performed 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 blastx program is used to determine the identity of the amino acid sequence. For example, in the BLAST web page of NCBI (National Center for Biothchnology Information) Calculation is performed with all filter settings such as “complexity” turned OFF (Altschul, SF et al. (1993) Nature Genet. 3: 266-272; Madden, TL et al. (1996) Meth. Enzymol. 266: 131 Altschul, SF et al. (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. & Madden, TL (1997) Genome Res. 7: 649-656). For example, the open gap cost is 5 for nucleotides and 11 for proteins, the cost for extend gaps is 2 for nucleotides and 1 for proteins, the penalty for nucleotide mismatch is -3, the reward for nucleotide match is 1, and the expect value is 10, wordsize is 11 nucleotides, protein is 2, Dropoff (X) for blast extensions in bits is 20 for blastn, 7 for other programs, X dropoff value for gapped alignment (in bits) is 15, other than blastn, final X The dropoff value for gapped alignment (in bits) is 50 for blastn and 25 for other programs. In comparing amino acid sequences, BLOSUM62 can be used as a matrix for scoring. The blast2sequences program (Tatiana A et al. (1999) FEMS Microbiol Lett. 174: 247-250), which compares two sequences, can create alignments of two sequences and determine sequence identity.
[0015]
In the protein (b), the total number of amino acids that are substituted, deleted, and / or added in the continuous partial amino acid sequence is preferably within 27%, more preferably within 25%, more preferably 20%. %, More preferably within 18%, more preferably within 15%, more preferably within 12%, more preferably within 10% (however, fractions of less than 1 are rounded down in terms of amino acids). Substitutions, deletions, and additions may be combined arbitrarily.
[0016]
In the amino acid substitution, it is considered that a protein substituted with an amino acid having similar properties is likely to maintain 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), β-branched amino acids (eg, threonine, valine, isoleucine), and aromatic amino acids (eg, tyrosine, phenylalanine) , Tryptophan, histidine) and the like. 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.
[0017]
In hybridization with the base sequence of SEQ ID NO: 1, 3, or 5, the target sequence is hybridized using this base sequence or its complementary strand, or a part thereof as a probe, and a string is obtained. It is confirmed whether the probe is significantly hybridized to the target sequence after washing under a gentle condition. As the 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 complementary strands, hybridization is performed in the same manner using only that sequence as a probe as a negative control. What is necessary is just to confirm that the probe does not hybridize significantly to the target sequence after washing. Hybridization can be performed by a conventional method using a nitrocellulose membrane or a nylon membrane. If the above stringent conditions are specifically illustrated, for example, 6 × SSC, 0.5% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, 5 × Denhardt solution (1 × Denhardt solution is 0.2% polyvinyl) In a solution containing pyrrolidone, 0.2% bovine serum albumin, and 0.2% ficoll), hybridization is performed overnight at 42 ° C., preferably 50 ° C., more preferably 60 ° C., more preferably 65 ° C. Is a condition in which 4 × SSC, 0.5% SDS, and 20 minutes are washed three times at the same temperature as the hybridization. More preferably, the post-hybridization washing is performed under the same conditions as washing at 4 × SSC, 0.5% SDS, 20 minutes twice, 2 × SSC, 0.5% SDS, 20 minutes once at the same temperature as the hybridization. is there. More preferably, the conditions are such that washing after hybridization is performed twice at 4 × SSC, 0.5% SDS, 20 minutes, followed by 1 × SSC, 0.5% SDS, 20 minutes at the same temperature as hybridization. . More preferably, post-hybridization washing is performed at the same temperature as the hybridization, 2 × SSC, 0.5% SDS, 20 minutes once, followed by 1 × SSC, 0.5% SDS, 20 minutes once, then 0.5 × SSC, 0.5% SDS, 20 minutes once.
[0018]
When a gene is isolated from a desired mammal by hybridization, for example, the base sequence of the mammalian AlkB homolog gene exemplified in the present specification (for example, SEQ ID NO: 1, 3, or 5) or a complementary strand thereof, or Using a part of the probe as a probe, a cDNA library prepared from an 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. Preferred is mammalian genomic DNA or cDNA. Specific examples include desired mammals such as mice, rats, and goats, primates such as monkeys, and humans.
[0019]
The target AlkB homolog protein in the present invention has an activity of removing a methyl group from 1-methyladenine and / or 3-methylcytosine in DNA and / or RNA in the presence of 2-oxoglutarate and divalent iron ions. is doing. The methyl group removal activity can be assayed by the method described below.
