WO2004029291A1 - Method of evaluationg canceration degree of mammal-origin specimen - Google Patents

Abstract

A method of evaluating the degree of canceration of a mammal-origin specimen characterized by comprising: (1) the first step of measuring the methylation frequency or an indication correlating thereto of HAND1 gene contained in the mammal-origin specimen; and (2) the second step of judging the degree of canceration of the specimen based on the difference obtained by comparing the methylation frequency or the indication correlating thereto thus measured with a control.

Description

 Specification

 TECHNICAL FIELD The present invention relates to a method for evaluating the degree of canceration of a mammal-derived specimen.

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

 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. It is shown that. It is clinically important to detect cancer at an early stage, to select an effective treatment method for the detected cancer, and to provide after-care such as confirmation of the recurrence of cancer after treatment.

 Therefore, based on the detection of genetic abnormalities, it is suitable for diagnosis methods for early detection of cancer, evaluation of the effectiveness of treatment methods for cancer, confirmation of cancer recurrence, etc. The development of a method is eagerly needed. Disclosure of the invention

 Under such circumstances, the present inventors have conducted intensive studies, and as a result, it has been found that the HAD1 gene is significantly more frequently found in the cancer cell line as compared with the healthy subject's itono-tori sample, and In this cancer cell line, the expression level of the HAND1 gene was found to be significantly lower than that in a tissue sample from a healthy subject. The present inventors have found that the expression level can be increased, and have led to the present invention.

 That is, the present invention

 1. A method for evaluating the degree of canceration of a mammal-derived specimen,

 (1) the first step of measuring the methyl methane frequency of the HA D1 gene contained in a specimen derived from a mammal or an index value having a correlation therewith, and

(2) a second step of determining the degree of canceration of the sample based on a difference obtained by comparing the measured methylation frequency or an index value correlated therewith with a control (Hereinafter, also referred to as the evaluation method of the present invention); 2. The evaluation method according to the above item 1, wherein the mammal-derived specimen is a cell; 2. The evaluation method according to the above item 1, wherein the specimen derived from an animal is a tissue (the term “tissue” has a broad meaning including body fluids such as blood, plasma, serum, and lymph, lymph nodes, etc.). ;

 4. A method for evaluating the degree of canceration of a mammal-derived specimen,

 (1) The first step of measuring the methyl methane frequency of the HAND1 gene contained in a mammal-derived specimen, and

 (2) a second step of determining the degree of canceration of the sample based on a difference obtained by comparing the measured methyl methane frequency with a control

An evaluation method characterized by having:

 5. The method according to item 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. 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 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 abundance of cancer cells in the mammalian-derived fibrous tissue; The evaluation method according to item 8, wherein the tissue and cancer are one or more types of tissue and cancer selected from the following:

 (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 knee tissue and the cancer is Teng's cancer; and

 (d) the tissue is kidney tissue and the cancer is kidney cancer;

10. The methylation frequency of a gene is determined by the presence of one or more 5'-CG-3's in the base sequence in the promoter, untranslated or translated regions of the gene. The evaluation method according to the above 1 or 4, wherein the methylation frequency of tosin is; 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) 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 a mucosal tissue and the cancer is a knee cancer; and

 (d) the tissue is kidney tissue and the cancer is kidney cancer;

 1 2. The methylation frequency of the gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence in the promoter region of the gene. The evaluation method described in 1 or 4 above, which is characterized by;

1 3. The methylation frequency of a gene is cytosine methylation in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence in the untranslated or translated region of the gene. The evaluation method according to the above 1 or 4, characterized in that the frequency is frequent;

 14. The methylation frequency of the gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence represented by SEQ ID NO: 1. The evaluation method described in the preceding paragraph 1;

 1 5. The evaluation method as described in the above item 14, wherein the cancer is one or more types of tissues and cancers selected from the following:

 (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 hepatic tissue and the cancer is Teng's cancer; and

 (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 methyl methane frequency of the HAND1 gene contained in a mammal-derived specimen, and

 (2) A second step of determining the degree of canceration of the sample based on a difference obtained by comparing an index value having a correlation with the measured methyl methane frequency with a control.

An evaluation method characterized by having:

1. The index value correlated with the methyl methane frequency of the HAND1 gene Item 16. The evaluation method according to Item 16, wherein the amount is an amount of the substance;

 18. The evaluation method according to the above item 17, wherein the amount of the expression product of the HAND1 gene is the amount of the transcript of the gene;

 19. The evaluation method according to the above item 17, wherein the amount of the expression product of the HA D1 gene is the amount of the translation product of the gene;

 20. A method for searching for a substance capable of promoting the expression of a HAND1 gene,

 (1) the first step of bringing the test substance into contact with the cancer cells,

 (2) The first step (1), followed by a second step of measuring the amount of the expression product of the HAND1 gene contained in the cancer cells,

 (3) The third step of determining the ability of the test substance to promote the expression of the HAND1 gene based on the difference obtained by comparing the measured amount of the expression product with the control

21. 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 described in 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 nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of HAND1, wherein the active ingredient is formulated in a pharmaceutically acceptable carrier;

24. Use of the methylated HAND1 gene as a cancer marker;

 25. The use according to the above item 24, wherein the cancer marker is at least one cancer marker selected from a stomach cancer marker, a colon cancer marker, a knee cancer marker and a renal adenocarcinoma marker;

 26. A method for suppressing cancer eh, 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 for suppressing canceration according to the above item 26, wherein:

And so on. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows that human normal gastric mucosal epithelial tissue (21N), two gastric cancer cell lines (MKN28 and MKN74), and 5Aza-dC, a methylidani inhibitor, were added at a concentration of ΙμΜ. FIG. 4 is a graph showing the amount of the HA D1 gene obtained by real-time PCR of DNA derived from the mRNA of the fraction D1 gene in two types of gastric cancer cell lines (28 and 74). The names of the cells used are shown at the bottom of the figure. (-) Indicates the absence of the methylation inhibitor 5Aza-dC, and (+) indicates the presence of 5Aza-dC. The vertical axis indicates the amount of HA D1 gene and the amount of PCNA gene.

 Figure 2 shows the results of PCR using genomic DNA prepared from human-derived normal gastric mucosal epithelial tissue (21N) and two types of gastric cancer cell lines (MK28 and MKN74) and treated with sodium bisulfite. FIG. 4 is a diagram (photograph) showing the results of analysis of the PCR reaction solution after PCR by agarose gel electrophoresis. The names of the cells used are shown at the bottom. The figure described as Sssl shows DNA obtained by treating 21N genomic DNA with the methylating enzyme Sssl. Further, the figure described as -Aza shows cells to which 5Aza-dC was added at the rate of Ι / iM during the culture of the cells. Lane U, PCR reaction solution using non-methylated specific primer; Lane # 1, PCR reaction solution using non-methylated specific primer.

 Figure 3 shows normal tissues from humans (colon, kidney, and knee), three types of knee cancer cell lines (HPAF-II, Mia PaCa-2 and HPAC), and three types of colon adenocarcinoma cells (HT-29). , C0L0205 and SW1116) and genomic DNA prepared from renal adenocarcinoma cell line (ACHN) and treated with sodium bisulfite were used as mirrors for PCR, and the PCR reaction solution after PCR was subjected to agarose gel electrophoresis. It is the figure (photograph) which showed the analysis result. 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 unmethylated specific primers; Lane # 1, PCR reaction solution for PCR using non-methylated specific primers. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention is described in detail below.

