JP2003093096A - Method for evaluating demethylation activity against dna of test specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening the demethylation activity against a DNA of a test specimen. SOLUTION: This method for evaluating the demethylation activity against the DNA of the test specimen comprises (1) culturing a cell in which a methylation DNA region on a chromosome has only one visualizable site, in the presence of the test specimen, (2) preparing a specimen by using the cell cultured at the step (1), (3) measuring a micronucleus frequency by using the specimen obtained at the step (2), and (4) discriminating the presence or absence of the demethylation activity based on the micronucleus frequency measured at the step (3).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for determining the presence or absence of a demethylation action on DNA of a test substance, for example, various biologically relevant substances.

[0002]

2. Description of the Related Art In today's genomic research, a gene is identified from nucleotide sequence information and the function and structure of the gene are pursued as in the past, that is, in addition to genetics, a mechanism for controlling gene expression is chromatin. Epigenetics, which is the research to be elucidated from the dynamic fluctuations of, has attracted attention.

With recent epigenetics, the role of methylation in the gene expression control mechanism, that is, "DN
Chromatin condenses after the methylation of A triggers, turning off the expression switch of genes in the vicinity. "
Or, conversely, the mechanism that “a gene whose expression was originally stopped due to the presence of methylated DNA is turned on by demethylation to start the expression of the gene” becomes clear. Came.

It has been elucidated that, for example, so-called tumor suppressor genes such as p16 function as follows. In normal cells, the expression of p16 usually suppresses the cells from becoming cancerous (ie, unmethylation of a sequence upstream of the p16 gene). On the other hand, in cancer cells, the expression of p16 is stopped (that is, stopped) by methylating the upstream sequence of the p16 gene.
In the medical field such as cancer treatment, a study to normalize the expression of tumor suppressor genes such as p16, whose expression was stopped due to carcinogenesis, by demethylation, and thereby to guide cells to normal directions Is being done. Therefore, in the future,
Development of new drugs that effectively exert demethylation is also expected. However, for that purpose, it is necessary to search for chemical substances and bio-related substances having a new demethylation action. However, cytogenetic methods for screening such demethylation effects have not yet been established.

For example, Fauth et al. (Mutageness 13:23
5-241, 1998), for the analysis of the demethylation action of 5-azacytidine, an analysis using a micronucleus test using human lymphocytes and FISH using a painting probe for all chromosomes is attempted. However, since this method requires a great deal of labor, it is not suitable for use as a screening method that requires quickness and convenience. Therefore, early establishment of a cytogenetic method for screening the demethylation effect is desired.

[0006]

The object of the present invention is to provide a method for screening for demethylation activity.

[0007]

The above-mentioned problems can be solved by the present invention as described below. That is; a method for evaluating the demethylation action of a test substance on DNA, comprising: (1) testing a cell characterized in that it has only one visible site in a methylated DNA region on a chromosome. Culturing in the presence of a substance; (2) preparing a specimen using the cells cultured in (1); (3) measuring the micronucleus frequency of the specimen obtained in (2); And (4) evaluating the presence or absence of a demethylation action based on the micronucleus frequency measured in (3).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION 1. Introduction The present invention provides a screening method for epigenetic action using such micronucleus as an index.
In particular, the present invention provides a cytogenetic screening method for determining the demethylation action of a test substance on DNA using chromatin disaggregation and DNA strand breaks in specific methylated DNA regions on the chromosome as indicators.

[0009] When various environmental mutagens cause morphological abnormalities such as cleavage, exchange and non-segregation in the chromosome, a part of the cell nucleus is separated and a minute nucleus is formed in the subsequent interphase. It is known to be done. Such minute nuclei are called micronuclei. By analyzing the appearance and frequency of such micronuclei, it is possible to detect chromosomal abnormalities due to DNA damage or mitotic inhibition. Today, the screening method for mutagenicity using micronucleus as an index is
It is widely used because of its advantages such as simple method and easy observation.

Recently, fluorescence in situ hyb
Attempts have also been made to analyze the specificity of chromosomes involved in the appearance of micronuclei by analyzing chromosomal information in micronuclei using ridization (FISH) method together. At that time, for the determination of the micronucleus derived from the chromosome structural abnormality, the DN for coloring the chromosome is used.
A probe (also called painting probe)
Is used. In addition, a kinetochore-specific DNA probe of a chromosome is used to determine a micronucleus derived from a numerical abnormality. However, such conventionally used micronucleus test is essentially a method for determining the chromosomal structural abnormality inducing property or the micronucleus abnormally inducing property of a chemical substance.

Therefore, the present inventor cannot utilize the micronucleus test in the method for screening the demethylation action of a test substance by simply applying the conventional micronucleus test. Therefore, we examined from various angles and found that the following are particularly important.
The first point is to select, as a material, a characteristic cell line that has only one methylated DNA region in the genome that can be visualized at the optical microscope level. The second point is to complement the methylated region. To determine whether the origin of the emerging micronucleus is the methylated region of interest using a conventional DNA probe.