[0020]
In cells expressing the above AlkB homolog gene, inhibition of the expression of this gene can increase the sensitivity of the cells to alkylating drugs. The alkylating agent used at this time is a compound that alkylates intracellular DNA or RNA to produce 1-methyladenine and / or 3-methylcytosine. Examples of such alkylating agents include SN such as methyl methanesulfonate (MMS). ₂ Many types of alkylating agents are known. In addition to MMS, cyclophosphamide and camlucitin can be exemplified. Furthermore, melphalan, chlorambucil, ifosfamide, busulfan, semustine, dacarbazine, nitromine, nimustine, ranimustine and the like used in clinical practice can be exemplified, but not limited thereto. The order of the step of administering the alkylating agent and the step of suppressing the expression or activity of the AlkB homolog is not particularly limited, and suppresses the expression or activity of the AlkB homolog before, simultaneously with, or after adding the alkylating agent to the cell. It's okay. The sensitivity of cells to alkylating drugs can be determined, for example, by measuring cell survival. For the measurement of cell viability, for example, MTT assay, or live cell count reagent SF (Nacalai Tesque) can be used.
[0021]
Suppression of the expression of the AlkB homolog gene may be suppression of the mRNA level and / or protein level of the gene. 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 contains a sense RNA that is homologous to a part of the mRNA sequence of the target gene and an antisense RNA that is complementary to the sense RNA. This refers to a phenomenon in which gene mRNA is induced to be destroyed and the expression of a target gene is inhibited (Genes Dev. 2001, 15: 188-200; Elbashir, SM et al. (2001) Nature 411: 494-498). When double-stranded RNA with RNAi effect is introduced into the cell, an enzyme called DICER (a member of the RNase III nuclease family) comes into contact with the double-stranded RNA, and the double-stranded RNA becomes a small interfering RNA (siRNA). ) Is broken down into small pieces called. In the present invention, in addition to the RNA generated by such intracellular processing, siRNA is also collectively referred to as an RNA molecule artificially synthesized or expressed in order to inhibit the expression of a target gene by RNAi. The RNA having an RNAi effect in the present invention includes these siRNAs. siRNA can suppress target gene expression in vivo (Anton P. et al., Nature Vol. 418 38-39 2002; David L. et al., Nature genetics Vol. 32 107-108, 2002) .
[0022]
The siRNA for a certain target gene is usually a sequence of 15 or more bases (more preferably a sequence of 16 or more, 17 or more, 18 or more, or 19 or more bases) in the transcription sequence (mRNA sequence) of this gene, And its complementary sequence, and these sequences hybridize to form a double strand. Preferably, a sequence of 19 to 30 bases, more preferably 20 to 25 bases, more preferably 21 to 23 bases or a complementary sequence thereof in one strand is included in one strand and forms a complementary pair therewith. RNA containing one 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 RNA corresponding to the full-length region or almost the full-length region of the target gene mRNA can be decomposed in advance with, for example, DICER, and the decomposition product can be used. This degradation product is expected to contain RNA molecules (siRNA) having the RNAi effect. According to this method, a region on mRNA that is expected to have an RNAi effect need not be selected. That is, the sequence having the RNAi effect of the target gene does not necessarily need to be precisely defined.
[0023]
Usually, a 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 desirably 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 (two thymines), UU (two uracils), and other double-stranded RNA having an overhang of bases (most preferably, a 19-pair double-stranded RNA portion and two bases) Molecules having (TT) overhangs) can be preferably used. The siRNA of the present invention includes a molecule in which the base that forms an overhang is DNA.
[0024]
Moreover, two strands forming a base pair in siRNA may be linked via a spacer. That is, RNA in which this spacer forms a loop and the RNA sequences before and after it 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.
[0025]
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 RNA 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 and the other strand of a double-stranded siRNA are expressed separately. Alternatively, two types of RNA 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 (forming a loop). RNA expressed from this vector forms an RNA stem with an RNAi effect and suppresses the expression of the target gene. The length of the stem is the same as that of the above siRNA, but may be, for example, 19 to 29 bases. The length of the spacer is not limited, but may be 3 to 23 bases, for example. It may or may not have an overhang of several bases at 5 ′ and / or 3 ′. These vectors can be easily produced by those skilled in the art using common genetic engineering techniques (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison 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 Natl 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, an RNA polymerase III promoter can be preferably used. Specifically, for example, U6 Pol III promoter and H1 RNA promoter (H1 RNA is one component constituting RNaseP) can be used.
[0026]
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 find the AA sequence and select 17-20 nucleotides following the AA (eg 19 nucleotides following AA). The base next to AA is not particularly limited, but 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%, and more preferably 35 to 65%. 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 preferable to search a public gene sequence database using a selected sequence as a query, and to confirm that there is no gene having the same sequence as a transcription sequence other than the target gene in the gene of the administered individual. The sequence is preferably selected from the protein coding sequence (CDS) of the target gene. A sequence containing a sequence excluding the first AA of the sequence thus selected (preferably having UU or TT added to 3 ′) and its complementary sequence (preferably having UU or TT at 3 ′ ) Is a suitable siRNA. Instead of searching for the sequence following AA, the sequence following CA may be searched in the same manner as described above. Alternatively, it may be a sequence other than AA or CA. It is also possible to appropriately select RNA having an optimal RNAi effect from a plurality of siRNAs prepared. Specific examples of suitable siRNAs include the RNAs shown in the examples. That is, siRNA containing the 1-19th sequence of SEQ ID NO: 7 and 8, siRNA containing the 1-19th sequence of SEQ ID NO: 9 and 10, and the 1-19th sequence of SEQ ID NO: 11 and 12 The siRNA to be contained (a base such as TT or UU may be added 3 ′ of each RNA) is preferably used. Similarly, siRNA containing the 1st to 19th sequences in each pair of SEQ ID NOs: 13 and 14, 15 and 16, 17 and 18 (bases such as TT or UU may be added to 3 ′ of each RNA ) Is preferably used.
