JP2003284599A - METHOD FOR EVALUATING Ah RECEPTOR ACTIVITY CONTROL ABILITY - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of evaluating Ah receptor activity control ability of a sample material with a small amount of the sample at a high sensitivity. <P>SOLUTION: The method for evaluating Ah receptor activity control ability comprises (1) a step for (a) realizing Ah receptor gene and (b) simultaneously or continuously contacting the sample and a protein synthesizing inhibitor with a cell containing a reporter gene having, at the downstream of a dioxin responsive sequence and a base sequence required for transcription initiation, a base sequence coding an inherently exogenous reporter protein of the dioxin responsive sequence, (2) a step for measuring translation product of the reporter gene of the cell contacted with the sample and the protein synthesizing material and (3) a step for evaluating Ah receptor activity control ability of the material based on the translation product measured in the step (2). <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for evaluating the ability to regulate aryl hydrocarbon receptor (Ah receptor) activity.

[0002]

2. Description of the Related Art Generally, polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) are collectively referred to as dioxins, and three types of compounds obtained by adding coplanar polychlorinated biphenyls (PCB) to them are dioxins. Are collectively called "like substances". Many of these dioxin-like substances have many structural isomers and homologues depending on the number and position of substituted chlorine, but some of them cause reproductive abnormalities, immunological abnormalities, carcinogenesis in vivo even in a very small amount. Therefore, attempts have been made to measure dioxin-like substances that are toxic as part of the safety assessment of the living environment and foods. Dioxin-like substances are intracellular intracellular aryl hydrocarbon receptors (Mol.Pharmaco
l., 39,13-19 (1991); Proc.Natl.Acad.Sci.USA, 89,8185-
8189 (1992). Hereinafter, it may be referred to as Ah receptor. ), Causing its toxicity [An
n.Rev.Pharmacol.Toxicol., 22,517-554 (1982), Ann.Re
v. Pharmacol. Toxicol., 26, 371-399 (1986)]. 2,3,7,8-
When a dioxin-like active substance such as tetrachlorodibenzo-p-dioxin (hereinafter referred to as TCDD) binds to the Ah receptor, the receptor is activated and translocates into the nucleus, and then Arnt [Ah receptor nuclear translocator. Scienc
e, 252, 954-958 (1991). ] Forms a heterodimer with a nuclear protein called. The dimer is a dioxin-responsive element [Dioxin responsive element (DRE); Xenob on the chromosome.
Sometimes called iotic responsive element (XRE). J. Biol. Chem., 263, 17221-17224 (1988)] activates transcription of a gene downstream of the sequence. Therefore, in order to detect a substance capable of inducing dioxin-like toxicity, a cell having a reporter gene in which a base sequence encoding a reporter protein is connected downstream of the dioxin-responsive sequence and the base sequence required for initiation of transcription. After contacting with the test substance and culturing, by measuring the expression level of the reporter gene, a method for evaluating the ability of the test substance to regulate Ah receptor activity has been proposed (Fund.Appl.Toxicol., 30,194- 203 (1996) etc.).

[0003]

When attempting to carry out the above-mentioned evaluation method using a reporter gene as part of the safety evaluation of living environment, food, etc., the usable sample amount may be limited. Therefore, it has been earnestly desired to develop a method capable of evaluating the ability of a test substance to regulate Ah receptor activity with higher sensitivity and a smaller sample amount.

[0004]

Means for Solving the Problems Under the circumstances, the present inventors have made extensive studies and as a result, in the method for evaluating the Ah receptor activity regulating ability for measuring the expression level of a reporter gene as described above, Of the test substance by adding not only the test substance but also the protein synthesis inhibitor to the medium of the cells having the cells, and measuring the amount of the translation product of the reporter gene in the cells contacted with the test substance and the protein synthesis inhibitor. It was found that the ability to regulate Ah receptor activity can be measured with higher sensitivity,
The present invention has been completed. That is, the present invention is: A method for evaluating the ability of a substance to regulate aryl hydrocarbon receptor activity, comprising: (1) (a) expressing an aryl hydrocarbon receptor gene;
A dioxin-responsive sequence and a base sequence necessary for initiation of transcription are connected to a cell containing a reporter gene in which a base sequence coding for a reporter protein originally foreign to the dioxin-responsive sequence is connected in parallel or before and after. And then contacting the test substance with the protein synthesis inhibitor, (2) the translation product amount of the reporter gene in the cell contacted with the test substance and the protein synthesis inhibitor, or an index value having a correlation with the amount. A step of measuring, and (3) a step of evaluating the ability of the substance to regulate aryl hydrocarbon receptor activity based on the amount of the translation product measured in the step (2) or an index value having a correlation with the amount. 2. A method characterized by (hereinafter sometimes referred to as the evaluation method of the present invention); 2. The method according to the above 1, wherein the cell is a cell containing the reporter gene on its chromosome; 3. The method according to the above item 1 or 2, wherein in the step (1), the contact between the cell and the test substance and the contact between the cell and the protein synthesis inhibitor are both performed concurrently for 20 hours or more; 4. The method according to any one of the above items 1 to 3, wherein the protein synthesis inhibitor is cycloheximide; The protein synthesis inhibitor to be brought into contact with the cells in the step (1) is 1 μg / ml in the medium with which the cells are brought into contact
5. The method according to the above item 4, which is a protein synthesis inhibitor contained at a concentration of less than 10 μg / ml. An arylhydrocarbon receptor comprising a step of selecting a substance having an arylhydrocarbon receptor activity-regulating ability based on the arylhydrocarbon receptor activity-regulating ability evaluated by the method according to any one of items 1 to 5 above. It is intended to provide a method for searching for a substance having activity regulating ability.

