JP2008102012A - Methods for identifying and isolating cancer stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a therapeutic method and a diagnostic method targeting cancer stem cells, a method for identifying cancer stem cells in cancer cells or cancer tissues and a method for separating cancer stem cells from cancer cells or cancer tissues are required.
According to the present invention, cancer cells or cancer tissues containing cancer stem cells are prepared, nucleostein-expressing cells are detected from the cancer cells or cancer tissues, and the nucleostemin-expressing cells are detected as cancer stem cells. There is provided a method for identifying cancer stem cells comprising identifying, and a method for separating cancer stem cells comprising separating cancer stem cells identified by the method from cancer cells or cancer tissues.
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Description

  The present invention relates to a method for identifying and separating cancer stem cells.

Cancer is the number one cause of death in Japan in recent years and causes more than 30,000 deaths each year. Despite advances in cancer detection and treatment, cancer mortality remains high. Despite significant advances in molecular analysis and understanding of cancer, it has not yet led to the development of effective treatments.
While existing therapies such as surgery, radiation therapy, chemotherapy, hormone therapy and immunotherapy are highly effective, their effects are limited by the emergence of treatment resistant and metastatic cancer cells. New approaches are needed to identify targets for treating such resistant and metastatic cancers.

  In various cancers, it has been known that cancer cells constituting cancer have heterogeneity (Non-patent Document 1), but cancer has been considered to originate from a single cell. (Non-Patent Documents 2 and 3). Assays using colony-forming ability in soft agar medium and colony-forming ability in spleen as an index have shown that cells having colony-forming ability exist only in trace amounts in the cell population (Non-Patent Documents 4 and 5). ). On the other hand, it was shown that a small number of cells have tumorigenic activity by transplantation in vivo (Non-patent Documents 6 and 7). From these results, it was shown that the majority of cancer cells in the cancer cell population do not have the ability to form tumors, and only some of the cancer cell populations that have only a trace amount have the ability to form tumors. Two models, stochastic model and hierarchical model, have been proposed as models to explain this (Non-Patent Document 8). The probabilistic model is that all cancer cells in a tumor have tumorigenic ability, but such ability is activated in some cells infrequently and asynchronously. On the other hand, in the hierarchical model, only a very small population of cells within a tumor exhibits a strong proliferation ability and a high tumorigenic ability, and further generates various progeny cells to form a hierarchical structure.

  The hierarchical model is consistent with a model that assumes the presence of cancer stem cells, and many findings that support this hypothesis have been reported recently. In other words, it is possible to use various antibodies for detecting cell surface markers, develop a flow cytometer for analyzing and separating cells, and non-suitable for transplanting and analyzing separated cancer cells. In conjunction with the development of immunodeficient mice such as obese diabetic / combined immunodeficiency (NOD / SCID) mice, it has become possible to identify and isolate cancer stem cells that are present in trace amounts in heterogeneous cancer cell populations. For example, in human acute myeloid leukemia cells, cancer stem cells were enriched in a CD34-positive / CD38-negative cell population that was present in a very small amount, like normal hematopoietic stem cells. The CD34 positive / CD38 negative cell population showed tumorigenic activity in NOD / SCID mice, and secondary transplantation was also possible (Non-patent Document 9). However, unlike normal hematopoietic stem cells, c-kit expression was lost (Non-patent Document 10). In human multiple myeloma, it was shown that cancer stem cells having tumorigenic potential in NOD / SCID mice were concentrated in a small number of CD138-negative fractions (Non-patent Document 11). Furthermore, the presence of cancer stem cells has been reported not only in blood cancer cells but also in certain solid cancers. For example, in human breast cancer, a small fraction of CD44 positive / CD24 negative (or low expression) cell fraction, CD44 positive / CD24 negative (or low expression) and epithelial-specific antigen (EPA) positive It was shown that cancer stem cells were concentrated in the cell fraction (Non-patent Document 12). In human glioblastoma and medulloblast (cell) tumor, it has been reported that cancer stem cells are concentrated in a small number of CD133 positive cell fractions and show tumorigenicity in NOD / SCID mice (Non-patent Document 13). ). Furthermore, in human prostate cancer, it is assumed that cancer stem cells are present in the fraction of CD44 positive / α2β1 highly expressed / CD133 positive cells, which is a very small cell population (Non-patent Document 14).

  In addition to this method of enriching cancer stem cells using cell surface markers, stem cells express a variety of ATP-binding cassette (ABC) transporters and have the ability to excrete the fluorescent dye Hoechst33342 (non-patented) A method of enriching cancer stem cells using literature 15) has been developed. That is, it has been reported that a cell population can be concentrated by separating a side population (SP) fraction using a cell sorter or a flow cytometer, from a cancer cell line. (Non-Patent Documents 16 and 17).

  In addition, breast cancer stem cells, glioblastoma and medulloblastoma cancer stem cells, melanoma cancer stem cells, etc., using the floating cell mass (sphere) culture method established as a method for culturing and concentrating neural stem cells (Non-patent Document 18) In a serum-free medium supplemented with basic fibroblast growth factor (bFGF), epithelial cell growth factor (EGF), platelet-derived growth factor (PDGF), etc., floating cell masses can be formed, maintained, and concentrated. Although reported, the proportion of cells having the properties of cancer stem cells is low (Non-Patent Documents 13, 16, 19, and 20).

From the above reports, cancer stem cells are characterized by the following (1) to (3) (Non-Patent Documents 21 and 22).
(1) Cancer stem cells possess self-renewal ability. Self-replication is not synonymous with cell proliferation. Self-renewal ability refers to the ability of one or both of two divided daughter cells to produce cells that have the same ability and degree of differentiation as the parent cell in the cell lineage. .
(2) Cancer stem cells can be differentiated into multiple types of cancer cells that constitute a tumor. A plurality of types of cancer cells differentiated from cancer stem cells have a hierarchical structure starting from the cancer stem cells in the cell lineage, similar to normal stem cells. Tumors having various characteristics are formed by gradually producing various types of cancer cells from cancer stem cells.
(3) Cancer stem cells possess high cell proliferation activity. Cancer stem cells allow excessive growth of a cancer cell population by repeating control division (self-renewal) and asymmetric division (differentiation).

Nucleostamine has been reported as a new nucleolar protein expressed in neural stem cells, cell populations containing hematopoietic stem cells (c-kit positive / Lineage negative), embryonic stem (ES) cells, and several human cancer cell lines. (Patent Document 1, Non-Patent Document 23).
Nucleostamine is thought to be necessary for the maintenance of stemness of stem cells derived from normal tissues due to its expression pattern, but nucleostemin is localized in the nucleolus. In order to detect nucleostemin expressed in cells, cell fixation is required, and cells that express nucleostein cannot be isolated in a living state. In addition, there have been no reports on expression control regions such as nucleostemin gene promoters and enhancers, and there has been no report on identification and isolation of cancer stem cells using a reporter system using the expression control regions.

It has already been reported that the expression of some genes expressed in stem cells and cancer cells is reduced in cancer stem cells (Patent Document 2). For example, the expression of genes such as ANKT, CKAP2, DLG7, and FANCG is decreased in cancer stem cells. However, there are no reports on the expression of nucleostemin in cancer stem cells, and the identification, isolation, enrichment and use of cancer stem cells by reporters using nucleostemin promoters.
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  Many treatments have been developed for the treatment of cancer, but recurrence and metastasis after treatment have become problems, and radical treatment methods are required. Since the knowledge that cancer stem cells exist not only in blood cancer but also in solid cancer has been reported, if treatment and diagnostic methods targeting cancer stem cells are established, Great improvement in life extension rate and quality of life is expected. Since cancer stem cells are contained in a very small proportion of heterogeneous cancer cell populations, practical cancer stem cell isolation / identification techniques are needed to establish therapeutic and diagnostic methods that target cancer stem cells. Is essential. As a method for identifying, isolating or enriching cancer stem cells, there is no effective method for various cancer types and cancer cell lines, and a method that can be widely applied to them is required.

  That is, an object of the present invention is to provide a method for identifying and separating cancer stem cells.

The present invention relates to the following (1) to (23).
(1) From preparing cancer cells or cancer tissue containing cancer stem cells, detecting cells expressing nucleostemin from the cancer cells or cancer tissues, and identifying cells expressing the nucleostemin as cancer stem cells A method for identifying cancer stem cells.
(2) A cancer cell or cancer tissue containing a cancer stem cell is prepared, a cell expressing nucleostemin is detected from the cancer stem cell or cancer tissue, and the cell expressing the nucleostemin is detected from the cancer cell or cancer tissue. A method for separating cancer stem cells, comprising separating.
(3) The method according to (1) or (2) above, wherein the nucleostemin is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5.
(4) The method according to (1) or (2) above, wherein the nucleostein comprises a polypeptide encoded by the nucleotide sequence represented by SEQ ID NO: 6 or 7.
(5) The method according to (1) to (4) above, wherein the method for detecting nucleostemin detects a messenger RNA of the nucleostemin in a cell.
(6) The method according to (5) above, wherein the method for detecting a nucleostemin messenger RNA uses in situ hybridization.
(7) The method according to (1) to (4) above, wherein the method for detecting nucleostemin detects the nucleostemin polypeptide in a cell.
(8) The method according to (7) above, wherein the method for detecting a nucleostamine polypeptide uses an antibody specific to the polypeptide.
(9) The method according to (8) above, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
(10) The method according to (7) to (9) above, wherein the method for detecting a cell expressing nucleostemin is an immunohistochemical method.
(11) The method according to (7) above, wherein the method for detecting a cell expressing nucleostemin is a method using an aptamer.
(12) The method according to (7) above, wherein the method for detecting cells expressing nucleostemin is a method using a cell sorter, a flow cytometer, or magnetic beads.
(13) A method for detecting a cell that expresses nucleostemin introduces a DNA having a sequence in which a DNA containing a promoter of the nucleostemin gene is operably linked to a reporter gene into the cell, thereby expressing the reporter gene. (7) The method according to (7) above, which is a method for detecting
(14) The method according to (13) above, wherein the nucleostemin gene promoter is DNA consisting of the base sequence represented by SEQ ID NO: 1, 2, or 3.
(15) The nucleostemin gene promoter comprises a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3, and has promoter activity in cancer stem cells. The method according to (13) above, which is a DNA having the DNA.
(16) The above, wherein the nucleostemin gene promoter is a DNA comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 and having promoter activity in cancer stem cells (13) The method described.
(17) A cancer stem cell that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to a DNA having a nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 as a promoter of a nucleostemin gene (13) The method according to (13) above, wherein the DNA has promoter activity.
(18) The method according to any one of (13) to (17) above, wherein the method for detecting the expression of the reporter gene is a method using a cell sorter, a flow cytometer, or magnetic beads.
(19) The method according to any one of (13) to (17) above, wherein the method for detecting the expression of the reporter gene is an immunohistochemical method using an antibody specific to the reporter protein or a method using Western blotting. The method described in 1.
(20) The method according to any one of (13) to (17) above, wherein the method for detecting expression of a reporter gene is a method for measuring messenger RNA of the reporter gene.
(21) The method according to any one of (1) to (20) above, wherein the cancer cells are blood cancer.
(22) The method according to any one of (1) to (20) above, wherein the cancer cells are derived from solid cancer.
(23) The method according to any one of (1) to (20) above, wherein the cancer cell is a cultured cancer cell line.

  The present invention provides a method for identifying and separating cancer stem cells.

1. Methods for identifying and isolating cancer stem cells using nucleostemin The nucleostemin used in the present invention is not particularly limited, but those derived from animals are preferred. For example, nucleostemin such as human, mouse, cow, horse, pig, dog, cat, goat, sheep or chicken can be used. In particular, nucleostemin derived from human is preferable. Specifically, Accession No. NM_014366, NM_206825, NM_206826, AF191018, AI028402, AK027514, AK027516, AY825265, BC001024, BF732788, CF272475, CR598596, CR607914, CR608648, AC104446, NT_005986, etc., registered in GenBank Nucleostemin, Accession No. Can be used. In addition, it is generally known that there are gene polymorphisms with deletion, substitution, or addition mutations in gene sequences within and between individual animals. Different nucleostemins can also be used.

A cancer stem cell is a cell necessary for the reconstruction of a continuously proliferative tumor and refers to a cell having the following capabilities (1) to (3).
(1) possesses self-replicating ability. Self-replication is not synonymous with cell proliferation. Self-renewal ability refers to the ability of one or both of two divided daughter cells to produce cells that have the same ability and degree of differentiation as the parent cell in the cell lineage. .
(2) Differentiate into multiple types of cancer cells that make up the tumor. A plurality of types of cancer cells differentiated from cancer stem cells have a hierarchical structure starting from the cancer stem cells in the cell lineage, similar to normal stem cells. Tumors having various characteristics are formed by gradually producing various types of cancer cells from cancer stem cells.
(3) possesses high cell proliferation activity. Cancer stem cells allow excessive growth of a cancer cell population by repeating control division (self-renewal) and asymmetric division (differentiation).

