JP4740621B2 - Method for detecting esophageal cancer and screening method for anti-esophageal cancer substance - Google Patents

Description

  The present invention relates to the detection of inactivation of the human Lipoprotein Receptor-Related Protein 1B (human LRP1B) gene in order to diagnose esophageal cancer at an early stage and further to examine the malignancy of esophageal cancer. . The present invention also provides an antitumor substance screening method using knowledge about the relationship between the human LRP1B gene and esophageal cancer.

Esophageal squamous cell carcinoma (ESC) affects more than 10,000 people every year in Japan, and the male-female ratio is about 6: 1, which is the most common in males and the sixth most common cancer in males. The annual number of deaths is 9,000-10,000 in Japan, accounting for 3% of all cancers. Therefore, esophageal cancer is one of the most common cancers, but it is one of the cancers with poor prognosis due to difficulty in early diagnosis and low response rate of chemotherapy (Pisani, P., Parkin). , DM, Bray, F. & Ferlay, J .: Estimates of the worldwide mortality from 25 cancers in 1990. Int. J. Cancer 83 , 18-29, 1999). Based on previous studies, esophageal cells are thought to undergo genetic changes in the course of cell differentiation and proliferation, resulting in canceration. However, it is not yet clear what kind of gene change induces canceration of esophageal cells. Therefore, there are no methods for detecting esophageal cancer and methods for examining the degree of malignancy of esophageal cancer.

  If the mechanism of esophageal cell canceration at the gene level is elucidated, it will be possible to detect esophageal cell canceration at the genetic level at an early stage and diagnose the malignancy of esophageal cancer. It should be possible to select anticancer substances. Specifically, it is considered that this problem can be solved by identifying a gene exhibiting a characteristic behavior in the early stage of esophageal cancer and conducting a technical examination centering on the gene.

Comparative Genomic Hybridization (CGH) is a sensitive and efficient method for detecting gene amplification on the genome. The present inventors used this CGH array method to amplify genes in esophageal cancer cells, that is, ATF3 gene and CENPF gene amplified in chromosome 1q32, GASC1 gene amplified in chromosome 9p23-24, chromosome 11q22 CIAP1 gene to be amplified, ZASC1 gene to be amplified by chromosome 3q26, etc. were identified (Pimkhaokaham, A., Shimada, Y., Fukuda, Y., Kurihara, N., Imoto, I., Yang, ZQ, Imamura, M. , Nakamura, Y., Amagasa, T., & Inazawa J .: Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF
genes in the 1q32 amplicon.Japanese Journal of Cancer Research, 91 , 1126-1133,
2000; Yang, ZQ, Imoto, I., Fukuda, Y., Pimkhaokham, A., Imamura, M., Sugano, S., Nakamura, Y., & Inazawa J .: Identification of a novel gene, GASC1, within an amplicon at 9p23-24 frequently detected in esophageal cancer cell lines, Cancer Research, 60 , 4735-4739, 2000; Imoto, I., Yang, XQ, Pimkhaokham, A., Tsuda, H., Shimada, Y., Imamura , M., Ohki, M. & Inazawa, J: Identification of cIAP1 as a candidate target gene within an amplicon at 11q22 in esophageal squamous cell carcinomas, Cancer Research, 61 , 6629-6634, 2001; Imoto, I., Yuki, Y., Sonoda, I., Ito, T., Shimada, Y., Imamura, M. & Inazawa J .: Identification of ZASC1 encoding a Kruppel-like zinc finger protein as a novel target for 3q26 amplification in esophageal squamous cell carcinomas , Cancer Research, 63 , 5691-5696, 2003).

In general, as a mechanism at the gene level of canceration, deletion of a tumor suppressor gene has been reported along with the amplification of multiple genes as described above, but identification of a specific gene deletion is usually performed by positional cloning or the like. When this method is used, it takes a lot of time and labor, and it is difficult to find the target gene. Also in the above CGH method, deletion of genomic DNA having a size smaller than 5-10 Mb was difficult to detect by a method using a normal metaphase chromosome (Bentz, M., Plesch, A., Stilgenbauer, S., Dohner, H. & Lichter, P .: Minimal sizes of deletions detected by comparative genomic hybridization, Genes Chromosomes Cancer, 21 , 172-175, 1998; Kirchhoff, M., Gerdes, T., Maahr, J , Rose, H., Bentz, M., Dohner, H. & Lundsteen, C .: Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervas, Genes Chromosomes Cancer, 25 , 410-413, 1999). For example, tumor suppressor genes such as DPC4 / SMAD4, RB1, PTEN, p16-INK4A, and RASSF1 narrow the homozygous deletion region by the chromosome mapping method, and finally the target tumor suppressor gene is identified. It was.

  In the present invention, the sensitivity and accuracy of the CGH method is improved, a high-throughput method is developed, and 800 types of BAC / PAC DNA loaded on the CGH array are selected to suppress cancer of esophageal cells. That is, the human Lipoprotein Receptor-Related Protein 1B (LRP1B) gene (hereinafter also referred to as human LRP1B gene) was successfully identified. Then, we elucidated the cause of inactivation of the human LRP1B gene, which causes canceration of esophageal cells, and the inactivation of the gene was 1) deletion of the gene, 2) CpG island of the gene And 3) suppression of histone H4 protein acetylation.

  Then, using such knowledge, the present inventors have succeeded in finding a means for detecting canceration of esophageal cells and a screening method for an antitumor substance having an inhibitory effect on the canceration, thereby completing the present invention.

  That is, the present invention provides an esophageal cancer detection method (hereinafter also referred to as the present detection method), which detects canceration of the esophageal cell by detecting inactivation of the human LRP1B gene in the esophageal cell. It is.

  The present invention also relates to the expression of the human LRP1B gene by contacting a test substance with an esophageal cancer cell line in which the expression of the human LRP1B gene is suppressed due to methylation of the CpG island of the human LRP1B gene. When the gene expression is detected and increased compared to a system that does not contact the test substance, the test substance can be activated by demethylating the CpG island of the human LRP1B gene. An esophageal cancer that provides a screening method for a substance that is selected as a tumor substance (hereinafter also referred to as screening method 1) and further suppresses the expression of human LRP1B gene due to suppression of acetylation of histone H4 protein The test substance is brought into contact with the cell line, the expression of the human LRP1B gene is detected, and the gene expression is increased compared to the system in which the test substance is not contacted. A screening method for the substance (hereinafter also referred to as the present screening method 2), wherein the test substance is selected as an antitumor substance capable of activating the human LRP1B gene by promoting acetylation of histone H4 protein. It is an invention to be provided.