[0027]
Those skilled in the art can appropriately prepare RNA having an RNAi effect based on the base sequence of the target gene. The base sequence of the target AlkB homolog gene can be easily obtained from a public gene database as described above, and the base sequence of the human AlkB homolog gene (SEQ ID NO: 1, 3, or 5) is used as a probe. It can be obtained by screening a mammalian cDNA library 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 description.
[0028]
The target gene sequence need not necessarily have a known full-length nucleotide sequence. 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. Is possible. The accession numbers of the EST sequences that showed high homology with human ABH2 and ABH3 in the GenBank database are listed below. However, these are only 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, the expression of the AlkB homolog gene may be inhibited using RNA having an RNAi effect in which a part of a continuous RNA region is one strand.
[0029]
(a) ABH2
BU857344, BM713799, BU621636, CB306784, BU535633, BU858046, CA425782, BU621592, AL528750, BU539036, BF812735, AW245078, AW246209, BI822466, BM790974, BM852409, AI201794, BE222844, AI078333, AI3747692, BI488628, AA602382A, 488628, AA602382A, 488628, AA602382A AI885651, AW206726, AW005870, AA782579, BG831183, BU542852, BG057635, AW971683, BE253917, AW952528, AI040632, H09745, AI124006, BM701520, AI138654, BQ013942, BE742854, AI253156, W73383, AI198796, BE02631485, AI198796, AA9314359W CB115208, BQ227530, AI278638, BF683378, BF591258, AI421482, AI417338, AI073924, BF183208, BM799140, AI675781, BU623121, BQ014087, AW071763, AW001162, AA550952, BE894785, D60111, BF751508, CB115574, BF6887791BI BM785502, BI666861, BI861994, BM546765, AW952001, BI767047, BF766222, BG751427, AI269764, BG284978, BE255140, CB118993, BI045022, AW005663, AI589576, AI284851, CB115220 AA333965, AI784306, AA911501, N92173, AI183736, AA865002, AW169802, BE857214
[0030]
(b) ABH3
BI860137, BX406644, AL570007, AL570007, BE250119, BE249876, AL571973, AL551946, BG283085, BF724816, BM824362, BM792655, BE293567, BG282971, BE789024, BE293665, BM545966, BF037645, BQ272207, AL543545, BM561898, AL551979, BX3906092 BM981193, BM985113, AL554302, BX406645, BM999346, CB048424, BI918447, BM694348, BU602469, AL577048, BQ939674, BM704858, BE408144, BU179848, BI258292, BQ233246, BG772137, BM548608, BE274419, BU553708, BI823026, BU1908356AA BE792222, CA445168, BU730492, BQ017150, BM971930, BM681387, BE891036, BE892721, BF062547, BE791860, BQ181579, BM011189, BU788104, BI222582, BF026256, BI908985, BE887345, AI636076, BU784435, BI561482, BG831972B, 933482, BG831972B BE296128, BF886167, BX361668, BQ375283, BX089006, BF823012, BE937862, BG823846, BQ375279, BI833740, AI916587, AI656885, BQ576341, BG187465, AW001947, BG191648 BG825625, BG370227, AA932508, AW451472, AI370748, AW247495, BM675933, BF763881, BE269759, AW860831
[0031]
When the synthesized siRNA is used clinically, the siRNA can be modified. For example, phosphorothioate type RNA can be used to reduce susceptibility to nuclease degradation and sustain the effect. It has been reported that substitution of siRNA phosphates with phosphorothioates does not change the RNAi effect and increases stability. It is also known that siRNA uridine and cytidine are converted to 2'-fluoro-2'-deoxyuridine and 2'-fluoro-2'-deoxycytidin, respectively. [Harborth J. et al. (2003) Nucleic Acid Drug Dev. 13: 83-105]. Therefore, siRNAs composed of such RNA analogs are suitable for clinical use. The siRNA of the present invention includes analogs of such RNA.
[0032]
In order to suppress the expression of the AlkB homolog gene, for example, the following nucleic acid (a) or (b) can be used.