In the present invention, cancer stem cells include any cells having the above-mentioned properties, preferably blood cancer stem cells, various solid cancer-derived stem cells, various cancer cell lines, more preferably blood cancer stem cells. It is done.
Hematological cancers include myeloma and lymphoma, specifically acute myeloid leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin disease, non-Hodgkin lymphoma Adult T-cell leukemia lymphoma, multiple myeloma, etc.

  Solid tumors include sarcomas and carcinomas, specifically fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, lubrication Membranous, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal Cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, sac adenocarcinoma, bone marrow cancer, bronchogenic cancer, renal cell cancer, ureteral cancer, liver cancer, bile duct cancer, choriocarcinoma, seminoma , Embryonic cancer, Wilms tumor, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, myeloblast, craniopharynx Cancer, laryngeal cancer, tongue cancer, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, black Tumor, peritoneal dissemination, teratoma, neuroblastoma, such as medulloblastoma and retinoblastoma, and the like.

Examples of cancer cell lines include U937, HL60, KG-1, KG-1a, K562, and other cancer cell lines that can be obtained from public cell banks such as ATCC, JCRB, and DSMZ.
In the present invention, blood cancer stem cells include, for example, CD34 positive / CD38 negative cells in acute myeloid leukemia, CD138 negative cells in multiple myeloma, and the like.
As solid cancer stem cells, CD44 positive / CD24 negative (or low expression) cells in breast cancer, CD44 positive / CD24 negative (or low expression) and epithelial-specific antigen (EPA) positive cells, Examples thereof include CD133 positive cells in neuroblastoma and CD44 positive / α2β1 high expression / CD133 + cells in prostate cancer.

  Cancer cell lines Cancer stem cells include acute myeloid leukemia-derived cell lines, CD34-positive / CD38-negative cells, multiple myeloma-derived cell lines, CD138-negative cells, breast cancer-derived cell lines, CD44-positive / CD24-negative (Or low expression) cell lines, CD44 positive / CD24 negative (or low expression) cells that are epithelial-specific antigen (EPA) positive, CD133 positive cells in neuroblastoma-derived cell lines And CD44 positive / α2β1 high expression / CD133 + cells in prostate cancer-derived cells.

In any of the above cancer cells or cancer cell lines, the side population cells having the high ability to excrete Hoechst33342 are also included in the cancer stem cells or cancer stem cell lines of the present invention.
Preparation Method of Cancer Cell and Cancer Tissue Containing Stem Cell In the present invention, the preparation method of cancer cell and cancer tissue containing cancer stem cell may be any method as long as the cancer cell or cancer tissue can be prepared safely and efficiently . Specifically, the following methods 1 .- (1) to 1 .- (7) are mentioned.

1 .- (1) Method for obtaining bone marrow cell mixture containing cancer stem cells from blood cancer by bone marrow puncture
Bone marrow puncture is performed from the sternum or iliac based on the method described in SE Haynesworth et al. Bone, 13, 81 (1992). Specifically, the skin surface where the bone marrow puncture is performed is disinfected and local anesthesia is performed. In particular, fully anesthetize the subperiosteum. Pull out the inner tube of the bone marrow puncture needle and attach a 10 mL syringe containing 5000 units of heparin to quickly aspirate the required amount of bone marrow fluid. On average, aspirate 10 mL to 20 mL of bone marrow fluid. Remove the bone marrow puncture needle and press and stop for about 10 minutes. After collecting the bone marrow from the obtained bone marrow fluid by centrifugation at 1,000 × g, the bone marrow cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the bone marrow cells are mixed with 10% FBS (fetal calf serum) α-MEM (α-modified MEM), DMEM (Dulbecco's modified MEM), IMDM (Isocove's modified Dulbecco's medium), etc. A bone marrow cell solution can be obtained by resuspending in a cell culture medium.

  The method for preparing a bone marrow cell mixture containing cancer stem cells from the bone marrow cell solution is not particularly limited as long as other cells mixed in the solution, such as blood cells and fibroblasts, can be removed. For example, MF Pittenger et al. Based on the method described in al. Science, 284, 143 (1999), the bone marrow cell solution is layered on a percoll with a density of 1.073 g / mL, and then centrifuged at 1,100 × g for 30 minutes to recover the cells at the interface. Thus, a bone marrow cell mixture containing cancer stem cells can be prepared.

1 .- (2) Method of obtaining blood cell mixture containing cancer stem cells by collecting peripheral blood from blood cancer Peripheral blood is collected from a vein. Specifically, the skin surface where peripheral blood is collected is disinfected. Pull out the inner cylinder of the injection needle and attach a 10 mL syringe containing 5000 units of heparin to quickly aspirate the required amount of peripheral blood. On average, aspirate 10 mL to 20 mL of peripheral blood. Remove the needle and press and stop for about 10 minutes. After collecting the peripheral blood from the obtained peripheral blood by centrifugation at 1,000 × g, the peripheral blood cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the peripheral blood cells are mixed with 10% FBS (fetal calf serum) α-MEM (α-modified MEM), DMEM (Dulbecco's modified MEM), IMDM (Isocove's modified Dulbecco's medium), etc. A peripheral blood cell solution can be obtained by resuspending in a cell culture medium.

  The method for preparing a blood cell mixture containing cancer stem cells from the peripheral blood cell solution is not particularly limited as long as other cells mixed in the solution, such as blood cells and fibroblasts, can be removed. For example, MF Pittenger et al. Based on the method described in al. Science, 284, 143 (1999), the peripheral blood cell solution was layered on a percoll with a density of 1.073 g / mL, and then centrifuged at 1,100 × g for 30 minutes to recover the cells at the interface. By doing so, a blood cell mixture containing cancer stem cells can be prepared.

1 .- (3) Method of obtaining cancer cell mixture containing cancer stem cells from solid cancer by cell puncture Cell puncture is performed from a tumor site. Specifically, the skin surface where cell puncture is performed is disinfected. Remove the inner tube of the cell puncture needle and attach a 10 mL syringe containing 5000 units of heparin to quickly collect the required amount of cancer tissue. On average, 0.1 g to 0.2 g of cancer tissue is aspirated. Remove the cell puncture needle and press and stop for about 10 minutes. The obtained cancer tissue is digested with a proteolytic enzyme such as trypsin, collagenase, or hyaluronidase, and then the cancer cells are collected by centrifugation at 1,000 × g, and then the cancer cells are washed with PBS (Phosphate Buffered Saline). After repeating this step twice, the cancer cells are treated with α-MEM (α-modified MEM), DMEM (Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbecco's medium) containing 10% FBS (fetal calf serum). A cancer cell mixture can be obtained by resuspending in a cell culture medium.

1 .- (4) Method of obtaining bone marrow cell mixture containing cancer stem cells from rat and mouse blood cancer Rats or mice were killed by cervical dislocation and thoroughly disinfected with 70% ethanol. The head muscles are removed. Remove scissors at the knee joint, remove the joint, and remove the muscles behind the femur. Insert the scissors into the hip joint, remove the joint, and remove the femur. After removing the muscles attached to the femur as much as possible with scissors, both ends of the femur are cut with scissors. Attach a needle of appropriate size according to the bone thickness to a 2.5 mL syringe, and add about 1.5 mL of cell culture medium such as α-MEM, DMEM, or IMDM containing 10% FBS (fetal calf serum). After filling the syringe, the tip of the injection needle is inserted into the stump on the knee joint side of the femur. Bone marrow cells are pushed out from the stump on the hip joint side by injecting the culture solution in the syringe into the bone marrow. The obtained bone marrow cells are suspended in the culture medium by pipetting. From the bone marrow fluid, a bone marrow cell mixture containing cancer stem cells can be prepared by the same method as the isolation of bone marrow cells from human bone marrow fluid described in 1 .- (1) above.

1 .- (5) Method of obtaining a cancer cell mixture containing cancer stem cells from solid tumors of rats and mice Rats or mice are killed by cervical dislocation, sufficiently disinfected with 70% ethanol, and then the tumor-forming part is opened and the tumor is incised. Remove the site. The obtained cancer cells are suspended in the culture solution using a proteolytic enzyme such as trypsin, collagenase, or hyaluronidase. From the cancer cell solution, a cancer cell mixture containing cancer stem cells can be prepared by the same method as the preparation of cancer cells from the human cancer cell solution described in 1 .- (3) above.

1 .- (6) Method for preparing tissue section containing cancer stem cells from human, rat and mouse solid tumors For example, based on the method described in JP-A-2005-261236, cancer from human, rat and mouse solid cancers Tissue sections containing stem cells can be made. The sample may be a part or all of cancer tissues derived from these organisms. Moreover, what cultured the cell and the structure | tissue may be used. In the case of cultured cells, the cells may be cultured on a slide glass and the slide glass may be used as a sample, or the cells cultured on a culture dish may be used as they are.

The sample does not necessarily need to be fixed, but can be used in a fixed manner. As a fixing method, the sample may be directly immersed in a fixing solution, or reflux fixed. The fixing solution is not particularly limited, but for example, glutaraldehyde, formaldehyde aqueous solution, formalin or a mixed solution thereof is used, and 4% formaldehyde aqueous solution is most preferably used.
In the case of tissue, the sample may be a section or a whole-mount. In the case of a section, any of a frozen section, a paraffin section, a plastic section using a resin or the like as an embedding agent may be used.

1 .- (7) Method for preparing cell specimens containing cancer stem cells from human, rat and mouse blood cancer or solid cancer cell mixtures and cancer cell lines Slide cells in solution or using cytospin Apply to a support such as glass. The cell may be a part or all of the cell. Moreover, the cell mixture prepared by the method described in 1 .- (1) to 1 .- (5) above may be used, or the cell mixture may be cultured.

  The sample does not necessarily need to be fixed, but can be used in a fixed manner. As a fixing method, the sample may be directly immersed in a fixing solution, or reflux fixed. The fixing solution may be anything, for example, glutaraldehyde, formaldehyde aqueous solution, formalin or a mixed solution thereof is used, and 4% formaldehyde aqueous solution is most preferably used.

Method for detecting cells expressing nucleostemin from cancer cells or cancer tissues In the present invention, the method for detecting cells expressing nucleostemin from cancer cells or cancer tissues is the above method 1 .- (1) to 1.-. Any method may be used as long as cancer stem cells can be detected using the expression levels of nucleostemin mRNA and protein in the cell mixture, tissue section, and cell specimen containing the cancer stem cells obtained in (7) as an index. Specifically, the following methods 1 .- (8) to 1 .- (9) are mentioned.

1 .- (8) Method for Detecting Cancer Cell Expressing Nucleostemin mRNA As a method for detecting nucleostemin mRNA, for example, Molecular Cloning: A Laboratory Manual Second Edition [T. Edited by T. Maniatis et al., Molecular Cloning: A Laboratory Manual 2nd ed., Published by Cold Spring Harbor Laboratory, (1989)], JP 2005-261236, etc. And a method of detecting by performing in situ hybridization based on the existing method. Specifically, it can be performed as follows.

  A cancer cell or cancer tissue specimen is prepared by the method described in 1 .- (6) and 1 .- (7) above. As pretreatment, fixed sections, cells or whole mount samples are treated with deparaffinization, rehydration, membrane permeability, such as trypsin, proteinase K, saponin, or streptolysin O. I do. Thereafter, the protein that binds to the mRNA is degraded, and proteolytic treatment is performed to expose the mRNA. Specific examples of the proteolytic agent used for the proteolytic treatment include thermolysin, proteinase K, or trypsin. Moreover, these proteolytic enzymes can also be mixed and used. The proteolytic treatment time is usually 5 to 30 minutes, and the treatment temperature is 37 to 50 ° C. It is preferable to perform treatment under several conditions including the concentration in advance to optimize the treatment time and treatment temperature. . Thereafter, treatments such as quenching with glycine and acetylation with acetic anhydride may be performed.

  After performing the above treatment, prehybridization and hybridization are performed. Prehybridization is performed in a solution containing, for example, SSC and formamide at 37 ° C. for 30 to 60 minutes. The concentration of each reagent is preferably 0.1-2 × SSC, 1-50% formamide. Hybridization is performed at 37 ° C. overnight in a solution containing, for example, SSC, SDS (sodium dodecyl sulfate) and formamide. Concentration of each reagent is 0.1 to 2 x SSC (composition of 1-fold concentration of SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate), 0.05 to 1% SDS, 1 to 50% Formamide is preferred. Probes used for hybridization are, for example, radioisotopes, digoxigenin, biotin, β-galactosidase, HRP (horse radish peroxidase), alkaline phosphatase, chloramphenicol acetyltransferase, growth hormone, luciferase, β-lactamase and derivatives thereof A protein selected from the group consisting of digoxigenin, and more preferably digoxigenin is used. The probe may be an oligonucleotide, a single-stranded DNA, a double-stranded DNA, or RNA. The probe may be synthesized by chemical synthesis, using a primer extension using a DNA-dependent DNA polymerase or an in vitro transcription system using a DNA-dependent RNA polymerase, and performing PCR (polymerase chain reaction). You may use it.