The human LRP1B gene and its transcript are already known (LRP1B gene: NM_018557) and are present on the 2q22.1 chromosome. The protein encoded by the human LRP1B gene is a protein similar to the low density lipoprotein (LDL) receptor, and the protein product is similar to the LDL receptor and is a large molecule (SEQ ID NO: 1) covering about 600 kDa. This protein is a multifunctional cell membrane receptor, and its ligand is known to be Urokinase plasminogen acitvator, Tissue-type plasminogen activator, and Plasminogen activator inhibitor type-I. When these ligands bind to the LRP1B receptor, internalization occurs and is led to degradation by the action of LRP1B domain IV minireceptor (mLRP1B) (Liu, CX, L
i, Y., Obermoeller-McCormick, LM, Schwartz, AL & Bu, G .: The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor -related protein, J. Biol. Chem., 276 , 28889-28896, 2001). However, it is not yet known that this human LRP1B gene is an important tumor suppressor gene involved in the development of human esophageal cancer.

A. This detection method As described above, this detection method is a method characterized by detecting inactivation of the human LRP1B gene in esophageal cells.

  The esophageal cells that are the target for detecting inactivation of the human LRP1B gene are preferably biopsy tissue cells of the specimen donor. This sample tissue cell may be a normal human esophageal cell or the cancer tissue of an esophageal cancer patient, but in reality, 1) As a result of endoscopy, canceration of the esophagus is suspected. The main target is the affected tissue when a lesion is found, or 2) an esophageal cancer tissue that has been confirmed to be esophageal cancer but its malignancy and progression must be determined. Can be.

  If inactivation of the human LRP1B gene in the `` the lesion tissue when a lesion that is suspected to be cancerous is found in the esophagus as a result of endoscopy, etc. '' The lesion tissue has progressed toward canceration or is already in the state of canceration, and it has been found that the malignancy is becoming high, and immediate full-scale treatment (removal of the lesioned part by surgery, etc. The need for conventional chemotherapy). In addition, when inactivation of the human LRP1B gene is observed in “a tissue of an esophageal cancer that has been confirmed to be esophageal cancer but its malignancy or progression needs to be determined”, the cancer It turns out that the malignancy of the tissue is getting higher, and the necessity of urgent full-fledged treatment (removal of lesions by surgery, full-scale chemotherapy, etc.) is indicated. The esophageal cell tissue collected as a specimen can be subjected to a necessary treatment, for example, DNA or RNA prepared from the collected tissue and subjected to this detection method.

  As described above, this detection method can identify canceration of the cells (particularly, initiation of canceration) by detecting inactivation of the human LRP1B gene in esophageal cells. Examples of the inactivation of the human LRP1B gene include 1) deletion of the human LRP1B gene, or 2) reduction of the gene expression level when the human LRP1B gene is not deleted.

1) Detection of human LRP1B gene deletion As representative methods capable of directly detecting the human LRP1B gene deletion, the CGH (Comparative Genomic Hybridization) method and the FISH method [fluorescence in situ hybridization (FISH) are used. : Fluorescence in situ hybridization): Yasui, K., Imoto, I., Fukuda, Y., Pimkahaokham, A., Yang, ZQ, Naruto, T., Shimada, Y., Nakamura, Y., and Inazawa: Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinoma. Genes Chromosomes Cancer, 32 , 112-118, 2001]. In this detection method of this embodiment, BAC (Bacterial Artificial Chromosome) DNA, YAC (Yeast Artificial Chromosome) DNA or PAC (Phage Artificial Chromosome) DNA (hereinafter also referred to as BAC DNA) having a human LRP1B gene is labeled, and FISH is used. In this way, the deletion part of the human LRP1B gene can be detected. Specifically, BAC DNA having a human LRP1B gene includes RP11-4C8, RP11-498P13, RP11-82P18, RP11-538A7, RP11-257N13, RP11-159P15, RP11-769M2, RP11-532L9, RP11-161A24, RP11-279M2, RP11-493I5, RP11-662P10, RP11-20E15, RP11-164E7, RP11-51L13, RP11-114B24, RP11-493I5, etc. can be mentioned.

  It is preferable and practical that the method of the above embodiment is performed using a genomic DNA fixing platform.

  Since normally obtained BAC DNA and the like are in a small amount for the production and practical use of a large number of genomic DNA fixing bases, it is necessary to obtain the DNA as a gene amplification product (this gene amplification process is also referred to as “infinite storage”). Say). In inexhaustible storage, first, BAC DNA or the like is digested with a 4-base recognition enzyme such as RsaI, DpnI, or HaeIII, and then ligated by adding an adapter. The adapter is an oligonucleotide consisting of 10 to 30 bases, preferably 15 to 25 bases. The double strand has a complementary sequence, and after annealing, the 3′-end oligonucleotide that forms the blunt end is linked to the oligonucleotide. It needs to be oxidized. Next, using a primer having the same sequence part as one of the oligonucleotides of the adapter, it can be amplified by PCR (Polymerase Chain Reaction) method and inexhaustible. On the other hand, a 50-70 base aminated oligonucleotide characteristic of each BAC DNA etc. can also be used as a detection probe.

  The desired DNA fixing substrate can be produced by fixing the inexhaustible BAC DNA or the like on the substrate, preferably a solid substrate. Examples of the solid substrate include glass, plastic, membrane, and three-dimensional array, and a glass substrate such as a slide glass is preferable. The solid substrate such as glass is more preferably coated by adhesion of poly-L-lysine, aminosilane, gold / aluminum, or the like.

  The concentration at which the inexhaustible DNA is spotted on the substrate is preferably 10 pg / μl to 5 μg / μl, more preferably 1 ng / μl to 200 ng / μl. The amount to be spotted is preferably 1 nl to 1 μl, more preferably 10 nl to 100 nl. Further, the size and shape of each spot fixed on the substrate are not particularly limited. For example, the size can be 0.01 to 1 mm in diameter, and the shape seen from the upper surface can be circular to elliptical. The thickness of the drying spot is not particularly limited, but is 1 to 100 μm. Further, the number of spots is not particularly limited, but is 10 to 50,000 per substrate to be used, more preferably 100 to 5,000. Each DNA is spotted in the range of Singular to Quadruplicate, but preferably spotted on Duplicate or Triplicate.