The present invention is an invention related to the use of a methylated HAND1 gene as a cancer marker 2 (for example, a gastric cancer marker, a colon cancer marker, a kidney cancer marker, a renal adenocarcinoma marker, etc.). Examples of the HAND1 gene used as a cancer marker in the present invention include, for example, an untranslated region and a translated region (coding region) which are a type II mRNA containing a nucleotide sequence encoding the amino acid sequence of HAND1, and a 5 ′ upstream thereof. And a human-derived gene containing a promoter region located at the same position. 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 nucleotide sequence encoding it are described in, for example, Genbank Accession No. Marauder-004821. Also, of the exons that are responsible for the untranslated region and the translated region (coding region) of the mRNA that includes the nucleotide sequence encoding the amino acid sequence of human-derived HAND1, the exon located at the 5'-upstream side is the most upstream. The base sequence of genomic DNA including exon 1) and its 5, upstream promoter region are described in, for example, Genbank Accession No. AC026688. In the nucleotide sequence described in Genbank Accession No. AC 026688, 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, and the salt of exon 1 The base sequence is shown at base numbers 1400 to 2198. The HAND1 gene used in the present invention includes, in addition to the above-mentioned gene having a known nucleotide sequence, a naturally occurring mutation in a powerful nucleotide sequence due to a species difference, an individual difference or a difference between organs and tissues of an organism. It also includes a gene having a base sequence in which a base is deleted, substituted or added by the above. In mammals, there is a phenomenon in which only cytosine is methylated among the four types of bases that make up 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. Then, the methylation modification of DNA is performed in the base sequence represented by 5′-CG-3 ′ (C represents cytosine, G represents guanine. Hereinafter, the base sequence may be referred to as CpG.) Limited to cytosine. The site to be methylated in cytosine is at position 5. At the time of DNA replication prior to cell division, immediately after replication, only cytosine in type II CpG is methylated, but cytosine in nascent chain CpG is immediately converted to methyl by the action of methyltransferase. I will be mad. Therefore, the state of DNA methylation is inherited by the new two sets of DNA even after DNA replication. In the first step of the evaluation method of the present invention, "methylidani frequency" refers to, for example, when the presence or absence of cytosine methylation in CpG to be prepared is examined for a plurality of haploids, the cytosine is methylated. Of haploids.

 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 transcription product of the gene). And the amount of the translation product of the gene). In the case of such an expression product, there is a negative correlation such that the higher the methylation frequency, the lower the methylation frequency. Examples of mammal-derived specimens in the first step of the evaluation method of the present invention include, for example, cancer cells such as stomach cancer, colon cancer, tsuzou cancer or renal adenocarcinoma cells or tissues containing the same, and stomach cancer, colon cancer, Cells that may contain DNA derived from cancer cells such as pancreatic cancer or renal adenocarcinoma cells, and tissues containing them (tissues such as blood, plasma, serum, lymph, and serum) In a broad sense, it includes biological fluids, lymph nodes, 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 muscle plate) collected from the test animal, the gastric submucosal tissue layer, the (proprietary) muscle And the serosal layer.

 These biological samples may be used as samples as they are, or biological samples prepared from such biological samples by various operations such as separation, fractionation, and immobilization may be used as samples.

When the mammal-derived specimen is blood, a blood sample collected by a periodic medical examination or a simple test can be used. In the first step of the evaluation method of the present invention, the method of measuring the methyl methane frequency of the HAND1 gene contained in a specimen derived from a mammal or an index value correlated therewith includes, for example, the following: You can do it like this.

 As a first method, after contacting DNA derived from a sample with a reagent that modifies non-methylated cytosine, the DNA is converted into a type II, and a primer capable of discriminating the presence or absence of cytosine to be analyzed is methylated. A method of performing a polymerase chain reaction (hereinafter, referred to as PCR) using, and examining the amount of amplification product obtained 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 sample, plasma or serum is prepared from blood according to a conventional method, and the prepared plasma or serum is used as a sample and free DNA (gastric cancer, colon cancer, kidney cancer or Analysis of DNA derived from cancer cells such as renal adenocarcinoma cells) revealed that DNA derived from cancer cells such as gastric, colon, spleen, or renal adenocarcinoma cells was avoided while avoiding blood cell-derived DNA. It can be analyzed to improve the sensitivity of detecting cancer cells such as gastric cancer, colon cancer, spleen cancer or renal adenocarcinoma cells, tissues containing the same, and the like.

 Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is used as a 铸 type using a primer capable of discriminating the presence or absence of cytosine methylation to be analyzed. Amplify by PCR and check the amount of amplification product obtained. The cytosine to be analyzed can be selected from cytosines in the nucleotide sequence of one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. it can.

Here, 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 includes exon 1 of the HAND1 gene derived from human. 5, the base sequence of genomic DNA containing the promoter region located upstream, and more specifically, the base sequence represented by SEQ ID NO: 1 (described in Genbank Accession No. AC026688). This corresponds to the complementary sequence of the base sequence represented by base numbers 24303 to 26500 in the base sequence.) 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 the nucleotide sequence The numbers are shown at 1400-2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, especially SEQ ID NO: 1 Cytosine in CpG present in a region where CpG is densely present in the nucleotide sequence represented by, for example, a high methylation frequency in cancer cells such as stomach cancer, colon cancer, spirae cancer or renal adenocarcinoma cells (ie, It shows a hypermethylation state. More specifically, cytosine having a high methylidation frequency in gastric, colon, knee or renal adenocarcinoma cells includes, for example, nucleotides 1153, 1160 1178 1187 1193 in the nucleotide sequence represented by SEQ ID NO: 1. Cytosine which is a base number represented by 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 the non-methylated cytosine, for example, bisulfite such as sodium hydrogen sulfite can be used. By the way, in principle, a reagent that specifically modifies only methylidyl cytosine may be used.

 To bring the reagent that modifies unmethylated cytosine into contact with the extracted DNA, for example, first denature the DNA with an alkaline solution (pH 914) and then use bisulfite such as sodium bisulfite. (Concentration in solution: 3M final concentration, etc.) and treat at 55 ° C for about 1016 hours (overnight). 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 peracyl, whereas methylated cytosine is not converted to peracyl and remains cytosine.

 Next, the DNA sequence treated with bisulfite or the like was converted to type II, and the base sequence when methylated cytosine was contained [the cytosine at the methylated position (cytosine in CpG) remains cytosine. Unmethylated cytosine (cytosine not included in CpG) has a base sequence of peracil] and a pair of methyl-specific primers selected from base sequences that are complementary to the base sequence. The PCR used (hereinafter also referred to as methylation-specific PCR), and the DNA sequence treated with bisulfite or the like as type III, and the base sequence when cytosine is not methylated (all PCR using a pair of non-methyl / Reich-specific plies selected from a nucleotide sequence complementary to the nucleotide sequence and a nucleotide sequence complementary to the nucleotide sequence (hereinafter, non-methyl nucleotide). Sometimes also referred to as le-specific P C R.) And performing.

In the above PCR, in the case of PCR using a methylation-specific ply (the former), DNA in which the cytosine to be analyzed is methylated is amplified, whereas in the case of PCR using unmethylation-specific primers (the latter), DNA in which the cytosine to be analyzed is not methylated is amplified. You. 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 nucleotide sequence containing a cytosine subjected to methylation is considered in consideration that cytosine not subjected to methylation is converted to peracyl, and cytosine subjected to methylation is not converted to peracyl. Design a PCR primer specific for DNA (a methylation-specific primer) and a PCR primer specific for a nucleotide sequence containing cytosine that has not been methylated (a non-methylation-specific primer). The ability to design based on DNA strands that have been chemically converted by bisulfite treatment and are no longer complementary is an important factor, based on each strand of DNA, which was originally double-stranded, and It is also possible to produce a primer specific for the methylation and a primer specific for the unmethylation. Such a primer is preferably designed 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, primers for measuring the methylation frequency of the HAND1 gene by methylation-specific PCR include, for example, a nucleotide sequence in the promoter region, untranslated region or translated region (coding region) of the HAD1 gene. It can be designed as described above based on a nucleotide sequence containing one or more cytosines in CpG existing in the 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, nucleotides 1153, 1160, and 11 in the nucleotide sequence represented by SEQ ID NO: 1 Design based on the nucleotide sequence containing at least one cytosine shown in 78, 1187, 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, etc. Can be. Examples of powerful 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>

 Ml: 5'-AATAGTTTAGGGCGTTGGTC-3 '(SEQ ID NO: 4)

M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5) As a reaction solution in methylidani-specific PCR, for example, 50 ng of 錄 -type DNA and 10 pmol / μΐ of each primer solution are used for each Ιμΐ. 4 μl of 2.5 raM dNTP, 2.5 μl of 10 × buffer (100 mM Tris-HCl pH 8.3, 500 raM KC1, 20 mM MgCl ₂ ), and 5 U / I 1 Is mixed with 0.2 μl, and sterilized ultrapure water is added thereto to obtain a reaction solution having a volume of 25 μl. As the reaction conditions, for example, after keeping the above-mentioned reaction solution at 95 ° C for 10 minutes, it is further heated at 95 ° C for 30 seconds, then at 55 to 65 ° C for 30 seconds, and further at 72 ° C. Conditions for performing heat retention for 30 to 40 cycles with one cycle per second are mentioned.