  The hybridized sample is washed, and mRNA is detected by selecting a detection method suitable for each label. For example, (1) In the case of a probe labeled with a radioisotope, development is performed using an emulsion. (2) When the label is biotin or digoxigenin, for example, a protein selected from the group consisting of β-galactosidase, HRP, alkaline phosphatase, chloramphenicol acetyltransferase, growth hormone, luciferase, β-lactamase, and derivatives thereof. Preferably, mRNA is detected by using an anti-biotin antibody or an anti-digoxigenin antibody to which an enzyme such as HRP, alkaline phosphatase, or β-galactosidase is bound, and coloring the substrate using a substrate for the enzyme. Furthermore, for example, mRNA may be detected by amplifying a signal by combining methods such as the ABC method. (3) FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescence When a probe is fluorescently labeled with a fluorescent dye or protein such as protein, yellow fluorescent protein, red fluorescent protein, or an anti-biotin antibody or anti-digoxigenin antibody, for example, a fluorescence microscope, a confocal laser microscope, or a laser scanning microscope Etc. can be used to detect the signal.

1 .- (9) Method for Detecting Cancer Cells Expressing Nucleostemin As a method for detecting nucleostemin, for example, Molecular Cloning: A Laboratory Manual Second Edition [T. Edited by T. Maniatis et al., Molecular Cloning: A Laboratory Manual 2nd ed., Published by Cold Spring Harbor Laboratory, (1989)], JP 2005-261236, etc. And an immunohistochemical method using an antibody against anti-nucleostemin, a method using an aptamer, and the like. This will be specifically described below.

1 .- (9-1) Immunohistochemical detection method using antibody against anti-nucleostemin (i) Method for producing anti-nucleostemin antibody As a method for producing an anti-nucleostemin antibody, nucleostemin is used. Any method may be used as long as it is a method for producing an antibody that is used as an immunogen and specifically recognizes nucleostamine. Specifically, the method of producing the polyclonal antibody and monoclonal antibody shown below is mentioned.

A. Preparation of antigen Nucleostemin or a purified fragment of the nucleostemin fragment, or a peptide having a partial amino acid sequence of the nucleostemin as an antigen, polyclonal antibody, monoclonal antibody, etc. Can be produced.

  The nucleostemin used in the present invention is not particularly limited, but is preferably derived from animals. Nucleostemin such as human, mouse, cow, horse, pig, dog, cat, goat, sheep or chicken can be used. In particular, nucleostemin derived from human is preferable. Specifically, Accession No. NM_014366, NM_206825, NM_206826, AF191018, AI028402, AK027514, AK027516, AY825265, BC001024, BF732788, CF272475, CR598596, CR607914, CR608648, AC104446, NT_005986, etc., registered in GenBank Nucleostemin, Accession No. Can be used. In addition, it is generally known that there are gene polymorphisms with deletion, substitution, or addition mutations in gene sequences within and between individual animals. Different nucleostemins can also be used.

A recombinant expression vector of the protein is constructed by inserting a DNA fragment encoding the nucleostemin or a full-length cDNA downstream of the promoter in the expression vector.
The recombinant protein expression vector is introduced into a host cell compatible with the expression vector.
Any host cell that can express the target DNA can be used, for example, Escherichia genus, Serratia genus, Corynebacterium genus, Brevibacterium genus, Bacteria belonging to the genus Pseudomonas, Bacillus, Microbacterium, Kluyveromyces, Saccharomyces, Shizosaccharomyces, Trichosporon Bacteria, yeasts, animal cells, insect cells and the like belonging to the genus, genus Schwanniomyces and the like can be used.

As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where nucleostemin gene DNA can be transcribed is used.
When a bacterium is used as a host cell, the recombinant expression vector for nucleostemin is capable of autonomous replication in the bacterium, and is composed of a promoter, a ribosome binding sequence, a DNA encoding nucleostemin, and a transcription termination sequence. A recombinant expression vector is preferred. A gene that controls the promoter may also be included.

  Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Amersham Pharmacia Biotech), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE -8 (manufactured by QIAGEN), pKYP10 [JP 58-110600], pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (Stratagene), pGEX (Amersham Pharmacia Biotech), pET-3 (Novagen), pTerm2 (USP4686191, USP4939094, USP5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)] and the like.

As the expression vector, it is preferable to use a vector in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
Any promoter can be used as long as it can be expressed in the host cell. For example, promoters derived from Escherichia coli and phages such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, T7 promoter, SPO1 promoter, SPO2 promoter, penP promoter and the like can be mentioned. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrpx2), a tac promoter, a letI promoter [Gene, 44, 29 (1986)] and a lacT7 promoter can also be used.

By substituting the base in the nucleotide sequence encoding the nucleostemin used in the present invention so as to be an optimal codon for host expression, the production rate of the target protein can be improved.
Although the transcription termination sequence is not necessarily required for the expression of the nucleostemin gene used in the present invention, it is preferable to place the transcription termination sequence immediately after the structural gene.

  Examples of host cells include Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Bacillus subtilisevi, liquelus subtilisevi Brevibacterium saccharolyticum ATCC14066, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110 and the like.

  As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], the protoplast method (Japanese Patent Laid-Open No. 63-248394), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

When yeast is used as a host cell, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.
Any promoter can be used as long as it can be expressed in yeast. For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter, CUP 1 A promoter etc. can be mention | raise | lifted.

  Examples of host cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulces, wan alluvius).

  As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, electroporation [Methods in Enzymol., 194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). )] Etc.

  When animal cells are used as host cells, examples of expression vectors include pCDNAI (manufactured by Invitrogen), pCDM8 (manufactured by Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], pAS3. -3 (JP-A-2-27075), pCDM8 [Nature, 329, 840 (1987)], pCDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987) )], PAGE210 and the like.

  Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, Examples include metallothionein promoter, heat shock protein promoter, SRα promoter and the like. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

Examples of host cells include human cells such as Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, and HBT5637 [JP-A-63-299]. it can.
As a method for introducing a recombinant vector, any method capable of introducing DNA into animal cells can be used. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075) Lipofection [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987), Virology, 52, 456 (1973)], etc. can be used. Transformants can be obtained and cultured according to the method described in Japanese Patent Application Laid-Open No. 2-227075 or Japanese Patent Application Laid-Open No. 2-257891.

  When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory Manual [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], Current Protocols Proteins can be expressed by the methods described in In-Molecular Biology Supplement 1-38 (1987-1997), Bio / Technology, 6, 47 (1988), etc.

That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (both manufactured by Invitrogen) and the like.

As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
Insect cells include, for example, Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, WHFreeman and Company, New York, (1992)], which is an ovarian cell of Spodoptera frugiperda, and High 5 (Invitrogen), an ovarian cell of Trichoplusia ni. Etc.) can be used.

Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method [JP-A-2-27075], the lipofection method [Proc. Natl. Acad Sci. USA, 84, 7413 (1987)].
As a gene expression method, in addition to direct expression, secretion according to the method described in Molecular Cloning, Second Edition [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] Production, fusion protein expression, etc. can be performed.

When expressed in yeast, animal cells or insect cells, a protein with a sugar or sugar chain added can be obtained.
By cultivating a transformant having a recombinant DNA into which nucleostemin gene DNA has been incorporated in a medium, producing and accumulating nucleostemin in the culture, and collecting the nucleostemin from the culture, Nucleostamine can be produced.

The method of culturing the transformant for producing nucleostemin in a medium can be performed according to a usual method used for culturing a host.
As a medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host, the medium contains a carbon source, a nitrogen source, an inorganic substance and the like that can be assimilated by the host. Either a natural medium or a synthetic medium may be used as long as the medium can be efficiently cultured.

Any carbon source may be used as long as each host can assimilate, for example, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, etc. Alcohols such as ethanol and propanol can be used.
Examples of the nitrogen source include ammonium salts of various inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof, and the like are used.

Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using, for example, an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter. Sometimes indoleacrylic acid (IAA) or the like may be added to the medium.

  As a medium for culturing a transformant obtained using an animal cell as a host cell, for example, RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [ Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] A medium supplemented with serum or the like can be used.

The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2.
Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.
As a medium for culturing a transformant obtained using insect cells as host cells, for example, a commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400 ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] and the like can be used.

The culture is usually carried out for 1 to 5 days under conditions of pH 6 to 7, 25 to 30 ° C.
Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.
Nucleostemin may be isolated and purified from the above-described transformant culture using conventional protein isolation and purification methods.

  For example, when nucleostemin is expressed in a dissolved state in the cells of the transformant, after completion of the culture, the cells are collected by centrifugation and suspended in an aqueous buffer, followed by an ultrasonic crusher, a French press, A cell-free extract is obtained by disrupting the cells with a Manton Gaurin homogenizer, dynomill or the like. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), cation exchange using resin such as S-Sepharose FF (Amersham Pharmacia Biotech) Chromatographic methods, hydrophobic chromatography methods using resins such as butyl sepharose and phenyl sepharose, gel filtration methods using molecular sieves, affinity chromatography methods, electrophoresis methods such as chromatofocusing methods, isoelectric focusing etc. A purified sample can be obtained by using the methods alone or in combination.

When nucleostemin is expressed in the form of insoluble matter in the transformant cells, the cells are recovered and then disrupted and centrifuged to recover the protein insoluble matter as a precipitate fraction.
The recovered insoluble matter of the protein is solubilized with a protein denaturant.
The solubilized solution is diluted or dialyzed to lower the concentration of the protein denaturant in the solubilized solution to return the protein structure to a normal three-dimensional structure, and then the same isolation and purification method as described above. A purified preparation of the protein is obtained.

When a derivative such as nucleostemin or a modified sugar thereof is secreted outside the cell, the derivative such as the protein or a sugar chain adduct thereof can be recovered from the culture supernatant. That is, the culture supernatant is recovered from the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
Specific examples of nucleostemin obtained in this way include proteins having the amino acid sequences represented by SEQ ID NOs: 4 and 5.

  Nucleostemin expressed by the above method can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). In addition, for example, peptide synthesizers manufactured by Advanced ChemTech, Perkin-Elmer, Amersham Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu, etc. Can also be synthesized.

B. Polyclonal antibody The polyclonal antibody against nucleostemin used in the present invention is the above 1 .- (9-1) A. The nucleostemin full-length polypeptide or partial fragment preparation prepared by the method described in 1. or a partial polypeptide having a partial amino acid sequence of the polypeptide as an antigen is used to immunize an animal, and a polyclonal antibody Can be produced.

As specific examples of animals to be administered, rabbits, goats, rats, mice, hamsters and the like can be used.
The dose of the antigen is preferably 50 to 100 μg per animal.
When a peptide is used, it is desirable to use a peptide covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as an antigen. The peptide used as an antigen can be synthesized with a peptide synthesizer.

  The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood was collected from the fundus venous plexus, and enzyme immunoassay (enzyme immunoassay (ELISA): published by the Shoin Shoin 1976, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) )] Etc. to measure the anti-nucleostemin antibody titer to confirm that the serum reacts with the antigen used for immunization.

Specific antibody titer measurement methods include various known techniques such as radioisotope immunoassay (RIA method), solid-phase enzyme immunoassay (ELISA method), fluorescent antibody method, passive hemagglutination method and the like. However, the RIA method and the ELISA method are preferable from the viewpoints of sensitivity, rapidity, accuracy, and possibility of automation.
The antibody titer measurement according to the present invention is not particularly limited, but can be performed by ELISA. Specifically, the antigen is adsorbed on the solid phase, and the solid surface on which the antigen is not adsorbed is covered with a protein unrelated to the antigen, for example, bovine serum albumin (BSA). The sample is brought into contact with a serially diluted sample (eg, mouse serum), the anti-nucleostemin antibody in the sample is bound to the antigen, and an antibody against a mouse antibody enzyme-labeled as a secondary antibody is added to bind to the mouse antibody. After washing, the antibody titer can be calculated by adding a substrate for the enzyme and measuring color development or the like based on substrate degradation.

Polyclonal antibodies can be obtained by obtaining serum from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
The method for separation and purification is not particularly limited, but centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)), or DEAE- Examples thereof include a method of treating a chromatography using a sepharose column, an anion exchange column, a protein A or G-column or a gel filtration column alone or in combination.

C. Monoclonal antibody A monoclonal antibody against nucleostemin used in the present invention is prepared, for example, as follows.

1 .- (9-1) A. An animal whose antiserum showed a sufficient antibody titer against the antigen prepared by the method described in 1) is used as a source of antibody-producing cells.
Specifically, the spleen of an animal showing a sufficient antibody titer is removed 3 to 7 days after the final administration of the antigen.
The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.

