JP2004135661A - Method for evaluating degree of canceration in sample originating from mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating a degree of canceration in a sample originating from a mammal based on detection of gene abnormality useful for evaluating a diagnostic method, a therapeutic method and the like for finding out cancers in an early stage. <P>SOLUTION: The method for evaluating the degree of canceration in the sample originating from the mammal comprises (1) a first process for measuring methylation frequency of HAND1 gene included in the sample originating from the mammal or an index-value relatively relating to the same and (2) a second process for judging the degree of canceration of the sample depending on difference given by comparing the methylation frequency or the index-value relatively relating to the same measured in the process (1) with a control. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a method for evaluating the degree of canceration of a specimen derived from a mammal, and the like.

Although it is becoming increasingly clear that cancer is a disease caused by genetic abnormalities, the mortality rate of cancer patients is still high, and currently available diagnostic and therapeutic methods are not always satisfactory. Is shown. It is clinically important to detect cancer at an early stage, select an effective treatment method for the detected cancer, and perform aftercare such as confirmation of the recurrence of cancer after treatment.

"Cancer as a Genetic Disease" (ISBN4-89553-447-2 C3047), edited by Toshio Kuroki and Tadao Kakizoe, published by Medical View Inc. (May 10, 1994)

Therefore, a method for evaluating the degree of canceration of a specimen derived from a mammal based on detection of a genetic abnormality, which is suitable for a diagnostic method for early detection of cancer, evaluation of the effectiveness of a treatment method for cancer, confirmation of the presence or absence of cancer recurrence, etc. The development of is eagerly awaited.

The present inventors have conducted intensive studies under such circumstances, and found that the HAND1 gene in cancer cell lines is methylated at a significantly higher frequency as compared to a tissue sample of a healthy subject, and In the strain, the expression level of the HAND1 gene was found to be significantly lower than that in a tissue sample of a healthy subject, and the expression level of such a gene was increased by applying a DNA methylation inhibitor to a cancer cell line. The present inventors have found that they can be performed, and have reached the present invention.
That is, the present invention
1. A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal or an index value correlated therewith, and (2) the measured methylation frequency or an index correlated therewith An evaluation method characterized by having a second step of determining the degree of canceration of the sample based on a difference obtained by comparing the value with a control (hereinafter, may be referred to as the present evaluation method). ;
2. 2. The method according to the above 1, wherein the mammal-derived specimen is a cell;
3. 2. The evaluation according to the above item 1, wherein the mammal-derived specimen is a tissue (the term "tissue" has a broad meaning including body fluids such as blood, plasma, serum, and lymph, lymph nodes, etc.). Method;
4. A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, and (2) the method based on a difference obtained by comparing the measured methylation frequency with a control. An evaluation method comprising a second step of determining the degree of canceration of the sample;
5. 2. The evaluation method according to the above 1, wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the degree of malignancy of the mammal-derived cell;
6. 5. The evaluation method according to the above item 4, wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the malignancy of the mammal-derived cell;
7. The evaluation method according to the above item 1, wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue;
8. The evaluation method according to the above item 4, wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue;
9. The evaluation method according to the above item 8, wherein the tissue and the cancer are one or more types of tissues and cancers selected from the following (a) to (d):
(A) the tissue is gastric tissue and the cancer is gastric cancer;
(B) the tissue is colon tissue and the cancer is colon cancer;
(C) the tissue is pancreatic tissue, and the cancer is pancreatic cancer;
(D) the tissue is kidney tissue and the cancer is kidney cancer;
10. The methylation frequency of a gene is the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5′-CG-3 ′ present in the nucleotide sequence in the promoter region, untranslated region or translated region of the gene. The evaluation method according to the above 1 or 4, characterized in that:
11. The evaluation method according to the above item 10, wherein the tissue and the cancer are one or more types of tissues and cancers selected from the following (a) to (d): (a) the tissue is a stomach tissue; Gastric cancer;
(B) the tissue is colon tissue and the cancer is colon cancer;
(C) the tissue is pancreatic tissue, and the cancer is pancreatic cancer;
(D) the tissue is kidney tissue and the cancer is kidney cancer;
12. The methylation frequency of the gene is characterized by being the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence in the promoter region of the gene. Evaluation method according to the above 1 or 4,
13. The methylation frequency of the gene is the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence in the untranslated or translated region of the gene The evaluation method according to the above 1 or 4, characterized by the following;
14． The preceding paragraph, wherein the methylation frequency of the gene is the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence represented by SEQ ID NO: 1 Evaluation method described in 1;
15. 15. The evaluation method according to the above 14, wherein the tissue and the cancer are one or more types of tissues and cancers selected from the following (a) to (d): (a) the tissue is gastric tissue, and the cancer is gastric cancer; Is;
(B) the tissue is colon tissue and the cancer is colon cancer;
(C) the tissue is pancreatic tissue, and the cancer is pancreatic cancer;
(D) the tissue is kidney tissue and the cancer is kidney cancer;
16. A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring an index value having a correlation with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, and (2) an index value having a correlation with the measured methylation frequency. An evaluation method, comprising: a second step of determining the degree of canceration of the sample based on a difference obtained by comparing with a control;
17. 17. The evaluation method according to the above item 16, wherein the index value having a correlation with the methylation frequency of the HAND1 gene is the amount of an expression product of the HAND1 gene;
18. Item 18. The evaluation method according to Item 17, wherein the amount of the expression product of the HAND1 gene is the amount of a transcript of the gene;
19. Item 18. The evaluation method according to Item 17, wherein the amount of the expression product of the HAND1 gene is the amount of the translation product of the gene;
20. A method for searching for a substance having an ability to promote expression of a HAND1 gene,
(1) a first step of bringing a test substance into contact with cancer cells;
(2) After the first step (1), a second step of measuring the expression product amount of the HAND1 gene contained in the cancer cells,
(3) A search method comprising a third step of determining the ability of a test substance to promote the expression of a HAND1 gene based on a difference obtained by comparing the measured amount of an expression product with a control. (Hereinafter, it may be described as the search method of the present invention.);
21. 20. The search method according to the above item 20, wherein the cancer cell is a gastric cancer cell;
22. An anticancer agent comprising, as an active ingredient, a substance having the ability found by the search method of the preceding item 20, wherein the active ingredient is formulated in a pharmaceutically acceptable carrier:
23. An anticancer agent comprising, as an active ingredient, a polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence of HAND1, wherein the active ingredient is formulated in a pharmaceutically acceptable carrier;
24. Use of a methylated HAND1 gene as a cancer marker;
25. The use according to the above item 24, wherein the cancer marker is one or more cancer markers selected from gastric cancer markers, colon cancer markers, pancreatic cancer markers and renal adenocarcinoma markers;
26. A method for suppressing canceration, comprising a step of administering a substance that reduces the frequency of methylation of the HAND1 gene to cells in the body of a mammal that can be diagnosed with cancer;
27. 27. The method according to item 26, wherein the cancer is gastric cancer;
And so on.

According to the present invention, a method for evaluating the degree of canceration of a mammal-derived specimen can be provided.

Hereinafter, the present invention will be described in detail.
The present invention relates to the use of a methylated HAND1 gene as a cancer marker (eg, a gastric cancer marker, a colon cancer marker, a pancreatic cancer marker, a renal adenocarcinoma marker, etc.).
The HAND1 gene used as a cancer marker in the present invention is, for example, an untranslated region and a translated region (coding region) that serve as templates for mRNA containing a nucleotide sequence encoding the amino acid sequence of HAND1, and are located 5 ′ upstream thereof. And a human-derived gene containing a promoter region. Specific examples include the human-derived HAND1 gene described in Gene, 224, 77-86 (1998). The amino acid sequence of human-derived HAND1 and the base sequence encoding it are described in, for example, Genbank Accession No. NM_004821. In addition, the exon (hereinafter referred to as exon) located at the 5'-upstream side among the exons carrying the untranslated region and the translated region (coding region) serving as the template of mRNA containing the nucleotide sequence encoding the amino acid sequence of human-derived HAND1 1) and a promoter region located 5 ′ upstream thereof are described in Genbank Accession No. AC026688 and the like, for example. In the nucleotide sequence described in Genbank Accession No. AC026688, a sequence complementary to the nucleotide sequence represented by nucleotide numbers 24303 to 26500 is shown in SEQ ID NO: 1. In the nucleotide sequence represented by SEQ ID NO: 1, for example, the ATG codon encoding methionine located at the amino terminus of the amino acid sequence of human-derived HAND1 protein is represented by nucleotide numbers 1656 to 1658, The sequence is shown at base numbers 1400-2198. The HAND1 gene used in the present invention includes, in addition to the above-described genes having a known nucleotide sequence, the nucleotide sequence of such a nucleotide sequence due to a naturally occurring mutation due to a species difference of an organism, an individual difference or an organ, a difference between tissues, and the like. Genes having a base sequence in which a deletion, substitution or addition has occurred are also included.

(4) In mammals, there is a phenomenon in which only cytosine is methylated among four types of bases constituting a gene (genomic DNA). For example, in the HAND1 gene derived from a mammal, a part of cytosine in the genomic DNA of the gene is methylated. The methylation modification of DNA is performed in the base sequence represented by 5′-CG-3 ′ (C represents cytosine, G represents guanine, and the base sequence may be referred to as CpG hereinafter). Limited to cytosine. The site of methylation in cytosine is at position 5. During DNA replication prior to cell division, only cytosine in the template chain CpG is methylated immediately after replication, but cytosine in the nascent chain CpG is also immediately methylated by the action of methyltransferase . Therefore, the state of DNA methylation is inherited by two new sets of DNA as they are even after DNA replication.