 After performing such PCR, the amounts of the obtained amplification products are compared. For example, an analysis method (denaturing polyacrylamide gel electrophoresis agarose gel) that can compare the amount of each amplification product obtained by PCR using a methylation-specific primer and PCR using a non-methylidation-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 concentration of the detected band is compared. Here, instead of DNA staining, a pre-labeled primer can be used, and the label concentration can be used as an index to compare the concentration of the band. 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, by monitoring PCR reaction products in real time and performing force kinetic analysis Can be used to compare the amount of each product. Examples of a method for performing real-time PCR include a method using a probe such as a type-dependent nucleic acid polymerase probe, and a method using an interpolator such as Cybergreen. As devices and reagents for the real-time PCR method, commercially available devices and kits can be used.

Such a method is also 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). To take advantage of the chemical differences between cytosine and 5-methylcytosine As a second method _c , after contacting DNA derived from a sample with a reagent for modifying unmethylated cytosine, the DNA containing cytosine to be analyzed as type II is amplified by PCR. And a method of directly analyzing the base sequence of the obtained amplification product. 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 sample, plasma or serum is prepared from blood according to a normal method, and the prepared plasma or serum is used as a sample to prepare free DNA (gastric cancer, colon cancer, knee cancer or Analysis of DNA derived from cancer cells such as renal adenocarcinoma cells) revealed that DNA derived from cancer cells such as stomach cancer, colon cancer, spleen cancer or renal adenocarcinoma cells was avoided while avoiding DNA derived from blood cells. Can be analyzed, and the sensitivity for detecting cancer cells such as gastric cancer, colon cancer, knee cancer or renal adenocarcinoma cells, tissues containing the same, and the like can be improved.

 Next, after contacting the extracted DNA with a reagent that modifies non-methylated cytosine, the DNA is converted into type III, and the base of the promoter region, untranslated region, or translated region (coding region) of the HAND1 gene is determined. Including the cytosine to be analyzed 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 present in the sequence DNA is amplified and the nucleotide sequence of the resulting amplification product is directly analyzed.

Here, 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 includes exon 1 of the HAND1 gene derived from human. 5, the base sequence of genomic DNA containing the promoter region located upstream, and more specifically, the base sequence represented by SEQ ID NO: 1 (described in Genbank Accession No. AC026688). This corresponds to the complementary sequence of the base sequence represented by base numbers 24303 to 26500 in the base sequence.) 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 the nucleotide number 1400-2198. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, especially SEQ ID NO: 1 In the nucleotide sequence shown in the above, cytos in CpG present in a region where CpG is densely present is, for example, a methyl ester which is high in cancer cells such as stomach cancer, colon cancer, victory cancer or renal adenocarcinoma cells. Indicates frequency (ie, hypermethylation). More specifically, cytosine having a high methylation frequency in stomach cancer, colon cancer, spleen cancer or renal adenocarcinoma cells includes, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, Cytosine which is a base number represented by 1187, 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 in the PCR, a primer pair capable of amplifying a DNA having a nucleotide sequence containing the cytosine based on the nucleotide sequence at the 5, 5 upstream and 3 ′ downstream of the cytosine to be analyzed is used. Good to design. The base system IJ for primer design is selected so as not to contain cytosine in CpG to be analyzed. If the nucleotide sequence selected for primer design does not contain cytosine at all, the selected nucleotide sequence and the nucleotide sequence complementary to the nucleotide sequence are used as they are in the primer nucleotide sequence. It can be. In addition, when the base sequence selected for primer design contains cytosine other than that to be analyzed, but the cytosine is not cytosine in CpG, design primers in consideration of the fact that these cytosines are converted to peracil. . That is, a pair of primers each having a base sequence in which all cytosines are peracil and a base sequence complementary to the base sequence are designed. Furthermore, when the base sequence selected for the primer design includes cytosine other than the analysis target and the cytosine is a cytosine in CpG, unmethylated cytosine is converted to peracyl, and Design primers in consideration of the fact that cytosine that has undergone shading is not converted to peracil. That is, the base sequence in the case where the methylated cytosine is contained [the cytosine at the methylated position (cytosine in CpG) remains cytosine, and the unmethylated cytosine (cytosine not included in CpG) is peracyl And a pair of methylation-specific primers each selected from a base sequence complementary to the base sequence, and a base sequence when cytosine is not methylated (all cytosines are And a pair of non-methyl lig specific primers each having a base sequence complementary to the base sequence. in this case In the above-mentioned PCR, a mixture of methylirido-specific primer pairs and non-methylirido-specific primer pairs is mixed and used in equal amounts.

 As a reagent for modifying the non-methylated cytosine, for example, bisulfite such as sodium hydrogen sulfite can be used. Incidentally, in principle, a reagent that modifies only the methylidyl cytosine may be used.

 In order to contact the extracted DNA with a reagent that modifies non-methylated cytosine, for example, first, the DNA is denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite is used. ) (Concentration in solution: 3M final concentration, etc.) and treat at 55 ° C for about 10-16 hours (overnight). 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 peracyl, whereas methylated cytosine is not converted to peracyl and remains cytosine.

 Next, the DNA treated with bisulfite or the like is subjected to PCR using a primer 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, primers for measuring the methylation frequency of the HAND1 gene by direct analysis of the nucleotide sequence include, for example, nucleotide sequences in the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. It can be designed as described above based on a nucleotide sequence containing one or more cytosines in CpG existing in the 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, nucleotides 1153, 1160, and 11 in the nucleotide sequence represented by SEQ ID NO: 1 78, 1187, 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, etc. can be designed as one or more cytosine analysis targets. For example, when the primers B1 and B2 shown below are used, the base sequence after the bisulfite treatment of the DNA having the base numbers 1234 to 1461 of the base sequence represented by SEQ ID NO: 1 DNA (228 bp) is amplified. The primer pair analyzes the methylation frequency of cytosine represented by nucleotide numbers 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 in the nucleotide sequence represented by SEQ ID NO: 1. You 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 the PCR, for example, 50 ng of と す る -type DNA, ρμο of each primer solution of 20ρηιο1 / μ1, μμΐ of 2 mM dNTP, 3 μl of 2 mM dNTP, 10X buffer (100 mM Tris- HCl pH 8.3, 500 mM KC1, 15 mM MgCl ₂ ) and 2.5 μl of a thermophilic DNA polymerase 5 U / μ1 with 0.2 μl, and add sterile ultrapure water to this. Reaction solution with a volume of 25 μl. As the reaction conditions, for example, after keeping the above-described reaction solution at 95 ° C for 10 minutes, the reaction solution is heated at 95 ° C for 30 seconds, then at 57 ° C for 30 seconds, and further at 72 ° C for 30 seconds. Conditions for performing the heat retention for one cycle for 30 to 40 cycles are mentioned.

 After performing such PCR, the base 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 the cytosine to be analyzed is cytosine or thymine (peracyl). In the 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 (peracyl), In addition, the frequency of methylation of cytosine to be analyzed can be measured. In addition, as a method for directly analyzing the nucleotide sequence, a cloned DNA was prepared from a plurality of clones obtained by cloning the amplification product obtained by PCR and using E. coli or the like as a host. Alternatively, the nucleotide sequence of the DNA may be analyzed. The frequency of cytosine methylated to be analyzed can also be measured by determining the percentage of the sample whose cytosine is detected at the position corresponding to the cytosine to be analyzed in the sample to be analyzed. . As a third method, after contacting DNA derived from a sample with a reagent that modifies nonmethylhistocytosine, a DNA that can be used to identify whether or not the DNA to be analyzed is cytosine methylated. One method is to hybridize one probe with another and examine the presence or absence of probe binding.

 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 sample, plasma or serum is prepared from blood according to an ordinary method, and the prepared plasma or serum is used as a sample and free DNA (gastric cancer, colon cancer, squamous cancer or Analysis of DNA derived from cancer cells such as renal adenocarcinoma cells) revealed that DNA derived from cancer cells such as stomach, colon, knee or renal adenocarcinoma cells was avoided while avoiding blood cell-derived DNA. Can be analyzed, and the sensitivity of detecting cancer cells such as gastric cancer, colon cancer, kidney cancer or renal adenocarcinoma cells, and tissues containing the same can be improved.