Treat the spleen cells of the obtained precipitate fraction with Tris-ammonium chloride buffer (pH 7.65) for 1-2 minutes to remove erythrocytes, and then wash with MEM medium 3 times. Used as
As myeloma cells used for the production of hybridomas, cell lines obtained from mice or rats are preferred. Specifically, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Curr. Topics Microbiol. Immunol., 81, 1 (1978), Eur. Immunol., 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) (Nature, 276, 269 (1978)), P3-X63-Ag8653 (653) (J. Immunol., 123, 1548 ( 1979)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] and the like. These cell lines consist of 8-azaguanine medium (RPMI-1640 medium with glutamine (1.5 mmol / L), 2-mercaptoethanol (5 × 10-5 mol / L), gentamicin (10 μg / mL) and fetal bovine serum. (FCS) [CSL (CSL), 10%] supplemented with medium (hereinafter referred to as normal medium) and further added with 8-azaguanine (15 μg / mL)] The cells are cultured in a normal medium 3 to 4 days before cell fusion, and 2 × 10 ⁷ or more of the cells are used for fusion.

The resulting antibody-producing cells and myeloma cells are washed thoroughly with MEM medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 L distilled water, pH 7.2), and antibody producing cells Number: Mix so that the number of myeloma cells is 5: 1 to 10: 1, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant.
Thoroughly disaggregate the cell group of the obtained precipitate fraction, and mix the cell group with 2 g of polyethylene glycol-1000 (PEG-1000) per 10 ⁸ antibody-producing cells at 37 ° C. with stirring at 37 ° C. 0.2 to 1 mL of a solution mixed with 0.7 mL of dimethyl sulfoxide (DMSO) is added, and 1-2 mL of MEM medium is added in several portions every 1 to 2 minutes.

After the addition, add MEM medium to prepare a total volume of 50 mL.
The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded. After loosening the cells of the obtained precipitate fraction gently, sucking with a pipette and blowing out gently, HAT medium [normal medium with hypoxanthine (10 ^-4 mol / L), thymidine (1.5 x 10 ^-5 mol) / L) and aminopterin (4 × 10 ⁻⁷ mol / L) added medium] suspended in 100 mL.

The suspension is dispensed at 100 μL / well into a 96-well culture plate and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator.
About the hole where the hybridoma grown in colony is observed, discard the half volume of the supernatant, add the same amount of HT medium (medium excluding aminopterin from HAT medium), and thereafter every 10 to 30 hours every 1-3 days. Conversion to HT medium is performed.

After culturing in HT medium for 3 to 4 days, a portion of the culture supernatant is taken and anti-nucleostemin is obtained by an enzyme immunoassay described in Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 14 (1988), etc. The antibody titer is measured, and a hybridoma that specifically reacts with the polypeptide used in the present invention is selected.
As the antibody titer measurement method used here, various known techniques such as radioisotope immunoassay (RIA method), solid phase enzyme immunoassay (ELISA method), fluorescent antibody method, passive hemagglutination reaction method and the like can be mentioned. However, the RIA method and the ELISA method are preferable from the viewpoints of sensitivity, rapidity, accuracy, and possibility of automation.

  Measurement of antibody titer by ELISA can be performed, for example, by the following procedure. That is, the antigen is adsorbed on the solid phase, and the solid surface on which the antigen is not adsorbed is covered with a protein unrelated to the antigen, such as bovine serum albumin (BSA), washed, and then serially diluted as a primary antibody. The anti-nucleostemin antibody in the sample is bound to the above antigen (for example, mouse serum), and an antibody against the mouse antibody that is enzyme-labeled as a secondary antibody is added to the mouse antibody. After washing, The antibody titer can be calculated by adding a substrate of the enzyme and measuring color development or the like based on the decomposition of the substrate.

In the above example, an 8-azaguanine resistant cell line was used, but a medium having an appropriate composition can be used by using other cell lines depending on the selection of the hybridoma.
The hybridoma that has been found to produce a specific antibody by measuring the antibody titer is transferred to another plate and cloned. Specific cloning methods include dilution by limiting dilution so that one hybridoma is contained in one well, soft agar method in which colonies are collected on a soft agar medium, and one cell by a micromanipulator. For example, a cell sorter, a flow cytometer, and a “sorter clone” that separates one cell by a magnetic bead, but the limiting dilution method is preferred.

For the plate hole in which the antibody titer is recognized, cloning is repeated 2 to 4 times by the limiting dilution method, and those in which the antibody titer is stably recognized are selected as the anti-nucleostemin antibody-producing hybridoma strain.
The hybridoma strain is cultured by replacing the HT medium with a normal medium. Mass culture is performed, for example, by rotary culture using a large culture bottle or spinner culture.

In addition, the hybridoma strain described above can also be grown in the same strain of mouse (for example, the above-described BALB / c) or the intraperitoneal cavity of a Nu / Nu mouse. For example, an 8-10 week old BALB / c female mouse treated with pristane is injected intraperitoneally with an anti-nucleostemin antibody-producing hybridoma strain at 2-4 × 10 ⁶ cells / mouse, and the hybridoma strain becomes ascites tumor. Let Ascites can be collected from the mouse and centrifuged to remove solids, which can then be used as a monoclonal antibody.

Examples of methods for identifying monoclonal antibodies and isotypes, subclasses and the like of the obtained monoclonal antibodies include the octterlony method, the ELISA method and the RIA method.
Protein quantification can be calculated, for example, by the Foreign Lowry method and absorbance at 280 nm [1.4 (OD280) = immunoglobulin 1 μg / mL].

  Monoclonal antibodies are, for example, centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)), or DEAE-Sepharose column, anion exchange column, Chromatography using a protein A or G-column or gel filtration column or the like can be used alone or in combination, and can be separated or purified by collecting IgG fractions.

(Ii) Method for detecting cells expressing nucleostemin using an immunohistochemical method An antibody against nucleostemin prepared by the method described in 1 .- (9) (i) above is used as a primary antibody. The sample is brought into contact with the sample pretreated by the method described in 1 .- (8) above. The primary antibody is a protein such as radioisotope, digoxigenin, biotin, β-galactosidase, HRP, alkaline phosphatase, chloramphenicol acetyltransferase, growth hormone, luciferase, β-lactamase and derivatives thereof, more preferably HRP or An antibody conjugated with alkaline phosphatase or the like is used. The reaction between the primary antibody and the sample is preferably 4 to 37 ° C. for 1 hour to several days, but the reaction conditions are not limited thereto.

  The sample is reacted with the primary antibody, washed with PBS or the like, and subjected to a crosslinking reaction using a crosslinking agent. As a cross-linking agent, EDC [0.1-10 mg / mL 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was converted to 0.1 mol / L female (2- (N-morpholino) ethanesulfonic acid monohydrate. Dissolved solution], SMPT [0.01-5 mmol / L 4-succinimidyloxycarbonyl-methyl-α- (2-pitidyldithio] toluene in PBS), BMH [0.5-20 mmol / L bis-maleimidohexane in PBS Solution], ABH [0.1-10 mmol / L p-azidobenzoyl hydrazide in PBS], SIAB [0.1-10 mmol / L N-succinimidyl [4-iodoacetyl] aminobenzoate in PBS], GMBS [0.1-10 mmol / L N- (g-maleimidobutyryloxy) succinimide ester in PBS], Sulfo-GMBS [0.1-10 mmol / LM sulfo-N- (g-maleimidobutyryloxy) succinimide ester in PBS], DMS [1-50 mmol / L Dimethylsuber PBS solution of midate dihydrochloride, BS3 [1-50 mmol / L bis (sulfosuccinimidyl suberate) in PBS), Sulfo-NHS [1-50 mmol / L sulfo-N-hydroxysulfosuccinimide] PBS solution], NHS [1-50 mmol / L N-hydroxysulfosuccinimide in PBS], ASBA [0.1-10 mmol / L 4- (p-azidosalicylamido) butylamine in PBS], etc. it can. The reaction is usually carried out at room temperature for 1-2 hours.

After the cross-linking reaction, the sample is washed with PBS or the like and reacted with a secondary antibody to which an enzyme such as HRP, alkaline phosphatase, β-Gal (β-galactosidase) or the like is bound. When using a primary antibody bound to a radioisotope, HRP or alkaline phosphatase, the reaction with the secondary antibody can be omitted.
The signal is detected by adding a substrate of the enzyme bound to the primary or secondary antibody and causing color development. Specific substrates include diaminobenzidine (DAB Tris Tablets, MUTO PURE CHEMICALS) for HRP, and NBT (Nitrotetrazolium Blue Chloride) and BCIP (5-Bromo-4-Chloro-) for alkaline phosphatase. For 3-indolyl-phosphate disodium salt) and β-Gal, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) can be mentioned. Further, for example, a signal can be amplified in combination with a method such as ABC method.

In the case of an antibody labeled with a radioisotope, it is developed using an emulsion.
FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescent protein, yellow If the primary or secondary antibody is fluorescently labeled with a fluorescent dye or protein such as fluorescent protein or red fluorescent protein, the signal may be detected using a fluorescent microscope, confocal laser microscope, or laser scanning microscope. it can.

1 .- (9-2) Method for Detecting Cells Expressing Nucleostemin Using Aptamer The aptamer used in the present invention is, for example, based on the method described in JP-A-2006-149302 as follows: In this way, aptamers can be produced.

  The aptamer in the present invention represents a nucleic acid molecule that recognizes various molecules such as proteins, amino acids, and antibiotics. In the detection method of the present invention, nucleostemin is specifically recognized and / or a nucleostemin organism. Any nucleic acid molecule having a function of inhibiting the biological activity may be used. Specifically, even RNA is DNA, and DNA is preferable. Furthermore, the aptamer may include a nucleic acid molecule in which the phosphate skeleton in the nucleic acid molecule structure is modified to increase the in vivo stability. Examples of the phosphate skeleton include a phosphate skeleton including a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidothioate bond, a phosphoramidate bond, a phosphordiamidate bond, and a methylphosphonate bond. Further, the aptamer has, as structural features, (1) at least one loop structure, (2) a primary structure including many deoxyguanosines (including guanosine and guanosine analogs), or (3) deoxyguanosine (guanosine, Also included are aptamers in which a guanosine analog (including guanosine analogs) forms a tetrameric cluster structure. The aptamer may be either a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule, but is preferably a single-stranded nucleic acid molecule.

  The number of bases of the aptamer used in the present invention is not particularly limited as long as it has a base length capable of specifically binding to nucleostemin. For example, it is 10 to 200 bases, preferably 20 to 150 bases. Bases, more preferably 30 to 150 bases, and even more preferably 50 to 150 bases, specifically 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 base aptamers. It is done.

The aptamer used in the present invention can also be used in a form in which other molecular species such as a fluorescent labeling dye is bound.
The aptamer used in the present invention may be any method that can produce an aptamer. For example, it can be produced using an in vitro selection method (Ellington et al., 1990; Tuerk et al., 1990).

In the present invention, the in vitro selection method refers to a method of selecting a molecule having affinity for nucleostein from a pool of nucleic acid molecules containing a random sequence and eliminating a molecule having no affinity.
Specifically, first, a single-stranded nucleic acid molecule containing a random base sequence of about 20 to 300 bases, preferably about 30 to 150, more preferably about 30 to 100 bases, such as DNA and RNA, is prepared. The nucleic acid molecule may be synthesized directly, or in the case of an RNA molecule, the DNA molecule may be synthesized first and then prepared by a transcription reaction. When the nucleic acid molecule is DNA, it preferably has base sequences serving as primers for enabling PCR amplification at both ends. The primer binding sequence portion may be prepared so as to have an appropriate restriction enzyme recognition sequence for excising the primer portion with a restriction enzyme after PCR amplification. The length of the primer binding sequence portion is not particularly limited, but is usually 20 to 50, preferably 20 to 30 bases. Further, in order to separate the single-stranded DNA after PCR amplification at the 5 ′ end of the primer by electrophoresis or the like, labeling may be performed using a radioactive substance, a fluorescent substance or the like. Further, when preparing an RNA molecule, it may be prepared by transcribing from a DNA molecule to an RNA molecule using a suitable promoter as a primer on the 5 ′ end side, for example, a nucleic acid molecule having a T7 promoter sequence. .

  Next, random nucleic acid molecules obtained by PCR amplification and nucleostemin or a peptide fragment encompassing a specific region of nucleostemin are mixed and incubated. After incubation, the mixture is subjected to electrophoresis to separate the nucleic acid molecule-nucleostemin complex and free nucleic acid molecule. Nucleic acid molecules forming a complex with nucleostemin are extracted according to a known method. When the recovered nucleic acid molecule is DNA, PCR amplification is further performed, and the amplified DNA is recovered as single-stranded DNA by heat denaturation or the like. When the recovered nucleic acid molecule is RNA, the RNA is reverse transcribed into cDNA, PCR amplified, and the amplified DNA is transcribed to prepare RNA.