  The dry spot can be prepared by, for example, placing BAC DNA or the like inexhaustible using a spotter on a substrate to form a plurality of spots and then drying the spots. As the spotter, an ink jet printer, a pin array printer, and a bubble jet (registered trademark) printer can be used, but an ink jet printer is preferably used. For example, GENESHOT (Nippon NGK Co., Ltd.) can be used.

  The desired DNA fixing substrate can be produced by fixing the inexhaustible BAC DNA or the like on the substrate, preferably a solid substrate.

  As one of means for directly detecting the deletion of the human LRP1B gene, Southern blotting can be mentioned. Southern blotting is a method for detecting the presence of a gene in a specimen by separating and fixing genomic DNA obtained from the specimen and detecting hybridization with the human LRP1B gene.

2) Detection of decrease in gene expression when the human LRP1B gene is not deleted The method of this embodiment quantifies the amount of polynucleotide or human LRP1B protein based on the human LRP1B gene (genomic DNA). Using this quantitative value as an index, a decrease in the expression of the human LRP1B gene can be detected. Examples of the polynucleotide include mRNA transcribed from the human LRP1B gene and cDNA obtained using the mRNA as a template.

  In addition, the amount of human LRP1B protein in a sample can be quantified by preparing an antibody against human LRP1B protein by a conventional method, etc., and the above quantitative value can be obtained. It is a “decrease” in the expression level of a gene, and the use of protein mass as an indicator in such negative detection must solve problems such as the presence of background and is not efficient. There is. Therefore, it is preferable to use a method for detecting the amount of polynucleotide based on the human LRP1B gene (genomic DNA) as the detection means in the method of this embodiment. Examples of the method for detecting the amount of polynucleotide include the following means.

  The most typical aspect is to detect the mRNA that is present in the sample indirectly by detecting cDNA in the sample using human LRP1B protein mRNA as a template, and to determine the extent to which the human LRP1B gene is expressed. (Hereinafter, the detection means of this embodiment is also referred to as LRP1B mRNA detection method. The detection method of this embodiment includes the above-mentioned real-time RT-PCR method).

  In the LRP1B mRNA detection method, typically, mRNA is selected from the total RNA of the specimen according to a known method (for example, a method using oligo dT). Then, from the obtained mRNA, a gene amplification capable of amplifying the gene using mRNA as a template, using a heat-resistant DNA polymerase using a nucleotide chain corresponding to the nucleotide sequence of a known human LRP1B gene as an amplification primer The presence of human LRP1B protein mRNA in the sample is detected by amplifying cDNA encoding the human LRP1B protein by a method such as RT-PCR and detecting the presence or absence of the amplification product by the gene amplification operation. Preferably, it can be detected in real time.

  Using the cDNA encoding human LRP1B protein amplified as described above as a template, a gene amplification operation using a heat-resistant DNA polymerase such as a PCR method is further performed to detect the presence or absence of the gene amplification product resulting therefrom. Thus, the presence of human LRP1B protein mRNA in the sample can be detected more accurately (the gene amplification primer used in the second gene amplification operation is the gene amplification used in the first gene amplification operation). It is necessary to use a nucleotide chain corresponding to the inside of the human LRP1B gene as a primer for gene amplification rather than a primer for gene amplification).

  The DNA chip method is one method for quantifying mRNA expressed in a sample cell. For example, a synthetic oligonucleotide having a characteristic sequence of the human LRP1B gene is immobilized on a substrate (substantially the same as the substrate that can be used in the CGH method described above) (cDNA can also be immobilized), The prepared RNA is labeled when cDNA is synthesized by reverse transcriptase. The expression level of mRNA can be measured by hybridizing the labeled cDNA and a synthetic oligonucleotide on the substrate and scanning the amount of bound label.

  Northern blotting is to isolate and fix mRNA obtained from a specimen, and to detect hybridization between the mRNA and a synthetic oligonucleotide having a characteristic sequence of the human LRP1B gene (cDNA can also be used). To detect the expression of the human LRP1B gene in the specimen.

  Causes other than the deletion of the human LRP1B gene that cause the expression level of the gene to decrease are a) inactivation by methylation of the CpG island of the human LRP1B gene, b) the degree of acetylation of the histone 4B protein. It can be roughly divided into two factors, inactivation due to the decrease. Hereinafter, these two factors will be briefly described.

a) Inactivation of human LRP1B gene by methylation of CpG island
Tight methylation of CpG rich promoter and exon region has been reported to cause transcriptional inactivation (Bird, AP & Wolffe, AP: Methylation-induced repression-belts, braces, and chromatin, Cell 99 , 451-454 1999). In cancer cells, CpG islands are densely methylated more frequently than other regions, and hypermethylation in the promoter region is deeply involved in inactivation of tumor suppressor genes in human cancer (Ehrlich,
M., Jiang, G., Fiala, E., Dome, JS, Yu, MC, Long, TI, Youn, B., Sohn, OS, Widschwendter, M., Tomlinson, GE, Chintagumpala, M., Champagne, M., Parham,
D., Liang, G., Malik, K. & Laird, PW: Hypomethylation and hypermethylation of
DNA in Wilms tumors, Oncogene, 21 , 6694-6702, 2002).

  As will be described later, a part of exon 1 of human LRP1B gene and CpG island present in intron 1 region have promoter activity, but it was found that promoter activity disappears when methylating this region in vitro. . In addition, the degree of methylation of this CpG island was strongly correlated with suppression of human LRP1B gene expression in some esophageal cancer cells. CpG islands can be demethylated by culturing esophageal cancer cells in the presence of 5-azadeoxycytidine (5-aza-dC), which is a demethylation reagent. As a result, human LRP1B gene Expression could be restored. From these results, it was found that high-density methylation of CpG islands (Hypermethylation) is one of the causes of frequent suppression of tumor suppressor gene expression in esophageal cancer cells.

b) It is known that the modification of inactivated histone protein by reducing the degree of acetylation of histone 4B protein is involved in the suppression of gene expression induced by DNA methylation (Cameron, EE, Bachman). , KE, Myohanen, S., Herman, JG & Baylin, SB: Synergy of demethylation and histone deacetylase inhibition in
the re-expression of genes silenced in cancer, Nat. Genet., 21, 103-107, 1999; Nguyen, CT, Gonzales, FA, & Jones, PA: Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation, Nucleic Acids Res., 29 , 4598-4606, 2001).