The `` methylation frequency '' in the first step of the evaluation method of the present invention refers to, for example, when the presence or absence of methylation of cytosine in CpG to be investigated is examined for a plurality of haploids, the cytosine is methylated. Expressed as a percentage of haploid.
In the first step of the evaluation method of the present invention, the “index value having a correlation with (methylation frequency)” refers to, for example, the amount of the expression product of the HAND1 gene (more specifically, the amount of the transcript of the gene). And the amount of translation product of the gene). In the case of such an amount of the expression product, there is a negative correlation such that the higher the methylation frequency is, the smaller the methylation frequency is.

Examples of the mammal-derived specimen in the first step of the evaluation method of the present invention include, for example, cancer cells such as stomach cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells or a tissue containing the same, and stomach cancer, colon cancer, pancreatic cancer Or a cell which may contain DNA derived from a cancer cell such as a renal adenocarcinoma cell, or a tissue containing the same (the term “tissue” refers to a body fluid such as blood, plasma, serum, lymph, pancreatic juice, lymph node, etc. ) Or biological samples such as body secretions (such as manure and milk). Specifically, for example, when the cancer is gastric cancer, the gastric mucosa layer (superficial epithelium, glandular tissue, lamina propria and mucosal muscularis), the gastric submucosal tissue layer, and the (proprietary) muscle layer And a serosal layer.
These biological samples may be used as they are as samples, or biological samples prepared by various operations such as separation, fractionation, and immobilization from such biological samples may be used as samples.
When the mammal-derived specimen is blood, a blood sample collected by a periodic medical examination, a simple test, or the like can be given.

方法 In the first step of the evaluation method of the present invention, a method for measuring the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal or an index value having a correlation therewith may be performed, for example, as follows.

As a first method, a DNA derived from a sample is brought into contact with a reagent for modifying unmethylated cytosine, and then the DNA is used as a template, and a polymerase is used using a primer capable of discriminating the presence or absence of cytosine methylation to be analyzed. A method of conducting a chain reaction (hereinafter referred to as PCR) and examining the amount of the obtained amplification product can be mentioned.
First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like. When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (gastric cancer, colon cancer, pancreatic cancer or kidney). (Including DNA derived from cancer cells such as adenocarcinoma cells), it is possible to analyze DNA derived from cancer cells such as stomach cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells while avoiding DNA derived from blood cells. In addition, the sensitivity for detecting cancer cells such as gastric cancer, colon cancer, pancreatic cancer, or renal adenocarcinoma cells, and tissues containing the same can be improved.
Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is used as a template and amplified by PCR using primers that can identify the presence or absence of cytosine methylation to be analyzed. Check the amount of amplification product obtained. The cytosine to be analyzed can be selected from cytosines in the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. .
Here, as the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene, exon 1 of the human-derived HAND1 gene, The base sequence of genomic DNA containing a promoter region located 5 ′ upstream can be mentioned. More specifically, the base sequence shown in SEQ ID NO: 1 (the base sequence of the base sequence described in Genbank Accession No. AC026688) can be used. Base sequence represented by base numbers 24303 to 26500). In the nucleotide sequence represented by SEQ ID NO: 1, the ATG codon encoding the amino-terminal methionine of human-derived HAND1 protein is represented by nucleotide numbers 1656 to 1658, and the nucleotide sequence of exon 1 is represented by nucleotide number 1400 ~ 2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, It shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells. More specifically, as a cytosine having a high methylation frequency in gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, 1187, Cytosine which is a base number represented by 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like can be mentioned.

As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium bisulfite can be used. Incidentally, in principle, a reagent that specifically modifies only methylated cytosine may be used.

In order to contact the extracted DNA with a reagent that modifies unmethylated cytosine, for example, the DNA is first denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite ( The solution is treated at 55 ° C. for about 10 to 16 hours (overnight) at a concentration of, for example, 3 M). To promote the reaction, denaturation at 95 ° C and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine.
Next, the DNA sequence treated with bisulfite or the like as a template, and the base sequence when the methylated cytosine is included [cytosine at the methylated position (cytosine in CpG) remains cytosine, Unmethylated cytosine (cytosine not included in CpG) is a uracil base sequence] and PCR using a pair of methylation-specific primers each selected from a base sequence complementary to the base sequence (hereinafter referred to as “ , And methylation-specific PCR.), And a base sequence in the case where DNA treated with bisulfite or the like is used as a template and cytosine is not methylated (all cytosines become uracils). PCR using a pair of unmethylation-specific primers each selected from a base sequence complementary to the base sequence and Sometimes also referred to as CR.) And perform.
In the above PCR, in the case of PCR using methylation-specific primers (the former), DNA in which cytosine to be analyzed is methylated is amplified, while in the case of PCR using non-methylation-specific primers ( In the latter case, DNA in which cytosine to be analyzed is not methylated is amplified. By comparing the amounts of these amplification products, the presence or absence of methylation of the target cytosine is examined. In this way, the methylation frequency can be measured.

Here, as a primer, a base containing a methylated cytosine in consideration of the fact that unmethylated cytosine is converted to uracil and that methylated cytosine is not converted to uracil. A PCR primer specific to a sequence (a methylation-specific primer) is designed, and a PCR primer specific to a nucleotide sequence containing unmethylated cytosine (a non-methylation-specific primer) is designed. Based on the DNA strand that has been chemically converted by bisulfite treatment and is no longer complementary, based on each strand of DNA that was originally double-stranded, a methylation-specific primer Unmethylated specific primers can also be made. Such a primer is preferably designed so as to contain cytosine in CpG near the 3 'end of the primer in order to increase the specificity of methyl and non-methyl. Further, one of the primers may be labeled to facilitate the analysis.

More specifically, a primer for measuring the methylation frequency of the HAND1 gene by methylation-specific PCR includes, for example, a nucleotide sequence in the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. It can be designed as described above based on the base sequence containing one or more cytosines in the existing CpG. For example, cytosine in CpG present in a region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, specifically, nucleotide numbers 1153, 1160, 1178, and 1187 in the nucleotide sequence represented by SEQ ID NO: 1 , 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like. Examples of such primers are shown below.
<Unmethylated specific primer>
U1: 5'-AATAGTTTAGGGTGTTGGTT-3 '(SEQ ID NO: 2)
U2: 5'-AATTTTACACTCAACCCA-3 '(SEQ ID NO: 3)
<Methylation-specific primer>
M1: 5'-AATAGTTTAGGGCGTTGGTC-3 '(SEQ ID NO: 4)
M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5)

As a reaction solution in the methylation-specific PCR, for example, 50 ng of DNA as a template, 1 μl of each primer solution of 10 pmol / μl, 4 μl of 2.5 mM dNTP, and 10 × buffer (100 mM Tris-HCl pH8 .3, 500 mM KCl, 20 mM MgCl ₂ ) 2.5 μl and heat-resistant DNA polymerase 5 U / μl 0.2 μl are mixed, and sterile ultrapure water is added to make the reaction volume 25 μl. Can be. As the reaction conditions, for example, after keeping the above-mentioned reaction solution at 95 ° C for 10 minutes, one cycle of 95 ° C for 30 seconds, then 55 to 65 ° C for 30 seconds, and further 72 ° C for 30 seconds The condition for performing the heat retention for 30 to 40 cycles is mentioned.
After performing such PCR, the amounts of the obtained amplification products are compared. For example, an analysis method (denaturing polyacrylamide gel electrophoresis or agarose gel) capable of comparing the amount of each amplification product obtained by PCR using a methylation-specific primer and PCR using an unmethylation-specific primer In the case of (electrophoresis), the gel after electrophoresis is stained with DNA to detect the band of the amplification product, and the concentrations of the detected bands are compared. Here, instead of DNA staining, a pre-labeled primer can be used to compare the band concentrations using the label as an index. When quantification is required, real-time PCR, a highly accurate quantification PCR method that can detect, for example, a two-fold difference in gene amount, can be performed by monitoring the PCR reaction product in real time and performing kinetic analysis. Can also be used to compare the amount of each product. Examples of the method for performing real-time PCR include a method using a probe such as a template-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. As devices and reagents for the real-time PCR method, commercially available devices and kits can be used.

Such a method is generally called methylation-specific PCR, and is a method reported by Herman et al. (Herman et al., Proc. Natl. Acad. Sci USA, 93, 9821-9826, 1996). This is a method utilizing the difference in chemical properties between cytosine and 5-methylcytosine.

As a second method, after contacting a DNA derived from a sample with a reagent for modifying unmethylated cytosine, a DNA containing cytosine to be analyzed is amplified by PCR using the DNA as a template, and the base of the resulting amplification product is amplified. A method for directly analyzing a sequence can be given.
First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like. When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (gastric cancer, colon cancer, pancreatic cancer or kidney). (Including DNA derived from cancer cells such as adenocarcinoma cells), it is possible to analyze DNA derived from cancer cells such as stomach cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells while avoiding DNA derived from blood cells. In addition, the sensitivity for detecting cancer cells such as gastric cancer, colon cancer, pancreatic cancer, or renal adenocarcinoma cells, and tissues containing the same can be improved.
Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is used as a template in the base sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. By performing PCR using primers designed as described below based on the base sequence containing cytosine in the base sequence represented by one or more CpGs, the DNA containing cytosine to be analyzed is amplified. The nucleotide sequence of the amplification product obtained is directly analyzed.
Here, as the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene, exon 1 of the human-derived HAND1 gene, The base sequence of genomic DNA containing a promoter region located 5 ′ upstream can be mentioned. More specifically, the base sequence shown in SEQ ID NO: 1 (the base sequence of the base sequence described in Genbank Accession No. AC026688) can be used. Base sequence represented by base numbers 24303 to 26500). In the nucleotide sequence represented by SEQ ID NO: 1, the ATG codon encoding the amino-terminal methionine of human-derived HAND1 protein is represented by nucleotide numbers 1656 to 1658, and the nucleotide sequence of exon 1 is represented by nucleotide number 1400 ~ 2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, It shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells. More specifically, as a cytosine having a high methylation frequency in gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, 1187, Cytosine which is a base number represented by 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like can be mentioned.