(A) An antisense nucleic acid for a transcription product of AlkB homolog gene or a part thereof
(B) A 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. There are several factors as described below for the action of the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing, translation initiation inhibition by hybridization with the translation initiation factor binding site, translation inhibition by hybridization with the ribosome binding site in the vicinity of the initiation codon, peptation by hybridization with the mRNA translation region and polysome binding site Elongation inhibition of de chain, and gene expression inhibition by hybrid formation with 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 (Hirashima and Inoue, Shinsei Kagaku Kogaku 2 Nucleic acid IV gene replication and expression, Japan Biochemical Society, Tokyo Chemical Doujin, 1993, p.319-347).
[0033]
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 or more nucleotides, preferably 14 or more nucleotides, and more preferably 15 or more nucleotides of a transcribed sequence of the AlkB homolog gene. For example, exon-intron boundary in initial transcription sequence, intron-exon boundary, region including translation initiation codon, untranslated region near 5 ′ end, or protein coding sequence (CDS) in mature mRNA of 13 or more consecutive nucleotides A nucleic acid containing an antisense sequence for 14 nucleotides or more, more preferably 15 nucleotides or more is preferred. 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, in order to reduce the sensitivity to nuclease degradation and maintain the activity as an antisense nucleic acid, use of S oligo (phosphorothioate-type oligonucleotide) in which O (oxygen) at the phosphate ester binding site is replaced with S (sulfur) Is generally known. This S oligo is currently undergoing 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 expression of the gene can be effectively suppressed. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. 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, more Preferably it is 30 bases or more, More preferably, it is 40 bases or more, More preferably, it is 50 bases or more, More preferably, it is 100 bases or more. Antisense RNA can also be expressed intracellularly. What is necessary is just to produce the vector which linked the nucleic acid which codes the target RNA downstream of the promoter active in a target cell, and just introduce | transduces this into a cell.
[0034]
As the vector, a viral vector such as a retrovirus vector, an adenovirus vector, and an adeno-associated virus vector, a non-viral vector such as a liposome, and the like can be used. Polymer carriers are known as non-viral vectors other than liposomes. Polyethylene glycol-polycation block co-polymers are used as delivery systems for nucleic acid drugs that exist stably in vivo and can circulate stably in the bloodstream. Examples include polymer nano micelles formed by self-association of nucleic acids and nucleic acids [Harada A. & Kataoka K. Science, 283, 65-67 (1999); Katayose S. & Kataoka K. Bioconjugate Chemistry, 8 (5) , 702-707 (1997)]. And this improved type, calcium phosphate micelle, an inorganic-organic composite nanostructure formed from polyethylene glycol-polyaspartic acid (PEG-PAsp) block copolymer and calcium phosphate [Kakizawa 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 KJ Amer. Chem. Soc., 121 (48), 11247-11248 (1999)], and these derivatives are considered to be effective as siRNA and nucleic acid carriers. Other polymer carriers include bioconjugate carriers [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.
[0035]
Moreover, inhibition of the expression of the AlkB homolog gene can also be carried out using a ribozyme or a vector encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. There are ribozymes having 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 group I intron type and M1 RNA contained in RNase P, but 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) have some active domains of about 40 nucleotides (Makoto Koizumi and Otsuka Eiko, protein nucleic acid enzyme, 1990, 35, 2191).
[0036]
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, and instead of C15, A15 or U15 However, it has been shown that it can be cut (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site, a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA can be created (Koizumi, M. et al. FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059.) .
[0037]
Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving 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 transcript of the target gene using a ribozyme.
[0038]
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 sensitivity of cells to alkylating drugs by inhibiting (reducing) 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
[0039]
Antibodies that bind to the AlkB homolog protein can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A small animal such as a rabbit is immunized with a natural or recombinant AlkB homolog protein, or a recombinant AlkB homolog protein expressed in a microorganism such as Escherichia coli as a fusion protein with GST, or a partial peptide thereof. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, an affinity column coupled with AlkB homolog protein or synthetic peptide, or the like. In the case of a monoclonal antibody, for example, an AlkB homolog protein or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate the cells, and the cells and mouse myeloma cells. Are fused using a reagent such as polyethylene glycol, and a clone producing an antibody that binds to the AlkB homolog protein is selected from the resulting fused cells (hybridoma). Next, the obtained hybridoma was transplanted into the abdominal cavity of the mouse, and ascites was collected from the mouse, and the obtained monoclonal antibody was obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, AlkB homolog protein. Or an affinity column coupled with a partial peptide thereof or the like. 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 a human, and particularly preferably a protein derived from a human.
[0040]
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 antibody modifications.
[0041]
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 protein partial peptide include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. In the present specification, the “antibody” generally means an antibody that reacts with the full length or fragment of a protein.
[0042]
In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having protein-binding activity. When the antibody of the present invention is used for the purpose of administering it to the human body (antibody therapy), a human antibody or a human type antibody is preferable in order to reduce immunogenicity.