  Trichostatin A (TSA) is a histone deacetylase inhibitor and an important tool for analyzing the relationship between acetylation and gene expression, but in the presence of TSA, some esophageal cancers When the cells were cultured, the expression of human LRP1B gene increased (described later). Therefore, it can be concluded that in the presence of TSA, the degree of acetylation of histone H4 protein bound to DNA in cells increases. Furthermore, it was found that the activation of human LRP1B gene expression induced by acetylation of histone H4 protein does not depend on methylation of CpG islands in esophageal cancer cells. On the other hand, it was also found that the increase in the degree of methylation in esophageal cancer cells was insufficient to suppress the expression of the human LRP1B gene induced by histone acetylation.

  c) For the sample cells in which the expression level of the gene is decreased by the above-mentioned means even though the human LRP1B gene is not deleted, the two inactivation factors of a) and b) above It is very important to classify them into the types in order to perform optimal treatment (selection of an anticancer drug to be administered) for the specimen provider. Specifically, a demethylating agent (5-aza) is used for a sample cell (primary cancer cell derived from cancer tissue) whose expression level of human LRP1B gene is found to be decreased by the detection means described above. Deoxycytidine: 5-aza-dC, etc.) or an acetylation promoter (trichostatin A, etc.) is allowed to act to study the recovery of the gene expression level, thereby classifying the above factors. it can.

That is, when the expression level of the human LRP1B gene is restored by allowing a demethylating agent to act on the sample cell, the suppressor of the gene in the sample cell is methylation of the CpG island. A corresponding antitumor effect is expected by administering a drug having a demethylating action. In addition, when the expression level of the human LRP1B gene is recovered by acting an acetylation promoter on the sample cell, the suppression factor of the gene in the sample cell is suppression of acetylation in the histone 4B protein. A corresponding antitumor effect is expected by administering a drug having an acetylation promoting action to a donor. If both of these reactions are observed, it can be concluded that the cause of the suppression of the expression level of human LRP1B gene in the sample cells is the above-mentioned methylation and suppression of acetylation. A corresponding antitumor effect is expected by administering both of the accelerating agents.

B. This screening method As described above, inactivation of the human LRP1B gene is a major factor for esophageal cancer, and a drug that normalizes the function of the gene can be used as an antitumor agent for esophageal cancer. It is thought that.

  In particular, this inactivation factor is not the deletion of the human LRP1B gene (particularly in the case of homotypic deletion), but the above-mentioned methylation of the CpG island of the gene and / or the acetylation of histone H4 protein. In the case of suppressing the above, a drug capable of eliminating or alleviating these factors is useful as an antitumor agent.

  Thus, as described above, the present invention provides the present screening method 1 which is a screening method for drugs having a demethylation action and the present screening method 2 which is a screening method for drugs having an acetylation promoting action. It is.

  As a premise for carrying out these screening methods, an esophageal cancer cell line in which the deletion of the human LRP1B gene (especially homotypic deletion) is not observed in the sample cells but the expression level of the gene is suppressed is secured. There is a need. That is, in this screening method 1, “an esophageal cancer cell line in which expression of the human LRP1B gene is suppressed due to methylation of the CpG island of the human LRP1B gene” is referred to as “histone H4 An esophageal cancer cell line in which the expression of the human LRP1B gene is suppressed due to suppression of protein acetylation is required. Establishing these cell lines can be performed according to conventional methods based on the above-described findings. For example, from the above-mentioned methods, for example, esophageal cancer cells confirmed by the CGH method or FISH method, it is confirmed that at least the deletion of human LRP1B gene (preferably homotypic deletion) is not observed. By acting on a demethylating reagent (for example, 5-azadeoxycytidine), a cell in which the level of the human LRP1B gene is restored is selected and subcultured to obtain a desired “human LRP1B gene CpG island. It can be established as an esophageal cancer cell line in which expression of the human LRP1B gene is suppressed due to methylation (hereinafter also referred to as methylated cancer cell line). In addition, an existing acetylation-promoting reagent (for example, trichostatin A) is selected from esophageal cancer cells that have been confirmed to have no human LRP1B gene deletion (preferably homotypic deletion). By selecting the cells in which the level of the human LRP1B gene is restored, the cells are subcultured, and the desired expression of the human LRP1B gene is suppressed due to the suppression of histone H4 protein acetylation. It can be established as “an esophageal cancer cell line” (hereinafter also referred to as a deacetylated cancer cell line).

In this screening method, it is necessary to bring the test substance into contact with the above-mentioned methylated cancer cell line or deacetylated cell line. Although the mode of this contact is not particularly limited, it is carried out by adding the test substance to the culture of the methylated cell line, preferably diluted at an appropriate dilution ratio, and subsequently culturing. And the expression level of human LRP1B gene in the methylated cancer cell line or deacetylated cell line before adding the test substance (as described above, real-time RT-PCR method, Southern blot method, Northern blot method, DNA chip method, etc. Preferably, it is detected as mRNA by a real-time RT-PCR method), and the expression level of the gene after addition is preferably quantified over time, and the difference in the expression level of the gene before and after quantification is determined in the test. If the expression level of the gene in the culture to which the test substance is added is higher than that of the control culture compared to the control culture cultured under the same conditions without adding the substance, The test substance is 1) “Anti-tumor substance capable of activating human LRP1B gene by demethylation of CpG island of human LRP1B gene” in a test using methylated cancer cell line (this sc Selected as leaning method 1), and 2) “Anti-tumor substance capable of activating human LRP1B gene by promoting acetylation of histone H4 protein” in tests using deacetylated cancer cell lines (this screening method) 2).

  Further, the present screening method is used to inoculate a substance screened as an antitumor component against a desired esophageal cancer, and further in vivo screening, for example, the above-mentioned methylated cancer cell line or deacetylated cell line It is preferable to finally narrow down the screening method using as an index the effect of suppressing the growth of the esophageal cancer cells in nude mice and improving the survival rate of the nude mice.