[0005]

The present invention will be described in more detail below. The cells that can be used in the method of the invention are Ah
A cell containing a reporter gene that expresses a receptor gene and has a base sequence encoding a reporter protein originally foreign to the dioxin response sequence connected downstream of the dioxin response sequence and the base sequence required for transcription initiation (hereinafter , Sometimes referred to as this cell). The present cells can be prepared, for example, by introducing DNA containing the above reporter gene into cells expressing the Ah receptor gene, and selecting cells into which the reporter gene has been introduced. Examples of cells that can be used to prepare the present cells include cells derived from mammals such as humans, mice, and rats, cells derived from insect animals,
Examples include eukaryotic cells such as yeast cells. Considering operability and reproducibility, cells that can be stably passaged are preferable. More specifically, for example, mouse-derived Hepa1clc7
Examples thereof include cells and cells endogenous to the Ah receptor gene such as human-derived HepG2 cells. Further, yeast cells, non-endogenous Ah receptor gene cells such as CV-1 cells or cells having a low expression level of Ah receptor gene, if the Ah receptor gene is introduced and expressed as described below into the cells, it can be used. Good.

Examples of the gene encoding the Ah receptor include a gene isolated from nature and an artificially modified gene. Specifically, for example,
Human-derived Ah receptor gene [GenBank Accession No.
L19872, Mol.Pharmacol.44,911-917 (1993)], mouse-derived Ah receptor gene [GenBank Accession No.M9462
3, Proc. Natl. Acad. Sci. USA, 89, 8185-8189 (1992)], or rat-derived Ah receptor gene [GenBank Accessi
on No.M94623, Nucleic Acids Res., 22,3038-3044 (199
4)] etc. The gene has its translation initiation codon A
Upstream of the TG, Kozak's consensus sequence (Nucleic Acids
Res., 12, 857-872 (1984)) may be linked. Such Ah receptor gene DNA is prepared, for example, by designing an oligonucleotide for amplifying the DNA encoding the receptor based on a known nucleotide sequence, and using the prepared oligonucleotide as a primer. Chain reaction (hereinafter PCR
Is written. ) And the like. Examples of the DNA used as a template in such PCR include commercially available cDNAs derived from various organisms. The obtained Ah receptor gene DNA is
For example, it is inserted into a vector so that it can be expressed and connected to a promoter, and the DNA of the obtained vector is introduced into cells. As the promoter, a promoter that can function in the introduced cell, that is, has a transcription initiation ability can be used. For example, if the cell is a eukaryotic cell, the Rous sarcoma virus (RSV) promoter,
Examples include cytomegalovirus (CMV) promoter and simian virus (SV40) early or late promoter. Examples of the vector include a plasmid having an origin of replication and a drug resistance gene capable of functioning in a microorganism suitable for genetic engineering techniques such as Escherichia coli. Specifically, a commercially available expression vector having the above promoter and a gene insertion site downstream thereof can be used. DNA containing such Ah receptor gene
Are introduced into cells as described below to obtain transformants. A stable transformant in which the introduced gene is introduced into the chromosome may be obtained. Also, in cells expressing the gene encoding the Ah receptor, in the same manner as described above,
The expression level of the receptor may be increased by introducing a gene encoding the receptor such that the gene can be expressed. In addition, for example, by introducing into a cell a gene encoding a protein having a function of increasing the transcription amount of a gene that is transcriptionally regulated by an Ah receptor, such as a coactivator of a nuclear hormone receptor, and expressing it, The ability to promote transcription may be enhanced.