Based on these findings, the inventor has further made earnest studies from various viewpoints, whereby the epigenetic action of a test substance (for example, a biologically relevant substance) using micronucleus frequency as an index. In particular, we have succeeded in providing a method for screening the demethylation effect.

2. Aspects of the invention Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a flowchart showing a conventional method for analyzing mutagenicity (that is, chromosomal aberration-inducing property) using micronuclei. FIG. 2 is a schematic diagram showing the flow of the analysis method according to the embodiment of the present invention.

FIG. 1 shows the flow of a micronucleus test for detecting chromosomal structural abnormalities and a micronucleus test for detecting numerical abnormalities by the conventional method. A chromosome-specific coloring DNA probe (also called a painting probe) is used to determine a micronucleus derived from a chromosome structural abnormality, and a chromosome centromere specific to a micronucleus derived from a numerical abnormality. DNA probes are used. In such a conventional micronucleus test, the cell type used was not so limited as long as it was a cell line capable of growing under certain conditions.

On the other hand, in the embodiment of the present invention, a cell having only one methylated DNA region in the genome which can be visualized under a microscope is used. The method for evaluating the demethylation action of a test substance on DNA, which is an aspect of the present invention, is as follows (FIG. 2).

First, cells having only one methylated DNA region in the genome which can be visualized under a microscope are cultured in a medium containing a test substance for a certain period of time. Cells that can be used in the present invention are cells that have only one methylated DNA region in the genome that can be visualized under a microscope. A highly repetitive methylated CpG sequence is located on chromosome 1
Cells that are concentrated in one location are preferred. Alternatively, a highly repetitive methylated CpG sequence is present at multiple sites on a chromosome, but methylated DN that can be selectively visualized at a desired site by staining or other identification means.
It may be a cell capable of obtaining the A region.

For example, male fetal lung fibroblast cell line TIG-
It is possible to use 7. The male fetal lung fibroblast cell line TIG-7 has a methylated DNA region (referred to as 15p +) specific to the short arm of chromosome 15.

Second, a sample is prepared using the cultured cells. As a method for preparing a specimen conveniently used in the present invention, generally, a method for preparing a specimen for a micronucleus test known per se can be used. For example, the cells may be fixed on a support such as a slide glass, or the cell suspension may be prepared by lysing the cytoplasm to form a naked nuclei. That is, such a sample may be a support-fixed sample or a floating cell sample.

Thirdly, a DNA probe having a sequence complementary to the above-mentioned methylated DNA region is added to the methylated DNA region.
Join to a region. Preferably such DNA
A fluorescent substance is added to the probe, and such a DNA probe (A ′) is used to perform fluorescence in situ hybridization on the sample.
hybridization, hereinafter referred to as FISH).
Alternatively, a luminescent substance, a radioisotope, a dye or the like can be used in place of the fluorescent substance, but the fluorescent substance is preferable from the viewpoint of easiness of detection and safety.

Fourthly, the demethylation action of the test substance on DNA is evaluated using the micronucleus as an index. The detection of micronuclei is carried out by visual observation using a microscope. Further, it is preferable to detect the fluorescent signal from the fluorescent substance applied to the DNA probe simultaneously or sequentially. Thereby, if it is detected that the fluorescent substance is contained in the induced micronucleus, it is a micronucleus induced by demethylation of the methylated DNA region hybridized with the DNA probe. Is determined. Therefore, the final judgment is that one test substance is tested in a plurality of specimens, the presence or absence of a FISH signal is confirmed in each specimen induced in each specimen, and it is judged that the signal is present. When the proportion of micronuclei is remarkably high, it is preferable to comprehensively judge that “the test substance has a demethylating action”.

The present invention also provides a male fetal lung fibroblast cell line TIG-7 for use in a method for evaluating the demethylating action of a test substance on DNA. Further, chromosome 15 of the cell line for use in the method for evaluating the demethylation action of a test substance on DNA is also included in the scope of the present invention. For example, the male fetal lung fibroblast cell line TIG-
Methylated CpG of chromosome 15 of 7 or 15
Alternatively, a DNA fragment containing a CpG island rich in (ie, mCpG) may be isolated and introduced into a cell containing almost no methylated CpG. [Example] 1. Cell Search First, cell lines that can be used in the present invention, namely
A human normal cell line having only one methylated DNA region in the genome that can be visualized under an optical microscope was searched.
As a result, on the short arm of chromosome 15 of the male fetal lung fibroblast cell line TIG-7, a specific methylated DNA region (ie,
15p +) was found. Furthermore, as a result of various cytogenetic analysis, this region was found to be ribosomal DNA.
It was revealed that it was formed by amplification of (rDNA).