  The present invention provides a screening kit for performing the screening method described above. The kit contains a methylated cancer cell line and / or a deacetylated cell line as a minimum component. In addition, other elements necessary for performing this screening method, such as reverse transcriptase, primer set, thermostable polymerase, nucleobase (4 types of dNTP mix), cell culture for real-time RT-PCR It is also possible to add a solution, a dilution buffer or the like.

  According to the present invention, it is possible to accurately grasp signs of canceration, cancer progression, and malignancy in esophageal cell specimens. In addition, it is possible to screen for therapeutic agents for esophageal cancer that occurs when the expression of the human LRP1B gene is inactivated by a specific mechanism.

[Example 1] Preparation of high-density CGH array carrying many cancer-related genes
800 BACs with genes or Sequence Tagged Site markers that are extremely important for canceration and cancer cell growth from the results of BLAST searches of National Cancer for Biotechnology and the University of California Santa Cruz Biotechnology and selected DNA. The / PAC clone was selected.

  BAC and PAC DNA were digested with DpnI, RsaI, and HaeIII, and then ligated with adapter DNA. Next, PCR was performed twice using a primer having an adapter sequence. One of the two primers is aminated at the 5 'end. This process is called inexhaustible storage, and the obtained DNA is defined as inexhaustible DNA. Inexhaustible DNA was covalently printed on an oligo DNA microarray (Matsunami Glass, Osaka) on Duplicate using an inkjet-type spotter (GENESHOT, NGK Insulators, Nagoya).

[Example 2] Deletion of human LRP1B gene in esophageal cancer cells In order to detect a novel homozygous deletion in esophageal cancer, using genomic DNA prepared from 44 types of esophageal cancer cells, CGH array analysis using 1 CGF array was performed.

  As a control, male healthy human genomic DNA was used and labeled with Cy5. Genomic DNA prepared from the above esophageal cancer cells was used as test DNA and labeled with Cy3. Specifically, DpnI-digested genomic DNA (0.5 μg) was added to 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 0.1 mM dCTP and 0.4 mM Cy3-dCTP (esophageal cancer cell) or 0.4 mM Cy5-dCTP (normal cell), respectively. In the presence, it was labeled by nick translation. Cy3 and Cy5 labeled dCTP were obtained from Amersham Biosciences (Tokyo). Both labeled genomic DNAs are precipitated by adding ethanol in the presence of Cot-1 DNA (Invitrogen), and 120 μl of hybridization mixture (50% formamide, 10% Dextran sulfate, 2X SSC (1xSSC: 150 mM NaCl / 15 mM Sodium Citrate) ), 4% sodium dodecyl sulfate, pH 7). After incubation at 37 ° C. for 30 minutes, the mixture was added to the CGH array set in the hybridization chamber, and incubated at 37 ° C. at a speed of 3 rpm (round per minute) for 48-72 hours. The CGH array was then washed in 50% formamide / 2x SSC (pH 7.0) solution at 50 ° C for 15 minutes, then washed in 2xSSC / 0.1% SDS at 50 ° C for 15 minutes, and then 0.1% The plate was washed with 0.1 M phosphate buffer (pH 8) containing Nonidet P-40 at room temperature for 15 minutes. After air drying, the CGH array was monitored for fluorescence derived from Cy3 and Cy5 using a GenePix 4000B scanner (Axon Instruments, CA, USA). The obtained results were analyzed using GenePix Pro4.1 imaging software (Axon Instruments). The average fluorescence intensity derived from Cy3 and the average fluorescence intensity derived from Cy5 were adjusted to the same value, and the ratio of Cy3 / Cy5 was determined. The Ratio value is 1 when there is no abnormality in the genome. It was determined that genomic amplification occurred when the Ratio value was 1.32 or more, and significant amplification was observed when the Ratio value was 4 or more. When the Ratio value was 0.75 or less, it was determined that the possibility of genomic heterozygote deletion was extremely high, and when the ratio value was 0.25 or less, the possibility of homozygote deletion was extremely high.

As a result, it was found that the gene of human LRP1B present in chromosome 2q22.1 was deleted in esophageal cancer (TE-6) cells. The Log ₂ Ratio at this time was −2.7 [FIG. 1A: Typical when CGH analysis was performed on the CGH array shown in Example 1 using TE-6 genomic DNA as a test cell. It is an array image. The decrease in the human LRP1B gene copy number in chromosome 2q22.1 is indicated by a clear signal (Log ₂ Ratio = -2.7) (indicated by the arrow in the right enlarged view). ]. On the other hand, 18 types of esophageal cancer cells had a Log ₂ Ratio in the range of -0.42 to -2.0. This result indicates that the human LRP1B gene is deleted in one of the two pairs of chromosomes (referred to as “Hemizygous deletion”). The deletion of the human LRP1B gene detected in TE-6 cells ranged from at least exon 3 to exon 30.

  When other cell types were studied, 6 cell types among 44 types of esophageal cancer cells [indicated by * in FIG. 1B]. FIG. 1B represents the detection of homozygous deletion of human LRP1B gene exons 1, 5, 7 and 10 in esophageal cancer cells (TE cell line and KYSE cell line). The asterisk indicates the cell type in which homozygous deletion was detected in any exon. N shows the result using DNA derived from a healthy person. PCR of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used as a control. ], A homozygous deletion of the human LRP1B gene was found. The detection frequency is 13.7%. Furthermore, in primary esophageal cancer subjected to laser-captured microdissection (LCM), 30 samples out of 70 samples [indicated by * in FIG. 1C]. FIG. 1C shows homozygous deletion of human LRP1B gene exons 5, 7 or 10 in Laser-captured microdissection (LCM) -treated primary esophageal cancer. The asterisk indicates the cell type in which homozygous deletion was detected in any exon. PCR of GAPDH gene was used as a control. ], A homozygous deletion of the human LRP1B gene was detected. The frequency was as high as 42.9%.

[Example 3] Study on suppression of human LRP1B gene expression in esophageal cancer cells Human LRP1B gene exon 8-9 and human LRP1B gene, which are frequently deleted regions of human LRP1B gene in esophageal cancer cells Two types of primers were designed from the sequence of exons 91-92 close to the 3 ′ end of each, and mRNAs derived from both regions were detected by RT-PCR.