[0043]
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, 1-methyladenine analogs and 3-methylcytosine analogs that interact with AlkB homologs but are not degraded can be suitably used. The 2-oxoglutarate analogs N-oxalylglycine, N-oxalyl-4S-alanine, N-oxalyl-4R-alanine, 2-thiono (N-oxalylglycine), and 2-mercaptoglutarate are known as inhibitors of AlkB. (Welford RW et al. (2003) J Biol Chem. 278: 10157-61). It is also conceivable to inhibit the activity of the AlkB homolog protein using these compounds and other 2-oxoglutaric acid analogs. In addition, for example, single compounds such as organic compounds, inorganic compounds, nucleic acids, proteins, peptides, sugars, or compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermentation microorganisms It may be a product, a marine organism extract, a plant extract or the like or a compound isolated and purified from the extract.
[0044]
The present invention also provides a screening method for candidate compounds that increase the sensitivity of mammalian cells to alkylating drugs. One embodiment thereof is a method using the binding between the test compound and an AlkB homolog protein or a partial peptide thereof as an index. Usually, a compound that binds to an AlkB homolog protein or a partial peptide thereof is expected to have an effect of inhibiting the function of the AlkB homolog protein.
[0045]
In this method, first, a test compound is contacted with a protein encoded by an AlkB homolog gene or a partial peptide thereof. The AlkB homolog protein or a partial peptide thereof is, for example, a purified form of the AlkB homolog protein or a partial peptide thereof, a form expressed intracellularly or extracellularly, depending on an indicator for detecting binding to a test compound, Alternatively, it may be in a form bound to an affinity column or other support. The test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label. Next, 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 AlkB homolog protein or its partial peptide and the test compound can be detected, for example, by a label added to the test compound bound to the AlkB 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 AlkB homolog protein expressed in or outside the cell or a partial peptide thereof as an indicator. Next, a test compound that binds to the protein encoded by the AlkB homolog gene or a partial peptide thereof is selected.
[0046]
There is no restriction | limiting in particular as a test compound used for a screening. For example, natural compounds, organic compounds, inorganic compounds, single compounds such as nucleic acids, proteins, peptides, sugars, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products , Marine organism extracts, plant extracts, human engineered compounds and the like, but are not limited thereto.
[0047]
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 removes 1-methyladenine and / or 3-methylcytosine contained in the substrate using DNA and / or RNA as a substrate. Therefore, the AlkB homolog protein is contacted with the substrate in the presence of the test compound, and the binding between the two is detected. A compound that suppresses the binding between the AlkB homolog protein and the substrate as compared to the absence of the test compound or the presence of a low dose (control) is selected. Such a compound becomes a compound that inhibits the activity of the AlkB homolog protein, and can be used as a drug that increases the sensitivity to an alkylating drug.
[0048]
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 contacted with a cell or cell extract expressing an AlkB homolog gene. 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), U2OS (osteosarcoma), C33A (cervical cancer), HT1080 (fibrosarcoma), PA-1 (ovarian teratocarcinoma), Tera2 (Fetal cancer), T24 (bladder cancer), K562 (chronic myeloid leukemia), Molt4 (acute lymphoblastic leukemia), A172 (glioblastoma), various tumor cells such as HeLa (cervical cancer) , And desired normal cells.
[0049]
The contact of the test compound with the cell expressing the AlkB homolog gene is usually performed by adding the test compound to the culture medium of the cell expressing the AlkB homolog gene, but is not limited to this method. When the test compound is a protein or the like, it can be brought into contact with the cell by introducing a DNA vector expressing the protein into the cell.
[0050]
Next, in this method, the expression of the AlkB homolog gene is measured. Here, the expression of a gene 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 Northern hybridization or RT-PCR. When measuring the translation level of a gene by detecting the protein, collect the protein fraction from the cell expressing the AlkB homolog gene and detect the expression of the AlkB homolog protein by electrophoresis such as SDS-PAGE. Can do. Further, 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.
[0051]
In this method, a compound that reduces the expression level is further selected as compared with the case where it is measured in the absence of a 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 alkylating 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 an alkylated anticancer agent (anticancer agent), for example.
[0052]
In yet another embodiment of the method of the present invention, a compound that decreases the expression level of the AlkB homolog gene of the present invention can be screened using a reporter gene. Specifically, such a screening method includes (a) a step of measuring the activity of an endogenous promoter of a mammalian AlkB homolog gene in the presence of a test compound, and (b) a decrease in the activity compared to a control. Selecting a compound to be produced. For this purpose, the test compound is first brought into contact 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, the term “functionally linked” means that the transcriptional regulatory region of the AlkB homolog gene and the reporter gene are induced so that expression of the reporter gene is induced by binding of a transcription factor to the transcriptional regulatory region of the AlkB homologous gene. Is 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 homolog gene, those skilled in the art can obtain the transcriptional regulatory region of the AlkB homolog gene present in the genome by a well-known method. In addition, if a knock-in animal or knock-in cell in which the coding region of the AlkB homolog gene is replaced with that of the reporter gene is prepared, the reporter gene can be expressed according to the promoter activity of the AlkB homolog gene, and screening is performed using this. It is also possible to perform. The expression level of the reporter gene is measured in the presence of the test compound, and a compound that reduces the expression level as compared with the case where it is measured in the absence of the test compound or in the presence of a low dose (control) is selected. The compound thus selected becomes a candidate compound for the alkylating agent combination.