 Then, 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 methylated as an analysis ¾ ^. Then, the presence or absence of binding between the DNA and the probe is examined. The cytosine to be analyzed can be selected from cytosines in the base sequence represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. it can.

Here, 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 includes exon 1 of the HAND1 gene derived from human, The base sequence of the genomic DNA containing the promoter region located 5 ′ upstream thereof can be mentioned, and more specifically, the base sequence represented by SEQ ID NO: 1 (the base sequence described in Genbank Accession No. AC026688) Corresponds to the complementary sequence of the 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 the 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, For example, cancer cells such as stomach cancer, colon cancer, pancreatic cancer, or renal adenocarcinoma cells show a high methylation frequency (ie, a hypermethylation state). More specifically, the stomach Examples of cytosine having a high methylation frequency in cancer, colon cancer, knee cancer or renal adenocarcinoma cells include, for example, nucleotides 1153, 1160, 1178, 1187, 1193, 1218, and 1232 in the nucleotide sequence represented by SEQ ID NO: 1. , 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, etc., and cytosine.

 The probe used in the hybridization is based on the base sequence containing cytosine to be analyzed, and cytosine that has not been subjected to methylation is converted to peracyl, and cytosine that has undergone methylation is converted to peracyl. You should design in consideration of not being done. That is, the base sequence when the methylated cytosine is contained [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 (all cytosines are Base sequence) or a non-methylation-specific probe having a base sequence complementary to such a base sequence. Such a probe may be used after being labeled in order to facilitate analysis of the presence or absence of a bond between the DNA and the probe. In addition, the probe may be used by immobilizing it on a carrier according to a usual method. In this case, it is preferable to label DNA extracted from a mammal-derived specimen in advance.

 As a reagent for modifying non-methyl histitocin, for example, bisulfite such as sodium hydrogen sulfite can be used. By the way, in principle, a reagent that specifically modifies only methylidyl 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 a bisulfite such as sodium bisulfite ( bisulfite) (concentration in the solution: 3M final concentration, for example), and treat at 55 ° C for about 10 to 16 hours (overnight). To accelerate the reaction, denaturation at 95 ° C and reaction at 50 can be repeated 10-20 times. In this case, the unmethylated cytosine is converted to peracyl, while the methylated cytosine is not converted to peracyl and remains cytosine.

If necessary, convert DNA treated with bisulfite, etc. to type II in the same manner as in the second method. The DNA may be amplified in advance by performing PCR.

 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 methylation of cytosine to be analyzed. By comparing the amount of DNA that binds to the methylation-specific probe with the amount of DNA that binds to the non-methionylation-specific probe, it is possible to determine the frequency of cytosine methylation to be analyzed. it can.

 More specifically, a probe for measuring the methylation frequency of the HA D1 gene is, for example, a CpG gene present in the nucleotide sequence in the promoter region, untranslated region or translated region (coding region) of the HA D1 gene. It can be designed as described above based on a base sequence containing one or more cytosines therein. 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, nucleotides 1153, 1160, and 1178 in the nucleotide sequence represented by SEQ ID NO: 1 , 1187, 1193, 1218, 1232, 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, etc. it can. An example of such a probe is shown below.

<Set 1>

Non-methylation specific probe: 5'-AAGTATGTAGTTTTTGTGTTTG-3 '(SEQ ID NO: 8) Methylation-specific probe: 5'-AAGTACGTAGTTTTCGTGTTCG-3' (SEQ ID NO: 9)

Non-methyl specific probe: 5'-TGTGGGTTGAGTGTAMATT-3 '(SEQ ID NO: 10)

Methylation-specific probe: 5'-CGCGGGTTGAGCGTAAAATT-3 '(SEQ ID NO: 11) Hybridization is described, for example, in Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition. The method can be carried out according to the usual method described in Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory press) or the like. Hybridization is usually performed under stringent conditions. Here, “stringent conditions J” include, for example, 6 XSSC (1.5 M NaCl, 0.15 M trisodium citrate containing After forming a hybrid at 45 ° C in a solution containing 10XSSC, the solution was washed at 50 ° C with 2XSSC (Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6) and the like. The salt concentration in the washing step is, for example, 50 in 2 × SSC. It can be selected from C conditions (low stringency conditions) to 0.2 XS SC to 50 ° C (high stringency conditions). The temperature in the washing step can be, for example, from room temperature (low stringency conditions) to 6

5 ° C (high stringency conditions) can be selected. Also, both salt concentration and temperature can be varied.

 After vigorous hybridization, the amount of DNA bound to the methylation-specific probe and the amount of DNA bound to the unmethylation-specific probe were compared to analyze (Ie, cytosine in the CpG contained in the base sequence on which the probe was designed) can be measured. A fourth method is to apply a restriction enzyme capable of discriminating the presence or absence of cytosine methylation to be analyzed to DNA derived from a sample, and then check for digestion by the restriction enzyme. You can also.

 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 blood according to a conventional method, and the prepared plasma or serum is used as a specimen to contain free DNA (stomach cancer, severe cancer, (Including DNA derived from cancer cells such as cancer or renal adenocarcinoma cells), it was found that DNA derived from cancer cells such as stomach cancer, colon cancer, knee cancer or renal adenocarcinoma cells was avoided while avoiding DNA derived from blood cells. NA can be analyzed, and the sensitivity for detecting cancer cells such as stomach cancer, conjunctive cancer, knee 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 should be selected from cytosines in the nucleotide sequence of one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the HAND1 gene. Can be. Here, 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 includes exon 1 of the HAND1 gene derived from human, 5, the base sequence of the genomic DNA containing the promoter region located upstream, and more specifically, the base sequence represented by SEQ ID NO: 1 (the base sequence described in Genbank Accession NO. AC026688). It corresponds to the complementary sequence of the base sequence represented by base numbers 24303 to 26500 in the sequence. 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 the 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, For example, cancer cells such as stomach cancer, colon cancer, spleen cancer or renal adenocarcinoma cells show a high methylation frequency (ie, hypermethylation state). More specifically, cytosine having a high methylation frequency in stomach cancer, colon cancer, knee cancer or renal adenocarcinoma cells includes, for example, in the nucleotide sequence represented by SEQ ID NO: 1, nucleotides 1153, 1160, 1178, Cytosine which is a base number represented by 1187, 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 sometimes referred to as “methylidani-sensitive restriction enzyme”) used in the method refers to digestion of a recognition sequence containing methylated cytosine. Means a restriction enzyme capable of digesting a recognition sequence containing cytosine that has not been methylated. In the case of DNA in which the cytosine contained in the recognition sequence is methylated, the DNA is not cleaved by the action of a methylation-sensitive restriction enzyme, whereas in the case of DNA in which the cytosine contained in the recognition sequence is not methylated The DNA is cleaved by the action of a methylation-sensitive restriction enzyme. Specific examples of methyl ligase-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, DNA treated with a methylation-sensitive restriction enzyme containing the cytosine to be analyzed in the recognition sequence is type II, and the cytosine to be analyzed is included. Including the recognition sequence, One method is to perform PCR using a primer pair that can amplify DNA that does not contain a recognition sequence, and to check for the presence or absence of DNA amplification (amplification product). If 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. In this way, by comparing the amount of the amplified DNA, the frequency of methylation of cytosine 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 force kinetic analysis. Can 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 type III-dependent nucleic acid polymerase probe, and a method using an internet power source 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 base numbers 1272 and 1377 in the base sequence represented by SEQ ID NO: 1, the cytosine is included in the recognition sequence of Hpall, and the methylation frequency of the cytosine is measured by the above method. can do.

Other methods for examining the presence or absence of digestion by the restriction enzyme include, for example, a method in which DNA that has been subjected to a methylation-sensitive restriction enzyme containing a cytosine to be analyzed in a recognition sequence is reacted with a HAND1 gene, In addition, there may be mentioned a method in which Southern hybridization is performed using a DNA not containing the recognition sequence of the restriction enzyme as a probe, and the length of the hybridized DNA is examined. When the cytosine to be analyzed is methylated, a longer DNA is detected than when the cytosine is not methylated. By comparing the amount of the detected long DNA with the amount of the short DNA, it is possible to measure the frequency of cytosine methylation to be analyzed. Using the various methods described above, the frequency of methyl ether of the HAND1 gene contained in a specimen derived from a mammal is measured. The measured methylation frequency and, for example, samples from healthy mammals that can be diagnosed as having no cancer cells such as stomach cancer, colon cancer, knee cancer and renal adenocarcinoma cells The methylation frequency of the contained HAND1 gene (control) is compared, and the degree of canceration of the specimen is determined based on the difference obtained by the comparison. For example, if the frequency of methylation 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 as compared to the control), the degree of canceration of the specimen is increased. Can be determined to be higher than the control.