As a primer used for the PCR, a primer pair capable of amplifying a DNA having a base sequence containing the cytosine is designed based on a base sequence 5 ′ upstream and a base sequence 3 ′ downstream of the cytosine to be analyzed. Good to do. The nucleotide sequence for primer design is selected so as not to contain cytosine in CpG to be analyzed. If the base sequence selected for primer design does not contain any cytosine, the selected base sequence and the base sequence complementary to such base sequence should be used as the base sequence of the primer as they are. Can be. In addition, when the base sequence selected for primer design contains cytosine other than the analysis target, but the cytosine is not cytosine in CpG, the primer is designed in consideration that these cytosines are converted to uracil. . That is, a pair of primers each having a base sequence in which all cytosines are uracil and a base sequence complementary to the base sequence are designed. Furthermore, when the base sequence selected for primer design contains cytosine other than the analysis target and the cytosine is cytosine in CpG, unmethylated cytosine is converted to uracil, and methylation is performed. The primers are designed in consideration that the cytosine undergoing the reaction is not converted to uracil. That is, when a methylated cytosine is contained, the base sequence [the cytosine at the methylated position (cytosine in CpG) remains cytosine, and the unmethylated cytosine (cytosine not included in CpG) is Uracil base sequence] and a base sequence complementary to the base sequence, and a pair of methylation-specific primers respectively selected from base sequences when cytosine is not methylated (all cytosines are uracil And a pair of unmethylation-specific primers each having a base sequence complementary to the base sequence. In this case, in the PCR described above, a methylation-specific primer pair and an unmethylation-specific primer pair are mixed and used in equal amounts.

試 薬 As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium bisulfite can be used. Incidentally, in principle, a reagent that modifies only methylated cytosine may be used.

In order to contact the extracted DNA with a reagent that modifies unmethylated cytosine, for example, the DNA is first denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite ( The solution is treated at 55 ° C. for about 10 to 16 hours (overnight) at a concentration of, for example, 3 M). To promote the reaction, denaturation at 95 ° C and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine.
Next, PCR is performed using DNA treated with bisulfite or the like as a template and using primers designed as described above. The nucleotide sequences of the obtained amplification products are compared, and the methylation frequency can be measured from the comparison.

More specifically, a primer for measuring the methylation frequency of the HAND1 gene by direct analysis of the nucleotide sequence is, for example, a primer in the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. Can be designed as described above on the basis of the nucleotide sequence containing one or more cytosines in CpG existing in CpG. For example, cytosine in CpG present in a region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, specifically, nucleotide numbers 1153, 1160, 1178, and 1187 in the nucleotide sequence represented by SEQ ID NO: 1 , 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like. For example, when the primers B1 and B2 shown below are used, a DNA (228 bp) having a base sequence obtained by bisulfite treatment of a base sequence represented by base numbers 1234 to 1461 of the base sequence shown by SEQ ID NO: 1 is amplified. Is done. The primer pair analyzes the methylation frequency of cytosine represented by base numbers 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, and 1434 in the base sequence represented by SEQ ID NO: 1. Can be used as primers.
<Primer>
B1: 5'-TAGAGTAGGGAGTTGAGTGGGAG-3 '(SEQ ID NO: 6)
B2: 5'-ACCCAAAAACCTATTTAACCCTTCTA-3 '(SEQ ID NO: 7)

As a reaction solution in PCR, for example, 50 ng of DNA as a template, 1 μl of each primer solution of 20 pmol / μl, 3 μl of 2 mM dNTP, and 10 × buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, A reaction solution in which 2.5 μl of 15 mM MgCl ₂ ) and 0.2 μl of 5 U / μl of heat-resistant DNA polymerase are mixed, and sterile ultrapure water is added to adjust the volume to 25 μl can be given. As the reaction conditions, for example, after keeping the above-mentioned reaction solution at 95 ° C. for 10 minutes, one cycle is performed at 95 ° C. for 30 seconds, then at 57 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds. Conditions for performing heat retention for 30 to 40 cycles can be given.
After performing such PCR, the nucleotide sequences of the obtained amplification products are compared, and the methylation frequency is measured from the comparison.
That is, by directly analyzing the base sequence of the amplification product, it is determined whether the base at the position corresponding to cytosine to be analyzed is cytosine or thymine (uracil). In a chart of peaks indicating bases in the obtained amplification product, by comparing the area of the peak indicating cytosine detected at the position corresponding to the cytosine to be analyzed with the area of the peak indicating thymine (uracil), The frequency of cytosine methylation to be analyzed can be measured. In addition, as a method of directly analyzing a nucleotide sequence, a cloned DNA is prepared from a plurality of clones obtained by once cloning an amplification product obtained by PCR using Escherichia coli or the like as a host, and The nucleotide sequence may be analyzed. The frequency of cytosine methylation to be analyzed can also be measured by determining the proportion of the sample in which the base detected at the position corresponding to cytosine to be analyzed in the sample to be analyzed is cytosine.

As a third method, after contacting the DNA derived from the sample with a reagent that modifies unmethylated cytosine, the DNA is hybridized with a probe capable of identifying the presence or absence of cytosine methylation to be analyzed, A method for examining the presence or absence of probe binding can also be mentioned.
First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like. When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (gastric cancer, colon cancer, pancreatic cancer or kidney). (Including DNA derived from cancer cells such as adenocarcinoma cells), it is possible to analyze DNA derived from cancer cells such as stomach cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells while avoiding DNA derived from blood cells. In addition, the sensitivity for detecting cancer cells such as gastric cancer, colon cancer, pancreatic cancer, or renal adenocarcinoma cells, and tissues containing the same can be improved.
Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is hybridized with a probe capable of discriminating the presence or absence of cytosine methylation to be analyzed, and The presence or absence of binding to the probe is examined. The cytosine to be analyzed can be selected from cytosines in the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. .
Here, as the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene, exon 1 of the human-derived HAND1 gene, The base sequence of genomic DNA containing a promoter region located 5 ′ upstream can be mentioned. More specifically, the base sequence shown in SEQ ID NO: 1 (the base sequence of the base sequence described in Genbank Accession No. AC026688) can be used. Base sequence represented by base numbers 24303 to 26500). In the nucleotide sequence represented by SEQ ID NO: 1, the ATG codon encoding the amino-terminal methionine of human-derived HAND1 protein is represented by nucleotide numbers 1656 to 1658, and the nucleotide sequence of exon 1 is represented by nucleotide number 1400 ~ 2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, It shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells. More specifically, as a cytosine having a high methylation frequency in gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, 1187, Cytosine which is a base number represented by 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like can be mentioned.

In the probe used for the hybridization, unmethylated cytosine is converted to uracil based on the base sequence containing cytosine to be analyzed, and methylated cytosine is not converted to uracil. It is good to design in consideration of this. That is, when a methylated cytosine is contained, the base sequence [the cytosine at the methylated position (cytosine in CpG) remains cytosine, and the unmethylated cytosine (cytosine not included in CpG) is Or a methylation-specific probe having a nucleotide sequence complementary to such a nucleotide sequence, and a nucleotide sequence when cytosine is not methylated (a nucleotide sequence in which all cytosines are uracil) ) Or a non-methylation-specific probe having a nucleotide sequence complementary to such a nucleotide sequence is designed. Such a probe may be used after being labeled in order to facilitate analysis of the presence or absence of binding between DNA and the probe. The probe may be used by immobilizing it on a carrier according to a usual method. In this case, DNA extracted from a mammal-derived specimen may be labeled in advance.

試 薬 As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium bisulfite can be used. Incidentally, in principle, a reagent that specifically modifies only methylated cytosine may be used.

In order to contact the extracted DNA with a reagent that modifies unmethylated cytosine, for example, the DNA is first denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite ( The solution is treated at 55 ° C. for about 10 to 16 hours (overnight) at a concentration of, for example, 3 M). To accelerate the reaction, denaturation at 95 ° C and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine.
If necessary, the DNA may be amplified in advance by performing PCR in the same manner as in the second method using DNA treated with bisulfite or the like as a template.
Next, hybridization is performed between DNA treated with bisulfite or the like or DNA previously amplified by the PCR and a probe capable of discriminating the presence or absence of cytosine methylation to be analyzed. By comparing the amount of DNA binding to the methylation-specific probe with the amount of DNA binding to the unmethylation-specific probe, the frequency of cytosine methylation to be analyzed can be measured.

More specifically, a probe for measuring the methylation frequency of the HAND1 gene is, for example, cytosine in CpG present in the base sequence in the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. Can be designed as described above based on a base sequence containing one or more of the following. For example, cytosine in CpG present in a region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, specifically, nucleotide numbers 1153, 1160, 1178, and 1187 in the nucleotide sequence represented by SEQ ID NO: 1 , 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like. Examples of such probes are shown below.
<Set 1>
Unmethylated specific probe: 5'-AAGTATGTAGTTTTTGTGTTTG-3 '(SEQ ID NO: 8)
Methylation-specific probe: 5'-AAGTACGTAGTTTTCGTGTTCG-3 '(SEQ ID NO: 9)
<Set 2>
Non-methyl specific probe: 5'-TGTGGGTTGAGTGTAAAATT-3 '(SEQ ID NO: 10)
Methylation-specific probe: 5'-CGCGGGTTGAGCGTAAAATT-3 '(SEQ ID NO: 11)

Hybridization is carried out, for example, by the usual method described in Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory press, etc. It can be performed according to. Hybridization is usually performed under stringent conditions. Here, “stringent conditions” refers to, for example, hybridization at 45 ° C. in a solution containing 6 × SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is referred to as 10 × SSC). Are formed and then washed with 2 × SSC at 50 ° C. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6). The salt concentration in the washing step can be selected, for example, from the condition of 2 × SSC at 50 ° C. (low stringency condition) to the condition of 0.2 × SSC up to 50 ° C. (high stringency condition). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). Also, both salt concentration and temperature can be varied.
After performing such hybridization, by comparing the amount of DNA bound to the methylation-specific probe with the amount of DNA bound to the unmethylation-specific probe, cytosine to be analyzed (that is, It is possible to measure the frequency of methylation of cytosine in CpG contained in the base sequence used as the basis for the design.