"A reporter gene comprising a nucleotide sequence encoding a reporter protein that is originally foreign to the dioxin responsive sequence connected downstream of the dioxin responsive element and a nucleotide sequence required for transcription initiation" (hereinafter referred to as responsive reporter gene). May be referred to as ").
Is, for example, a DNA having a dioxin response sequence and a DN having a nucleotide sequence necessary for initiation of transcription in order from the upstream.
It can be prepared by connecting these DNAs so that A and DNAs having a nucleotide sequence encoding a reporter protein that is originally foreign to the dioxin response sequence are aligned. The “dioxin responsive element” is a specific nucleotide sequence existing in the transcriptional regulatory region of a target gene whose expression level is regulated by Ah receptor, and a complex formed by binding a ligand, Ah receptor and Arnt is Recognizing a sequence and binding to it promotes transcription of the target gene located downstream thereof. Specifically, for example, Xenobiotic responsive elemen
A nucleotide sequence also called t, which is a cytochrome P4501A1 gene [cyp1A1, J. Biol. Chem., 263, 17221-17224 (198
8), Nucleic Acids Res., 15, 4179-4191 (1987)], Glutathione S-transferase Ya subunit gene
[Proc. Natl. Acad. Sci. USA, 87,3826-3830 (199
0)], UDP-glucuronosyl transferase gene [J.
Biol. Chem., 271, 3952-3958 (1996)] and the like, include the nucleotide sequence of the 5'upstream region of a mammalian gene. In addition, the consensus sequence of dioxin response sequence
[Core sequence: 5 '-(T / A) GCGTG, J. Biol. Chem., 271, 395
2-3958 (1996)] can be mentioned as a base sequence containing one or more times. In addition, in order to obtain sufficient transcription control ability, it is preferable that the above-mentioned consensus sequence is usually linked in tandem in about 2 to 5. DNA having such a base sequence
Can be prepared by chemical synthesis, amplification by PCR or the like and cloning. The "base sequence required for transcription initiation" is a base sequence having a TATA box and a leader sequence for transcription initiation,
For example, the nucleotide sequence of the 5'upstream region of the thymidine kinase gene (tk), the 5'upstream region of the glutathione S-transferase Ya subunit gene [eg, with the transcription start point as 1, the −164th base to + 66th base Region containing, Pr
oc. Natl. Acad. Sci. USA, 87, 3826-3830 (1990)], the 5'upstream region of the cytochrome P4501A1 gene [eg, the transcription start point is 1, the −70th base to the + 120th base Region containing base, Eur. J. Biochem., 159, 219-225 (198
6)] and the like. The DNA having such a base sequence is prepared, for example, by designing an oligonucleotide for amplifying a DNA encoding the above-mentioned region based on a known base sequence, and using the prepared oligonucleotide as a primer. It can be prepared by performing PCR used as. Examples of the "base sequence encoding a reporter protein that is originally foreign to the dioxin responsive sequence" include, for example, "base sequence encoding a reporter protein" originally derived from a gene different from the "dioxin responsive sequence". I can give you. The reporter protein encoded by such a nucleotide sequence is preferably a protein whose expression level can be easily measured based on the enzyme activity of the protein, and examples thereof include firefly luciferase, Renilla luciferase, β-galactosidase, and chloramphenicol acetyl. Examples include transferase and alkaline phosphatase. A DNA having a nucleotide sequence encoding such a reporter protein can be obtained, for example, by subjecting a commercially available plasmid DNA containing the DNA to restriction enzyme digestion to isolate the desired DNA.

A DNA containing a responsive reporter gene is inserted into a vector such as a plasmid and introduced into the above-mentioned cells,
This cell can be prepared by selecting a cell into which a responsive reporter gene has been introduced. At this time,
To facilitate selection of cells into which a responsive reporter gene has been introduced, an expression plasmid for a selectable marker gene such as a drug resistance gene may be introduced at the same time. Examples of the selectable marker gene that can be used in this manner include neomycin resistance (aminoglycoside phosphotransferase) gene, blasticidin S resistance gene, hygromycin resistance gene and the like. To introduce the plasmid DNA into which the responsive reporter gene is integrated as described above, for example, in order to introduce it into cells of mammalian origin, first, the cells are seeded in a culture vessel (for example,
10 ⁵ to 10 ⁷ cells / diameter 6 cm to 10 cm Petri dish), 5% (v / v) to 10
5% C using αMEM medium containing serum (% / v / v)
Incubate at 37 ° C for several hours to overnight under O ₂ and saturated humidity conditions. The plasmid DNA having the responsive reporter gene incorporated therein is introduced into the cells thus cultured. Examples of the method for introducing DNA into cells include general lipofection method, DEAE-dextran method, calcium phosphate method, electroporation method, and the like. Specifically, for example, when using commercially available lipofectin (manufactured by GIBCO-BRL), the operation is performed according to the attached manual, and the amount of plasmid DNA to be introduced, the amount of lipofectin, the type of cells, the number of cells, etc. It is advisable to conduct a preliminary study to find the optimum conditions. When a plasmid containing a drug resistance gene is introduced at the same time as a plasmid containing a responsive reporter gene, the amount of plasmid DNA containing a drug resistance gene should be 1/100 of the amount of DNA in the plasmid containing a responsive reporter gene. It may be good to set it to about 5 to 1/10. As the plasmid DNA, for example, DNA purified by the CsCl density gradient centrifugation method or DNA having the same degree of purity can be used. A linearized plasmid DNA may be used which has been previously digested with a restriction enzyme that does not have a recognition site in the region required for preparation of the present cells (responsive reporter gene, selectable marker gene, etc.). In order to obtain the present cell containing a responsive reporter gene on its chromosome, for example, a cell into which a plasmid having a responsive reporter gene and a plasmid containing a selectable marker gene have been introduced is introduced into the cell. The cells are cultured in a medium under the conditions corresponding to the selected selectable marker gene, and the culture is continued until the non-transformed cells die and the colonies derived from the transformed cells have an appropriate size. During this period, the medium is exchanged once or twice / week if necessary. By carrying out such an operation, it is possible to obtain the present cell in which the introduced responsive reporter gene is integrated into the chromosome and the responsive reporter gene is stably retained in the cell. In order to confirm that the introduced responsive reporter gene was integrated into the cell chromosome, the genomic DNA of the cell was prepared according to a general genetic engineering method, and the introduced genomic DNA was introduced from the prepared genomic DNA. The presence of the responsive reporter gene may be detected by using a method such as PCR or Southern hybridization in which a DNA having a partial base sequence of the reporter gene is used as a primer or a probe. The cells thus prepared can be cryopreserved and can be used in a sleeping state if necessary, so that it is possible to save the labor of preparing transformants for each experiment and to prepare them in advance. It becomes possible to carry out a test using the present cells whose handling conditions are confirmed. This is also useful, for example, in performing large scale screening with automated robots.