 Here, the term "degree of canceration" has the same meaning as generally used in the art. Specifically, for example, when a mammal-derived specimen is a cell, it means the degree of 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.

The expression of the HAND1 gene is lower in cancer cells such as gastric cancer cells than in samples 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 expressed normally. As a result, the amount of the expression product of the gene (more specifically, 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) having a correlation with the frequency of methylation of the HAD1 gene contained in a specimen derived from a mammal is measured, and the measured methylation level is measured. The degree of canceration of the sample can be determined based on a difference obtained by comparing an index value (for example, the amount of an expression product) having a frequency correlation with a control. In the first step of the evaluation method of the present invention, a method for measuring an index value having a correlation with the methylation frequency of the HA D1 gene contained in a specimen derived from a mammal includes, for example, a transcript of a HAND1 gene. A method for measuring the amount of mRNA can be given. The measurement includes, for example, RT (Reverse Transcription) -PCR, Northern blotting [Molecular Cloning, Cold Spring Harbor Laboratory (1989)], in situ RT-PCR [Nucleic Acids Res., 21, 3159-3166. (1993)], in situ hybridization method, NASBA method [Nucleic acid sequence-based amplification, nature, 350, 9-92 (1991)] Any suitable method may be used.

 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 the Northern plot method is used to measure the amount of raRA contained in the prepared sample, the detection probe is a HAND1 gene or a part thereof (restriction enzyme cleavage of HAND1 gene, base of HAND1 gene, Or about 100 bp to about 100 bp, which are chemically synthesized based on the sequence, and can be detected under the detection conditions used in the hybridization with the mRNA contained in the sample. There is no particular limitation as long as it gives specificity.

 When the RT-PCR method is used to measure the amount of mRNA contained in the prepared sample, the primer used may be any primer 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). Using these primers, the amount of the transcript by the RT-PCR method can also be measured 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 force kinetic analysis. Can be used to compare the amount of each product. Examples of a method for performing real-time PCR include a method using a probe such as a type III-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) In the first step of the evaluation method of the present invention, an index value having a correlation with the methyl methane frequency of the HAND1 gene contained in a specimen derived from a mammal is measured. Another method is to measure the amount of HAND1 protein, which is a translation product of the HAND1 gene. it can. For this measurement, for example, using an antibody specific to the HAND1 protein (monoclonal antibody, polyclonal antibody), an immunoblot method described in Cell Engineering Handbook, Yodosha, 207 (1992), and a separation method 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.

 Using various methods as described above, an index value having a correlation with the frequency of methylation of the HAND1 gene contained in a specimen derived from a mammal is measured. There is a correlation between the index value that correlates with the measured methylation frequency and the methylation frequency of the HAND1 gene contained in a sample from a healthy mammal 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 an index value positively correlated with the methylation frequency of the HAND1 gene contained in a mammal-derived specimen is higher than the control, or an index value negatively correlated with the HAND1 gene is lower than the control If the HAND1 gene is in a highly methylated state compared to the control, it can be determined that the degree of canceration of the sample is higher than 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 methyl ane frequency of the HAND1 gene or an index value correlated therewith include gastric cancer, colon cancer, renal 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 relates to the detection of cancer cells such as gastric cancer, colon cancer, Teng cancer, bladder cancer, renal adenocarcinoma, chorioadenocarcinoma, adrenal cortical adenocarcinoma cells containing these primers, probes or specific antibodies as reagents. Cancer, gastric cancer, consequently cancer, knee cancer, bladder cancer, renal adenocarcinoma, chorioadenocarcinoma, adrenal cortical adenocarcinoma in which these primers, probes or specific antibodies are immobilized on a carrier We also provide chips for detecting cancer cells such as cells, and the scope of the rights of the present invention evaluation method is similar to the above-mentioned detection kit ゃ detection chip, which utilizes the substantial principle of the method. This includes use in various forms.

The expression of the HAND1 gene is higher than that in samples of cells or tissues derived from healthy mammals. Low in cancer cells such as gastric cancer cells. On the other hand, as shown in the examples described below, the amount of the expression product of the gene can be increased by causing a substance that inhibits DNA methylation associated with the HAND1 gene to act on cancer cells such as gastric cancer cells. it can. This is a substance that can compensate for the decreased expression level of HAND1 gene in cancer cells such as gastric cancer cells or the accompanying decrease in function, for example, non-methyl chick (or does not have the abnormal methylation 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 The first is the treatment of gastric cancer and other cancers, the gastric mucosa layer (superficial epithelium, glandular tissue, lamina propria and mucosal muscularis), the gastric submucosal tissue layer, (proprietary) muscle It is useful for suppressing canceration of normal tissues such as the stratum corneum and the serosal layer.

 For example, canceration may be suppressed by administering a substance that decreases the methyl methane frequency of the HAND1 gene to cells in the body of a mammal that can be diagnosed with cancer. For example, by providing a substance that inhibits DNA methyl hydride related to the HAND1 gene to cancer cells such as gastric cancer cells, the cytosine in CpG present in the nucleotide sequence in the promoter region or coding region of the HAND1 gene can be normalized. As in the case of tissue, it can be hypomethylated to increase the expression level of HAND1 gene transcript mRNA and, consequently, increase the expression level of HAND1 gene translation product HAND1 protein. . Also, for example, by introducing a cDNA comprising a base sequence encoding the amino acid sequence of the HAND1 gene or HAND1 protein into cancer cells such as gastric cancer cells, the expression level of HAND1 protein in cancer cells such as gastric cancer cells is increased. Will be able to.

 That is, the present invention is characterized in that (1) the active ingredient contains a substance having the 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 nucleic acid having a nucleotide sequence encoding an amino acid sequence of HAD1 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. The agent can be produced by mixing the active ingredient with a pharmaceutically acceptable carrier such as a water-soluble solvent, a water-insoluble solvent, a buffer, a solubilizing agent, an isotonic agent, a stabilizer and the like. . If necessary, auxiliary agents such as preservatives, suspending agents and emulsifiers may be added. In the case of parenteral administration (in general, injection or the like is preferable), the anticancer agent can be used in the form of an ordinary 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 (for example, cells in a mammal that can be diagnosed with cancer). For example, parenteral administration includes, for example, injection (subcutaneous, intravenous, topical) and the like.

 The dosage 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 patient's cells. 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 nucleic acid having a nucleotide sequence encoding the amino acid sequence of HAND1 into a cell, a gene transfer method using a viral vector, a gene transfer method using a non-viral vector (Nikkei Science, April 1994, pp. 20-45, Special Issue on Experimental Medicine, 12 (15) (1994), Experimental Medicine Supplement “Basic Technology for Gene Therapy”, Yodosha (1996)), etc. it can.

Examples of the former gene transfer method include, for example, a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a box virus, a polyvirus, a symbiswinores, or other DNA virus or an RNA virus. Examples include a method of introducing a DNA encoding TR4 or mutant TR4 by incorporating the DNA. Examples of the method of gene transfer using a non-viral vector include a method of directly administering an expression plasmid into muscle (DNA vaccine method), a ribosome method, a ribofectin method, a microinjection method, a calcium phosphate method, and an electoral poration. Law. Methods for using a nucleic acid having a nucleotide sequence encoding the amino acid sequence of HAND1 as an active ingredient of a gene therapy drug as an anticancer drug include an in vivo method in which the nucleic acid is directly introduced into the body, and a method using cells specific to humans. An ex vivo method in which the nucleic acid is introduced into the cells outside the body after removal, and the cells are returned 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 nucleic acid 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 ribosome preparation such as a suspension, a cryogen, or a centrifugal concentrated cryogen. Such a preparation may be pharmaceutically acceptable, for example, a carrier such as a water-soluble solvent, a non-water-soluble solvent, a buffer, a solubilizing agent, 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 (in general, injection or the like is preferable), the anticancer agent can be used in the form of a normal liquid such as a solution. The search method of the present invention is a method for searching for a substance capable of promoting the expression of the HAND1 gene, comprising: (1) a first step of bringing a test substance into contact with cancer cells; (2) a first step after the first step (1); A second step of measuring the amount of the expression product of the HAND1 gene contained in the cancer cells, (3) the 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 cells in the first step of the search method of the present invention are not particularly limited, and may be cancer cells isolated from a cancer tissue derived from a mammal, or may be derived from a mammal established as a cell line. It may be a cancer cell line. Examples of the mammal include human, monkey, mouse, rat, hamster and the like. Preferred types of cancer include gastric cancer. Specifically, for example, MKN28 (available from JCRB), MKN 74 (available from JCRB), KAT0-ΙΠ (available from ATCC), AGS (available from ATCC) And known human gastric cancer cell lines such as Hs746T (available from ATCC).