[0053]
The reporter 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. 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 was introduced. Examples thereof include cells. Those skilled in the art can produce the above-mentioned vector by a general genetic engineering technique. Introduction of the vector into the cells can be carried out by a general method, for example, calcium phosphate precipitation method, electric pulse perforation method, lipophetamine method, microinjection method 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. DNA can be integrated into a chromosome by a method generally used by those skilled in the art, for example, a gene introduction method using homologous recombination.
[0054]
When screening 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, cells contained in a commercially available in vitro transcription translation kit An 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 to each other can be used.
[0055]
In screening, a test compound can be added by adding to a cell or cell extract, but 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 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 the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.
[0056]
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, or a cell or a cell extract expressing the protein. Next, the activity of the protein is measured. A compound that reduces the activity of the AlkB homolog protein is then selected as compared to when 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 in cells, or may be a protein expressed by introducing an exogenous AlkB homolog gene. A cell expressing 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 general genetic engineering techniques. The AlkB homolog protein used for this screening is preferably a full-length protein that does not contain mutations, but a protein in which a part of the amino acid sequence is substituted and / or deleted as long as it has an activity equivalent to that protein. It may be. Further, it may be a fusion protein with another protein.
[0057]
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. To measure this activity, for example, N- [ ^Three DNA and RNA are modified with a radiolabeled methyl group using H] methyl-N-nitrosourea or the like. AlkB homologue protein is reacted with this substrate, DNA and RNA are ethanol precipitated, and radioactivity is measured with a liquid scintillation counter [Aas PA et al. (2003) Nature. 421: 859-63.], Or vice versa Activity can be quantified by analysis by high performance liquid chromatography using a phase column [Falnes PO et al. (2002) Nature. 419: 178-82., Trewick SC (2002) Nature. 419: 174- 8.].
[0058]
Alternatively, AlkB homolog protein is reacted with DNA and RNA treated with an alkylating agent such as methyl sulfonate as a substrate. The reacted DNA and RNA are heat-treated at 90 ° C to 180 ° C under acidic conditions to release 1-methyladenine and 3-methylcytosine and analyze by high performance liquid chromatography using a reverse phase column or ion exchange column May be assayed. [Falnes PO et al. (2002) Nature. 419: 178-82., Trewick SC (2002) Nature. 419: 174-8.]
[0059]
Alternatively, DNA and RNA treated with an alkylating agent such as methyl sulfonate and RNA are reacted with AlkB homolog protein, and the consumed oxygen is measured with an oxygen electrode [Trewick SC (2002) Nature. 419: 174 -8.], A method in which AlkB homologue protein is reacted with DNA or RNA treated with an alkylating agent such as methyl methylsulfonate as a substrate, formaldehyde produced is reacted with a Nash reagent, and fluorescence is measured [Falnes PO et al. (2002) Nature. 419: 178-82.], produced by reacting AlkB homolog protein with 2-oxoglutaric acid radiolabeled with DNA or RNA treated with an alkylating agent such as methyl methylsulfonate. The method of measuring radiolabeled carbon dioxide with a liquid scintillation counter [Welford RW et al. (2003) J Biol Chem. 278: 10157-61.] it can.
[0060]
Further, the activity of the AlkB homolog protein includes a function of complementing an Escherichia coli AlkB mutant. Escherichia coli alkB mutants are sensitive to alkylating agents (methyl methanesulfonate) at low concentrations. By transforming this Escherichia coli with the AlkB homolog gene, resistance to the alkylating agent is restored. Complementary genes can be screened using resistance to methyl methanesulfonate as an indicator. [Wei Y. F et al. (1995) J Bacteriol. 177: 5009-15.]
[0061]
The present invention also relates to a kit comprising a compound that inhibits the expression of an AlkB homolog gene or the activity of an AlkB homolog protein, and an alkylating agent. Such a kit is used when an alkylating agent is administered to a cell or a living body. Sensitivity to an alkylating agent can be increased by using a compound that inhibits the expression or activity of an AlkB homologue in combination with the alkylating agent. Each drug can be appropriately combined with a pharmaceutically acceptable carrier.