Further, more preferable cells for measuring the activity of the test substance with respect to the transcription promoting ability of the Ah receptor are obtained by using the ligand responsiveness of the expression level of the responsive reporter gene in the thus obtained cells as an index. You can also choose. Specifically, first, the colony of the present cell obtained as described above is divided into a plurality of pieces and re-planted to grow the cells. Add a solution of Ah receptor ligand to a part of it (ligand addition group) for 4 hours to 2
After culturing for about one day, the expression level of the responsive reporter gene is measured. Further, as a control, the solvent used for the preparation of the ligand solution is added to another part of the cells (control group) and the cells are similarly cultured, and the expression level of the responsive reporter gene is measured. The method for measuring the expression level of a responsive reporter gene depends on the type of reporter gene used, but in general, it is obtained by adding a cell lysing agent to the cells to be measured to prepare a cell extract. The amount of the reporter protein encoded by the responsive reporter gene contained in the extract is measured. For example, when the reporter protein encoded by the responsive reporter gene has an enzyme activity, after reacting a substrate specific to the enzyme with a cell extract containing the protein, the amount or reaction of the remaining substrate The amount of product is defined by its luminescence amount, fluorescence absorbance,
The absorbance is used as an index for measurement. Specifically, for example,
When the luciferase gene is used as the reporter gene, when luciferin, which is a substrate for luciferase, is reacted with the cell extract, light is emitted with an intensity proportional to the amount of luciferase in the cell extract. Therefore, by measuring this luminescence intensity with a measuring device such as a luminometer, the amount of luciferase in the cell extract, and by extension,
The expression level of the luciferase gene can be known. By subtracting the expression level of the responsive reporter gene in the control section from the expression level of the responsive reporter gene in the ligand-added section, the increase in the expression level of the responsive reporter gene due to contact with the ligand is determined. And
A cell is selected which has an increased expression level of the responsive reporter gene due to contact with a ligand that is at least 2 times or more, preferably 10 times or more that of the control group.
When the colony thus obtained does not consist of a single transformed cell, the cell is diluted and further cultured to select a colony consisting of a single cell.

By carrying out the evaluation method of the present invention using the present cells obtained as described above, the ability of the test substance to regulate the transcription promoting ability of the Ah receptor can be measured. First, a test substance and a protein synthesis inhibitor are brought into contact with the present cells [step (1)]. The contact between the present cell and the test substance, and the contact between the present cell and the protein synthesis inhibitor are maintained at the same time in parallel, are maintained in parallel for a period of time, or are staggered at all. For example, they are maintained one after another. The contact time between the present cell and the test substance and the contact time between the present cell and the protein synthesis inhibitor may be the same length or may be different. For example, if both substances are brought into contact with the cells at the same time by simultaneously adding the test substance and the protein synthesis inhibitor to the medium of the cells, and if this contact is carried out in parallel and maintained for the same time, it will be troublesome for medium preparation and medium replacement. Can be reduced. In addition, contact between the cells and the test substance should be about 20
More than an hour, specifically, about 16 hours to about 50 hours
If it is maintained for about a time, the amount of the translation product of the reporter gene, which will be described later, reaches almost saturation, so that the occurrence of measurement error due to the difference in measurement time can be reduced. In a more specific embodiment, for example,
About 10 ³ cells per well to about 5 x 10 ⁴ per well plate
Inoculate about cells and add 100 μL to 200 μL of serum-containing medium (αMEM etc.) to 5% (v / v) to 10% (v / v), and add 5%
Incubate at 37 ° C for several hours to 1 day under CO ₂ and saturated humidity conditions. Then, the solution of the test substance and the solution of the protein synthesis inhibitor are added to the medium of the present cells in culture at the same time or at a staggered time. Alternatively, the medium of the present cells may be replaced with a medium containing a test substance and / or a protein synthesis inhibitor. As the solvent for dissolving the test substance and the protein synthesis inhibitor, for example, dimethyl sulfoxide (DMSO), ethanol, distilled water and the like are used. To reduce the effect of the solvent on the cells, the volume ratio of the test substance solution and the protein synthesis inhibitor solution added to the medium should be about 0.5% (v
/ v) to about 2% (v / v) or less.
When the solution of the test substance or the protein synthesis inhibitor is an aqueous solution, for example, it is sterilized by filtration with a filter having a pore size of 22 μm and then added to the above medium. Protein biosynthesis inhibitors include cycloheximide, puromycin, anisomycin, blasticidin S,
Examples include chlortetracycline (aureomycin) and emetine. The concentration of the protein synthesis inhibitor added may be, for example, when the substance is cycloheximide, about 1 μg or more and less than about 10 μg per 1 ml of the medium.