As a fourth method, a method may be used in which a restriction enzyme capable of identifying the presence or absence of methylation of cytosine to be analyzed is allowed to act on DNA derived from a sample, and then the presence or absence of digestion by the restriction enzyme is examined.
First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like. When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (gastric cancer, colon cancer, pancreatic cancer or kidney). (Including DNA derived from cancer cells such as adenocarcinoma cells), it is possible to analyze DNA derived from cancer cells such as stomach cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells while avoiding DNA derived from blood cells. In addition, the sensitivity for detecting cancer cells such as gastric cancer, colon cancer, pancreatic cancer, or renal adenocarcinoma cells, and tissues containing the same can be improved.
Next, a restriction enzyme capable of discriminating the presence or absence of cytosine methylation to be analyzed is allowed to act on the extracted DNA, and then the presence or absence of digestion by the restriction enzyme is examined. The cytosine to be analyzed can be selected from cytosines in the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. .
Here, as the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene, exon 1 of the human-derived HAND1 gene, The base sequence of genomic DNA containing a promoter region located 5 ′ upstream can be mentioned. More specifically, the base sequence shown in SEQ ID NO: 1 (the base sequence of the base sequence described in Genbank Accession No. AC026688) can be used. Base sequence represented by base numbers 24303 to 26500). In the nucleotide sequence represented by SEQ ID NO: 1, the ATG codon encoding the amino-terminal methionine of human-derived HAND1 protein is represented by nucleotide numbers 1656 to 1658, and the nucleotide sequence of exon 1 is represented by nucleotide number 1400 ~ 2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, It shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells. More specifically, as a cytosine having a high methylation frequency in gastric cancer, colon cancer, pancreatic cancer or renal adenocarcinoma cells, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, 1187, Cytosine which is a base number represented by 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like can be mentioned.

The “restriction enzyme capable of discriminating the presence or absence of cytosine methylation” (hereinafter, also referred to as a methylation-sensitive restriction enzyme) used in the method refers to a digestion sequence that does not digest a recognition sequence containing methylated cytosine. , A restriction enzyme capable of digesting a recognition sequence containing unmethylated cytosine. In the case of DNA in which cytosine contained in the recognition sequence is methylated, the DNA is not cleaved even when a methylation-sensitive restriction enzyme is acted on, while in the case of DNA in which cytosine contained in the recognition sequence is not methylated If a methylation-sensitive restriction enzyme is allowed to act, the DNA is cleaved. Specific examples of methylation-sensitive enzymes include, for example, HpaII, BstUI, and the like.

As a method for examining the presence or absence of digestion by the restriction enzyme, for example, using a DNA that has been subjected to a methylation-sensitive restriction enzyme containing a cytosine to be analyzed in the recognition sequence as a template, a recognition sequence containing the cytosine to be analyzed is used as a template. A method may be mentioned in which PCR is performed using a primer pair capable of amplifying a DNA that does not contain a recognition sequence of the enzyme other than the recognition sequence, and the presence or absence of DNA amplification (amplification product) is examined. When the cytosine to be analyzed is methylated, an amplification product is obtained. On the other hand, if the cytosine to be analyzed is not methylated, no amplification product can be obtained. Thus, by comparing the amount of the amplified DNA with the amount of DNA, the frequency of cytosine methylation to be analyzed can be measured.
When quantification is required, real-time PCR, a highly accurate quantification PCR method that can detect, for example, a two-fold difference in gene amount, can be performed by monitoring the PCR reaction product in real time and performing kinetic analysis. Can also be used to compare the amount of each product. Examples of the method for performing real-time PCR include a method using a probe such as a template-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. As devices and reagents for the real-time PCR method, commercially available devices and kits can be used.
For example, in the case of cytosine represented by nucleotide numbers 1272 and 1377 in the nucleotide sequence represented by SEQ ID NO: 1, the cytosine is included in the recognition sequence of HpaII, and the methylation frequency of the cytosine is measured by the above method. be able to.

In addition, as another method for examining the presence or absence of digestion by the restriction enzyme, for example, a DNA that has been subjected to a methylation-sensitive restriction enzyme containing a cytosine to be analyzed in a recognition sequence is derived from the HAND1 gene, and Alternatively, Southern hybridization using a DNA not containing the recognition sequence of the restriction enzyme as a probe to determine the length of the hybridized DNA may be used. When the cytosine to be analyzed is methylated, a longer DNA is detected than when the cytosine is not methylated. By comparing the detected amount of long DNA with the amount of short DNA, the frequency of cytosine methylation to be analyzed can be measured.

Using the various methods described above, the methylation frequency of the HAND1 gene contained in a mammal-derived specimen is measured. The measured methylation frequency and, for example, the methylation frequency of the HAND1 gene contained in a sample derived from a healthy mammal that can be diagnosed as having no cancer cells such as gastric cancer, colon cancer, pancreatic cancer, and renal adenocarcinoma cells ( Control), and the degree of canceration of the sample is determined based on the difference obtained by the comparison. For example, if the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal is higher than that of the control (if the HAND1 gene is hypermethylated compared to the control), the degree of canceration of the specimen is reduced. It can be determined to be high by comparison with the control.
Here, the term "degree of canceration" has the same meaning as generally used in the art, and specifically means, for example, when a mammal-derived specimen is a cell, means the malignancy of the cell. In addition, for example, when the mammal-derived specimen is a tissue, it means the amount of cancer cells present in the tissue, and the like.

The expression of the HAND1 gene is lower in cancer cells such as gastric cancer cells than in specimens of cells and tissues derived from healthy mammals. This is because the frequency of methylation of the gene is high in cancer cells such as gastric cancer cells, and the gene cannot be normally expressed. As a result, the amount of the expression product of the gene (more specifically, the amount of the transcript And the amount of translation products). As described above, in the evaluation method of the present invention and the like, instead of the methylation frequency, an index value having a correlation with the methylation frequency (in the above case, an index value indicating the amount of the expression product and having a negative correlation). ) May be measured.
That is, in the evaluation method of the present invention, an index value (for example, the amount of an expression product) correlated with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal is measured, and the index value is correlated with the measured methylation frequency. The degree of canceration of the sample can be determined based on a difference obtained by comparing a relevant index value (eg, the amount of an expression product) with a control.

In the first step of the evaluation method of the present invention, as a method of measuring an index value that is correlated with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, for example, the amount of mRNA that is a transcript of the HAND1 gene Can be mentioned. For the measurement, for example, RT (Reverse Transcription) -PCR method, Northern blot method [Molecular Cloning, Cold Spring Harbor Laboratory (1989)], in situ RT-PCR method [Nucleic Acids Res., 21, 3159-3166 (1993) )], In situ hybridization, and NASBA [Nucleic acid sequence-based amplification, nature, 350, 91-92 (1991)].
A sample containing mRNA which is a transcription product of the HAND1 gene contained in a specimen derived from a mammal may be prepared by extraction, purification, or the like, from the specimen according to an ordinary method.
When Northern blotting is used to measure the amount of mRNA contained in the prepared sample, the detection probe is a HAND1 gene or a part thereof (restriction enzyme cleavage of the HAND1 gene, based on the nucleotide sequence of the HAND1 gene). A chemically synthesized oligonucleotide of about 100 bp to about 1000 bp) that gives detectable specificity under the detection conditions used in hybridization with mRNA contained in the sample. There is no particular restriction.
When the RT-PCR method is used to measure the amount of mRNA contained in the prepared sample, the primer used may be any one that can specifically amplify only the HAND1 gene. There are no particular restrictions on the region to be amplified or the base length. Examples of such primers include the following primers (S: sense, A: antisense) and the like. Using these primers, the amount of the transcript can also be measured by the RT-PCR method as described in Examples below.
When quantification is required, real-time PCR, a highly accurate quantification PCR method that can detect, for example, a two-fold difference in gene amount, can be performed by monitoring the PCR reaction product in real time and performing kinetic analysis. Can also be used to compare the amount of each product. Examples of the method for performing real-time PCR include a method using a probe such as a template-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. As devices and reagents for the real-time PCR method, commercially available devices and kits can be used.
S: 5'-GTGAGAGCAAGCGGAAAAG-3 '(SEQ ID NO: 12)
A: 5'-GTGCGTCCTTTAATCCTCTTC-3 '(SEQ ID NO: 13)

Further, in the first step of the evaluation method of the present invention, as another method for measuring an index value having a correlation with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, for example, a translation product of the HAND1 gene A method for measuring the amount of the HAND1 protein can be given. For the measurement, for example, using a specific antibody (monoclonal antibody, polyclonal antibody) against the HAND1 protein, immunoblotting described in Cell Engineering Handbook, Yodosha, 207 (1992), and separation by immunoprecipitation A known method such as an indirect competitive inhibition method (ELISA method) may be used.
Incidentally, a specific antibody against the HAND1 protein can be produced according to a usual immunological method using the protein as an immunizing antigen.

指標 Using various methods as described above, an index value having a correlation with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal is measured. An index value that is correlated with the measured methylation frequency is, for example, a correlation between the methylation frequency of the HAND1 gene contained in a healthy mammal-derived specimen that can be diagnosed as having no cancer cells such as gastric cancer cells. By comparing with a certain index value (control), the degree of canceration of the sample is determined based on the difference obtained by the comparison. If the index value that has a positive correlation with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal is higher than the control or the index value that has a negative correlation is lower than the control, (If the HAND1 gene is hypermethylated as compared to the control), it can be determined that the degree of canceration of the specimen is higher than that of the control.