For in the first step of the present searching method of contacting a test substance to cancer cells, as the amount of cancer cells, usually sufficient if about 1 0 ⁴ -1 0 ⁸ cells, about 1 0 ⁵ to 1 0 ⁷ cells Is preferred. In addition, the concentration of the test substance is usually about 0.1 ng / m1 to about 100 g Zm1, and preferably about 1 ng / m1 to about 50 μg / m1. The time for bringing the test substance into contact with the cancer cells is usually 1 hour to 5 days, preferably several hours to 2 days. The number of times the test substance is brought into contact with the cancer cells may be one or more.

 The environment in which the test substance is brought into contact with the cancer cells is preferably an environment that maintains the life activity of the cancer cells, and examples thereof include an environment in which the 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, the amount of the expression product of the HAND1 gene contained in the cancer cells is measured by the method described in the first step of the present invention. Method of Measuring Index Value Correlated to Met / Reich Frequency of Gene ”.

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 the control, the method described above is used. Thus, 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, And the amount of HAND1 gene expression product (when the test substance is not brought into contact with the cancer cells) (control), and promotes the expression of the HAND1 gene of the test substance based on the difference obtained by the comparison. Determine your ability to do so. Assuming that the amount of HAD1 gene expression product in cancer cells contacted with the test substance is compared with the control (in this case, the amount of HAND1 gene expression product in cancer cells not contacted with the test substance) If the test substance is high, it can be determined that the test substance has the ability to promote the expression of the HAD1 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.In this case, the expression of the HAND1 gene of the other test substance in advance may be used. Preferably, the ability to promote present is known.

 In this way, it is possible to search for a substance capable of promoting the expression of the HAND1 gene. As a background or a control, the amount of the HAND1 gene expression product contained in a normal cell line such as a normal gastric cell line or a sample derived from a healthy mammal that can be diagnosed as having no cancer cells such as gastric cancer cells is determined. It is preferable to measure both when the test substance is contacted and when it is not. Example

 EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited thereto.

Example 1 Test for confirming the methylated state of HAND1 gene in gastric cancer cell lines (Part 1) Two human gastric cancer cell lines [MKN28 (JCRB) and MKN74 (JCRB)] were cataloged by JCRB (Japan Cancer Research Resources Bank). described, was cultured to Konfuruento dedicated media Me other each cell line, they were collected each about Iotakaiiotaomikuron ⁷ cells. The collected cells or human-derived normal gastric mucosal epithelial tissue [obtained from a patient by obtaining an informed consent] were added to SEDTA buffer [10 mM Tris-HCl (pH 8.0), lOmM EDTA (pH 8.0). , LOOmM NaCl], and then homogenized. To the resulting mixture was added proteinase K (Sigma) to 500; Ug / ml and sodium dodecyl sulfate to 1% (w / v), and the mixture was shaken at 55 ° C for about 16 hours. I'm sorry. After completion of the shaking, the mixture was subjected to phenol [saturated with 1 M Tris-HCl (pH 8.0)]. 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 is dried in TE buffer (10 mM Tris, ImM EDTA, pH 8.0), and RNase A (Sigma) is added thereto at 40 / ig / ml. Incubated. The incubated mixture was subjected to phenol-cloth form extraction. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, followed by ethanol precipitation to recover 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. R es., 22, 2990-2997, 1994; treated with sodium bisulfite according to the method described in Herman et al., Pro. Natl. Acad. Sci. USA, 93, 982 1-9826, 1996. That is, genomic DNA (about 1 g) after treatment with the restriction enzyme was dissolved in distilled water to prepare a 20 μl genomic DNΑ solution, and about 6 μM of 6M sodium hydroxide was added thereto. The mixture was left at room temperature for 15 minutes. 120 μl of [0.6 mM hydroquinone (Sigma) and 3.6 N sodium bisulfite (Sigma)] was added to the standing mixture, and the mixture was added at 95 ° C. for 30 seconds and then at 50 ° C. for 15 minutes. Insulation was performed for 15 cycles. 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 6M7 sodium chloride 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 standing mixture. The collected precipitate was suspended in 20 μl of ΤΕ buffer.

 The obtained DNA was used as a mirror, and a DNA (228 bp) having the nucleotide sequence represented by nucleotides 1234 to 1461 in the nucleotide sequence represented by SEQ ID NO: 1 was subjected to bisulfite treatment, and then the DNA having the nucleotide sequence was amplified by PCR. For this purpose, 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 reaction solution for PCR, 50 ng of DNA to be 铸, 20 μmol / 1 of each of the above-mentioned primer solutions (ΐμΐ), 3 mM of each 2 mM dNTP, 3 × 1 and 10 × buffer solution (100 mM Tris-HCl pH 8. 3, 500 mM KC1, 15 mM MgCl ₂ ) and 2.5 μl of the thermophilic DNA polymerase 511/1 and 0.21 are mixed with sterile ultrapure water to make a volume of 25/1. xl was used. After incubating the reaction solution at 95 ° C for 10 minutes, perform 40 cycles of 95 ° C for 30 seconds, followed by 57 ° C for 30 seconds and 72 ° C for 30 seconds as one cycle. PCR was performed under the conditions. After PCR, DNA amplification was confirmed by 2% agarose gel electrophoresis.

After the PCR product was ligated into pGEM-T-Easy (Promega), it was introduced into E. coli XLlBlue strain. Transformed E. coli colonies were picked up by 10 cells per stomach cancer cell line. The obtained E. coli lysate was subjected to PCR under the same conditions as above, and the PCR reaction solution was subjected to agarose genole electrophoresis in the same manner as above. After the application, a gel portion containing 228 bp DNA was cut out, and DNA was extracted therefrom. The nucleotide sequence of the extracted DNA was analyzed using a DNA sequencer (ABI310, PE Biosystems). Based on the sequence results of DNA prepared from each colony, it was determined whether cytosine in CpG existing in the region represented by base numbers 1234 to 1461 in the base sequence represented by SEQ ID NO: 1 was replaced with peracyl. The frequency of DNA in which cytosine was not substituted with uracil /, that is, the methylation frequency of the cytosine, was measured among the DNAs prepared from the 10 tested knees.

 The results are shown in Table 1. In the case of DNA prepared from normal gastric mucosal epithelial tissue (21N), 12 cytosines in CpG existing in the region represented by base numbers 1234 to 1461 (base numbers 1¾6, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434) showed little DNA not substituted with peracyl (ie, methylated DNA). 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 DNAs not substituted with peracyl (that is, methylated DNA).

Cell Cytosine methylation frequency *

 Base number

 1266 1272 1292 1305 1307 1316

 21N 0 0 1 0 3 2

 Picture 28 5 8 10 10 10 10

 MKN74 8 4 10 10 8 8

 Cell Cytosine methylation frequency *

 Base number

 1356 1377 1399 1401 1422 1434

 21N 2 1 4 2 2 5

 MKN28 6 9 10 7 7 6

Wisteria 74 8 3. 7 5 9 8 * For each cytosine, indicates the number of DNAs in which the cytosine is not substituted with peracil, among DNA prepared from 10 colonies. The cytosine to be analyzed is represented by the base number in the base sequence shown in SEQ ID NO: 1. Example 2 Test for confirming the expression status of HAND1 gene in gastric cancer cell lines and the effect of methyl eh inhibitor on the expression of the gene

 Two human gastric cancer cell lines (MKN28 and MKN74) were cultured in a special medium until they reached 70% confluence, and then each cell was collected. Each of the collected cells (about 100 mg wet weight) and human-derived normal stomach mucosal epithelial tissue (21N: about 50 mg) were mixed with 1 ml of an IS0GEN solution (Nitsubion Gene) and homogenized. The suspension was added with 2 ml of black-mouthed form. After the suspension, the mixture was centrifuged (4 ° C, 15000xg, 15 minutes) to recover the supernatant. The collected supernatant was suspended by adding 0.5 ml of isopropanol, and then centrifuged (4 ° C, 15000xg, 15 minutes) to recover a precipitate (RNA). The collected precipitate is 75 ° /. After rinsing with ethanol, it was dissolved in DEPC (getyl pyrocarbonate) treated water.