The step of mixing the above-described nucleic acid molecule and nucleostemin, the step of separating the nucleic acid molecule bound to nucleostemin, the step of PCR amplification (in the case of RNA, reverse transcription after amplification), the amplified nucleic acid molecule The operation consisting of the steps used for binding with nucleostemin is repeated several times. The obtained nucleic acid molecule can be sequenced according to a known method.
The synthesis of the aptamer may be performed by chemical synthesis, in vitro transcription system using DNA-dependent RNA polymerase or DNA-dependent DNA polymerase, and amplification synthesis method by PCR. Based on the sequence of the nucleic acid molecule obtained by the above operation, the phosphate skeleton is modified, phosphorothioate bond, phosphorodithioate bond, phosphoramidothioate bond, phosphoramidate bond, phosphoramidate bond An aptamer composed of a phosphate skeleton containing a methylphosphonate bond and the like can also be synthesized.

The pretreated sample is subjected to a nucleostemin binding reaction with an aptamer by the method described in 1 .- (8) above. The aptamer is preliminarily heated in a phosphate buffer containing 1 mmol / L MgCl ₂ (hereinafter referred to as a binding buffer) at 94 ° C. for 3 minutes and then cooled at 4 ° C. for 30 minutes to regenerate the higher-order structure. Make up. The binding reaction is performed, for example, in a solution containing a binding buffer at 4 ° C. to 37 ° C. for 0.5 hours to several days. The aptamer used for the binding reaction is more preferably a protein such as a radioisotope, digoxigenin, biotin, β-galactosidase, HRP, alkaline phosphatase, chloramphenicol acetyltransferase, growth hormone, luciferase, β-lactamase, and derivatives thereof. Is labeled with digoxigenin.

  After the binding reaction, the sample is washed and a detection reaction suitable for each label is performed. For example, (1) in the case of an aptamer labeled with a radioisotope, development is performed using an emulsion. (2) When the label is biotin or digoxigenin, proteins such as β-galactosidase, HRP, alkaline phosphatase, chloramphenicol acetyltransferase, growth hormone, luciferase, β-lactamase and derivatives thereof, more preferably HRP, alkali Detection is carried out using an anti-biotin antibody or anti-digoxigenin antibody to which an enzyme such as phosphatase or β-galactosidase is bound, and coloring with a substrate for the enzyme. Furthermore, it is also possible to amplify and detect signals by combining methods such as the ABC method. (3) FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescence When aptamers labeled with fluorescent dyes or proteins such as proteins, yellow fluorescent proteins, red fluorescent proteins, or anti-biotin antibodies or anti-digoxigenin antibodies are used, for example, fluorescence microscopy, confocal laser microscopy, or laser A signal can also be detected using a scanning microscope or the like.

Method for separating cancer stem cells expressing nucleostemin When a sample identified as a cancer stem cell as described above is applied to a slide glass, a laser microdissection described in 1 .- (10) below, etc. Can be used to isolate cancer stem cells expressing nucleostemin. On the other hand, when the sample identified as a cancer stem cell is a solution, the cancer stem cell can be separated using a cell sorter, a flow cytometer, or a magnetic bead described in 1 .- (11) below.

1 .- (10) Method for separating cancer stem cells expressing nucleostemin using laser microdissection Cancer tissue specimen or cancer cells prepared in the above 1 .- (6) or 1 .- (7) To separate cancer cells expressing nucleostein from a specimen under a microscope, the laser microdissection method (M. Emmert-Buck et al: Science 274 : 998-1001 ( 1996), RF. Bonner et al: Science 278 : 1481-1483 (1997)). For example, cancer stem cells can be isolated using laser capture microdissection system LM100 (Olympus).

1 .- (11) A method using a flow cytometer having a sorting function, or a method of separating cancer stem cells expressing nucleostemin using magnetic beads A flow site having a sorting function used in the present invention The meter detects the expression of nucleostemin by converting the fluorescence intensity emitted from the antibody, probe, and aptamer, more preferably the antibody, bound to the nucleostemin molecule expressed in cancer stem cells into an electrical signal. Any cancer stem cells can be isolated. For example, there are flow cytometers such as a water droplet charging method and a cell capture method (flow cytometer freely, pp. 14-23, Shujunsha, 1999). In addition, cancer stem cells may be separated using a plurality of antigens by combining the types of fluorescence used. Specific examples of fluorescent dyes include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed , Green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, Hoechst33342, etc. (Flow cytometer freely, pp. 3-13, Shujunsha, 1999).

Specifically, for example, cancer stem cells can be separated by a flow cytometer as follows.
The cells are first subjected to membrane permeabilization treatment such as trypsin, proteinase K, saponin or streptridine O treatment, and then the primary antibody recognizing the antigen and the target cell sample are mixed, and then on ice for 30 minutes to 1 hour. Incubate. When the primary antibody is made into the aforementioned liposome or modified with a peptide or sugar chain, the primary antibody that directly recognizes the antigen and the target cell sample can be mixed. When the primary antibody is labeled with fluorescence, separation is performed with a flow cytometer after washing. When the primary antibody is not fluorescently labeled, after washing, the fluorescently labeled secondary antibody having binding activity to the primary antibody and the cells reacted with the primary antibody are mixed, and again on ice for 30 minutes to 1 hour Incubate. After washing, the cells stained with the primary antibody and the secondary antibody are separated with a flow cytometer. When the primary antibody and the secondary antibody are not fluorescently labeled and the secondary antibody is labeled with biotin, the fluorescently labeled streptavidin that has binding activity to the secondary antibody after washing, the primary antibody and the secondary antibody The cells reacted with the next antibody are mixed and incubated again on ice for 30 minutes to 1 hour. After washing, primary cells, secondary antibodies, and cells stained with fluorescently labeled streptavidin are separated with a flow cytometer.

  Hoechst 33342 is a DNA-binding dye that can stain living cells. Since the majority of bone marrow cells are actively dividing, they stain very brightly, but immature cells stain darker. It is known that immature cells have a higher dye-exclusion ability by ABC (ATP binding cassette) transporter (Nakauchi Hiromitsu, Protein Nucleic Acid Enzyme, Vol.45, No.13, pp.2056-2062, 2000, Goodell , MA et al. J Exp Med, 183, 1797-1806 (1996), http://www.bcm.tmc.edu/genetherapy/goodell/new_site/index2.html). Therefore, cancer stem cells can be separated with higher purity by separating cells stained with an anti-nucleostemin antibody and not taking up Hoechst 33342 with a flow cytometer.

  Methods for labeling nucleostemin of cancer stem cells include, for example, fluorescently labeled anti-nucleostemin antibodies (FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome , Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, etc.], or anti-nucleostemin antibody and fluorescent labeled secondary antibody [FITC (fluorescein isothiocyanate ), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red Labeled with fluorescent protein, etc.] or protein-labeled anti-nucleostemin antibody (β-galactosidase, alkaline phosphatase, chloramphenicol acetyltran Labeled with proteins such as ferrase, growth hormone, luciferase, β-lactamase and derivatives thereof) and fluorescently labeled secondary antibodies (FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed) , Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein], or biotin-labeled anti-nucleostemin antibody and fluorescent labeled streptavidin [ FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Red670, PerCP, TRI-Color, QuantumRed, green fluorescent protein, blue fluorescent protein, yellow Fluorescent protein, red fluorescent protein] can be used.

  Further, fluorescently labeled anti-nucleostemin antibody or fluorescently labeled nucleostemin specific aptamer (FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allo-phycocyanin), TR (TexasRed), Cy3, Cy5, CyChrome, Red613, Labeled with Red670, PerCP, TRI-Color, QuantumRed, Green Fluorescent Protein, Blue Fluorescent Protein, Yellow Fluorescent Protein, Red Fluorescent Protein, etc.], or peptides that promote cell adhesion and cellular uptake such as fibronectin, For example, peptides such as HIV-tat, D-tat, Rg-tat, or Antennapedia and partial peptides thereof, membrane-permeable peptides such as 11R (11-mer Arg), Octa (8-mer Arg), ODD, SynB1, Synthetic peptides such as SynBX, XMTM, DPV, and Chariot, or sugar chains can be used for labeling of nucleostemin after modification.

The method using magnetic beads can separate a large amount of cells expressing the target surface antigen. For example, it is performed as follows.
After the primary antibody is reacted with the cells, the primary antibody that has not reacted with the cells is removed, and the magnetic beads to which the secondary antibody that specifically binds to the primary antibody is added are bound. After washing away the remaining secondary antibody, the cells specifically bound to the magnetic bead added antibody can be separated by a stand provided with a magnet. The materials and equipment necessary for these operations are available from DYNAL.

The method using magnetic beads can be used similarly when removing unnecessary cells from a cell sample. The StemSep method sold by Stem Cell Technologies Inc (Vancouver, Canada) can be used to remove unnecessary cells more efficiently.
Cancer stem cells can be further enriched by appropriately combining a separation method using an anti-nucleostamine antibody and antibodies against other cell markers.

  Examples of antibodies against other cell markers of cancer stem cells include antibodies that recognize blood cancer stem cell surface antigens CD13, CD34, CD38, CD33, CD44, CD117, CD123, CD133, CD138, Flt-3, Sca-1 CD44, CD24, CD19, CD133, ESA (Epithelial-Specific Antigen), antibodies that recognize integrin α2β1, surface antigens of hematopoietic cells, CD34, CD117, CD14, CD45, CD90, solid cancer stem cell surface antigens Antibodies that recognize CD150, Sca-1, Ly6c, Ly6g, antibodies that recognize the surface antigens of vascular endothelial cells Flk-1, Flt-1, CD31, CD105, CD144, CD44 that is a surface antigen of mesenchymal cells And antibodies that recognize CD140, antibodies that recognize the integrin surface antigens CD49b, CD49d, CD29, and CD41, and those that recognize the matrix receptors CD54, CD102, CD106, and CD44. By combining these antibodies and techniques, cancer stem cells can be concentrated with higher purity.

2. Identification or isolation of cancer stem cells using nucleostemin promoter Identification or isolation of cancer stem cells using the nucleostemin promoter of the present invention can be performed by, for example, the following 2 .- (1) to 2 .- (6 ).

2 .- (1) Nucleostamine promoter A promoter includes an expression regulatory region capable of associating with RNA polymerase in a cell, and initiates transcription of a coding sequence existing downstream (toward the 3 ′ end) of the region. It refers to a region on DNA including a region having a function to be caused. In the present invention, functionally binding a promoter to a gene refers to a state in which the promoter is linked so as to be able to induce transcription of the gene connected downstream.

  The nucleostamine promoter used for identification or isolation of cancer stem cells of the present invention is not particularly limited as long as it has promoter activity in cancer stem cells. For example, human, mouse, rat, cow, horse, pig, dog Nucleostamine promoters such as cat, goat, sheep or chicken can be used. Specifically, DNA consisting of the base sequence represented by SEQ ID NO: 1, 2 or 3 can be mentioned.

The nucleotide sequence represented by SEQ ID NO: 1 is a sequence of a region consisting of about 7 kb from the 6th g downstream of the translation start point (1st atg) of the mouse nucleostemin gene to the 7078th g upstream of the translation start point. It is.
The nucleotide sequence represented by SEQ ID NO: 2 is a sequence of a region consisting of about 10 kb from the 6th g downstream of the translation start point (1st atg) of the mouse nucleostemin gene to the 10058th g upstream of the translation start point. It is.

The nucleotide sequence represented by SEQ ID NO: 3 is a sequence of a region consisting of about 10 kb from the third a downstream from the translation start point (1st atg) of the human nucleostemin gene to the 9998th a upstream from the translation start point. is there.
The sequence of the mouse nucleostemin gene includes Genbank Accession Number NM_153547, NM_178846, AI785289, AK034825, AK077523, AK087757, AK167094, AK167183, AY181025, AY185498, BC018560, BC037996, BF021177, BG065812, BG079080, 0000 Genbank Accession Numbers NM_014366, NM_206825, NM_206826, AF191018, AI028402, AK027514, AK027516, AY825265, BC001024, BF732788, CF272475, CR598596, CR607914, CR608648, AC104446, NT_005986 can be used as the sequence of the human nucleostemin gene.

  The method for measuring the promoter activity of the DNA used in the present invention may be any measuring method as long as the promoter activity of the DNA can be measured. For example, it can be measured by a reporter gene assay system. Specifically, it can be performed by the following steps (1) to (3).

(1) Downstream of the subject promoter, for example, luciferase gene, alkaline phosphatase gene, chloramphenicol resistance gene, β-galactosidase gene, green fluorescent protein gene, blue fluorescent protein gene, yellow fluorescent protein gene, red fluorescent protein Genes for activity measurement comprising a sequence in which reporter genes such as genes, β-lactamase genes and derivatives thereof are operably linked are prepared.
(2) A cancer stem cell and a control cell are transformed with the activity measuring plasmid of (1). Here, the control cell refers to a differentiated cancer cell derived from a cancer stem cell (having lost its properties as a cancer stem cell) or a normal cell of a tissue from which the cancer stem cell is derived.
(3) The presence or absence of reporter gene expression in the cells obtained in (2) above is detected, and the expression intensity is measured.