In the evaluation method of the present invention, primers, probes or specific antibodies that can be used in various methods for measuring the methylation frequency of the HAND1 gene or an index value correlated therewith include gastric cancer, colon cancer, pancreatic cancer, and bladder cancer. It is useful as a reagent for a kit for detecting cancer cells such as renal adenocarcinoma, chorioadenocarcinoma, and adrenal cortical adenocarcinoma cells. The present invention provides a kit for detecting cancer cells such as stomach cancer, colon cancer, pancreatic cancer, bladder cancer, renal adenocarcinoma, villous adenocarcinoma, adrenal cortical adenocarcinoma cells, which contain these primers, probes or specific antibodies as reagents. Or detection of cancer cells such as gastric cancer, colon cancer, pancreatic cancer, bladder cancer, renal adenocarcinoma, villous adenocarcinoma, adrenal cortical adenocarcinoma cells in which these primers, probes or specific antibodies are immobilized on a carrier. The present invention also provides a chip for use, and the scope of the evaluation method of the present invention includes use in the form of a detection kit or a detection chip as described above utilizing the substantial principle of the method. .

The expression of the HAND1 gene is lower in cancer cells such as gastric cancer cells than in specimens of cells and tissues derived from healthy mammals. On the other hand, as shown in the examples described later, the amount of the expression product of the gene can be increased by causing a substance that inhibits DNA methylation related to the HAND1 gene to act on cancer cells such as gastric cancer cells. This is a substance capable of compensating for a decrease in the expression level of the HAND1 gene in a cancer cell such as a gastric cancer cell or a concomitant decrease in the function-for example, non-methylation (or does not cause methylation abnormality as observed in cancer) ) HAND1 gene [Gene, 224, 77-86 (1998)], an expression product of the gene, a substance capable of promoting the expression of the gene (for example, a substance that inhibits DNA methylation related to the HAND1 gene, a HAND1 gene A substance that reduces the frequency of methylation of gastric cancer)-for treating cancer such as gastric cancer, gastric mucosa (surface epithelium, glandular tissue, lamina propria and mucosal muscularis), gastric submucosa, (proprietary) muscle It is useful for suppressing canceration of normal tissues such as the stratum corneum and the serosal layer.
For example, canceration will be suppressed by administering a substance that reduces the frequency of methylation of the HAND1 gene to cells in the body of a mammal that can be diagnosed with cancer. Further, for example, by providing a substance that inhibits DNA methylation related to the HAND1 gene to cancer cells such as gastric cancer cells, cytosine in CpG present in the nucleotide sequence in the promoter region or coding region of the HAND1 gene can be converted to normal tissue. In the same manner as described above, it is possible to increase the expression level of mRNA, which is a transcription product of the HAND1 gene, and thereby increase the expression level of the HAND1 protein, which is a translation product of the HAND1 gene, by hypomethylation. Further, for example, by introducing a cDNA comprising a nucleotide sequence encoding the amino acid sequence of the HAND1 gene or the HAND1 protein into a cancer cell such as a gastric cancer cell, the expression level of the HAND1 protein in a cancer cell such as a gastric cancer cell can be increased. right.

That is, the present invention is characterized in that (1) the active ingredient contains a substance having an ability to promote the expression of the HAND1 gene, and the active ingredient is formulated in a pharmaceutically acceptable carrier. An anticancer agent comprising: (2) a polynucleotide having a base sequence encoding the amino acid sequence of HAND1 as an active ingredient, wherein the active ingredient is formulated in a pharmaceutically acceptable carrier. (Hereinafter sometimes collectively referred to as the anticancer agent of the present invention).

The dosage form of the anticancer agent of the present invention is not particularly limited as long as it is a normal preparation, but such a preparation is pharmaceutically acceptable, for example, a water-soluble solvent, a non-water-soluble solvent, a buffer, a solubilizing agent. It can be produced by blending an active ingredient with a carrier such as an isotonic agent and a stabilizer. If necessary, auxiliary agents such as preservatives, suspending agents and emulsifiers may be added. In the case of parenteral administration (generally, injection or the like is preferable), the anticancer agent can be used in the form of a usual liquid such as a solution.
An effective amount of the anticancer agent of the present invention can be parenterally administered to a mammal such as a human (eg, cells in a mammal that can be diagnosed with cancer). For example, parenteral administration methods include, for example, injection (subcutaneous, intravenous, topical) and the like.
The dose varies depending on the age, sex, body weight, degree of disease, type of the anticancer agent of the present invention, dosage form, etc. of the mammal to be administered, but usually, the active ingredient works effectively in the cells of the patient. It is sufficient to administer an amount of the active ingredient that produces an intracellular level equal to the appropriate concentration level. In addition, the above-mentioned daily dose can be administered once or in several divided doses.

Here, as a method for introducing a polynucleotide having a nucleotide sequence encoding the amino acid sequence of HAND1 into a cell, a method for introducing a gene using a viral vector and a method for introducing a gene using a non-viral vector (Nikkei Science, 1994 April issue, pp. 20-45, Experimental Medicine Special Edition, 12 (15) (1994), Experimental Medicine Separate Volume, “Basic Technology for Gene Therapy,” Yodosha (1996), and the like.
The former gene transfer method includes, for example, encoding TR4 or mutant TR4 into a DNA virus or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, and simbis virus. And a method of incorporating and introducing DNA to be used. Examples of a gene transfer method using a non-viral vector include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. Is raised.
In addition, as a method of using a polynucleotide having a base sequence encoding the amino acid sequence of HAND1 as an active ingredient of a gene therapy agent as an anticancer agent, an in vivo method of directly introducing the polynucleotide into the body, a specific cell from humans, Ex vivo method to introduce the polynucleotide into the cells outside the body and return the cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 ( 1994), Experimental Medicine Special Edition, 12 (15) (1994)).
In the case of the former in vivo method, a polynucleotide having a nucleotide sequence encoding the amino acid sequence of HAND1 can be administered by an appropriate route depending on the disease, condition, and the like. For example, it can be administered by injection into gastric cancer cells, veins, arteries, subcutaneous, intradermal, intramuscular, and the like.
The dosage form of the gene therapy agent as the anticancer agent may be an injection, or may be a liposome preparation such as a suspension, a freezing agent, or a centrifugal concentrated frozen agent. Such a preparation may be pharmaceutically acceptable, for example, a carrier such as a water-soluble solvent, a water-insoluble solvent, a buffer, a solubilizer, an isotonic agent, a stabilizer, or the like. Or a plasmid-type gene). If necessary, auxiliary agents such as preservatives, suspending agents and emulsifiers may be added. In the case of parenteral administration (generally, injection or the like is preferable), the anticancer agent can be used in the form of a usual liquid such as a solution.

The search method of the present invention is a method for searching for a substance having the ability to promote the expression of the HAND1 gene, wherein (1) a first step of bringing a test substance into contact with cancer cells, (2) a first step after (1), A second step of measuring the amount of the expression product of the HAND1 gene contained in the cancer cells, (3) expression of the HAND1 gene of the test substance based on the difference obtained by comparing the measured amount of the expression product with a control A third step of determining the ability to promote
The cancer cell in the first step of the search method of the present invention is not particularly limited, and may be a cancer cell isolated from a cancer tissue derived from a mammal, or a cancer derived from a mammal established as a cell line. It may be a cell line. Examples of the mammal include humans, monkeys, mice, rats, hamsters, and the like. Preferred types of cancer include gastric cancer. Specifically, for example, MKN28 (available from JCRB), MKN74 (available from JCRB), KATO-III (available from ATCC), AGS (available from ATCC), Hs746T (available from ATCC) and the like Known human gastric cancer cell lines can be mentioned.
The amount of cancer cells for bringing the test substance into contact with the cancer cells in the first step of the search method of the present invention may be generally about 10 ⁴ to 10 ⁸ cells, preferably about 10 ⁵ to 10 ⁷ cells. The concentration of the test substance may be generally about 0.1 ng / ml to about 100 μg / ml, and preferably about 1 ng / ml to about 50 μg / ml. The time for bringing the test substance into contact with the cancer cells is usually about 1 hour to about 5 days, and preferably about several hours to about 2 days. The number of times the test substance is brought into contact with the cancer cells may be one time or plural times.
The environment in which the test substance is brought into contact with the cancer cells is preferably an environment in which the life activity of the cancer cells is maintained, and examples thereof include an environment in which an energy source of the cancer cells coexists. Specifically, it is advantageous that the first step is performed in a medium.
In the second step of the search method of the present invention, in order to measure the amount of the expression product of the HAND1 gene contained in the cancer cells, the above-mentioned `` In the first step of the evaluation method of the present invention, the HAND1 gene contained in a mammal-derived specimen Method for Measuring Index Value Correlating to Methylation Frequency ”.
To determine the ability of the test substance to promote the expression of the HAND1 gene based on the difference obtained by comparing the amount of the expression product measured in the second step of the search method of the present invention with a control, as described above. Then, when the amount of the measured expression product and the concentration of the test substance for bringing the test substance into contact with the cancer cells in the first step of the search method of the present invention are set to zero (that is, the test substance is brought into contact with the cancer cells). The expression of the HAND1 gene (when not performed) is compared with the amount of the HAND1 gene expression control (control) to determine the ability of the test substance to promote the expression of the HAND1 gene based on the difference obtained by the comparison. Suppose that the amount of the expression product of the HAND1 gene contained in the cancer cells contacted with the test substance is compared with the control (in this case, the amount of the expression product of the HAND1 gene contained in the cancer cells not contacted with the test substance). If it is higher, it can be determined that the test substance has the ability to promote the expression of the HAND1 gene. Of course, as a control, the amount of the expression product of the HAND1 gene when the cancer cell is contacted with another test substance may be used, and in this case, the ability of the other test substance to promote the expression of the HAND1 gene in advance. Is preferably known.
In this way, it is possible to search for a substance having the ability to promote the expression of the HAND1 gene. Incidentally, as a background or control, normal cell lines such as normal stomach cell line, the expression product amount of the HAND1 gene contained in a sample derived from a healthy mammal that can be diagnosed as having no cancer cells such as gastric cancer cells, It is preferable to measure both when the test substance is brought into contact and when it is not brought into contact.