[0062]
The present invention also relates to an alkylating agent sensitizer and a concomitant alkylating agent comprising a compound that inhibits the expression or activity of an AlkB homolog. These agents can be formulated as pharmaceutical compositions, for example. 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 to enhance the anticancer effect of alkylating drugs in cancer treatment. When formulated 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, buffering agents, suspending agents, isotonic agents, binders, disintegrating agents, lubricants. Agents, fluidity promoters, flavoring agents, and the like, but are not limited to these, and other commonly used carriers can be used as appropriate.
[0063]
In formulating the drug of the present invention, the above-mentioned carrier can be added as necessary according to a conventional method. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.
[0064]
Examples of the dosage form of the drug include tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coating agents, pellets, sublingual agents, pastes, etc. Examples of parenteral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and liquids for external use. Those skilled in the art can select the optimal dosage form according to the route of administration and the administration target. . In addition, when a vector that expresses a protein or nucleic acid (such as siRNA or antisense) that inhibits the expression or activity of an AlkB homolog is administered in vivo, a viral vector such as a retrovirus, adenovirus, or Sendai virus, or a liposome Non-viral vectors can be used. Polymer carriers are known as non-viral vectors other than liposomes. Polyethylene glycol-polycation block co-polymers are used as delivery systems for nucleic acid drugs that exist stably in vivo and can circulate stably in the bloodstream. Examples include polymer nano micelles formed by self-association of nucleic acids and nucleic acids [Harada A. & Kataoka K. Science, 283, 65-67 (1999); Katayose S. & Kataoka K. Bioconjugate Chemistry, 8 (5) , 702-707 (1997)]. And this improved type, calcium phosphate micelle, an inorganic-organic composite nanostructure formed from polyethylene glycol-polyaspartic acid (PEG-PAsp) block copolymer and calcium phosphate [Kakizawa 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 KJ Amer. Chem. Soc., 121 (48), 11247-11248 (1999)], and these derivatives are considered to be effective as siRNA and nucleic acid carriers. Other polymer carriers include bioconjugate 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 can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, etc. .
[0065]
【Example】
The invention described in the present specification can be easily devised by those skilled in the art, but such embodiments are included in the scope of the present invention. References cited in this specification are incorporated as a part of this specification. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
[0066]
[Example 1]
In this example, a method for increasing the sensitivity to an alkylating drug by suppressing the expression of the AlkB homolog gene in human cells is exemplified.
[0067]
[Cell culture, transfer cushion]
As human cells, HeLa (human cervical cancer cells) and A549 (human lung cancer cells) were used. All human cells are 10% fetal bovine serum, Dulbecco's modified Eagle's medium containing 50 μg / ml gentamicin, 37 ° C, 5% CO ₂ Cultured under conditions. SiRNA was used to suppress the expression of the AlkB homolog gene. The synthesis of siRNA was performed at Fasmac Co., Ltd. Twenty-four hours prior to siRNA transfer cushion, cells were seeded in a 24-well plate and transfer cushioned in a 20-50% confluent state. The transfer cushion of siRNA (20 pmol each) to each cell was performed according to the manual using Oligofectoamine (Invitrogen) and Lipofectoamine2000 (Invitrogen). As a control siRNA (NS), a double-stranded polynucleotide comprising uucuccgaacgugucacgudTdT (SEQ ID NO: 19) and acgugacacguucggagaadTdT (SEQ ID NO: 20) was used.
[0068]
[Quantification of mRNA]
Total RNA was extracted from cells 48 hours after siRNA transfer cushion using RNeasy Mini Kit (Qiagen). Quantitative PCR was performed using ABI PRISM 7000 Sequence Detection System (Applied Biosystems). ABH2 gene (SEQ ID NO: 3) (XM — 058581), ABH3 gene (SEQ ID NO: 5) (NM — 139178), RT-PCR primers for β-actin gene, and TaqMan probe were purchased from Applied Biosystems. RT-PCR reaction was performed using TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosysytems) according to the manual. Quantitative comparison was performed using β-actin as a standard.
[0069]
[Alkylating agent sensitivity test]
As alkylating agents, methyl methanesulfonate, cyclophosphamide, and carmustine were used. Each drug was added 24 hours after siRNA transfer cushion, and the cells after 24 hours were further measured with a living cell count reagent SF (Nacalai Tesque).
[0070]
The sequence of siRNA used in the experiment is shown below.

[Result]
An experiment was conducted using siRNA against ABH2 and ABH3 genes to determine whether cancer cells are sensitive to alkylating drugs when the expression of ABH2 and ABH3 genes is suppressed. First, ABH2 and ABH3 gene expression suppression effects were examined using ABH2 and ABH3 siRNAs using HeLa cells, which are human cervical cancer cells. As a result, expression was suppressed in all siRNAs against ABH2 and ABH3 described above. Hereinafter, in ABH2 and ABH3, experiments were performed using siRNAs composed of RNAs represented by 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 (FIG. 1). In A549 cells, which are human lung cancer cells, when siRNA was similarly treated, ABH2 suppressed gene expression to 14% and ABH3 to 8% (FIG. 2). Suppressing the expression of ABH2 and ABH3 genes did not affect the growth and proliferation of HeLa cells and A549 cells.