2. Effect of Demethylation The cell line TIG-7 was treated with 5-azacytidine (hereinafter referred to as 5azaC), which is a typical demethylating substance. As a result, the chromatin decondensation reaction (that is, demethylation reaction) was clearly observed only in the relevant region in the untreated group.
Was observed (Fig. 3). In each image schematic diagram shown in FIG. 3, a black-painted portion indicates a portion where a green fluorescence signal was observed, and the green fluorescence signal indicates a centromeric probe of chromosome 15. The shaded area indicates the area where the red fluorescence signal was observed, and the red fluorescence signal indicates the rDNA probe. a and a ′ are untreated groups, and b and b ′ are 5azaC treated groups (here, 15
There is p + chromatin decondensation). Also, C and C'are 5
It is an enlarged view of 15p + in an azaC processing group. Further, a, b, and c show black and white inverted images of DAPI staining, and a ′, b ′, and c ′ are schematic views of FISH images (FIG. 3).

When 5azaC was treated at a final concentration of 0 to 1.0 μM for 24 hours, the maximum concentration of 1.0 μM was
Despite the extremely low cell growth inhibition rate of about 20%, it is efficient in almost 100% of metaphase cells.
Chromatin disaggregation of p + was observed (Fig. 4).

3. Micronucleus induction experiment Using male fetal lung fibroblast cell line TIG-7,
Micronucleus induction experiments with FISH on DNA were performed.
That is, mitomycin C (MMC), which is a typical chromosomal structural abnormality inducing substance, and 5-, which is a typical demethylating substance.
Treated with azacitidine (5azaC). The results of comparative analysis of the frequency of micronuclei induced by treating them are shown below.

FIG. 5 shows F of micronucleus observed by a microscope.
The schematic diagram of an IDSH image is shown. Where a to k are rDN
A (+) micronucleus and l is rDNA (−) micronucleus.
Each FISH signal is the same as in FIG. That is, FIG.
In the schematic diagram of each of the images, a black-painted portion indicates a portion where a green fluorescence signal is observed, and the green fluorescence signal indicates a centromeric probe of chromosome 15. The shaded area indicates the area where the red fluorescence signal was observed, and the red fluorescence signal indicates the rDNA probe.

FIG. 6 is a graph showing the appearance frequency of micronuclei. Further, in FIG. 7, rDNA (+) and rDNA
The micronucleus appearance rate of (-) was shown. At each treatment concentration and treatment time, most of the micronuclei induced in the 5azaC treatment group were rDNA (+) (Fig. 5a-).
k), the rMC (-) micronuclei were predominant in the MMC-treated group (Fig. 5l). Here, 5azaC is 0.5μ
It was revealed that the micronucleus appearance rate reaches about 20% by the treatment with the concentration of M for 24 hours.

Under the conditions of the present experiment, the appearance frequency of rDNA (+) micronuclei was 70% or more in the 5azaC-treated group (Fig. 7, 5azaC-treated group). That is, it was proved that treatment with 5azaC on the male fetal lung fibroblast cell line TIG-7 induces micronuclei by demethylation. On the other hand, in the MMC-treated group, the appearance frequency of micronuclei is 2
0% or less, which is a clearly low value (Fig. 7,
MMC treatment group). Therefore, it was suggested that in the MMC-treated group, DNA strand breakage due to DNA damage may have occurred. From these results, the effectiveness of the method of the present invention became clear.

It has become gradually clear that demethylation is involved in various diseases such as cancer and injury due to aging, and all life phenomena such as cell differentiation, senescence, immortalization and carcinogenesis. Therefore, the present invention is intended for further study of various diseases and life phenomena, and
It is considered to be an extremely effective means for determining the demethylation action of biological substances. It is also considered to be extremely effective for screening preventive and therapeutic agents for various diseases such as cancer and injury due to aging. Besides this, the application range of the present invention is extremely wide.

Since the present invention is a novel method for evaluating demethylation activity and useful for cancer research and clinical studies, it is not limited to the above description, and various modifications, additions and diversions are possible. Needless to say. Further, although the above-mentioned example provides a method for DNA on a chromosome,
The conditions required by the user (for example, economics, processing capacity, required space, etc.) are similarly obtained by carrying out the artificially prepared nucleic acid molecule similarly using a genetic material introduced or activated by recombination. It is possible to provide an evaluation method using cells that are advantageous in terms of man-hours, etc.). Further, the measurement in the present invention is also included in the microscope in the present invention as long as it has a configuration capable of appropriately magnifying and imaging similar to various known microscopes, and minute measurement data can be optically measured. It is not necessary to have a portion (for example, an eyepiece lens) other than the configuration that can be acquired in a physical manner. Further, the means for performing the measurement process and the means for analyzing or utilizing the measurement result do not have to be provided in the same device or place, and for example, an evaluation device installed at a remote place (for example, a screen or a printer). By connecting with a display device such as a display device, a data analysis device, etc.) that can be sent and received, it is possible to quickly support the site such as bedside, operating room, remote facility, small-scale facility, etc. It can also contribute to the efficiency of secondary use based on the above.