Next, as described above, the translation product amount of the responsive reporter gene in the present cell contacted with the test substance and the protein synthesis inhibitor or the index value having a correlation with the amount is measured [step (2 )]. When the Ah receptor expressed by the present cells is activated by binding of a test substance (for example, a dioxin-like active substance), transcription of a responsive reporter gene is promoted and a reporter protein which is a translation product of the reporter gene. Are either accumulated in the cells or secreted into the medium. By measuring the amount of the reporter protein or the index value having a correlation with the amount thereof, the translation product amount of the reporter gene per cell of the present cell or the index value having a correlation with the amount is measured. Specifically, for example, when a luciferase gene is used as a reporter gene, a crude cell extract prepared from the present cells contacted with a test substance and a protein synthesis inhibitor is treated with luciferin, which is a substrate for luciferase. When added, it emits light with an intensity proportional to the amount of luciferase in the crude cell extract. Therefore, the amount of luciferase, that is, the amount of translation product of the luciferase gene can be known by measuring the luminescence intensity with a measuring device such as a luminometer. Similarly, the amount of the translation product of the reporter gene in the present cell which is brought into contact with the protein synthesis inhibitor and not brought into contact with the test substance or an index value having a correlation with the amount may be measured.

Next, the ability of the test substance to regulate Ah receptor activity is evaluated based on the amount of translation product measured in the step (2) or an index value having a correlation with the amount [step (3)]. The ability to regulate Ah receptor activity includes, for example, agonist activity for Ah receptor and antagonist activity for Ah receptor. For example, the translation product amount of the reporter gene measured in the present cell contacted with the test substance and the protein synthesis inhibitor, or an index value having a correlation with the amount, and the test substance may be contacted with the protein synthesis inhibitor. The amount of the translation product or the index value measured in the cells that are not contacted is compared. If the translation product amount of the reporter gene in the present cell contacted with the test substance and the protein synthesis inhibitor or the index value having a correlation with the amount thereof is higher, the test substance is evaluated to have an agonist activity to the Ah receptor. can do. On the other hand, for example, in the present cell contacted with a dioxin-like active substance such as TCDD, a test substance and a protein synthesis inhibitor, and in each of the present cells contacted with the dioxin-like active substance and a protein synthesis inhibitor and not contacted with the test substance The amount of the translation product of the reporter gene or the index value having a correlation with the amount is measured by the same method as described above. The dioxin-like active substance, the test substance, and the protein synthesis inhibitor are compared with the translation product amount of the reporter gene in the present cell contacted with the dioxin-like active substance and the protein synthesis inhibitor or an index value having a correlation with the amount. If the amount of the translation product of the reporter gene in the present cells contacted with the above or the index value having a correlation with the amount thereof is lower, it can be evaluated that the test substance has an antagonist activity to the Ah receptor.

Based on the agonistic activity of the test substance against the Ah receptor evaluated by the evaluation method of the present invention as described above, the TCDD-equivalent amount of the test substance can be determined. For example, TCDD is added as a test substance at various concentrations to the above measurement system, and the translation product amount of the reporter gene at each concentration or an index value having a correlation with that amount is measured. Then, the obtained measured value and the concentration of TCDD are plotted to create a calibration curve. By applying to the obtained calibration curve the translation product amount of the reporter gene measured for each test substance or an index value having a correlation with the amount thereof, the TCDD equivalent amount of the test substance can be determined. The TCDD equivalent calculated in this way is GC-M
It has the same meaning as "toxic equivalent (TEQ)" in dioxins analysis method by S measurement. Further, it becomes possible to select / search for a substance having the ability to regulate Ah receptor activity based on the ability to regulate Ah receptor activity evaluated by the evaluation method of the present invention.

[0014]