As a result, mRNA derived from exon 91-92 could not be detected in 6 cell lines in which homozygote deletion occurred, but mRNA derived from exon 8-9 was detected. Furthermore, RT-PCR products derived from exon 8-9 and exon 91-92 could not be detected in 14 of 38 cell lines in which homozygous deletion did not occur [Figure 1D by RT-PCR analysis. Human LRP1 in detected esophageal cancer cells
It shows the expression of B exons 8-9 and exons 91-92. Cell types showing homozygous deletions indicated by an asterisk in FIG. 1B were similarly marked with an asterisk. Among 38 cell types that do not show homozygous deletion, 14 cell types (TE-1, -5, -8, -9, -11 and KYSE30, 150, 170, 770, 790, 960, 1250, 2400, 2650), human LRP1B mRNA expression disappeared at a frequency of 36.8%. ]. This frequency was 36.8%. Therefore, the reason why human LRP1B mRNA cannot be detected is considered to be other mechanisms in addition to gene deletion on the genome.

[Example 4] Examination of methylation of CpG island of human LRP1B gene
CpG island methylation is one of the mechanisms that suppress gene expression. As a result of analyzing the CpG island of the human LRP1B gene using CpGPLOT, a 828 bp fragment containing a part of the 3 ′ side of exon 1 of the human LRP1B gene and the 5 ′ side of intron 1 was identified. The CpG sites in this region and the CpG islands in which CpG sites are densely displayed are displayed. [FIG. 2A: Schematic map of CpG islands consisting of 828 bp including exon 1 and intron 1 of human LRP1B gene. Exon 1 is indicated by a white box, and the transcription start point is indicated by +1. The arrow indicates the transcription start direction. CpG islands were identified by CpGPLOT (http://www.ebi.ac.uk/emboss/cpgplot/) and displayed with a black bar, and the position of CpG was shown as a vertical line in the enlarged view. The CpG island is composed of the human LRP1B gene from +718 to +1545. Regions analyzed by the promoter assay (Regions 1 to 5), regions subjected to bisulfite-PCR analysis, and regions subjected to bisulfite sequencing are indicated by horizontal lines. ].

  In order to examine the promoter activity of the CpG island of the human LRP1B gene identified here, it was divided into five fragments (Fig. 2A Region 1-5) including the periphery of this CpG island, and these fragments were divided into luciferase reporter plasmid (pGL3-Basic vector: Promega, WI, USA) and cervical cancer (HeLa) cells as well as esophageal cancer (TE-4) cells were transiently co-transfected with the pRL-hTK internal standard vector (Promega). After culturing for 36 hours, the luciferase activity was measured according to the manual using the Dual-Luciferase reporter assay system (Promega), and the firefly luciferase activity derived from the pGL3 vector having each region was measured using the urine derived from the pRL-hTK internal standard vector. RLA (Relative luciferase activity), which is a value divided by shiita luciferase activity, was displayed. As a result, Regions 1, 2 and 3 showed 19, 12-14 and 8-10 RLA activities, respectively, while Regions 4 and 5 showed no activity [FIGS. 2A and B: FIG. 2B shows the human LRP1B gene CpG. It shows suppression of promoter activity by methylating the promoter activity of the island and its region in vitro. Regions 1 to 5 of the human LRP1B gene CpG island were methylated in vitro with SssI (CpG) methylase in the presence and absence of S-adenosylmethionine (SAM). The former is a condition in which CpG is methylated, and the latter is a condition in which methylation is not performed. Two sets of Region 1-5 DNA prepared here were inserted into a pGL3 basic vector to construct a firefly luciferase expression plasmid. These expression plasmids were transfected into Hela cells and TE-4 cells, and after culture, the target firefly luciferase activity was measured by standardizing both cell extracts with Renilla luciferase as an internal control. The obtained results were each expressed as an average value ± SE of three experiments. ]. Therefore, it was found that the region containing the CpG island has promoter activity, and that the promoter activity increases as the size of the sequence upstream of this region is increased.

  Next, CpG islands were methylated using SssI (CpG) methylase (New England Biolabs, MA, USA) using S-adenosylmethionine as a substrate to examine promoter activity. Methylation occurred almost completely by confirming that it was not digested with methylation sensitive restriction enzymes. The activity disappeared when the plasmids from Regions 1, 2 and 3 were methylated (FIGS. 2A and B: above). Therefore, it was found that even when the human LRP1B gene is not deleted, the expression of the human LRP1B gene is almost completely suppressed when the CpG island is methylated.

  Furthermore, we investigated whether the CpG island of the human LRP1B gene was actually methylated in esophageal cancer cells. Using the EZ DNA methylation kit (ZYMO REAEARCH, CA, USA), genomic DNA (2 µg) derived from esophageal cancer cells is treated overnight at 50 ° C in sodium bisulfite to amplify the desired methylated DNA PCR was performed using primers designed as described above. The obtained amplification product was digested with TaqI restriction enzyme (New England BioLabs). TaqI did not digest sequences modified with unmethylated sodium bisulfite, but monitored the degree of methylation using the property of digesting sequences not modified with methylated sodium bisulfite. After electrophoresis of the PCR fragment, the concentration ratio of the methylated fragment band to the non-methylated fragment band was measured by densitometry using MultiGauge2.0 (Fujifilm, Tokyo). The degree of methylation was expressed in%. It is considered that the promoter activity is significantly suppressed when it is methylated by 20% or more. This sequence was subcloned into TOPO TA cloning vector (Invitrogen), and the nucleotide sequence was determined.

  Degree of methylation of human LRP1B gene CpG island in esophageal cancer cells (KYSE170, 770, 790, 960 & TE-1, -8) in which homozygous deletion of human LRP1B gene is not detected but this gene is not expressed [Figure 2C shows the bisulfite-PCR analysis of the human LRP1B gene CpG island of esophageal cancer cells digested with the methylation sensitive restriction enzyme TaqI. Results are shown. M represents a band obtained when a methylated CpG island was used as a template, and% represents the percent methylated. N1 and N2 show the results when using genomic DNA derived from a healthy person. ]. On the other hand, significant methylation in this region was not detected in cells expressing the human LRP1B gene (KYSE110, 1260, 70) or normal white blood cells.