The process of culturing cells and the test substance, the process of hybridization with a DNA probe, and the measurement of micronuclei are not limited to the above-mentioned processing conditions (particularly, the processing container), and can be used for various purposes. It may be changed according to. For example, the reaction with the DNA probe as the reaction nucleic acid may be carried out not on the slide glass but in other reaction vessels such as wells, porous arrays and flow cells.

Based on the above description, the present invention provides a pharmaceutical product (for example, a diagnostic drug, a therapeutic drug, a prophylactic drug, etc.) produced by the above evaluation method (particularly a screening method), an apparatus for executing the above evaluation method, and the above. It also includes a program (or software) for causing the execution device to execute the evaluation method, and a report document (for example, inspection table, chart, prescription, manufacturing request form, pharmaceutical quality table, etc.) output by the evaluation method. is there.

[0033]

INDUSTRIAL APPLICABILITY The present invention provides a method for screening the demethylation effect.

[Brief description of drawings]

FIG. 1 is a flowchart of a conventional mutagenicity screening method using micronuclei.

FIG. 2 is a flow chart showing a method for evaluating the demethylation action on DNA of a test substance using micronuclei as an index, which is one embodiment of the present invention.

FIG. 3 is a diagram of a chromosome showing a methylated chromosomal region and chromatin decondensation due to demethylation in the present invention.

FIG. 4 is a graph showing chromatin decondensation and cell viability by treatment with 5azaC.

FIG. 5 shows interphase nuclei containing micronuclei induced by 5azaC and MMC treatment.

FIG. 6 is a graph showing the appearance rate of micronuclei by treatment with 5azaC and MMC.

FIG. 7 is a graph showing a comparison of appearance rates of rDNA positive micronuclei.

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Claims (10)

[Claims] 1. A method for evaluating the demethylation action of a test substance on DNA, comprising: (1) a cell characterized in that it has only one visible site in a methylated DNA region on a chromosome. , Culturing in the presence of a test substance; (2) preparing a specimen using the cells cultured in (1); (3) measuring micronucleus frequency in the specimen obtained in (2) And (4) evaluating the presence or absence of a demethylation action on the basis of the micronucleus frequency measured in (3). 2. A method for evaluating the demethylation action of a test substance according to claim 1 on DNA, which further comprises (a) the preparation in (2) above prior to the measurement in (3) above. Hybridizing with a DNA probe complementary to a specific methylated region in the chromosome of cells contained in the prepared sample; (b) simultaneous or sequential detection with the measurement of micronucleus frequency in (4) above. Determining whether the DNA probe is contained in the formed micronucleus. 3. A method for evaluating the demethylation action of the test substance according to any one of claims 1 and 2 on DNA, wherein the preparation of the specimen of (2) supports the cells. An evaluation method comprising fixing to a body surface. 4. A method for evaluating the demethylation action of the test substance according to claim 1 on DNA, wherein the preparation of the specimen in (2) above leaves the cells naked. An evaluation method comprising nucleating and floating in a solution. 5. A method for evaluating the demethylation action of a test substance according to any one of claims 2 to 4 on DNA, comprising: (i) the DNA probe according to (a) being fluorescent. (Ii) the hybridization described in (a) above is performed by fluorescence in situ hybridization; (iii) the micronucleus frequency described in (4) above is measured using a microscope. The evaluation method is performed by detecting micronuclei, and the presence or absence of the DNA probe described in (b) above is determined by optically detecting the signal of the fluorescent substance. 6. A method for evaluating the demethylation action of a test substance on a nucleic acid molecule, comprising the steps of (1) culturing predetermined cells in the presence of the test substance, and (2) (1) Reacting a reactive nucleic acid molecule that specifically binds to a methylatable specific site of the cultured cell under conditions capable of hybridizing with the nucleic acid molecule of the cultured cell, and (3) depending on specific measurement conditions. And a step of detecting only methylation of the specific site in the cultured cells. 7. The method according to claim 6, wherein the reactive nucleic acid molecule is labeled with a measurable labeling substance, and the specific measurement condition measures the presence or absence of binding of the labeling substance at a specific site of the cell. The method described. 8. The method according to claim 7, wherein the detecting step includes a step of measuring the presence of the labeling substance in the micronucleus. 9. The method according to claim 6, wherein the intracellular nucleic acid molecule has only one measurable methylation site under specific measurement conditions. 10. The method according to claim 6, wherein the detecting step comprises measuring a plurality of cells under the same conditions and statistically processing information on methylation.