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

Example 1 (Confirmation test of methylation status of HAND1 gene in gastric cancer cell line (Part 1))
Two human gastric cancer cell lines [MKN28 (JCRB) and MKN74 (JCRB)] were cultured in a dedicated medium for each cell line described in the catalog of JCRB (Japan Cancer Research Resources Bank) until confluence. After that, about 1 × 10 ⁷ cells were collected. SEDTA buffer [10 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 100 mM NaCl] was added to the collected cells or human normal gastric mucosal epithelial tissue [obtained by obtaining informed consent from the patient]. After adding 10 times the volume, this was homogenized. After adding 500 μg / ml of proteinase K (Sigma) and 1% (w / v) of sodium dodecyl sulfate to the obtained mixture, the mixture was shaken at 55 ° C. for about 16 hours. After the shaking, the mixture was extracted with phenol [saturated with 1 M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, and the precipitate was recovered by ethanol precipitation. The collected precipitate was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), RNase A (Sigma) was added thereto to a concentration of 40 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour. The incubated mixture was subjected to phenol / chloroform extraction. The aqueous layer was collected, and NaCl was added thereto to a concentration of 0.5 N, and then the precipitate was collected by ethanol precipitation to collect a precipitate (genomic DNA). The recovered precipitate was rinsed with 70% ethanol to obtain genomic DNA.
After digesting the obtained genomic DNA with the restriction enzyme BamHI, Clark et al., Nucl. Acids. Res., 22, 2990-2997, 1994; Herman et al., Pro. Natl. Acad. Sci. USA, 93, 9821-9826, 1996, and treated with sodium bisulfite. That is, genomic DNA (about 1 μg) after restriction enzyme treatment was dissolved in distilled water to prepare a 20 μl genomic DNA solution, and about 1 μl of 6 M sodium hydroxide was added thereto, and the mixture was left at room temperature for 15 minutes. did. After adding 120 μl of [0.6 mM hydroquinone (Sigma) and 3.6 N sodium bisulfite (Sigma)] to the left mixture, the mixture was incubated at 95 ° C. for 30 seconds and then at 50 ° C. for 15 minutes for 1 cycle. Cycled. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). The purified DNA was dissolved in 50 μl of TE buffer, and 2.5 μl of 6 M sodium hydroxide was added thereto, and the mixture was left at room temperature for 5 minutes. Then, the precipitate (DNA) was recovered by ethanol precipitation of the left mixture. The collected precipitate was suspended in 20 μl of TE buffer.

Using the obtained DNA as a template, a DNA having the nucleotide sequence represented by nucleotide numbers 1234 to 1461 (228 bp) in the nucleotide sequence represented by SEQ ID NO: 1 and subjected to bisulfite treatment to amplify the DNA having the nucleotide sequence by PCR Then, a primer having the following nucleotide sequence was synthesized.
B1: 5'-TAGAGTAGGGAGTTGAGTGGGAG-3 '(SEQ ID NO: 6)
B2: 5'-ACCCAAAAACCTATTTAACCCTTCTA-3 '(SEQ ID NO: 7)

As a PCR reaction solution, 50 ng of DNA as a template, 1 μl of each of the above primer solutions of 20 pmol / μl, 3 μl of each 2 mM dNTP, and 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ) was mixed with 2.5 μl and 0.2 μl of 5 U / μl of a heat-resistant DNA polymerase, and sterilized ultrapure water was added thereto to make a liquid volume of 25 μl. After incubating the reaction solution at 95 ° C. for 10 minutes, PCR is performed under the condition that the temperature is maintained at 95 ° C. for 30 seconds, then at 57 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds as one cycle of 40 cycles. Was. After PCR, DNA amplification was confirmed by 2% agarose gel electrophoresis.
After ligation of the PCR product into pGEM-T-Easy (Promega), this was introduced into E. coli XL1Blue strain. Transformed Escherichia coli colonies were picked up by 10 cells per gastric cancer cell line and lysed. Using the obtained E. coli lysate as a template, PCR was performed under the same conditions as described above. After subjecting the PCR reaction solution to agarose gel electrophoresis in the same manner as described above, a gel portion containing 228 bp DNA was cut out, and the DNA was extracted therefrom. The nucleotide sequence of the extracted DNA was analyzed using a DNA sequencer (ABI310, PE Biosystems). For the sequencing results of DNA prepared from each colony, to determine whether cytosine in CpG present in the region represented by base numbers 1234 to 1461 in the base sequence represented by SEQ ID NO: 1 has been replaced with uracil, Of the DNAs prepared from the 10 colonies tested, the frequency of DNA in which cytosine was not substituted with uracil, that is, the methylation frequency of the cytosine was measured.
The results are shown in Table 1. In the case of DNA prepared from normal gastric mucosal epithelial tissue (21N), 12 cytosines (base numbers 1266, 1272, 1292, 1305, 1307, 1316) in CpG present in the region indicated by base numbers 1234 to 1461 , 1356, 1377, 1399, 1401, 1422, 1434), almost no DNA not substituted with uracil (ie, methylated DNA) was found. On the other hand, in the case of DNA prepared from two types of gastric cancer cell lines (MKN28 and MKN74), most of the cytosines in CpG present in the region are not substituted with uracil (that is, methylated DNA). ).

＊各シトシンについて、10コロニーから調製されたDNAのうち、当該シトシンがウラシルに置換されていないＤＮＡの数を示す。また解析対象のシトシンは、配列番号１で示される塩基配列における塩基番号で示す。

* For each cytosine, indicates the number of DNAs in which the cytosine has not been replaced with uracil among DNAs prepared from 10 colonies. The cytosine to be analyzed is represented by a base number in the base sequence represented by SEQ ID NO: 1.

Example 2 (Confirmation test of HAND1 gene expression state in gastric cancer cell line and effect of methylation inhibitor on the expression of the gene)
After two human-derived gastric cancer cell lines (MKN28 and MKN74) were cultured in a dedicated medium until they reached 70% confluence, each cell was collected. 1 ml of ISOGEN solution (Nippon Gene) was mixed and homogenized with each of the collected cells (about 100 mg wet weight) and human-derived normal gastric mucosal epithelial tissue (21 N: about 50 mg), and 0.2 ml of chloroform was added thereto. Was added and suspended. After the suspension, the mixture was centrifuged (4 ° C., 15000 × g, 15 minutes) to collect the supernatant. The collected supernatant was suspended by adding 0.5 ml of isopropanol and then centrifuged (4 ° C., 15000 × g, 15 minutes) to collect a precipitate (RNA). The collected precipitate was rinsed with 75% ethanol, and then dissolved in DEPC (diethyl pyrocarbonate) -treated water.
Two human gastric cancer cell lines (MKN28, MKN74) were inoculated at a density of about 3 × 10 ⁵ cells / 10 cm plate, and cultured using a dedicated medium. One day after the inoculation, 5-aza-2'-deoxycytidine (manufactured by Sigma) as a methylation inhibitor (hereinafter referred to as 5Aza-dC) was added to the medium to a concentration of 1 μM. Twenty-four hours after the addition of 5Aza-dC, the medium was replaced with the above medium to which 5Aza-dC was not added, and the culture was continued. Then, 3A and 5 days after inoculation, 5Aza-dC was similarly added to the medium. Six days after inoculation, the cells were collected, and RNA was extracted and collected from the collected cells in the same manner as described above.
After treating the thus obtained RNA with DNaseI (Ambion), cDNA was synthesized using this as a template and using SuperscriptII (Invitrogen) according to the protocol attached to the enzyme. DNA (88 bp) derived from HAND1 gene mRNA was amplified by performing Real Time PCR using the synthesized cDNA as a template and the following HAND1 S and HAND1 A as a primer pair. At this time, as a control, PCR was performed using the above cDNA as a template and the following PCNA S and PCNA A as a primer pair to thereby derive from the mRNA of the Proliferating cell nuclear antigen (PCNA) gene. DNA was amplified.
<Primer (S: sense, A: antisense)>
HAND1 S: 5'-GTGAGAGCAAGCGGAAAAG-3 '(SEQ ID NO: 12)
HAND1 A: 5'-GTGCGTCCTTTAATCCTCTTC-3 '(SEQ ID NO: 13)
PCNA S: 5'-ATGTCGATAAAGAGGAGGAA-3 '(SEQ ID NO: 14)
PCNA A: 5'-AGAGTGGAGTGGCTTTTGTA-3 '(SEQ ID NO: 15)

As a PCR reaction solution, 50 ng of cDNA as a template, 1 μl of each of the two primer solutions of 10 pmol / μl, 4 μl of each 2.5 mM dNTP, 4 μl of 5 mM dUTP, and 10 × SYBR Green PCR Buffer were used. 5 μl, 6 μl of 25 mM MgCl ₂ , 0.3 μl of 5 U / μl of heat-resistant DNA polymerase (AmpliTaq Gold), and 0.5 μl of AmpEraseUNG, and sterilized ultrapure water was added to make a volume of 50 μl. Using. Real Time PCR was performed using iCycler Thermal Cycler (Bio-Rad Laboratories). When amplifying DNA derived from mRNA of HAND1 gene, the reaction solution was kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 60 ° C for 30 seconds, and then at 72 ° C for 30 seconds. Was performed as one cycle to perform Real Time PCR to quantify the HAND1 gene. When amplifying DNA derived from PCNA gene mRNA, the reaction solution was incubated at 95 ° C. for 10 minutes, and then subjected to 95 ° C. for 30 seconds followed by 55 ° C. for 60 seconds as one cycle. Was performed to quantify the PCNA gene.
The result is shown in FIG. In the case of human-derived normal gastric mucosal epithelial tissue (21N), DNA derived from the mRNA of the HAND1 gene was detected, whereas the DNA was not detected in any of the two gastric cancer cell lines. Was. That is, expression of the HAND1 gene was confirmed in human-derived normal gastric mucosal epithelial tissue (21N), whereas expression of the HAND1 gene was not observed in any of the two gastric cancer cell lines.
In the case of MKN28 and MKN74 cultured in the presence of 1 μM 5Aza-dC, DNA derived from HAND1 gene mRNA was detected. The DNA derived from the PCNA gene mRNA was cultured in the absence of 5Aza-dC in the case of normal gastric mucosal epithelial tissue (21N), and cultured in the presence of 1 μM 5Aza-dC in the case of gastric cancer cell lines MKN28 and MKN74 cultured in the absence of 5Aza-dC. The same was detected in both cases of MKN28 and MKN74. That is, in the case of the gastric cancer cell lines MKN28 and MKN74, the expression of the HAND1 gene was observed in the presence of the methylation inhibitor.
The above results revealed that the methylation of cytosine in gastric cancer cell lines was inhibited by a methylation inhibitor, and that the HAND1 gene was expressed in the presence of the methylation inhibitor.