In addition, 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 special medium. One day after the inoculation, 5-methyl-2'-deoxycytidine (hereinafter referred to as 5Aza-dC) (manufactured by Sigma), a methylidani inhibitor, was added to the medium to a concentration of l ^ uM. 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, 5 days after the inoculation, 5Aza-dC was similarly added to the medium 3 days after the inoculation. 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 RNA thus obtained with DNasel (Ambion), the RNA was converted into type III, and cDNA was synthesized using Superscriptll (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 type I and the following HAND1 S and HANDl A as a primer pair. . In this case, as a control, perform PCR using the above-mentioned cDNA as type III and PCNAS and PCNAA shown below as a primer pair. As a result, DNA derived from mRNA of the proliferating cell nuclear antigen (PCNA) gene was amplified.

Kufuimaichi (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 PCR reaction solutions, 50 ng of type III cDNA, each of the above two lOpmol / 1 primer solutions, 4 μl of each 2.5 mM dNTP, 4 μl of 5 mM dUTP, and 5 μl of 10XSYBR Green PCR Buffer , 25raM MgCl ₂ with 6 ^ 1, thermophilic DNA polymerase (AmpliTaq Gold) 5 11 // 21 with 0.31, AmpEraseUNG with 0.5μ1, and add sterile ultrapure water to make a volume of 50μ. 1 was used. Real Time PCR was performed using iCycler Thermal Cycler (Bio-Rad Laboratories). When amplifying DNA derived from the mRNA of the HA D1 gene, incubate the reaction solution 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. Real time PCR was performed using C for 30 seconds as one cycle, and the HAD1 gene was quantified. When amplifying DNA derived from PCNA gene mRNA, incubate the reaction solution at 95 ° C for 10 minutes, and then cycle at 95 ° C for 30 seconds and then at 55 ° C for 60 seconds. Real Time PCR was performed to quantify the PCNA gene.

 The results are shown in FIG. In the case of human-derived normal gastric mucosal epithelial tissue (21N), DNA derived from the mRNA of the HAN D1 gene was detected, whereas in both gastric cancer cell lines, the DNA was detected. Did not. That is, expression of the HAD1 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 ΜΚ28 and ΜΚΝ74 cultured in the presence of ΙμΜ5Aza-dC, DNA derived from HAND1 gene mRNA was detected. The DNA derived from the PCNA gene mRNA was expressed in the presence of 5Aza-dC in the case of normal gastric mucosal epithelial tissue (2IN) and in the presence of 5μA-dC in the case of gastric cancer cell lines MKN28 and MKN74 cultured in the absence of 5Aza-dC. In both cases of ΜΚΝ28 and ΜΚΝ74 cultured in Detected similarly. That is, in the case of the gastric cancer cell lines MKN28 and N74, expression of the HAH gene was observed in the presence of the methylation inhibitor.

 From the above results, it became clear that the methylation of cytosine in the gastric cancer cell line was inhibited by the methylation inhibitor, and that the HAD1 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 (part 2) and effect of methylation inhibitor on expression of the gene

Two human-derived gastric cancer cell lines (MKN28, MKN74) were inoculated at a density of about 3 × 10 ⁵ cells / 10 cm plate and cultured using a special medium. On the first day after the inoculation, 5Aza-dC, a methylidani inhibitor, was added to the medium to a concentration of 1M. 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. Next, on the 3rd and 5th days after the inoculation, 5Aza-dC was added to the medium in the same manner. Six days after the 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 type III, PCR was carried out using the following non-methylidani-specific primers U1 and U2 or the methylidani-specific primers Ml and M2. When the unmethylation-specific primers U1 and U2 are used, and when the methyleic-specific primers Ml and M2 are used, the nucleotide sequence represented by nucleotide numbers 1141 to 1324 of the nucleotide sequence represented by SEQ ID NO: 1 A 184 bp DNA having a base sequence after the DNA having the above-mentioned is treated with bisulfite is amplified. <Unmethylated specific primer>

U1: 5'-AATAGTTTAGGGTGTTGGTT-3 '(SEQ ID NO: 2)

U2: 5 '-AATTTTACACTCMCCCA-3' (SEQ ID NO: 3)

<Methylation specific primer>

Ml: 5 '-AATAGTTTAGGGCGTTGGTC-3' (SEQ ID NO: 4)

M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5) Methylation-specific primer and unmethylation-specific primer have specificity First, genomic DNA [genomic DNA (l)] was extracted from normal gastric mucosal epithelial tissue (21N) by a conventional method, and a part of this was treated with methylase Sssl (NEB). All 5'-CG-3 of the genomic DNA was methylated to obtain DNA [DNA (2)]. The obtained DNA (l) and DNA (2) were also subjected to the above-mentioned methylation-specific PCR and non-methylation-specific PCR.

As a reaction solution for PCR, 50 ng of the DNA to be type II, 20 μmol /; U1 of each of the above-mentioned primer solutions in ΐμ each, 2.51 of each 2 mM dNTP, 2.51 and 10X buffer (lOOraM Tris- HCl _P 8.3, 500raM KC1, 20mM MgCl ₂ ;) 51 and ff thermophilic DNA polymerase 5U / μ1 0.2μ1 are mixed, and sterile ultrapure water is added to the mixture. Was used at 25 μl. When using the above unmethylation-specific primer, incubate the reaction solution at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 56 ° C for 30 seconds, and then at 72 ° C. The PCR was carried out under the condition that 33 cycles of incubation were performed with 30 seconds as one cycle. When the above-mentioned methylation-specific primer is used, the reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 61 ° C for 30 seconds, and then at 72 ° C. PCR was performed under the condition that 33 cycles of incubation were performed at C for 30 seconds 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 results are shown in FIG. In the case of human-derived normal gastric mucosal epithelial tissue (21N), when non-methylation-specific primers were used (lane U), a band of amplified DNA was observed, and methylation-specific primers were used. In this case (lane M), no band of amplified DNA was detected. Therefore, in the case of human-derived normal gastric mucosal epithelial tissue (21N), at least cytosines represented by nucleotide numbers 1153, 1160, 1307, and 1316 of the nucleotide sequence represented by SEQ ID NO: 1 are methylated. It was determined not to be. In addition, in the case of two types of gastric cancer cell lines (ΜΚΝ28, ΜΚΝ74) cultured in the absence of a methylation inhibitor (5Aza-dC 0 μΜ), the use of non-methyl eh specific primers (lane U) No amplified DNA band was detected in the sample, and when a methylation-specific primer was used (the amplified DNA fragment was observed in the lane. Therefore, under the above conditions, SEQ ID NO: 1 The cytosines represented by base numbers 1153, 1160, 1307, and 1316 in the base sequence were determined to be methylated.