  The methylation degree of each CpG dinucleotide in the human LRP1B gene CpG island was analyzed by the bisulfite sequencing method. As a result, the CpG island of the human LRP1B gene is particularly highly methylated at sites 39-67 in cells without homozygous deletion (KYSE170, 770, 790, 980 & TE-1, -8). In cells expressing the human LRP1B gene (KYSE110, 1260, 70), it was not methylated [FIG. 2D shows the bisulfite genomic sequence of the human LRP1B gene CpG island. When the transfer start point is displayed as +1, CpG islands occupy from +718 to +1545. There are 67 CpG sites in this region, which are represented by squares. Open squares are unmethylated CpG sites, and black squares are methylated CpG sites. There are 6 to 9 cells, but these are the numbers of genome sequencing, and each result is shown. The cells used were human LRP1B gene-expressing cells (expressed as Expression (+)) KYSE110, KYSE1260 and KYSE70, and cells not expressing the human LRP1B gene (expressed as Expression (-)) KYSE170, KYSE770, KYSE790, KYSE960 , TE-1 and TE-8. ]. Therefore, human LRP1B gene expression is not detected or very low when esophageal cancer cells have a homozygous deletion of the human LRP1B gene and when the CpG island of the human LRP1B gene is highly methylated I understood. These two cases strongly suggest a relationship between human LRP1B gene expression and canceration of esophageal cells.

[Example 5] Methylation of human LRP1B gene CpG island in primary esophageal cancer So far, methylation of human LRP1B gene CpG island in esophageal cancer cells has been analyzed, but the same phenomenon actually occurs in esophageal cancer tissues. I examined whether or not. As a result, it was found that methylation of human LRP1B gene CpG island occurred in 5 out of 34 primary esophageal cancer [Fig. 2E shows that human LRP1B gene CpG island of primary esophageal cancer is expressed with methylation sensitive enzyme TaqI. The analysis by bisulfite-PCR after digestion is shown. M represents a band obtained when a methylated CpG island was used as a template, and% represents the percent methylated. ]. Its frequency is 14.7%. Compared with established esophageal cancer cells, the frequency of methylation of the human LRP1B gene CpG island is lower in cancer tissues, but this analysis requires a certain amount of DNA. It can be mixed. From the above results, inactivation of the human LRP1B gene by methylation of the human LRP1B gene CpG island is presumed to play an important role in canceration of esophageal cells.

[Example 6] Expression of human LRP1B gene when demethylated using 5-aza-dC Human LRP1B gene expression when human LRP1B gene CpG island was demethylated in esophageal cancer cells was examined. Specifically, human LRP1B mRNA induction ability was measured when esophageal cancer cells were treated for 5 days in the presence of various concentrations of 5-aza-dC, which is a methyltransferase inhibitor. As a result, human LRP1B mRNA-inducing ability was found by performing 5-aza-dC treatment in cells in which human LRP1B gene was not deleted but human LRP1B mRNA was not detected. In cells with homozygous deletion of the human LRP1B gene, the ability to induce human LRP1B mRNA was not detected by the same treatment [FIG. 2F shows humans in esophageal cancer cells in the presence and absence of 5-aza-dC. The LRP1B gene expression analysis is shown. RT-PCR was performed using two kinds of specific primers for amplifying human LRP1B gene exon 8-9, and the band obtained as a result of electrophoresis was shown. GAPDH was used as an internal standard. The genome used for the template is a cell type that does not express the human LRP1B gene (KYSE170, 770, 790, 960 & TE-1, -5, -8, -11) and is not homozygous. A cell type in which loss was detected (TE-6) was prepared after culturing for 5 days in the presence and absence of 5-aza-dC. ].

[Example 7] Examination of the relationship between CpG methylation and histone acetylation
It has become clear that the suppression of gene expression associated with methylation of CpG islands is due to changes in the chromosome structure of the genome. Similarly, hypoacetylation of histone proteins that interact with the genome also changes the genome structure and affects gene expression. In this study, we investigated the relationship between CpG methylation and histone acetylation using trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-dC, a methyltransferase inhibitor.

  When KYSE170 cells in which the human LRP1B gene CpG island was densely methylated were incubated in the presence of TSA, higher expression of the human LRP1B gene was detected than when incubated with 5-aza-dC alone [FIG. 3 shows the expression of LRP1B mRNA in human LRP1B gene non-expressing esophageal cancer cells (KYSE170) after culturing in the presence of 5-aza-dC and TSA. KYSE170 cells were cultured in the presence of 5-aza-dC for 5 days and in the presence of TSA or in the presence of 5-aza-dC and TSA for 12 hours. Using the genomic DNA prepared from each cell as a template, RT-PCR was performed using two types of specific primers for amplifying human LRP1B gene exon 8-9, and the bands obtained as a result of electrophoresis were shown. . GAPDH was used as an internal standard. ]. When KYSE170 cells were incubated in the presence of TSA and 5-aza-dC, higher human LRP1B gene expression was observed.