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

Reference Example Preparation of this cell Using the rat genomic DNA as a template, the nucleotide sequence
Oligonucleotide consisting of 5'-gcgctagcatggtagcgccttgtcagcc-3 '(SEQ ID NO: 1) and base sequence 5'-gcaagctt
Rat glutathione S-transferase Y was obtained by performing PCR using an oligonucleotide consisting of gagtactgacctagcgagag-3 '(SEQ ID NO: 2) as a primer.
Promoter region derived from the 5'upstream region of the a subunit gene (region consisting of bases from -164th to + 53th with the transcription start point as 1) (Proc. Natl. Acad. Sci. US
A, 87, 3826-3830 (1990)) is amplified and cloned. The cloned DNA
Reporter plasmid pGV-P (sold by Toyo Ink Co .;
Contains the firefly luciferase gene. The plasmid pGV-Ya is obtained by inserting the plasmid pGV-Ya between the HindIII site and the NheI site. On the other hand, rat glutathione S-transferase Y
Dioxin response element existing in the 5'upstream region of a subunit gene [Proc. Natl. Acad. Sci. USA, 87, 3826
~ 3830 (1990)], the following two kinds of oligonucleotides having a nucleotide sequence are synthesized. Oligonucleotide A; 5'-ctcaggcatgttgcgtgcatccctg
aggccagccgagct-3 '(SEQ ID NO: 3) oligonucleotide B; 5'-cggctggcctcagggatgcacgcaa
catgcctgagagct-3 '(SEQ ID NO: 4) Next, T4 is added to each of the oligonucleotides A and B.
Polynucleotide kinase is allowed to act to phosphorylate the 5'end and then annealed to form double-stranded DNA. The double-stranded D
After purifying NA by agarose gel electrophoresis, a ligation reaction is performed to obtain a DNA (hereinafter referred to as XREx5) in which five double-stranded DNAs are tandemly ligated. The DNA XREx
5 was inserted into the SacI cleavage site of the above-mentioned plasmid pGV-Ya to form a responsive reporter gene in which a base sequence encoding firefly luciferase was connected downstream of the dioxin response sequence and the base sequence required for transcription initiation. The resulting plasmid pGV-Ya-XREx5 is obtained. 1 of Hepa1clc7 cells (ATCC)
× 10 ⁶ pieces were seeded on a petri dish with a diameter of 10 cm, and a medium was prepared by adding 10% (v / v) fetal bovine serum to αMEM medium (hereinafter,
It may be referred to as αMEM medium containing 10% serum. ) Medium 37
Incubate overnight at 5 ° C in the presence of 5% CO ₂ . Incidentally, in the examples the present reference example and described below, culture All 37 ℃, 5% CO ₂
Perform in the presence. The plasmid pGV-Ya-XREx5 (plasmid containing responsive reporter gene) is digested with BamHI.
In addition, a plasmid containing the neomycin resistance (aminoglycoside phosphotransferase) gene pRc / RSV (INVIT
ROGEN) is digested with HindIII. Then these two digested plasmids, namely BamHI digested p
GV-Ya-XREx5 2.8 μg, and HindIII digested pRc / RSV
0.5 μg and 300 μl of αMEM medium are mixed (mixture A). On the other hand, 36 μl of lipofectin (manufactured by GIBCO-BRL) and 300 μl of αMEM medium are mixed (mixed solution B). After each of these mixed solutions is left at room temperature for 30 minutes, mixed solution A and mixed solution B are mixed and left at room temperature for 10 minutes. Next, the Heapa1clc7 cells cultured overnight as described above were cultured in αMEM medium.
After washing once, add 5.4 ml of αMEM medium to the above mixture (A + B) and mix,
Incubate for hours. Then, the medium is replaced with 10% serum-containing αMEM medium, and the cells are further cultured for 1 day. Subsequently, the cells were peeled from the dish and collected, and the collected cells were suspended in αMEM medium containing 10% serum to make 100 ml in total, and the resulting cell suspension was added to a 96-well plate at a rate of 100 μl / well. Sow. After incubating this plate overnight, geneticin was added to a concentration of 1.6 mg / ml, containing 10% serum α.
The medium dissolved in MEM medium was added at a rate of 100 μl / well (final concentration of geneticin was 800 μg / ml). The culture is further continued, during which the medium is replaced with αMEM containing 10% serum containing 800 μg / ml of geneticin once every 4 to 5 days. The volume of medium is 200 μl / well.
After 3 weeks of culturing, cells are peeled from the wells where colonies are found and collected, and the collected cells are divided into 3 pieces, seeded on separate plates, and further cultured for 1 day. Next, DMSO alone was added to the first plate (solvent control), and 3-MC dissolved in DMSO was added to the second plate to a final concentration of 2 μM, and the culture was continued overnight. . The third plate is used as a master plate and culture is continued. Remove the medium from each well of each plate containing DMSO or 3-MC, and add PBS (-) [in 1L,
Sodium chloride: 8000 mg, potassium chloride: 200 mg, sodium monohydrogen phosphate (anhydrous): 1150 mg, sodium dihydrogen phosphate (anhydrous): 200 mg, sold by Nissui Pharmaceutical Co., Ltd.] 2
Wash wells once. Then, the cell lysate of PicaGene Dual Kit (sold by Toyo Ink Co., Ltd.) is diluted 5-fold, 15 μl / well is added, and the mixture is left at room temperature for 1 hour or more to lyse the cells to obtain a cell extract. The plate is set in a luminometer equipped with an enzyme substrate automatic injector, and 75 μl of a luciferin substrate solution is automatically dispensed to measure the amount of luminescence. A clone having a high rate of increase in the expression level of the luciferase gene due to the addition of 3-MC and a large measured value of luminescence is selected and used in the following experiment.

Example 1 The present cells were seeded on a 96-well plate at a rate of 2 × 10 ⁴ cells / well, and the medium was prepared by adding 10% (v / v) fetal bovine serum to αMEM medium for 1 day. Cultured. Cycloheximide (manufactured by Wako Pure Chemical Industries, Ltd.) in DMSO was added to each well so that the final concentration in the medium was 0 to 10 μg / mL [0.5% (v / v) in volume], and at the same time, TCDD 0.2 A ng / mL DMSO solution was added so that the final concentration in the medium was 1 pg / mL [0.5% (v / v) in volume] (TCDD addition group). For some wells,
DMSO was added in place of the DMSO solution of TCDD (solvent control group). Continue culturing, remove the medium after about 24 hours, and add PBS (-).
The cells were washed twice. Dilute cell lysate from PickaGene Dual Kit (sold by Toyo Ink Co., Ltd.) 5 times to 15 μL /
The wells were added, and the cells were lysed by leaving them at room temperature for 1 hour or more to obtain a cell extract. A 96-well plate containing the cell extract was set in a luminometer (TR717, manufactured by ABI) with an automatic enzyme substrate injector, and luciferin substrate solution [20 mM Tricine (pH7.8), 2.67 mM MgSO _4, 0.1 mM EDTA, 3
3.3 mM DTT, 270 μM Coenzyme A, 530 μM ATP, 470 μM L
[uciferin] at a rate of 75 μL / well
Inj .: 1.6 seconds, Meas. Int .: The luminescence amount was measured at 5 seconds. The results are shown in Table 1.