The nucleostemin promoter DNA used in the present invention has one or more bases deleted, substituted or added (hereinafter sometimes referred to simply as “mutation”) in the base sequence represented by SEQ ID NO: 1, 2, or 3. And DNA having a promoter sequence specific to cancer stem cells.
The nucleostemin promoter DNA is obtained by Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons ( 1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985) etc. It can be obtained by introducing a site-specific mutation into DNA consisting of the base sequence represented by SEQ ID NO: 1, 2, or 3.

The number and position of mutation may be any as long as the mutated DNA has cancer stem cell-specific promoter activity. Examples of the method for measuring cancer stem cell-specific promoter activity include the reporter assay system described above.
One or more bases are deleted, substituted or added in the DNA of the present invention when one or more bases are deleted, substituted or added at any position in one or more base sequences in the same sequence. It means that deletions, substitutions or additions may occur simultaneously. Furthermore, the downstream side from the translation start point or transcription start point on the 3 ′ side of the base sequence represented by SEQ ID NO: 1, 2, or 3 may be deleted.

The number of bases to be deleted, substituted or added is not particularly limited, but is such a number that can be deleted, substituted or added by a known method such as the above site-directed mutagenesis, and is 1 to 1000, preferably 1 The number is ˜500, more preferably 1 to 100, particularly preferably 1 to 50, including 1, 5, 10, 20, 30, 40 and 50.
In addition, in order for the DNA of the present invention to have cancer stem cell-specific promoter activity, at least 60% or more, preferably 80% or more, more preferably, the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 It is preferable to have a homology of 90% or more, more preferably 95% or more.

  Nucleotide sequence homology is determined using the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul. be able to. Based on this algorithm BLAST, a program called BLASTN has been developed [J. Mol. Biol., 215, 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

The DNA of the present invention hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 and has a cancer stem cell-specific promoter activity. Includes DNA.
DNA that can hybridize under stringent conditions refers to DNA having the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 as a probe using colony hybridization, plaque hybridization, or Southern blot hybridization. It means DNA obtained by using a hybridization method or the like. Specifically, using a filter on which colony or plaque-derived DNA is immobilized, in the presence of 0.7 to 1.0 mol / l sodium chloride, 65 After hybridization at 0 ° C., a 0.1 to 2-fold concentration SSC solution (composition of a 1-fold concentration SSC solution consists of 150 mmol / l sodium chloride and 15 mmol / l sodium citrate) was used. DNA that can be identified by washing the filter under conditions can be mentioned. Hybridization is performed according to the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be done according to this. Specifically, the DNA capable of hybridizing is DNA having at least 60% or more homology with the base sequence represented by SEQ ID NO: 1, 2, or 3 when calculated using the above-mentioned BLAST or the like, preferably May be DNA having 80% or more homology, more preferably DNA having 90% or more homology, and more preferably DNA having 95% or more homology.

2 .- (2) Vector containing nucleostemin promoter The vector used in the present invention is the nucleotide sequence represented by the above-mentioned nucleostemin promoter DNA, for example, SEQ ID NO: 1, 2 or 3, upstream of the foreign gene sequence. There is no particular limitation as long as a foreign gene can be expressed specifically in cancer stem cells.

  Examples of the foreign gene include β-galactosidase gene, alkaline phosphatase gene, chloramphenicol acetyltransferase gene, growth hormone gene, luciferase gene, green fluorescent protein gene, blue fluorescent protein gene, yellow fluorescent protein gene, red fluorescent protein gene, Examples thereof include reporter genes such as β-lactamase genes and derivatives thereof. The derivatives include artificially produced mutants.

  As a vector for introducing a gene having the nucleostemin promoter functionally linked to a reporter gene into an animal cell or a fertilized egg of a non-human mammal, a vector that can replicate autonomously in a host cell or can be integrated into a chromosome, or one Those capable of transient expression are used. Any vector such as a plasmid vector or a viral vector can be used. Alternatively, the vector portion can be cleaved and removed using an appropriate restriction enzyme, and the region containing the promoter and reporter gene can be introduced into cells or fertilized eggs.

  When animal cells are used as host cells, examples of expression vectors include plasmids, phages, cosmids, and the like. Examples thereof include pCDNAI (manufactured by Invitrogen), pCDM8 (manufactured by Invitrogen), pAGE107 [JP-A-3-22979; Cytotechnology; , 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pCDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [ J. Biochem., 101, 1307 (1987)], pAGE210, pNASS β, pGL3, pCAT3, pβgal, pEGFP-1, pEGFP-N1, pEYFP-1, pSEAP2, pGeneBLAzer-TOPO and the like.

  As a method for introducing a recombinant vector into animal cells, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (JP-A-2-27075), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], Virology, 52, 456 (1973), and the like.

It can also be inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. The recombinant viral vector plasmid is introduced into packaging cells compatible with the viral vector plasmid.
As the packaging cell, any cell can be used as long as it can replenish the deficient protein of the recombinant viral vector plasmid deficient in at least one gene encoding a protein necessary for virus packaging. it can. For example, HEK293 cells derived from human kidney, mouse fibroblast NIH3T3, and the like can be used.

  Proteins to be replenished in packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retroviral vectors, and HIV virus-derived gag, pol, env, vpr, vpu, for lentiviral vectors. Proteins such as vif, tat, rev, nef, adenovirus vectors such as E1A and E1B, adeno-associated viruses such as Rep (p5, p19, p40), and Vp (Cap) Can be used.

Viral vector plasmids include MFG [Proc. Natl. Acad. Sci. USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)], LL-CG, CL-CG CS-CG, CLG [Journal of Virology, 72, 8150-8157 (1998)], pAdex1 [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like.
A recombinant viral vector can be produced by introducing the recombinant viral vector plasmid into the packaging cell.

  Examples of the method for introducing the viral vector plasmid into the packaging cell include the calcium phosphate method [JP-A-2-27075] and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. I can give you.

2 .- (3) Identification or isolation of cancer stem cells from cells introduced with a vector containing a nucleostemin promoter The above-mentioned cancer cells introduced with a vector having a reporter gene linked downstream of the nucleostemin promoter are derived from animals. Any cancer cell can be used. Specifically, the tumor-derived cancer cells or cancer cell lines described in 1. above can be mentioned. As animals, humans, mice, rats, cows, horses, pigs, dogs, cats, goats, sheep, or chickens, and as cancer cells, cancer cells derived from blood cancer, solid cancer, cultured cancer cell lines, etc. Can do.

In addition, after introducing a vector in which a reporter gene is bound downstream of a nucleostemin promoter into normal cells, as described in 2- (4) below, oncogene introduction, chemical carcinogen treatment, radiation Cells that have become cancerous by irradiation of can also be used.
The identification or isolation of cancer stem cells is preferably performed using a detection method according to the characteristics of each reporter gene, and the expression level of the reporter gene in the cell is determined by a known method (edited by the Department of Cancer Control, University of Tokyo). New Cell Engineering Protocol, Shujunsha (1993), Biotechniques, 20, 914 (1996), J. Antibiotics, 49, 453 (1996), Trends in Biochemical Sciences, 20, 448 (1995), Cell Engineering, 16, 581 (1997), etc.]. Specific to the reporter gene according to the method described in 1 .- (8) above, a method using a cell sorter, a flow cytometer, and magnetic beads according to the method described in 1 .- (11) above. In situ hybridization using a simple probe, an immunohistochemical detection method using an antibody specific to the reporter protein according to the method described in 1 .- (9-1) above, or the above 1 .- ( The fluorescently labeled aptamer showing specific binding to the reporter protein according to the method described in 9-2), or the enzyme activity of the reporter protein according to the method described in 2 .- (1) above. For example, a method for measuring a fluorescent substance to be generated can be used.

  Whether the cell identified or isolated by the method of the present invention has the property of a cancer stem cell has the ability to generate various cancer cells constituting the tumor from which the cell is derived, the ability to replicate itself, and the ability to form tumors. It can be checked by confirming. Compared with differentiated normal cancer cells, when transplanted into non-human immunodeficient animals, preferably immunodeficient mice, such as NOD / SCID mice, NOG mice, etc., for example, 1/2 or less, preferably It can be examined by confirming that the recipient mouse has the ability to form a tumor with a cell number of 1/5 or less, more preferably 1/10 or less.

2 .- (4) Identification or isolation of cancer stem cells from transgenic non-human mammal carcinogenic animals into which a vector containing a nucleostemin promoter has been introduced Non-human mammals as recipient animals such as mice, rats, rabbits, pigs Mammals such as dogs, sheep and goats. In particular, rodents such as mice and rats are preferred.

Production of the transgenic non-human animal used in the present invention is carried out, for example, according to a mouse embryo operation manual (Modern Publishing, 1989), a molecular biology protocol (Nanedo, 1994), Gene Targeting (Yodosha, 1995), etc. Can do.
Specifically, first, a superovulated female and male are mated. The oviduct is removed from the female that has passed about 12 hours after mating, and a fertilized egg at the 1-cell stage (pronuclear stage) is collected and placed in an appropriate culture medium. Next, the vector of the present invention is microinjected into the pronucleus of a fertilized egg. On the other hand, a male that has undergone vasectomy is mated with a female that has reached sexual maturity to produce a pseudopregnant female. After microinjection, the surviving fertilized egg is transplanted into the oviduct of the pseudopregnant female. Thereafter, the fetus generated from the fertilized egg transplanted into the fallopian tube is removed by natural delivery or incision. Genomic DNA is prepared from a part of the tail of the resulting pup. The obtained DNA is analyzed to determine whether the resulting pup is a transgenic animal. Transgenic animals are crossed to establish a line.

In the transgenic non-human mammal used in the present invention, the vector may be integrated into the chromosome of the mammal.
For example, a tumor can be formed in a transgenic non-human mammal by the following method, and cancer stem cells can be identified, enriched, and separated using reporter gene expression as an indicator.
Methods for forming tumors in non-human mammals include oncogene introduction, radiation (eg, but not limited to, heavy particle radiation, X-rays, gamma rays, ultraviolet rays, microwaves, etc.), and chemical carcinogens (eg, limited thereto). N-ethyl-N-nitrosourea, N-Methyl-N-nitrosourea, 3,4-benzopyrene, 3-methylcholanthrene, 2-acetylaminofluorene, 7,12-dimethylbenz [a] anthracene, N-nitroso-N-buthylurea , 7,8,12-trimethylbenz (a) anthracene, etc.) can be used.

  Examples of oncogenes include, but are not limited to, Meis1, HoxA9, HoxD13, HoxB3, HoxB8, HoxA10, MLL-ENZ, MLL-AF9, AML-ETO, MOZ-TIF2, CDX2, Bcr-Abl , BCL6, maf-B, FGFR, c-maf, SCL, Hox11, LMO2, LMO1, E2a-Pbx1, TEL-Abl, myc, etc. for solid tumors, ENS-WT1, k-ras, Bcrp1, ras, Examples include src, jun, met, fos, and ret. Any oncogene can be used as long as it forms a tumor in the target organ or tissue. Moreover, each can also be used in combination.

  As a vector used for introducing an oncogene into a non-human mammal, any vector can be used as long as it can express the gene. -As described in (2), any vector such as plasmid, phage, cosmid, virus can be used. Alternatively, the vector portion can be cleaved and removed using an appropriate restriction enzyme or the like, and a fragment containing a region necessary for expression of the gene can be prepared and used. In addition, as a method for introducing the gene into a recipient, a known method can be used as long as the gene can be expressed. Although the case where a viral vector is used is demonstrated below, it is not limited to this.

  As a method of constructing a recombinant viral vector plasmid by inserting an oncogene gene DNA fragment or full-length cDNA downstream of the promoter in the viral vector plasmid, the target genetic DNA is located downstream of the promoter in the viral vector plasmid. Although it will not specifically limit if it can insert, For example, a recombinant viral vector plasmid can be constructed | assembled based on the method described in Nature, 423, 255-260 (2003).

  The recombinant viral vector plasmid is introduced into packaging cells compatible with the viral vector plasmid. As the packaging cell, any cell can be used as long as it can replenish the deficient protein of the recombinant viral vector plasmid deficient in at least one gene encoding a protein necessary for virus packaging. it can. For example, HEK293 cells derived from human kidney, mouse fibroblast NIH3T3, and the like can be used.

  Proteins to be replenished in packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retroviral vectors, and HIV virus-derived gag, pol, env, vpr, vpu, for lentiviral vectors. Proteins such as vif, tat, rev, nef, adenovirus vectors such as E1A and E1B, adeno-associated viruses such as Rep (p5, p19, p40), and Vp (Cap) Can be used.