This result suggests that methylation of CpG islands and changes in chromosomal conformation are intricately related and affect the expression of the human LRP1B gene. To quantitatively analyze histone protein acetylation, ChIP assay was performed on five types of esophageal cancer cells (KYSE110, 1260, 70, 170, 960). The ChIP assay can amplify the target genome sequence by immunoprecipitation of the genome using an antibody against acetylated histone H4 and then diluting it to about 1/100 to perform PCR. The PCR product was subjected to 3% agarose gel electrophoresis, and the amount of DNA forming a complex with acetylated histone was monitored. The method was performed according to the manual of this kit (Upstate Biotechnology, NY, USA). As a result, in cells expressing the human LRP1B gene (KYSE110, 1260, 70), it was found that the CpG island of the human LRP1B gene forms a complex with excess acetylated histone H4. On the other hand, in cells having a CpG island of the human LRP1B gene that is excessively methylated (KYSE170, 960), only a few hyperacetylated histones H4 were detected [FIG. 2H shows humans in esophageal cancer cells. The status of histone acetylation of LRP1B gene is shown. Human LRP1B obtained by immunoprecipitation of human LRP1B gene-expressing cells (KYSE110, 1260, 70) and non-human LRP1B gene-expressing cells (KYSE170, 960) by chromatin ChIP assay using an antibody against acetylated histone H4 The densely methylated region of the gene (FIG. 2D) was amplified by PCR and detected. The sample before immunoprecipitation was amplified by PCR (indicated as Input) and compared with the result after immunoprecipitation. Further, in the DNA after immunoprecipitation, the 5 ′ end region of the GAPDH gene was amplified and used as a control. The obtained band was scanned with a densitometer, and the ratio of the value of the LRP1B band to the value of the GAPDH band was displayed in the lower column. ]. KYSE170 cells were incubated in the presence of 5-aza-dC, TSA, or 5-aza-dC + TSA, and the degree of histone acetylation of the CpG island of the human LRP1B gene was monitored by ChIP assay. 5-aza-dC was found to synergistically enhance histone acetylation [Figure 2I shows the effects of 5-aza-dC and TSA on acetylation of histone H4 protein in human LRP1B gene CpG island in esophageal cancer cells Shows the effect of. KTSE170 cells were cultured in the presence of 5-aza-dC for 5 days and in the presence of TSA or in the presence of 5-aza-dC and TSA for 12 hours. Similarly to FIG. 2H, immunoprecipitation was performed using an antibody against acetylated histone H4, and then PCR was performed to monitor the degree of acetylation of acetylated histone. The band obtained by ChIP-PCR was quantified with a densitometer, and the ratio between the value calculated from the human LRP1B gene and the value calculated from the GAPDH gene is shown in the figure below. The presence or absence of TSA and 5-aza-dC at the time of culture was indicated by + and −. ]. Therefore, the degree of histone acetylation of the human LRP1B gene CpG island is directly correlated with the expression of human LRP1B mRNA, and conversely, the degree of methylation of this island is not correlated with the expression of human LRP1B mRNA. It became clear to show.

[Example 8] Inhibition of growth of esophageal cancer cells after recovery of human LRP1B gene expression To further clarify the role of human LRP1B gene in canceration of esophageal cells, when the expression of this gene was recovered, Whether cancer cell growth was suppressed was examined. First, a plasmid (pBICEP-CMV-2-mLRP1B) that expresses the FLAG tag mini-receptor of the human LRP1B gene was constructed. This can be used to monitor the role of the full-length human LRP1B gene. This plasmid was prepared by inserting human LRP1B cDNA amplified by RT-PCR into a pBICEP-CMV-2 expression vector (Sigma, MO, USA) so that the FLAG epitope and the translation frame matched. As a control, an empty vector (pBICEP-CMV2-mock) into which no human LRP1B gene was inserted was used. These expression plasmids were mixed with FuGENE6 (Roche Diagnostics, Tokyo) as a transfection reagent to transfect TE-8 cells and KYSE170 cells. Three weeks later, cells that grew in the presence of the neomycin-based drug G418 were fixed with 70% ethanol and counted by staining with crystal violet. As a result, the number of colonies was significantly reduced in cells transfected with pBICEP-CMV-2-mLRP1B compared to cells transfected with the empty vector [Figure 3 shows the expression of human LRP1B gene on the growth of esophageal cancer cells. It is drawing which showed the influence of recovery. (A and B): KYSE170 cells (A) and TE-8 cells using an expression plasmid (pBICEP-CMV-2-mLRP1B) and an empty vector (pBICEP-CMV-2-mock) having the FLAG tag LRP1B mini-receptor (B) was transfected. In both cells, the human LRP1B gene expression is not detected because the CpG island of the human LRP1B gene is methylated. After transfection and culturing in the presence of G-415 for 3 weeks, the colonies formed in 6-well plates were compared with those using a mini-receptor expression plasmid (indicated as mLRP1B) and an empty vector (indicated as Mock). Illustrated. By introducing the human LRP1B gene, the number of colonies formed was drastically reduced. (C and D): The results of quantification of the number of colonies generated in KYSE170 cells (C) and TE-8 cells (D) are shown. The number of colonies formed in the above experiments A and B was counted, and the result was shown by a bar graph. Colonies larger than 2 mm were counted, and the results of three experiments were displayed as mean values ± SE. ]. As a control, TE-8 cells transfected with an empty vector produced a significantly lower number of colonies than KYSE170 cells, which reflects the suppression of human LRP1B gene expression in the former. This result clearly shows that human LRP1B gene expression suppresses the growth of esophageal cancer cells, and is presumed to function as a tumor suppressor gene.

It is the photograph which showed the homozygote deletion of the human LRP1B gene and the expression level of this gene in an esophageal cancer cell (TE-6). It is one of the drawings which showed the methylation degree of the human LRP1B gene CpG island in an esophageal cancer cell and a primary esophageal cancer. It is the other figure which showed the degree of methylation of the human LRP1B gene CpG island in an esophageal cancer cell and a primary esophageal cancer. It is the photograph which showed the influence of the human LRP1B gene expression recovery on the proliferation of esophageal cancer cells.

Claims (6)

By detecting inactivation by methylation of CpG island of human LRP1B gene in esophageal cells or inactivation by lowering the degree of acetylation of histone 4B protein of the gene , canceration of the esophageal cells is detected. A method for detecting esophageal cancer. The method for detecting a gene according to claim 1, wherein the gene is genomic DNA, cDNA, or mRNA. In the detection method of inactivation of human LRP1B gene, the gene in the sample is detected using DNA chip method, Southern blot method, Northern blot method, real-time RT-PCR method, FISH method, or CGH method. The gene detection method according to claim 1 or 2 . A test substance is contacted with an esophageal cancer cell line in which the expression of the human LRP1B gene is suppressed due to methylation of the CpG island of the human LRP1B gene, and the expression of the human LRP1B gene is detected. The test substance is selected as an antitumor substance capable of activating the human LRP1B gene by demethylation of the CpG island of the human LRP1B gene. Substance screening method. A test substance is contacted with an esophageal cancer cell line in which expression of the human LRP1B gene is suppressed due to suppression of histone H4 protein acetylation, and the expression of the human LRP1B gene is detected. When the test substance is increased compared to the system that does not contact the test substance, the test substance is selected as an antitumor substance capable of activating human LRP1B gene by promoting acetylation of histone H4 protein. Method. The method for screening a substance according to claim 4 or 5 , wherein the expression of the human LRP1B gene is detected as the amount of mRNA by a real-time RT-PCR method.

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