On the other hand, in the case of gastric cancer cell lines MKN28 and MKN74 cultured in the presence of ΙμΜ5Aza-dC, When the non-methylated specific primer was used (lane U), a band of the amplified DNA was recognized, and when the methylated specific primer was used (lane M), a weak band was detected. It was only. Therefore, under these conditions, it was determined that in many genomic DNAs, the cytosines represented by base numbers 1153, 1160, 1307, and 1316 in 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 knee cancer cell lines (HPAF-II, Mia PaCa-2 and HPAC), three colon adenocarcinoma cell lines (HT-29, C0L0205 and SW1116), and renal adenocarcinoma cell line (ACHN) [ Both were obtained from the American Type Culture Collection (ATCC)]. After culturing until confluence in a dedicated medium described in the ATCC catalog for each cell line, about lxlO ⁷ cells were collected. After adding a 10-fold volume of SEDTA buffer [lOraM Tris-HCl (pH 8.0), lOmM EDTA (pH 8.0), lOOniM NaCl] to the collected cells, the mixture was homogenized. To the resulting mixture was added proteinase K (Sigma) at 500 / _ig / ml and sodium dodecyl sulfate at 1% (w / v), and the mixture was shaken at 55 ° C for about 16 hours. . After shaking, the mixture was subjected to phenol [saturated with 1 M Tris-HCl (pH 8.0)] · chloroform extraction. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, followed by ethanol precipitation to recover the precipitate. Dissolve the collected precipitate in TE buffer (10 mM Tris, ImM EDTA, H8.0), and add it to ^^ g / ml: Add Rase A (Sigma) for 1 hour at 37 ° C. Incubated. The incubated mixture was subjected to phenol-mouth opening extraction. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, followed by ethanol precipitation to recover 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, knee) was obtained from Clark et al., Nucl. Acids. Res., 22, 2990-2. 997, 1994; Herman et al., Pro. Natl. Acad. Sci. USA, 93, 9821-9826, 1996. Treated with sodium bisulfite according to the method described. That is, genomic DNA (about 1 μg) is dissolved in distilled water to prepare a 201 genomic DNA solution, 6 M of sodium hydroxide is added thereto to about Ι μΐ, and the mixture is allowed to stand at room temperature for 15 minutes. I left it. After adding [0.6 mM hydroquinone (Sigma) and 3.6N sodium bisulfite (Sigma)] 1201 to the mixture left as it is, this was added to 95 ° C for 30 seconds and then to 50 ° C for 15 minutes for one cycle. Insulation was performed for 15 cycles. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). The purified DNA was dissolved in 50 / TE buffer, and 2.5 ^ 1 6M 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 standing mixture. The collected precipitate was suspended in 50 μΐ of TE buffer or ultrapure water.

 The obtained DNA was designated as type III, and PCR was carried out in the same manner as in Example 3 using the non-methyl-specific primers U1 and U2, or the methyl-specific specific primers Ml and # 2. When using unmethylation-specific primers U1 and U2, and when using methylation-specific primers Ml and M2, the base sequence represented by base numbers 1141 to 1324 of the base sequence represented by SEQ ID NO: 1 A 184 bp DNA having a base sequence after the biisulfite treatment of the DNA having the above is amplified.

Unmethylation-specific primer>

U1: 5'-AATAGTTTAGGGTGTTGGTT-3 '(array No. 2)

U2: 5, -AATTTTACACTCAACCCA-3 '(SEQ ID NO: 3)

Methylation-specific primers>

 Ml: 5'-AATAGTTTAGGGCGTTGGTC-3 '(SEQ ID NO: 4)

M2: 5'-AATTTTACGCTCAACCCG-3 '(SEQ ID NO: 5)

As a PCR reaction solution, 20 ng of type III DNA (derived from a cancer cell line) or 40 ng (derived from normal tissue), 20 ρπιο1 / ν プ ラ イ マ ー of each of the above primer solutions 11 and 2 mM dNTP were used. Mix 5 μl, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KC1, 20 mM MgCl ₂ ) with 5 ^ 1, and heat-resistant DNA polymerase 51Ι / μ1 with 0.2 / i1. Then, sterilized ultrapure water was added to this to make a liquid volume of 25 μl. When using the above non-methylidani specific primer Incubate the reaction solution 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 as one cycle. PCR was performed under cyclic conditions. When the above methylation-specific primer is used, the reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds and then at 61 ° C for 30 seconds. The PCR was performed under the conditions of performing 40 cycles of incubation at 72 ° C for 30 seconds as one cycle. In each case, after performing PCR, the PCR reaction solution containing the amplification product was subjected to 2% agarose gel electrophoresis. Figure 3 shows the results. In the case of human-derived normal tissues, colons and kidneys showed a broadened DNA band when non-methyl eh specific primers were used (lane U) and when methylation-specific primers were used (lane U) No band of amplified DNA was detected in the case M). In the spleen, 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 knee, 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. In addition, among the various cancer cell lines tested, in the case of cell lines other than the renal gland bladder cancer cell line (5637ACH), the band of amplified DNA was observed when a non-methylation-specific primer was used (lane ϋ). No detection was observed, and 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 the cell lines other than the renal adenocarcinoma cell line (ACH), 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. Renal adenocarcinoma cell line

 With respect to (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 meth / Leich. Industrial potential

According to the present invention, it is possible to provide a method for evaluating the degree of cancer in a specimen derived from a mammal. Sequence listing free text

SEQ ID NO: 2

 Oligonucleotide primers designed for PCR SEQ ID NO: 3

 Oligonucleotide primer designed for PCR SEQ ID NO: 4

 Oligonucleotide primer designed for PCR SEQ ID NO: 5

 Oligonucleotide primer designed for PCR SEQ ID NO: 6

 Oligonucleotide primers designed for PCR SEQ ID NO: 7

 Oligonucleotide primer designed for PCR SEQ ID NO: 8

 Oligonucleotide designed for probe SEQ ID NO: 9

 Oligonucleotide designed for probe SEQ ID NO: 10

 Oligonucleotide designed for probe SEQ ID NO: 1 1

 Oligonucleotide designed for probe SEQ ID NO: 1 2

 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

The scope of the claims  1. A method for evaluating the degree of cancer in a mammal-derived specimen,  (1) the first step of measuring the methylation frequency of the HAND1 gene contained in a mammal-derived specimen or an index value having a correlation therewith, and  (2) a second step of determining the degree of canceration of the sample based on the difference obtained by comparing the measured methylation frequency or an index value correlated therewith with a control. Evaluation method. 2. The method according to claim 1, wherein the mammal-derived specimen is a cell. 3. The evaluation method according to claim 1, wherein the specimen derived from a mammal is a tissue. 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 HA D1 gene contained in a specimen derived from a mammal, and  (2) a second step of determining the degree of canceration of the sample based on a difference obtained by comparing the measured methylation frequency with a control An evaluation method comprising: 5. The evaluation method according to claim 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. 6. The evaluation method according to claim 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 claim 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 claim 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 claim 8, wherein the cancer is one or more types of tissues and cancers selected from the following:  (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 Teng's organ and the cancer is Teng's cancer; and  (d) The tissue is kidney tissue, and the cancer is kidney cancer. 10. Cytosine in the nucleotide sequence of one or more 5'-CG-3's present in the nucleotide sequence in the promoter region, untranslated region or translated region of the gene whose methylation frequency is 5. The evaluation method according to claim 1, wherein the methylation frequency is: 11. The evaluation method according to claim 10, wherein the tissue cancer is one or more types of tissues and cancers selected from the following:  (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 a spleen tissue and the cancer is spleen cancer; and  (d) The tissue is kidney tissue, and the cancer is kidney cancer. 12. The methylation frequency of a gene is the frequency of cytosine methyl cycling in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence in the promoter region of the gene. The evaluation method according to claim 1 or 4, wherein 13. The methylation frequency of a gene is determined by the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence in the untranslated region or translated region of the gene. The evaluation method according to claim 1, wherein the evaluation method is a degree. 14. The methylation frequency of the gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence represented by SEQ ID NO: 1. The evaluation method according to claim 1, wherein 15. 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) 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 a knee tissue, and the cancer is a knee cancer; and  (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) a second step of determining the degree of canceration of the sample based on a difference obtained by comparing an index value having a correlation with the measured methyl methane frequency with a control. An evaluation method comprising: 17. The evaluation method according to claim 16, wherein the index value power S correlated with the methylation frequency of the HAND1 gene is the amount of an expression product of the HAND1 gene. 18. The evaluation method according to claim 17, wherein the amount of the expression product of the HA D1 gene is the amount of the transcript of the gene. 19. 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. 20. A method for searching for a substance capable of promoting the expression of a HAND1 gene, (1) the first step of bringing the test substance into contact with the cancer cells,  (2) The first step (1), followed by a second step of measuring the amount of the expression product of the HAND1 gene contained in the cancer cells,  (3) The third step of determining the ability of the test substance to promote the expression of the HAND1 gene based on the difference obtained by comparing the measured amount of the expression product with the control A search method comprising: 21. The search method according to claim 20, wherein the cancer cells are gastric cancer cells. 22. An anticancer drug 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. . 23. An anticancer agent comprising, as an active ingredient, a nucleic acid having a nucleotide sequence encoding the amino acid sequence of HAD1, and the active ingredient is formulated in a pharmaceutically acceptable carrier. 24. Use of the 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 a stomach cancer marker, a colon cancer marker, an enzyme cancer marker, and a renal adenocarcinoma marker. 26. A method for suppressing canceration, comprising a step of administering a substance that reduces the frequency of methylation of the HAD1 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.