Example 3 (Confirmation test of methylation status of HAND1 gene in gastric cancer cell line (No. 2) and effect of methylation inhibitor on expression of the gene)
Two human gastric cancer cell lines (MKN28, MKN74) were inoculated at a density of about 3 × 10 ⁵ cells / 10 cm plate and cultured using a dedicated medium. One day after inoculation, 5Aza-dC, a methylation inhibitor, was added to the medium to a concentration of 1 μM. Twenty-four hours after the addition of 5Aza-dC, the medium was replaced with the above medium to which 5Aza-dC was not added, and the culture was continued. Then, 3A and 5 days after inoculation, 5Aza-dC was similarly added to the medium. Six days after inoculation, the cells were collected, and genomic DNA was extracted and collected from the collected cells in the same manner as in Example 1.
The extracted and recovered genomic DNA was treated with sodium bisulfite in the same manner as in Example 1. Using the obtained DNA as a template, PCR was performed using the following unmethylation-specific primers U1 and U2 or methylation-specific primers M1 and M2. When using unmethylation specific primers U1 and U2, when using methylation specific primers M1 and M2, the base sequence represented by base numbers 1141 to 1324 of the base sequence represented by SEQ ID NO: 1 The 184 bp DNA having the nucleotide sequence after the DNA having the DNA is subjected to biisulfite treatment is amplified.
<Unmethylated specific primer>
U1: 5'-AATAGTTTAGGGTGTTGGTT-3 '(SEQ ID NO: 2)
U2: 5'-AATTTTACACTCAACCCA-3 '(SEQ ID NO: 3)
<Methylation-specific primer>
M1: 5'-AATAGTTTAGGGCGTTGGTC-3 '(SEQ ID NO: 4)
M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5)
First, genomic DNA [genomic DNA (1)] was extracted from normal gastric mucosal epithelial tissue (21N) by a conventional method to confirm that the methylation-specific primer and the unmethylation-specific primer had specificity. Then, a part of this was treated with a methylating enzyme SssI (NEB) to methylate all 5′-CG-3 ′ of genomic DNA to obtain DNA [DNA (2)]. The obtained DNA (1) and DNA (2) were also subjected to the above-mentioned methylation-specific PCR and non-methylation-specific PCR.

As a PCR reaction solution, 50 ng of DNA as a template, 1 μl of each of the above primer solutions of 20 pmol / μl, 2.5 μl of each 2 mM dNTP, and 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl , 20 mM MgCl ₂ ) and 0.2 μl of 5 U / μl of heat-resistant DNA polymerase, and sterilized ultrapure water was added thereto to adjust the volume to 25 μl. When the above-mentioned unmethylation-specific primer is used, the reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 56 ° C for 30 seconds, and further at 72 ° C for 30 seconds. Was performed under the condition that the heat retention was performed 33 times in a cycle of 1 cycle. When the above-mentioned methylation-specific primer was used, the reaction solution was kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 61 ° C for 30 seconds, and further at 72 ° C for 30 minutes. The PCR was performed under the condition that the temperature was maintained at 33 cycles with one second as one cycle. In each case, after performing the PCR, the PCR reaction solution containing the amplification product was subjected to 2% agarose gel electrophoresis. The result is shown in FIG. In the case of human-derived normal gastric mucosal epithelial tissue (21N), when an unmethylated specific primer is used (lane U), an amplified DNA band is observed, and when a methylated specific primer is used No band of amplified DNA was detected in (lane M). Therefore, in the case of human-derived normal gastric mucosal epithelial tissue (21N), at least cytosines represented by base numbers 1153, 1160, 1307, and 1316 of the base sequence represented by SEQ ID NO: 1 are not methylated. It was judged. In the case of two gastric cancer cell lines (MKN28 and MKN74) cultured in the absence of a methylation inhibitor (5Aza-dC 0 μM), when unmethylation-specific primers were used (lane U) No amplified DNA band was detected, and when a methylation-specific primer was used (lane M), an amplified DNA band was observed. Therefore, under these conditions, it was determined that cytosines represented by base numbers 1153, 1160, 1307, and 1316 of the base sequence represented by SEQ ID NO: 1 were methylated.
On the other hand, in the case of gastric cancer cell lines MKN28 and MKN74 cultured in the presence of 1 μM 5Aza-dC, an amplified DNA band was observed when the unmethylated specific primer was used (lane U). When a methylation specific primer was used (lane M), only a weak band was detected. Therefore, under these conditions, it was determined that in many genomic DNAs, cytosines represented by base numbers 1153, 1160, 1307, and 1316 of the base sequence represented by SEQ ID NO: 1 were not methylated.
From the above results, it was revealed that the methylation of cytosine in the gastric cancer cell line was inhibited by a methylation inhibitor.

Example 4 (Confirmation test of methylation status of HAND1 gene in various cancer cell lines)
Three human-derived pancreatic cancer cell lines (HPAF-II, Mia PaCa-2 and HPAC), three colon adenocarcinoma cell lines (HT-29, COLO205 and SW1116), and renal adenocarcinoma cell line (ACHN) Were also obtained from the American Type Culture Collection (ATCC)]. After culturing until confluence in a dedicated medium for each cell line described in the catalog of the ATCC, about 1 × 10 ⁷ cells were collected. A 10-fold volume of a SEDTA buffer [10 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 100 mM NaCl] was added to the collected cells, and the mixture was homogenized. After adding 500 μg / ml of proteinase K (Sigma) and 1% (w / v) of sodium dodecyl sulfate to the obtained mixture, the mixture was shaken at 55 ° C. for about 16 hours. After the shaking, the mixture was extracted with phenol [saturated with 1 M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, and the precipitate was recovered by ethanol precipitation. The collected precipitate was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), RNase A (Sigma) was added thereto to a concentration of 40 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour. The incubated mixture was subjected to phenol / chloroform extraction. The aqueous layer was collected, and NaCl was added thereto to a concentration of 0.5 N, and then the precipitate was collected by ethanol precipitation to collect a precipitate (genomic DNA). The recovered precipitate was rinsed with 70% ethanol to obtain genomic DNA.
Genomic DNA obtained from the above cancer cell lines or genomic DNA (BioChain) derived from normal tissues (colon, kidney, pancreas) was purified by Clark et al., Nucl. Acids. Res., 22, 2990-2997, 1994; Herman et al., Pro. Natl. Acad. Sci. USA, 93, 9821-9826, 1996. That is, genomic DNA (about 1 μg) was dissolved in distilled water to prepare a 20 μl genomic DNA solution, about 1 μl of 6M sodium hydroxide was added thereto, and the mixture was left at room temperature for 15 minutes. After adding 120 μl of [0.6 mM hydroquinone (Sigma) and 3.6 N sodium bisulfite (Sigma)] to the left mixture, the mixture was incubated at 95 ° C. for 30 seconds and then at 50 ° C. for 15 minutes for 1 cycle. Cycled. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). The purified DNA was dissolved in 50 μl of TE buffer, and 2.5 μl of 6 M sodium hydroxide was added thereto, and the mixture was left at room temperature for 5 minutes. Then, the precipitate (DNA) was recovered by ethanol precipitation of the left mixture. The collected precipitate was suspended in 50 μl of TE buffer or ultrapure water.