As the viral vector plasmid, those capable of producing a recombinant virus in the packaging cell are used.
Viral vector plasmids include MFG [Proc. Natl. Acad. Sci. USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Research, 18, 3587-3596 (1990)], LL-CG, CL-CG CS-CG, CLG [Journal of Virology, 72, 8150-8157 (1998)], pAdex1 [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like.

  Any promoter can be used as long as it can express the protein in animal tissues. For example, cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus Promoters, metallothionein promoters, heat shock protein promoters, SRα promoters, and the like. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

  A recombinant viral vector can be produced by introducing the recombinant viral vector plasmid into the packaging cell. Examples of the method for introducing the viral vector plasmid into the packaging cell include the calcium phosphate method [JP-A-2-27075] and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)]. I can give you.

By administering the viral vector to a transgenic non-human mammal containing the vector of the present invention, a tumor can be formed in the animal.
Tumors can also be formed in the animals by non-viral gene transfer methods. Non-viral gene transfer methods include calcium phosphate coprecipitation (Virology, 52, 456 (1973); Science, 209, 1414 (1980)), microinjection (Proc. Natl. Acad. Sci. USA, 77, 5399 ( Proc. Natl. Acad. Sci. USA, 77, 7380 (1980); Cell, 27, 223 (1981); Nature, 294, 92 (1981)], liposome-mediated membrane fusion-mediated transfer method [ Proc. Natl. Acad. Sci. USA, 84, 7413 (1987); Biochemistry, 28, 9508 (1989); J. Biol. Chem., 264, 12126 (1989); Hum. Gene Ther., 3, 267 ( 1992); Science, 249, 1285 (1990); Circulation, 83, 2007 (1992)] or direct DNA uptake and receptor-mediated DNA transfer [Science, 247, 1465 (1990); J. Biol. Chem., 266, 14338 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655 (1991); J. Biol. Chem., 264, 16985 (1989); BioTechniques, 11, 474 (1991); Proc. Natl Acad. Sci. USA, 87, 3410-3414 (1990); Proc. Natl. Acad. Sci. USA, 88, 4255 (1991); Proc. Natl. Acad. Sci. USA, 87, 4033 (1990) .... Proc Natl Acad Sci USA, 88, 8850 (1991);.. Hum Gene Ther, 3, 147 (1991)] and the like.

Identification or separation of cancer stem cells from cancer cells or cancer tissues can be performed by the methods described in 2 .- (5) and 2 .- (6) below.
Further, after separating cells derived from the transgenic non-human mammal, irradiation with radiation (for example, heavy particle beam, X-ray, gamma ray, ultraviolet ray, microwave, etc.), chemical carcinogen (for example, N-ethyl-N-nitrosourea) , N-Methyl-N-nitrosourea, 3,4-benzopyrene, 3-methylcholanthrene, 2-acetylaminofluorene, 7,12-dimethylbenz [a] anthracene, N-nitroso-N-buthylurea, 7,8,12-trimethylbenz (a ) anthracene) treatment or oncogene is introduced and the resulting cells are introduced into the recipient non-human mammal. In addition, the cells can be cultured and used.

  Examples of oncogenes include, but are not limited to, Meis1, HoxA9, HoxD13, HoxB3, HoxB8, HoxA10, MLL-ENZ, MLL-AF9, AML-ETO, MOZ-TIF2, CDX2, Bcr-Abl , BCL6, maf-B, FGFR, c-maf, SCL, Hox11, LMO2, LMO1, E2a-Pbx1, TEL-Abl, myc, etc. for solid tumors, ENS-WT1, k-ras, Bcrp1, ras, Examples include src, jun, met, fos, and ret. Any oncogene can be used as long as it has the ability to cancerize cells derived from a target organ or tissue. Moreover, each can also be used in combination.

  As the cells derived from the transgenic non-human mammal, cells derived from any tissue can be used. For example, bone marrow cells, peripheral blood cells, hematopoietic stem cells, cells derived from various organs, and the like can be used. Non-human mammals used as recipients include mammals other than humans, for example, mammals such as mice, rats, rabbits, pigs, dogs, sheep, and goats. Rodents are preferred. Furthermore, immunodeficient animals such as NOD / SCID mice and NOG mice are preferable, and it is more preferable to use them after radiation treatment.

2 .- (5) Identification or isolation of cancer stem cells from blood cancer of transgenic non-human mammalian carcinogenic animals As a method for obtaining bone marrow cells having cancer stem cells from transgenic non-human mammalian carcinogenic animals, for example, the following procedure is used. Can be acquired.

  A transgenic non-human mammal carcinogenic animal is killed by cervical dislocation and thoroughly disinfected with 70% ethanol, and then the femur skin and quadriceps are excised. Remove scissors at the knee joint, remove the joint, and remove the muscles behind the femur. Insert the scissors into the hip joint, remove the joint, and remove the femur. After removing the muscles attached to the femur as much as possible with scissors, both ends of the femur are cut with scissors. Attach a needle of appropriate size according to the bone thickness to a 2.5 mL syringe, and add about 1.5 mL of cell culture medium such as α-MEM, DMEM, or IMDM containing 10% FBS (fetal calf serum). After filling the syringe, the tip of the injection needle is inserted into the stump on the knee joint side of the femur. Bone marrow cells are pushed out from the stump on the hip joint side by injecting the culture solution in the syringe into the bone marrow. The obtained bone marrow cells are suspended in the culture medium by pipetting. From the bone marrow fluid, a bone marrow cell mixture having cancer stem cells can be prepared by the same method as the preparation of bone marrow cells from human bone marrow fluid described in 1 .- (1) above.

  Identification or separation of cancer stem cells from the bone marrow cell mixture is performed as described in 2. above. -It can be carried out by the method described in (3).

2 .- (6) Identification or isolation of cancer stem cells from solid tumors of transgenic non-human mammalian carcinogenic animals The method for obtaining cancer cells having cancer stem cells from transgenic non-human mammalian carcinogenic animals is not particularly limited. It can be obtained by the procedure.

  The transgenic non-human mammal carcinogenic animal is killed by cervical dislocation and sufficiently disinfected with 70% ethanol, and then the tumor forming part is opened and the tumor site is removed. The obtained cancer cell is not particularly limited as long as it is a proteolytic enzyme. For example, it is suspended in the culture solution with trypsin, collagenase, or hyaluronidase. From the cancer cell solution, a cancer cell mixture having cancer stem cells can be prepared by the same method as the preparation of cancer cells from the human cancer cell solution described in 1 .- (3) above.

Identification or separation of cancer stem cells from the cancer cell mixture can be performed by the method described in 2 .- (3) above.
Hereinafter, the present invention will be described more specifically with reference to examples.

Identification and isolation of cancer stem cells using nucleostemin (1)
A retroviral vector expressing the leukemia oncogene HoxA9 / Meis1 was prepared according to literature methods [Nature, 423, 255-260 (2003)]. On the other hand, the FACS sorter (BD) was used for blood cells obtained from the bone marrow of the transgenic mouse containing the upstream sequence of the nucleostemin gene and the EGFP (enhanced green fluorescent protein) gene prepared in Reference Example using a known method. FACS Aria, BD Biosystems) was used to isolate a lineage negative cell population (Lin-) using cell surface antigen expression as an index, and c-kit positive Sca-1 positive Cell population (KSL) was sorted. As antibodies against cell surface antigens, anti-c-kit antibodies (BD Parmingen, 2B8), anti-Sca-1 antibodies (BD Parmingen, D7), anti-CD4 antibodies (BD Parmingen, L3T4), Anti-CD8 antibody (BD Parmingen, 53-6.72), anti-B220 antibody (BD Parmingen, RA3-6B2), anti-TER-119 antibody (BD Parmingen), anti-Gr-1 antibody (BD Parmingen) RB6-8C5) and anti-Mac-1 antibody (BD Parmingen, M1 / 70) were used. CD4 and CD8 are T cell markers, B220 is a B cell marker, TER-119 is a red blood cell marker, Gr-1 is a granulocyte marker, and Mac-1 is a macrophage marker. Positive cells were defined as lineage positive cells, and all negative cells were defined as lineage negative cells.

Using a known method [Nature, 431, 997-1002 (2004)], the KSL cell is infected with a retroviral vector that expresses HoxA9 / Meis1, and a KSL cell that expresses HoxA9 / Meis1 is prepared. Mice were transplanted. In other words, KSL cells expressing HoxA9 / Meis1 from the tail vein after irradiating a lethal dose of X-rays (neurite 9.5 Gy, 0.5 Gy / min) using C57BL / 6 mice (Ly5.1) as recipient mice Injected. At that time, 4 × 10 ⁵ C57BL / 6 mouse-derived bone marrow mononuclear cells were simultaneously injected. Recipient mice developed acute myeloid leukemia approximately 50 days after transplantation and died. Bone marrow cells were collected after onset and the expression of EGFP was examined. About 90% of the cells were EGFP positive (FIG. 1). The bone marrow cells were divided into 4 according to the expression of EGFP, and the 1st, 2nd, 3rd and 4th were assigned from the weakest expression (FIG. 1). The expression of nucleostemin messenger RNA in these fractions correlated with the expression of EGFP. Examination of cell surface markers of leukemia in 4 fractions revealed that the higher the expression of EGFP, the greater the population of c-kit positive cells.

  Therefore, when EGFP expression and c-kit expression were examined simultaneously, c-kit-EGFP-/ + (No. 1), c-kit-EGFP ++ (No. 2), c-kit + EGFP ++ (No. 3), It was divided into 4 fractions of c-kit + EGFP +++ (No. 4) (FIG. 2A). Morphologically No. 1 is a cell with a strong tendency to differentiate into small neutrophils, while No. 2 and No. 3 show a slightly undifferentiated myeloblast morphology, whereas No. 4 with strong EGFP is It was a large undifferentiated cell population.

  Each cell population was added with SCF (stem cell factor), TPO (thrombopoietin), IL-6, IL-3 and G-CSF and cultured by the methylcellulose method. It was high (FIG. 4B). In addition, colonies derived from EGFP strong positive population (c-kit + EGFP +++) are composed of EGFP positive undifferentiated cells and negative differentiated cell populations, whereas EGFP positive positive cell population (c-kit From + EGFP +), only differentiated cells weakly positive for EGFP were observed. Therefore, it was suggested that the leukemia cells were induced to be differentiated and produced from the EGFP strong positive population, and it was shown that the EGFP strong positive cells are a population of leukemia sources, that is, cancer stem cells.

  Furthermore, c-kit-EGFP-/ + (No.1), c-kit-EGFP ++ (No.2), c-kit + EGFP ++ (No.3), c-kit + EGFP +++ (No.4) 10 When transplanted to irradiated C57BL / 6 mice together with × 105 C57BL / 6 mouse-derived bone marrow mononuclear cells, only c-kit + EGFP +++ developed leukemia (FIG. 3). Since this EGFP strong positive leukemia population produces other fractions of cancer cells in the body, the high nucleostemin expressing cell population is a cancer stem cell in the acute myeloid leukemia model by HoxA9 / Meis1 It was shown that.

Identification and isolation of cancer stem cells using nucleostemin (2)
A retroviral vector expressing leukemia oncogene Bcr-abl was prepared according to literature methods [J. Exp. Med., 189, 1399-1412 (1999)]. On the other hand, the FACS sorter (BD FACS Aria, BD Biosystems) was used for the blood cell line obtained from the bone marrow of a transgenic mouse containing the upstream sequence of the nucleostemin gene and the EGFP gene prepared in Reference Example. The KSL cell population was prepared in the same manner as in Example 1. The KSL cell population was infected with a retroviral vector expressing Bcr-abl using a known method, and KSL cells expressing Bcr-abl were prepared and transplanted into recipient mice. That is, KSL cells expressing Bcr-abl from the tail vein after irradiation with a lethal dose of X-rays (neurite 9.5 Gy, 0.5 Gy / min) using C57BL / 6 mice (Ly5.1) as recipient mice Injected. At that time, 4 × 10 ⁵ C57BL / 6 mouse-derived bone marrow mononuclear cells were simultaneously injected.

  Recipient mice developed leukemia and died in about 20 days. The leukemia cells in the bone marrow of this mouse were divided into No. 1, No. 2, No. 3, and No. 4 fractions based on the expression levels of EGFP and c-kit (FIG. 4A). When cells # 1, # 2, # 3 and # 4 were cultured in methylcellulose with cytokines (SCF, TPO, IL-6, IL-3 and G-CSF), the EGFP strong positive cell population was the most Colonies formed with high frequency (FIG. 4B). From the above, it was shown that also in the chronic myelogenous leukemia model by Bcr-abl, the nucleostamine highly expressing cell population is a cancer stem cell.