[0072]
ABH2 and ABH3 siRNA-treated HeLa cells and A549 cells were examined for their sensitivity to alkylating drugs. Sensitivity was examined by measuring the number of viable cells using methyl methanesulfonate (MMS), which is known as a compound that efficiently methylates DNA and RNA as an alkylating agent. As a result, as shown in Fig. 3, when ABH2 and ABH3 expression was suppressed, compared to NS in both HeLa cells and A549 cells (the double-stranded RNA used as a control does not show expression suppression) It was confirmed that the sensitivity to MMS increased.
[0073]
In addition to MMS, the effects on alkylating drugs used clinically as anticancer agents were investigated. Cyclophosphamide and carmuscitin were used as anticancer agents. I c ₅₀ The results of comparison by value are shown in Table 1. MMS IC in HeLa cells ₅₀ The value is about 1/3 of that of NS, and cyclophosphamide and carmuscitin have lower IC than NS. ₅₀ The value was confirmed. Similar results were obtained with A549 cells.
From the above results, it was proved that ABH2 and ABH3 are completely new targets as concomitant drugs with alkylating drugs, and siRNAs of ABH2 and ABH3 are useful concomitant drugs with alkylating drugs.
[0074]
Table 1 Sensitivity to various alkylating drugs

[0075]
【The invention's effect】
The present invention has made it possible to increase the sensitivity of cells to alkylating drugs. Alkylating drugs are widely used in cancer treatment and the like, and the method of the present invention can enhance the effect of alkylating drugs in cancer treatment. Compounds that inhibit the expression or activity of mammalian AlkB homologs are extremely useful as alkylating agent combinations.
[0076]
[References]
1. Dinglay S, Trewick SC, Lindahl T, Sedgwick B.
Defective processing of methylated single-stranded DNA by E. coli AlkB mutants.
Genes Dev. 2000. 14 (16): 2097-105.
2. Dinglay S, Gold B, Sedgwick B.
Repair in Escherichia coli alkB mutants of abasic sites and 3-methyladenine residues in DNA.
Mutat Res. 1998. 407 (2): 109-16.
3. Wei YF, Carter KC, Wang RP, Shell BK.
Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.
Nucleic Acids Res. 1996. 24 (5): 931-37.
4.Wang G, Palejwala VA, Dunman PM, Aviv DH, Murphy HS, Rahman MS, Humayun MZ.
Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli.
Genetics. 1995. 41 (3): 813-23.
5. Wei YF, Chen BJ, Samson L.
Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes.
J Bacteriol. 1995. 177 (17): 5009-15.
6. Chen BJ, Carroll P, Samson L.
The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity.
J Bacteriol. 1994. 176 (20): 6255-61.
7. Volkert MR, Gately FH, Hajec LI.
Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents.
Mutat Res. 1989. 217 (2): 109-15.
8. Rebeck GW, Coons S, Carroll P, Samson L.
A second DNA methyltransferase repair enzyme in Escherichia coli.
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Adaptive response of Escherichia coli to alkylation damage.
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10.Kondo H, Nakabeppu Y, Kataoka H, Kuhara S, Kawabata S, Sekiguchi M. Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA.
J Biol Chem. 1986. 261 (33): 15772-7.
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Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.
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12. Teo I, Sedgwick B, Kilpatrick MW, McCarthy TV, Lindahl T.
The intracellular signal for induction of resistance to alkylating agents in E. coli.
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Molecular cloning and characterization of the alkB gene of Escherichia coli.
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[0077]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows suppression of gene expression by siRNA of ABH2 and ABH3 in HeLa cells. Total RNA was extracted from cells 48 hours after transfection of siRNA, and expression of ABH2 and ABH3 genes was measured by semi-quantitative RT-PCR. NS is a control RNA. 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 expression of ABH2 and ABH3 genes was measured by semi-quantitative RT-PCR. NS is a control RNA. The expression of ABH2 and ABH3 genes is shown relative to NS.
FIG. 3 shows the effect of ABH2 and ABH3 treatment on sensitivity to alkylating drugs by siRNA treatment.

Claims (9)

  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.   The method according to claim 1, wherein the mammalian AlkB homolog gene is ABH2 or ABH3.   The method according to claim 1, wherein the expression of a mammalian AlkB homolog gene is inhibited by RNAi.   The method of claim 1, wherein the mammalian cell is a cancer cell.   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.   The alkylating agent combination agent according to claim 5, wherein the mammalian AlkB homolog gene is ABH2 or ABH3.   The alkylating agent combination agent according to claim 5, wherein the compound is siRNA or a vector expressing siRNA. A method for 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.   A method for producing an alkylating agent combination comprising a step of mixing a compound selected by the method according to claim 8 with a pharmaceutically acceptable carrier.

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