PCR was performed using the obtained DNA as a template and non-methylation-specific primers U1 and U2 or methylation-specific primers M1 and M2 in the same manner as in Example 3. When using unmethylation specific primers U1 and U2, when using methylation specific primers M1 and M2, the base sequence represented by base numbers 1141 to 1324 of the base sequence represented by SEQ ID NO: 1 The 184 bp DNA having the nucleotide sequence after the DNA having the DNA is subjected to biisulfite treatment is amplified.
<Unmethylated specific primer>
U1: 5'-AATAGTTTAGGGTGTTGGTT-3 '(SEQ ID NO: 2)
U2: 5'-AATTTTACACTCAACCCA-3 '(SEQ ID NO: 3)
<Methylation-specific primer>
M1: 5'-AATAGTTTAGGGCGTTGGTC-3 '(SEQ ID NO: 4)
M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5)

As a PCR reaction solution, 20 ng of DNA as a template (derived from a cancer cell line) or 40 ng (derived from normal tissue), 1 μl of each of the above primer solutions of 20 pmol / μl, 2.5 μl of each 2 mM dNTP, and 10 × 5 μl of a buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 20 mM MgCl ₂ ) and 0.2 μl of 5 U / μl of a heat-resistant DNA polymerase were mixed, and sterile ultrapure water was added thereto to reduce the volume to 25 μl. Was used. When the above-mentioned unmethylation-specific primer is used, the reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 56 ° C for 30 seconds, and further at 72 ° C for 30 seconds. Was performed under the condition that the temperature was maintained at 40 cycles for one cycle. When the above-mentioned methylation-specific primer was used, the reaction solution was kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 61 ° C for 30 seconds, and further at 72 ° C for 30 minutes. The PCR was performed under the condition that the temperature was maintained at 40 cycles, each cycle being one cycle. In each case, after performing the PCR, the PCR reaction solution containing the amplification product was subjected to 2% agarose gel electrophoresis. The result is shown in FIG. In the case of normal tissues derived from humans, amplified DNA bands were observed in colon and kidney when unmethylated specific primers were used (lane U), and when methylated specific primers were used (lane U) In M), no amplified DNA band was detected. In the pancreas, DNA bands were observed in both lane U and lane M. Therefore, in the case of colon and kidney, it was determined that at least cytosines represented by base numbers 1153, 1160, 1307, and 1316 of the base sequence represented by SEQ ID NO: 1 were not methylated. In the pancreas, it was found that a part of cytosine represented by base numbers 1153, 1160, 1307, and 1316 in the base sequence represented by SEQ ID NO: 1 was methylated. In addition, among the various cancer cell lines tested, in the case of cell lines other than renal gland bladder cancer cell line (5637ACHN), amplified DNA bands were detected when non-methylation specific primers were used (lane U). However, when a methylation-specific primer was used (lane M), an amplified DNA band was observed. In the renal adenocarcinoma cell line (ACHN), DNA bands were observed in both lane U and lane M. Therefore, under these conditions, in cell lines other than the renal adenocarcinoma cell line (ACHN), cytosines represented by nucleotide numbers 1153, 1160, 1307, and 1316 of the nucleotide sequence represented by SEQ ID NO: 1 are methylated. Was determined to be. In the renal adenocarcinoma cell line (ACHN), it was found that some of the cytosines represented by base numbers 1153, 1160, 1307, and 1316 in the base sequence represented by SEQ ID NO: 1 were methylated.

According to the present invention, a method for evaluating the degree of canceration of a mammal-derived specimen can be provided.

Human-derived normal gastric mucosal epithelial tissue (21N), two gastric cancer cell lines (MKN28 and MKN74), and two gastric cancer cell lines (MKN28 and MKN74) to which 5Aza-dC, a methylation inhibitor, was added at a concentration of 1 μM. 2) is a diagram showing the amount of the HAND1 gene obtained by amplifying DNA derived from the mRNA of the HAND1 gene by Real Time PCR. The names of the cells used are indicated at the bottom of the figure. (-) Indicates the absence of methylation inhibitor 5Aza-dC, and (+) indicates the presence of 5Aza-dC. The vertical axis indicates the amount of HAND1 gene and the amount of PCNA gene. PCR is performed using genomic DNA prepared from human-derived normal gastric mucosal epithelial tissue (21N) and two types of gastric cancer cell lines (MKN28 and MKN74) and treated with sodium bisulfite as templates, and a PCR reaction solution after PCR FIG. 3 is a diagram (photograph) showing the results of analyzing agarose gel electrophoresis. The names of the cells used are shown at the bottom. The figure described as SssI shows DNA obtained by treating 21N genomic DNA with the methylating enzyme SssI. Further, the figure described as -Aza shows cells to which 5Aza-dC was added at a rate of 1 μM during the culture of the cells. Lane U, PCR reaction solution for PCR using unmethylation-specific primers; Lane M, PCR reaction solution for PCR using unmethylation-specific primers. Human normal tissues (colon, kidney, pancreas) and three pancreatic cancer cell lines (HPAF-II, Mia PaCa-2 and HPAC), three colon adenocarcinoma cell lines (HT-29, COLO205 and SW1116) and kidney A diagram showing the results of performing PCR using genomic DNA prepared from an adenocarcinoma cell line (ACHN) and treated with sodium bisulfite as a template, and analyzing the PCR reaction solution after PCR by agarose gel electrophoresis (photograph) ). The names of the cells used and the type of cancer are shown at the bottom of the photo. Lane U, PCR reaction solution for PCR using unmethylation-specific primers; Lane M, PCR reaction solution for PCR using unmethylation-specific primers.

SEQ ID NO: 2
Oligonucleotide primer designed for PCR SEQ ID NO: 3
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primers designed for PCR SEQ ID NO: 5
Oligonucleotide primers designed for PCR SEQ ID NO: 6
Oligonucleotide primers designed for PCR SEQ ID NO: 7
Oligonucleotide primers designed for PCR SEQ ID NO: 8
Oligonucleotide SEQ ID NO: 9 designed for probe
Oligonucleotide SEQ ID NO: 10 designed for probe
Oligonucleotide SEQ ID NO: 11 designed for probe
Oligonucleotide SEQ ID NO: 12 designed for probe
Oligonucleotide primers designed for PCR SEQ ID NO: 13
Oligonucleotide primers designed for PCR SEQ ID NO: 14
Oligonucleotide primers designed for PCR SEQ ID NO: 15
Oligonucleotide primers designed for PCR

Claims (27)

A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal or an index value correlated therewith, and (2) the measured methylation frequency or an index correlated therewith An evaluation method, comprising: a second step of determining a degree of canceration of the sample based on a difference obtained by comparing a value with a control. (4) The method according to the above (1), wherein the mammal-derived specimen is a cell. (4) The evaluation method according to (1), wherein the mammal-derived specimen is a tissue. A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, and (2) the method based on a difference obtained by comparing the measured methylation frequency with a control. An evaluation method comprising a second step of determining a degree of canceration of a sample. (4) The evaluation method according to (1), wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the malignancy of the mammal-derived cell. (5) The evaluation method according to (4), wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the malignancy of the mammal-derived cell. (4) The evaluation method according to (1), wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue. (5) The evaluation method according to (4), wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue. The evaluation method according to claim 8, wherein the tissue and cancer are one or more types of tissue and cancer selected from the following (a) to (d):
(A) the tissue is gastric tissue and the cancer is gastric cancer;
(B) the tissue is colon tissue and the cancer is colon cancer;
(C) the tissue is pancreatic tissue, and the cancer is pancreatic cancer;
(D) The tissue is kidney tissue, and the cancer is kidney cancer. The methylation frequency of a gene is the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5′-CG-3 ′ present in the nucleotide sequence in the promoter region, untranslated region or translated region of the gene. The evaluation method according to claim 1, wherein: The method according to claim 10, wherein the tissue and the cancer are one or more types of tissues and cancers selected from the following (a) to (d):
(A) The tissue is gastric tissue, and the cancer is gastric cancer.
(B) The tissue is colon tissue, and the cancer is colon cancer.
(C) The tissue is pancreatic tissue, and the cancer is pancreatic cancer.
(D) The tissue is kidney tissue, and the cancer is kidney cancer. The methylation frequency of the gene is characterized by being the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence in the promoter region of the gene. The evaluation method according to claim 1. The methylation frequency of the gene is the methylation frequency of cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence in the untranslated or translated region of the gene The evaluation method according to claim 1 or 4, wherein: The methylation frequency of the gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'present in the nucleotide sequence represented by SEQ ID NO: 1. Item 1. The evaluation method according to Item 1. The evaluation method according to claim 14, wherein the tissue and the cancer are one or more types of tissues and cancers selected from the following (a) to (d).
(A) The tissue is gastric tissue, and the cancer is gastric cancer.
(B) The tissue is colon tissue, and the cancer is colon cancer.
(C) The tissue is pancreatic tissue, and the cancer is pancreatic cancer.
(D) The tissue is kidney tissue, and the cancer is kidney cancer. A method for evaluating the degree of canceration of a mammal-derived specimen,
(1) a first step of measuring an index value having a correlation with the methylation frequency of the HAND1 gene contained in a specimen derived from a mammal, and (2) an index value having a correlation with the measured methylation frequency. And a second step of judging the degree of canceration of the sample based on a difference obtained by comparing with a control. 17. The evaluation method according to claim 16, wherein the index value having a correlation with the methylation frequency of the HAND1 gene is the amount of the expression product of the HAND1 gene. 18. The evaluation method according to claim 17, wherein the amount of the expression product of the HAND1 gene is the amount of a transcript of the gene. 18. The evaluation method according to claim 17, wherein the amount of the expression product of the HAND1 gene is the amount of the translation product of the gene. A method for searching for a substance having an ability to promote expression of a HAND1 gene,
(1) a first step of bringing a test substance into contact with cancer cells;
(2) After the first step (1), a second step of measuring the expression product amount of the HAND1 gene contained in the cancer cells,
(3) A search method comprising a third step of determining the ability of a test substance to promote the expression of a HAND1 gene based on a difference obtained by comparing the measured amount of an expression product with a control. . 21. The search method according to claim 20, wherein the cancer cells are gastric cancer cells. 抗 An anti-cancer agent comprising, as an active ingredient, a substance having the ability found by the search method according to claim 20, wherein the active ingredient is formulated in a pharmaceutically acceptable carrier. (4) An anticancer agent comprising, as an active ingredient, a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence of HAND1, and the active ingredient is formulated in a pharmaceutically acceptable carrier. 使用 Use of methylated HAND1 gene as a cancer marker. 25. The use according to claim 24, wherein the cancer marker is one or more cancer markers selected from gastric cancer markers, colon cancer markers, pancreatic cancer markers, and renal adenocarcinoma markers. (4) A method for suppressing canceration, comprising a step of administering a substance that reduces the frequency of methylation of the HAND1 gene to cells in the body of a mammal that can be diagnosed with cancer. 27. The method according to claim 26, wherein the cancer is gastric cancer.

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