Identification and isolation of cancer stem cells using nucleostemin (3)
p53 − / − Ptch1 +/− mice were prepared based on the literature method [Cancer Res. 2001 Jan 15; 61 (2): 513-6]. It was confirmed that medulloblastoma derived from the cerebellum occurred after 3 months. This mouse was bred with a transgenic mouse containing the upstream sequence of the nucleostemin gene and the EGFP gene prepared in Reference Example. Using medulloblastoma cells, the expression level of EGFP was used as an index, and EGFP strong positive, positive and weak positive cells were collected. The sorted cells were injected into the basal ganglia of nude mice (purchased from SLC) at 1.5 × 10 ⁵ cells per mouse. Two months later, when the brain of the recipient mouse was observed, EGFP-positive cancer cells were observed in the mouse injected with EGFP strong-positive cells. On the other hand, in mice injected with EGFP positive and weak positive cells, tumor development could not be visually observed. Based on the above findings, it was shown that cancer stem cells of brain tumors can be identified.

(Reference Example 1)
Cloning of transcriptional regulatory sequence of nucleostemin gene Cloning of the upstream sequence of the mouse nucleostemin gene First, the genomic sequence containing the mouse nucleostemin gene was cloned.
A clone (RP23-84O17, Invitrogen) containing a mouse nucleostemin gene (Genbank Accession No. BC037996) was selected from the BAC clone into which the mouse genome was inserted (BACPAC Resources Center) and subjected to the following Southern analysis.
The probe for Southern analysis was specific for the mouse nucleostemin gene represented by SEQ ID NOs: 12 and 13, using genomic DNA purified from the tail of 6-week-old male C57Bl / 6 mice (CLEA Japan) as a template. Amplified with primers and DIG-labeled with PCR DIG Probe Synthesis Kit (Roche) was used.

A nylon membrane (Hybond N +, Amersham) transcribed with BAC clone RP23-84O17 treated with restriction enzymes (KpnI, BamHI, EcoRI or HindIII, TaKaRa) was probed with the DIG-labeled mouse nucleostemin gene. Hybridization was performed using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche).
As a result, it was confirmed that RP23-84O17 contained a nucleostemin gene sequence having a length predicted from the mouse genome sequence (Genbank Accession No. NW — 000091).

  Next, a library was prepared by cloning the fragment obtained by enzymatic treatment of RP23-84O17 with KpnI into pBlueScriptIISK (-) (Stratagene), and the above probe and DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Was used to confirm a subclone of the KpnI fragment (12.2 kb) containing the upstream sequence of the nucleostemin gene. As a result of sequence analysis, the cloned fragment contained a sequence containing 10 kb upstream from the translation start point (1st ATG) of the nucleostemin gene.

2. Construction of reporter vector containing upstream sequence of mouse nucleostemin gene Enhanced green fluorescent protein (EGFP) gene reporter vector (pEGFP-N1, BD Biosciences Clontech) was cleaved with AseI and BglII and converted to a blunt end Thereafter, a reporter vector (pPLE) in which the promoter and enhancer regions were excised was constructed by rebinding. A 10 kb sequence from the restriction enzyme site KpnI of the above KpnI fragment (12.2 kb) to StuI present downstream of the translation start point (ATG) is inserted from the restriction enzyme site KpnI of pPLE into BamHI (smoothed), An EGFP reporter vector pPLEmNSgKSB10 (FIG. 7) containing a genomic sequence 10 kb upstream from the translation start position of the stemin gene was constructed. The genomic sequence 10 kb upstream from the translation start position of the nucleostemin gene contained in pPLEmNSgKSB10 is shown in SEQ ID NO: 2.

3. Transcriptional regulatory ability of the 10 kb sequence upstream of the nucleostemin gene In order to analyze the transcriptional regulatory ability of the 10 kb genomic sequence upstream of the nucleostemin gene, the reporter activity of the constructed EGFP reporter vector pPLEmNSgKSB10 was examined.
PPLEmNSgKSB10 was introduced by electroporation (GenePulser, BioRad) into embryonic stem cells (ES cells) [Cell, 94, 339-352 (1998)] in which the expression of the nucleostemin gene was reported, and 300 μg / mL A pPLEmNSgKSB10 stably transfected ES cell line was established by culturing in the presence of G418 (Geneticin, Invitrogen).
As a result of observing the established cell line under a fluorescence microscope (ECLIPSE TE300, Nikon), EGFP is expressed in many cells, and the genomic sequence of 10 kb upstream of the nucleostemin gene shows transcriptional activity in ES cells. It became clear.

Furthermore, as a result of inducing differentiation by culturing the cell line in the absence of LIF (leukemia inhibitory factor), it was revealed that the reporter activity decreases.
Next, with respect to the cell line, a cell population having a high expression level of EGFP (EGFP ++) and a cell population having a low expression level (EGFP +) are separated using a FACS sorter (BD FACSVantage SE System, BD Biosystems). The expression level of was examined.

  That is, cDNA was synthesized from the sorted cells in the same manner as described above, and quantitative RT-PCR was performed using ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The sequence of the mouse nucleostemin gene specific primer is SEQ ID NO: 8 and 9, the sequence of the mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene specific primer is SEQ ID NO: 10 and 11, and the sequence of the EGFP gene specific primer Are shown in SEQ ID NOs: 14 and 15, respectively.

Moreover, what cloned the PCR product amplified by each PCR primer set to pT7Blue (TaKaRa) was prepared, the calibration expression was created from what diluted these serially, and the expression level of the gene in each sample was quantified.
The expression level of the nucleostemin gene in each sample was expressed as a relative amount with respect to the expression level of the GAPDH gene.

  The results are shown in FIG. It was revealed that the expression level of the EGFP reporter gene in the cell population is correlated with the expression of the endogenous nucleostemin gene. From this result, it was shown that the genomic sequence of about 10 kb upstream of the cloned nucleostemin gene contains the positive and negative transcriptional regulatory regions of the nucleostemin gene.

Reference example 2
Generation of EGFP reporter gene transgenic mice
The genomic sequence of 10 kb upstream of the nucleostemin gene contained in pPLEmNSgKSB10 was cleaved with KpnI and then with NdeI, and then deleted stepwise from the 5 ′ side using the Erase-a-Base System (Promega).
The plasmid was recircularized and then introduced into E. coli to obtain pPLE-N1 mNSgKSB10 AU7078. SEQ ID NO: 1 shows the base sequence upstream of the approximately 7 kb nucleostemin gene contained in pPLE-N1 mNSgKSB10 AU7078.
The plasmid NS promoter7-EGFP / pCMV-SPORT6 was constructed by ligating the SalI / NotI fragment containing the 7-kb nucleostemin gene upstream sequence and EGFP gene contained in pPLE-N1 mNSgKSB10 AU7078 to pCMV-SPORT6 (Invitrogen). did. Thereafter, the SV40polyA portion contained in pPLE-N1 mNSgKSB10 AU7078 was amplified by PCR. At that time, a NotI cleavage site was inserted into the 5 ′ primer (SEQ ID NO: 16), and a NotI-SalI cleavage site was inserted into the 3 ′ primer (SEQ ID NO: 17). After digesting the PCR product with NotI, it was inserted into the NotI site of NS promoter7-EGFP / pCMV-SPORT6 to prepare pNS Promoter7-EGFP (FIG. 7).

  At Wyeth Laboratories Co., Ltd., a fragment obtained by excising only the nucleostemin gene transcription regulatory sequence-EGFP-PolyA portion was purified using the restriction enzyme SalI, and injected into the pronuclei of fertilized eggs derived from C57BL / 6. Introduced fertilized eggs were transplanted into the oviduct of a foster parent according to a conventional method and developed into individuals. The resulting primary transgenic mouse (F0) was crossed with C57BL / 6 mice to establish a strain.

It is the figure which showed the expression level in the mouse | mouth bone marrow cell which developed acute myeloid leukemia of EGFP which is a reporter gene. The horizontal axis represents the fluorescence intensity of EGFP, and the vertical axis represents the count number. A is a figure which showed the expression level in the mouse | mouth bone marrow cell which developed acute myeloid leukemia of EGFP and c-kit which are reporter genes. The horizontal axis represents the fluorescence intensity of EGFP, and the vertical axis represents the fluorescence intensity of c-kit. FIG. 2B is a diagram showing colony-forming ability in methylcellulose of cell fractions classified into four categories (No. 1, No. 2, No. 3, No. 4) in FIG. 2A. The vertical axis represents the number of colonies per 1000 cells. It is the figure which transplanted each cell fraction classified into 4 classifications (1st, 2nd, 3rd, 4th) in FIG. 2A to the recipient mouse | mouth, and showed the mouse survival rate and survival time accompanying leukemia onset. The horizontal axis represents time (days), and the vertical axis represents survival rate (%). A is a graph showing the expression levels of EGFP and c-kit, which are reporter genes, in mouse bone marrow cells that have developed chronic myeloid leukemia. The horizontal axis represents the fluorescence intensity of EGFP, and the vertical axis represents the fluorescence intensity of c-kit. FIG. 4B is a diagram showing colony-forming ability in methylcellulose of cell fractions classified into four categories (No. 1, No. 2, No. 3, No. 4) in FIG. 4A. The vertical axis represents the number of colonies per 5000 cells. It is the figure which showed the restriction enzyme map of pPLEmNSgKSB10. ES cell lines stably transfected with pPLEmNSgKSB10 were separated into EGFP high expression cell population (EGFP ++) and EGFP low expression cell population (EGFP +) by FACS using reporter activity as an index, and endogenous NS genes (□ column) in each. And EGFP gene expression levels (■ column) were measured by RT-PCR and compared. The vertical axis represents the relative expression level of the gene. It is the figure which showed the restriction enzyme map of pNS Promoter7-EGFP.

SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 9—Description of artificial sequence: synthetic DNA
SEQ ID NO: 10—Description of artificial sequence: synthetic DNA
SEQ ID NO: 11—Description of artificial sequence: synthetic DNA
SEQ ID NO: 12—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 13—Description of artificial sequence: synthetic DNA
SEQ ID NO: 14—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 15—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 16—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of artificial sequence: synthetic DNA

Claims (23)

A cancer stem cell comprising preparing a cancer cell or cancer tissue containing a cancer stem cell, detecting a cell expressing nucleostemin from the cancer cell or cancer tissue, and identifying the cell expressing the nucleostemin as a cancer stem cell Identification method. Preparing cancer cells or cancer tissues containing cancer stem cells, detecting cells expressing nucleostemin from the cancer cells or cancer tissues, and separating cells expressing the nucleostemin from the cancer cells or cancer tissues A method for separating cancer stem cells. The method according to claim 1 or 2, wherein the nucleostemin is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5. The method according to claim 1 or 2, wherein the nucleostemin is a polypeptide consisting of an amino acid sequence encoded by the base sequence represented by SEQ ID NO: 6 or 7. The method according to claim 1, wherein the method for detecting a cell expressing nucleostemin is detecting a messenger RNA of the nucleostemin in the cell. The method according to claim 5, wherein the method for detecting nucleostemin messenger RNA is in situ hybridization. 5. The method according to claim 1, wherein the method for detecting a cell expressing nucleostemin detects the nucleostemin in the cell. The method according to claim 7, wherein the method for detecting a cell expressing nucleostemin uses an antibody specific to the nucleostemin. The method according to claim 8, wherein the antibody is a monoclonal antibody or a polyclonal antibody. The method according to any one of claims 7 to 9, wherein the method for detecting a cell expressing nucleostemin is an immunohistochemical method. The method according to claim 7, wherein the method for detecting cells expressing nucleostemin is a method using an aptamer. The method according to claim 7, wherein the method for detecting a cell expressing nucleostemin is a method using a cell sorter, a flow cytometer, or a magnetic bead. A method for detecting a cell that expresses nucleostemin introduces a DNA having a sequence in which a DNA containing a promoter of the nucleostemin gene is operably linked to a reporter gene, and detects the expression of the reporter gene The method according to claim 7, which is a method. The method according to claim 13, wherein the promoter of the nucleostemin gene is DNA consisting of the base sequence represented by SEQ ID NO: 1, 2, or 3. A DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 and having promoter activity in cancer stem cells. The method of claim 13. The promoter of a nucleostemin gene is a DNA comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3, and having promoter activity in cancer stem cells. the method of. DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 and having promoter activity in cancer stem cells The method according to claim 13. The method according to any one of claims 13 to 17, wherein the method for detecting the expression of a reporter gene is a method using a cell sorter, a flow cytometer, or magnetic beads. The method according to any one of claims 13 to 17, wherein the method for detecting the expression of a reporter gene is an immunohistochemical method using an antibody specific to the reporter protein or a method using Western blotting. The method according to any one of claims 13 to 17, wherein the method for detecting the expression of a reporter gene is a method for measuring messenger RNA of the reporter gene. The method according to any one of claims 1 to 20, wherein the cancer cell is a hematological cancer. The method according to any one of claims 1 to 20, wherein the cancer cells are derived from a solid cancer. The method according to any one of claims 1 to 20, wherein the cancer cell is a cultured cancer cell line.

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