[0017]

[Table 1] *: Net value of luminescence amount = luminescence amount of TCDD addition group-luminescence amount of solvent control group The luminescence amount of 4 wells was averaged. SD: Standard deviation

Example 2 Cells were seeded in the same manner as in Example 1 and cultured for about 1 day. To the obtained cells, a solution of cycloheximide in DMSO was added at a final concentration of 0, 1.5, or 2.0 μg / mL in the medium [0.5% (v / v by volume).
v)], and at the same time, a DMSO solution of TCDD was added so that the final concentration in the medium was 0 to 1 pg / mL [0.5% (v / v) in volume] (TCDD addition group). Further, DMSO was added to some wells in place of the DMSO solution of TCDD (solvent control group). The culture was continued, and after about 24 hours, the medium was removed and the cells were washed twice with PBS (-). A 5-fold diluted cell lysing agent was added at 15 μL / well, and left at room temperature for 1 hour or more to lyse the cells to obtain a cell extract. Similar to Example 1,
Add 96-well plate containing cell extract to luminometer (TR
717), luciferin substrate solution was added automatically at a rate of 75 μL / well, Delay Inj .: 1.6 sec, Meas. In
t .: The amount of light emission was measured at 5 seconds. Statistical processing of the obtained measured values was performed using the t-test of MS Excel. The results are shown in Table 2.

[0019]

[Table 2] CHX: Cycloheximide luminescence amount Net value = TCDD addition group luminescence amount-solvent control group luminescence amount The luminescence amount was the average value of 4 wells. #: Minimum T that is significantly higher than the solvent control in the t test
CDD concentration group (P <0.05) *:> 3σ (σ = SD of solvent control group) minimum TCDD concentration group

Example 3 (1) In the same manner as in Example 1, 1 × 10 ⁴ or 2 × 10 ⁴ cells were seeded per well in a 96-well plate and cultured for about 1 day. A cycloheximide solution in DMSO was added to the obtained cells at a final concentration of 0, 2.5 or 10.0 μg / mL [in the volume]
0.5% (v / v)], and at the same time, DMSO of TCDD
The concentration of the solution in the medium is 2.0 pg / mL [0.5% (v / v) by volume]
(TCDD addition section). Further, DMSO was added to some wells in place of the DMSO solution of TCDD (solvent control group). The culture was continued, and the medium was removed after 2, 4, 6, 8, 12, and 24 hours, and the cells were washed twice with PBS (-). A 5-fold diluted cell lysing agent was added at 15 μL / well, and left at room temperature for 1 hour or more to lyse the cells to obtain a cell extract. As in Example 1, a 96-well plate containing a cell extract was placed in a luminometer with an enzyme substrate automatic injector (LB96V,
(Beltold), and the substrate luciferin solution was automatically added at a rate of 50 μL / well. Delay Inj .: 1.6
Second, Meas. Int .: The light emission was measured at 5 seconds. The value obtained by subtracting the luminescence amount of the solvent control group from the luminescence amount of the TCDD-added group was defined as the luminescence amount Net value. The results are shown in FIGS. 1 and 2. (2) As in Example 1, 1 × 10 ⁴ or 2 × 10 ⁴ cells were seeded per well in a 96-well plate and cultured for about 1 day. A cycloheximide solution in DMSO was added to the obtained cells at a final concentration of 0, 2.5 or 10.0 μg / mL [in the volume]
0.5% (v / v)], and at the same time, DMSO of TCDD
The concentration of the solution in the medium was 2.0 pg / mL ([0.5% (v / v
v)] was added. Continue culturing, 21.5, 24, 2
After 6.5, 29 and 31.5 hours, the medium was removed and the cells were washed twice with PBS (-). A 5-fold diluted cell lysing agent was added at 15 μL / well, and left at room temperature for 1 hour or more to lyse the cells to obtain a cell extract. As in Example 1, a 96-well plate containing a cell extract was set in a luminometer equipped with an enzyme substrate automatic injector, and a substrate luciferin solution was automatically added at a rate of 50 μL / well. Delay Inj .: 1.6 seconds, Meas. In
t .: The amount of light emission was measured at 5 seconds. The value obtained by subtracting the luminescence amount of the solvent control group from the luminescence amount of the TCDD-added group was defined as the luminescence amount Net value. The results are shown in FIGS. 3 and 4.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method capable of evaluating the ability of a test substance to regulate Ah receptor activity with higher sensitivity and a smaller sample amount.

[Sequence Listing Free Text] SEQ ID NO: 1 Oligonucleotide primer designed for PCR SEQ ID NO: 2 Oligonucleotide primer designed for PCR SEQ ID NO: 3 To prepare DNA containing dioxin responsive sequence Designed oligonucleotide SEQ ID NO: 4 Oligonucleotide designed to produce DNA containing dioxin-responsive sequence

[0023]

[Sequence list] <110> Sumitomo Chemical Co. Ltd.    <120> Method for analyzing transactivation control activity to Ah recept or    <130> P154090    <160> 4    <210> 1 <211> 28 <212> DNA <213> Artificial Sequence    <220> <223> Designed oligonucleotide primer for PCR <400> 1 gcgctagcat ggtagcgcct tgtcagcc 28 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence    <220> <223> Designed oligonucleotide primer for PCR <400> 2 gcaagcttga gtactgacct agcgagag 28 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence    <220> <223> Designed oligonucleotides to synthesize DNA containing dioxin resp onsive element <400> 3 ctcaggcatg ttgcgtgcat ccctgaggcc agccgagct 39 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence    <220> <223> Designed oligonucleotides to synthesize DNA containing dioxin resp onsive element <400> 4 cggctggcct cagggatgca cgcaacatgc ctgagagct 39

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measuring the ability of TCDD to regulate Ah receptor activity using the present cells. 1 per well
× 10 ⁴ cells were seeded and cultured for about 1 day, then TCDD
Then, cycloheximide was added to the medium, and after the lapse of time shown on the horizontal axis, the luciferase activity was measured. The vertical axis represents the light emission amount Net value. The white bar is the section where cycloheximide was added to 2.5 μg / mL, the shaded bar is
A group in which cycloheximide was added so as to be 10.0 μg / mL,
The black bar shows the section without addition of cycloheximide.

FIG. 2 is a diagram showing the results of measuring the ability of TCDD to regulate Ah receptor activity using the present cells. After seeding 2 × 10 ⁴ cells per well and culturing for about 1 day, TC
DD and cycloheximide were added to the medium, and after the lapse of time shown on the horizontal axis, the luciferase activity was measured.
The vertical axis represents the light emission amount Net value. White bar is 2.5 μg / mL
To which the cycloheximide was added, the shaded bar represents the group to which cycloheximide was added so that the concentration was 10.0 μg / mL, and the black bar represents the group to which cycloheximide was not added.

FIG. 3 is a diagram showing the results of measuring the ability of TCDD to regulate Ah receptor activity using the present cells. 1 per well
× 10 ⁴ cells were seeded and cultured for about 1 day, then TCDD
Then, cycloheximide was added to the medium, and after the lapse of time shown on the horizontal axis, the luciferase activity was measured. The vertical axis represents the light emission amount Net value. The white bar is the section where cycloheximide was added to 2.5 μg / mL, the shaded bar is
A group in which cycloheximide was added so as to be 10.0 μg / mL,
The black bar shows the section without addition of cycloheximide.

FIG. 4 is a diagram showing the results of measuring the ability of TCDD to regulate Ah receptor activity using the present cells. After seeding 2 × 10 ⁴ cells per well and culturing for about 1 day, TC
DD and cycloheximide were added to the medium, and after the lapse of time shown on the horizontal axis, the luciferase activity was measured.
The vertical axis represents the light emission amount Net value. White bar is 2.5 μg / mL
To which the cycloheximide was added, the shaded bar represents the group to which cycloheximide was added so that the concentration was 10.0 μg / mL, and the black bar represents the group to which cycloheximide was not added.

Claims (6)

[Claims] 1. A method for evaluating the ability of a substance to regulate aryl hydrocarbon receptor activity, which comprises (1)
(A) an aryl hydrocarbon receptor gene is expressed, and (b) a base sequence encoding a reporter protein originally foreign to the dioxin responsive sequence is connected downstream of the dioxin responsive sequence and a base sequence required for transcription initiation. Contacting a test substance with a protein synthesis inhibitor in parallel or in tandem with a cell containing the reporter gene, (2) the reporter gene in the cell contacted with the test substance and the protein synthesis inhibitor And (3) the amount of the translation product measured in step (2) or the index value having a correlation with the amount, and the substance And a step of evaluating the ability of the aryl hydrocarbon receptor activity to be regulated. How. 2. The method according to claim 1, wherein the cell is a cell containing the reporter gene on its chromosome. 3. In the step (1), the contact between the cell and the test substance and the contact between the cell and the protein synthesis inhibitor are both maintained concurrently for 20 hours or more.
Or the method described in 2. 4. The method according to claim 1, wherein the protein synthesis inhibitor is cycloheximide. 5. The protein synthesis inhibitor to be brought into contact with cells in the step (1) is added to the medium with which the cells are brought into contact with 1 μm
The method according to claim 4, wherein the protein synthesis inhibitor is contained at a concentration of g / ml or more and less than 10 μg / ml. 6. A step of selecting a substance having an aryl hydrocarbon receptor activity-regulating ability based on the aryl hydrocarbon receptor activity-regulating ability evaluated by the method according to any one of claims 1 to 5. And a method of searching for a substance having the ability to regulate aryl hydrocarbon receptor activity.